Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Two potential applications of bacterial cellulose produced by A. xylinum Vallejo-Cordoba, Belinda 1984

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1984_A6_7 V34.pdf [ 16.97MB ]
Metadata
JSON: 831-1.0096177.json
JSON-LD: 831-1.0096177-ld.json
RDF/XML (Pretty): 831-1.0096177-rdf.xml
RDF/JSON: 831-1.0096177-rdf.json
Turtle: 831-1.0096177-turtle.txt
N-Triples: 831-1.0096177-rdf-ntriples.txt
Original Record: 831-1.0096177-source.json
Full Text
831-1.0096177-fulltext.txt
Citation
831-1.0096177.ris

Full Text

TWO POTENTIAL APPLICATIONS OF BACTERIAL CELLULOSE PRODUCED BY A. XYLINUH by BELINDAlvALLEOO CORDOBA B . S c , UniversidacT Iberoamericana, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (The Department of Food Science) We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1984 ©Belinda Va l le jo Cordoba, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f FOOD SCIENCE The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main M a l l V a n c o u v e r , C a n a d a V6T 1Y3 D a t e OCTOBER 15, 1984 ABSTRACT The foca l point of t h i s research project was the development of two app l i ca t ions of c e l l u l o s e produced from b a c t e r i a l c u l t u r e s of Acetobacter xyl inum. The f i r s t part of t h i s study re l a te s to the c u l t u r e method. Se l ec t ion of i n f l u e n t i a l factors for maximum c e l l u l o s e production i n defined medium was c a r r i e d out by using Taguchis ' L2y (3 ) F r a c t i o n a l F a c t o r i a l Design. Sucrose and peptone concentrat ions and pH were found to be s i g n i f i c a n t sources of v a r i a t i o n on c e l l u l o s e y i e l d s . S t a b i l i t y of three s t r a i n s of ^ . xylinum were assessed over s e r i a l t rans fers of s t a t i c and swir led c u l t u r e s . It was concluded that a l l the s t r a i n s tested were unstable under swir led c o n d i t i o n s , ATCC 1*4-851 being the most s table of the three when grown under s t a t i c c o n d i t i o n s . Growth curves of the three s t r a i n s were studied and c e l l u l o s e y i e l d s , pH values , sugar u t i l i z a t i o n , sugar conversion and nitrogen content in dry c e l l u l o s e were determined over a 4-0 day incubat ion per iod . Comparison of the growth curves of ^ . xylinum s t r a i n s showed that t h i s organism var ied widely i n i t s e f f i c i e n c y of convert ing sugar to c e l l u l o s e . The degree of polymerizat ion (D.P.) values of the c e l l u -lose synthesized by two of the s t r a i n s were followed over an incubat ion period of 32 days. The D.P. values of both s t r a i n s appeared to decrease with incubat ion time. - i i i -The second aspect of t h i s study was to develop a process that i n t e g r a t e s c e l l u l o s e f i b r i l s i n t o a c o t t o n - l i k e f i b r e . Since A^ . xylinum n a t u r a l l y produces c e l l u l o s e i n the form of hi g h l y f i b r i l l a r r ibbons, a process was devised to mechanically d i r e c t b a c t e r i a l c e l l s to spin these ribbons i n t o o r i e n t e d p a r a l l e l f i l a m e n t s by c u l t i v a t i n g the organisms i n a s t r a i g h t - l i n e flow path. Aeration rates i n the system appeared to i n f l u e n c e growth and c e l l u l o s e y i e l d s . Flow mode ( i n t e r m i t t e n t and continuous) and ^ . xylinum s t r a i n were shown to be h i g h l y s i g n i f i c a n t sources of v a r i a t i o n on the t e n s i l e strength of f i b r e s . Optimum condi-t i o n s f o r the me r c e r i z a t i o n treatment of f i b r e s f o r achieving maximum t e n s i l e strength were determined by using a mapping super simplex o p t i m i z a t i o n technique. T e n s i l e p r o p e r t i e s of mercerized and unmercer-i z e d f i b r e s were studied by conducting load-elongation t e s t s to specimen f a i l u r e . L i g h t and e l e c t r o n microscopy were employed to observe the f i n e f i b r e s t r u c t u r e . F i n a l l y , the t h i r d aspect of t h i s study c o n s i s t e d of the develop-ment of a process f o r the p u r i f i c a t i o n and h y d r o l y s i s of A. xylinum c e l l u l o s e f o r the manufacture of m i c r o c r y s t a l l i n e c e l l u l o s e (MCC). Chemical composition and p h y s i c a l p r o p e r t i e s of the spray d r i e d MCC were determined. The flow behaviour of A_. xylinum MCC d i s p e r s i o n s was examined and compared to commercial A v i c e l PH-101 MCC. Rheograms of both MCC d i s p e r s i o n s d i s p l a y e d non-Newtonian pseudoplastic behaviour. A. xylinum MCC d i s p e r s i o n s were found to have r h e o l o g i c a l behaviour of the t h i x o t r o p i c type, that i s , there was a r e v e r s i b l e change i n v i s c o -s i t y with time at a constant r a t e of shear. - i v -Results of t h i s study ind ica ted that i t i s t e c h n i c a l l y f e a s i b l e to produce synthet ic c o t t o n - l i k e f ib re s by s t imulat ing /\. xylinum c e l l s to extrude t h e i r c e l l u l o s e ribbons i n a p a r a l l e l f a sh ion . xylinum c e l -lu lo se appeared to be a good source of raw mater ia l in the manufacture of m i c r o c r y s t a l l i n e c e l l u l o s e because i t i s a h ighly p u r i f i e d form of c e l l u l o s e which can be made ava i l ab l e a l l year around at any l o c a t i o n . - V -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES x i ACKNOWLEDGEMENTS xv INTRODUCTION 1 LITERATURE REVIEW 3 A. ^. Xylinum C e l l u l o s e Production 3 1. D e s c r i p t i o n of Acetobacter xylinum 3 2. Acetobacter xylinum c u l t u r e s 5 3. C e l l u l o s e b i o s y n t h e s i s processes 13 a) I n t r a c e l l u l a r 13 b) E x t r a c e l l u l a r 16 B. I n d u s t r i a l A p p l i c a t i o n s of B a c t e r i a l C e l l u l o s e 19 C. M i c r o c r y s t a l l i n e C e l l u l o s e 23 MATERIALS AND METHODS 28 A. Test Organisms 28 B. C u l t i v a t i o n Method 28 1. Cu l t u r e propagation and c o n d i t i o n s 28 2. S e l e c t i o n of i n f l u e n t i a l f a c t o r s f o r maximum c e l l u l o s e production i n defined medium 29 C. Cu l t u r e S t a b i l i t y 31 D. Growth Curves 32 1. pH measurement 32 2. Nitrogen determination ( c e l l - n i t r o g e n i n dry c e l l u l o s e ) 32 3. C e l l u l o s e determination 33 4. Sugar u t i l i z a t i o n and conversion 33 E. Degree of P o l y m e r i z a t i o n vs. Incubation Time 34 F. C e l l u l o s i c F i b r e Production and C h a r a c t e r i z a t i o n 35 1. D e s c r i p t i o n of the f i b r e production apparatus 35 2. Process f o r the production of f i b r e s 41 - v i -Page 3. Tens i l e propert ies determination 46 4. Mapping super simplex opt imiza t ion of mercer izat ion treatment 49 5. S t a t i s t i c a l analyses 51 a) Ef fect of A. xylinum s t r a i n and flow mode on t e n s i l e strength 51 b) Ef fect of batch on t e n s i l e propert ies 51 c) E f fec t of mercer izat ion on t e n s i l e propert ies . . . . 52 6. F ibre microstructure 52 a) L ight microscopy 52 b) Scanning e lec t ron microscopy 53 7. C r y s t a l l i n i t y index, c r y s t a l s i ze and degree of polymerizat ion determination 54 G. M i c r o c r y s t a l l i n e C e l l u l o s e Production 54 1. C e l l u l o s e production 54 2. C e l l u l o s e p u r i f i c a t i o n . NaOH extrac t ion 54 3. Bleaching 56 4. Hydro lys i s 57 5. Spray drying 58 H. M i c r o c r y s t a l l i n e C e l l u l o s e Charac te r i za t ion 59 1. Phys i ca l property tes t s 59 a) C r y s t a l l i n i t y and c r y s t a l s i ze 59 b) Degree of polymerizat ion and p a r t i c l e s i ze 59 c) Moisture adsorption 59 d) Zeta p o t e n t i a l 60 e) Colour determination 60 2. Chemical composition 60 a) C e l l u l o s e determination 60 b) Nitrogen determination 61 c) Ash determination 62 3. Rheologica l propert ies 62 RESULTS AND DISCUSSION 66 A. Se lec t ion on I n f l u e n t i a l Factors in Defined Medium 66 B. Cul ture S t a b i l i t y 74 C. Growth Curves 78 D. Degree of Polymerizat ion vs . Incubation Time 81 E . C e l l u l o s i c F ibres Production 85 1. F ibre production apparatus 85 - v i i -Page 2. Process for the production of f ibre s 86 a) E f fec t of A. xylinum s t r a i n and flow mode on t e n s i l e strength 86 b) Influence of aerat ion rate on growth and c e l l u l o s e y i e l d 90 F . C e l l u l o s i c F ibre Charac ter i za t ion 94 1. Tens i l e propert ies 94 a) E f fec t of moisture content on t e n s i l e strength . . . 94 b) Mapping super simplex opt imizat ion of mercer izat ion treatment 97 c) Ef fect of mercer izat ion on t e n s i l e propert ies . . . . 105 d) E f fec t of batch on t e n s i l e propert ies 109 2. F ibre Micros tructure 112 a) L ight microscopy 112 b) Scanning e lec t ron microscopy 115 3. C r y s t a l l i n i t y index, c r y s t a l s i ze and degree of polymerizat ion 122 G. M i c r o c r y s t a l l i n e C e l l u l o s e (MCC) Production 122 1. C e l l u l o s e production and p u r i f i c a t i o n 122 2. E f fec t of acid hydro ly s i s on weight lo s s and degree of polymerizat ion 134 H. M i c r o c r y s t a l l i n e C e l l u l o s e Charac te r i za t ion 139 1. Chemical composit ion. C e l l u l o s e , ni trogen and ash content 139 2. Phys i ca l propert ies 141 a) C r y s t a l l i n i t y , c r y s t a l s i z e , degree of polymerizat ion and p a r t i c l e s i ze determination . . . 141 b) Moisture adsorption 143 c) Zeta p o t e n t i a l determination 145 d) Colour determination 146 3. Rheologica l propert ies 146 CONCLUSIONS 176 LITERATURE CITED 180 - v i i i -LIST OF TABLES Page Table 1. Ana ly s i s of variance (Taguchi L 2 7 3 ) for c e l l u -lo se y i e l d s obtained from 27 nutr ient media 67 Table 2. Mean values of percentage of c e l l u l o s e " d e f i c i e n t " CFU i n l i q u i d swir led and s t a t i c c u l t u r e s , as a funct ion of number of t rans fers (n=3) 75 Table 3. The average degree of polymerizat ion of b a c t e r i a l c e l l u l o s e produced by incubat ion of the P h i l i p p i n e s t r a i n of A. xylinum (n=4) 83 Table 4-. The average degree of polymerizat ion of b a c t e r i a l c e l l u l o s e produced by incubat ion of ATCC 14851 s t r a i n of _A. xylinum (n=4) 84 Table 5. T e n s i l e strength of c e l l u l o s e f i b r e s produced by two s t r a i n s of JK. xylinum c u l t i v a t e d under cont in-uous or in termi t tent flow (Mean ± SD) 87 Table 6. Ana ly s i s of variance of mean t e n s i l e strength for c e l l u l o s e f i b r e s produced by two s t r a i n s of A^. xylinum c u l t i v a t e d under continuous or in termi t tent flow 89 Table 7. Mercer iza t ion treatments and mean t e n s i l e strength values obtained with mapping super simplex opt imi-zat ion (n=3) 102 Table 8. Mean values of t e n s i l e proper t ie s of mercerized and unmercerized f i b r e s (n=8) 107 Table 9. Ana ly s i s of variance for t e n s i l e strength of mer-c e r i z e d and unmercerized f i b r e s 107 Table 10. Ana lys i s of variance for e longat ion of mercerized and unmercerized f i b r e s 108 Table 11. Ana lys i s of variance for modulus of e l a s t i c i t y of mercerized and unmercerized f i b r e s 108 Table 12. Mean values of t e n s i l e propert ies of c e l l u l o s e f i b r e s produced from two batches (n=8) 110 Table 13. Ana ly s i s of variance for t e n s i l e strength of f i b r e s produced i n two d i f f e r e n t batches 110 Table 14. Ana lys i s of variance for elongation of f i b r e s pro-duced i n two d i f f e r e n t batches 111 - ix -Page Table 15. Ana lys i s of variance for elongation of f i b r e s pro-duced i n two d i f f e r e n t batches 111 Table 16. E f fect of NaOH ext rac t ion of swollen and freeze dr ied c e l l u l o s e on DP, C r l , c r y s t a l s i ze and n i t r o -gen content (Mean values ± SD) 128 Table 17. Ana lys i s of variance of ni trogen content of NaOH-extracted c e l l u l o s e samples 130 Table 18. Ana lys i s of variance of c r y s t a l l i n i t y index of NaOH-extracted c e l l u l o s e samples 130 Table 19. Ana lys i s of variance of degree of polymerizat ion of NaOH-extracted samples 131 Table 20. Ana lys i s of variance of c r y s t a l s ize of NaOH-extracted samples 131 Table 21. Chemical composition of m i c r o c r y s t a l l i n e c e l l u l o s e s (Mean ± SD) 140 Table 22. Mean values of c r y s t a l l i n i t y index (CRI), c r y s t a l s i z e , degree of polymerizat ion ( D P ) , c r y s t a l s i ze and p a r t i c l e s ize for ^ . xylinum MCC and A v i c e l PH-101 MCC 142 Table 23. Colour of A. xylinum MCC and A v i c e l PH-101 MCC 147 Table 24. Consistency c o e f f i c i e n t s and flow behaviour ind ice s for the upcurve and downcurve of xylinum MCC at 2 5 ° C and various concentrat ions 149 Table 25. Consistency c o e f f i c i e n t s and flow behaviour ind ice s for the upcurve and downcurve of A v i c e l PH-101 MCC at 2 5 ° C and various concentrat ions 150 Table 26. Gel strength r a t i o ( G o / G 1 0 ) of 6, 5, 4 and 3 % d i sper s ions of A. xylinum MCC at d i f f e r e n t tempera-tures 152 Table 27. Gel strength r a t i o ( G o o / G 1 0 ) of 6, 5, 4 and 3 % d i sper s ions of A v i c e l PH-101 MCC at d i f f e r e n t temperatures 153 Table 28. Consistency c o e f f i c i e n t s and flow behaviour ind ice s of ^ . xylinum MCC di spers ions at various tempera-tures and concentrat ions 155 Table 29. Consistency c o e f f i c i e n t s and flow behaviour ind ice s of A v i c e l PH-101 MCC d i spers ions at various temper-atures and concentrat ions 156 - X -Page Table 30. Consistency c o e f f i c i e n t s and flow behaviour ind ice s fo r A . xylinum MCC and A v i c e l PH-101 MCC di spers ions at 25 C and various concentrat ions 160 Table 31. Apparent v i s c o s i t y (at shear rate = 100 s " 1 ) - c o n -cent ra t ion r e l a t i o n s h i p for /\. xylinum MCC disper-sions from 25 to 45 °C 163 Table 32. Apparent v i s c o s i t y (at shear rate = 100 s _ 1 ^ c o n -cent ra t ion r e l a t i o n s h i p for A v i c e l MCC di spers ions from 25 to 4 5 ° C 163 Table 33. Apparent v i s c o s i t y (at shear rate = 100 s - ^ - t e m p -erature r e l a t i o n s h i p for / \ . xylinum MCC di spers ions at d i f f e r e n t concentrat ions 167 Table 34-. Apparent v i s c o s i t y (at shear rate = 100 s - 1 ) - t emp-erature r e l a t i o n s h i p for A v i c e l PH-101 MCC di sper-sions at d i f f e r e n t concentrat ions 167 Table 35. Consistency c o e f f i c i e n t s and flow behaviour ind ice s fo r a 3% d i sper s ion of / \ . xylinum MCC at 2 5 ° C and at d i f f e r e n t pH 170 Table 36. Consistency c o e f f i c i e n t s and flow behaviour ind ice s fo r a 3% d i sper s ion of A v i c e l PH-101 MCC at 2 5 ° C and at d i f f e r e n t pH 170 Table 37. Consistency c o e f f i c i e n t s and flow behaviour ind ice s fo r a 3% d i sper s ion of A v i c e l PH-101 MCC or A. xylinum MCC + 0.*% NaCl at 2 5 ° C 174 - x i -LIST OF FIGURES Page Figure 1. Proposed model of normal c e l l u l o s e assembly 20 Figure 2. F i b r e production apparatus (prel iminary design) 36 Figure 3. F i b r e production apparatus. Side view 38 Figure 4. Grooves or channels i n the f i b r e production apparatus 39 Figure 5. Ramp with carved grooves or channels providing a s t r a i g h t l i n e flow path 40 Figure 6. pH c o n t r o l l e r Speedomax equipped with a pH e lec-trode 42 Figure 7. Polycarbonate p l a s t i c chamber provided with a i r f i l t e r , cotton plug ( a i r vent) and NaOH feeder 43 Figure 8. Polycarbonate p l a s t i c cover with a i r f i l t e r attached 44 Figure 9. E f f ec t curves r e s u l t i n g from the s e l e c t i o n of i n -f l u e n t i a l f ac tors i n defined media Figure 10. C e l l u l o s e production and pH changes during growth of three Acetobacter xylinum s t r a i n s i n 100 mL of defined medium 79 Figure 11. Sugar conversion of t o t a l sugar and nitrogen con-tent per 100 g of c e l l u l o s e of three Acetobacter  xylinum s t r a i n s 80 Figure 12. Sugar u t i l i z a t i o n and conversion of u t i l i z e d sugar by three Acetobacter xylinum s t r a i n s 82 Figure 13. Influence of a i r flow rate on sugar u t i l i z a t i o n and pH of _A. xylinum ATCC 14-851 grown i n defined medium . . 91 Figure 14. Influence of a i r flow rate on c e l l u l o s e y i e l d , sugar convers ion, ni trogen content and d i s so lved oxygen of _A. xylinum ATCC 14851 grown i n defined medium 93 Figure 15. Mean t e n s i l e strength of / \ . xylinum c e l l u l o s e f i b r e s e q u i l i b r a t e d over f i v e saturated s a l t so lu-t ions (n=3) 95 - x i i -Page Figure 16. Mapping r e s u l t s of experiments to maximize t e n s i l e s t r e n g t h of b a c t e r i a l c e l l u l o s e f i b r e s . Response values p l o t t e d against f a c t o r l e v e l s . a) NaOH, % 99 b) Heating temperature, °C 100 c) Heating time, min 101 Figure 17. T y p i c a l load-elongation curve of a ^ . xylinum c e l -l u l o s e yarn conducted to specimen f a i l u r e 106 Figure 18. Li g h t micrograph of st a i n e d c e l l u l o s e f i b r e matrix embedded with b a c t e r i a (pw =130 ym) 113 Figure 19. Li g h t micrograph of c e l l u l o s e f i b r e s i n an organ-i z e d p a r a l l e l f a shion (pw = 130 ym) 113 Figure 20. U n i d i r e c t i o n a l l y o r i e n t e d c e l l u l o s e f i b r e s under p o l a r i z e d l i g h t (pw = 130 ym) 114 Figure 21. Scanning e l e c t r o n micrograph of unmercerized c e l l u -l o s e f i b r e s produced under u n i d i r e c t i o n a l flow showing a loose f i b r e s t r u c t u r e (pw = 10 ym) 116 Figure 22. Scanning e l e c t r o n micrograph of mercerized c e l l u -l o s e f i b r e s produced under u n i d i r e c t i o n a l flow (pw = 247 ym) 116 Figure 23. Scanning e l e c t r o n micrograph of mercerized c e l l u -l o s e f i b r e s produced under u n i d i r e c t i o n a l flow showing a compact f i b r e s t r u c t u r e (pw = 11 ym) 117 Figure 24. Scanning e l e c t r o n micrograph of unmercerized c e l l u -l o s e f i b r e s produced under s t a t i c c o n d i t i o n s show-ing a network of entangled loose d i s o r i e n t e d f i b r e s (pw = 11 ym) 118 Figure 25. Scanning e l e c t r o n micrograph of unmercerized c e l l u -l o s e f i b r e s produced under s t a t i c c o n d i t i o n s show-ing a bacterium entrapped w i t h i n the matrix (pw = 12 ym) 118 Figure 26. Scanning e l e c t r o n micrograph of unmercerized c e l l u -l o s e f i b r e s poroduced under s t a t i c c o n d i t i o n s (pw = 28 ym) 119 Figure 27. Scanning e l e c t r o n micrograph of unmercerized c e l l u -l o s e f i b r e s produced under s t a t i c c o n d i t i o n s (pw = 208 ym) 119 Figure 28. Scanning e l e c t r o n micrograph of mercerized c e l l u -l o s e f i b r e s produced under s t a t i c c o n d i t i o n s (pw = 11 ym) 120 - x i i i -Page Figure 29. Scanning electron micrograph of mercerized c e l l u -l o se f i b r e s produced under s t a t i c conditions show-ing a compact structure (pw = 22 ym) 120 Figure 30. Scanning electron micrograph of mercerized c e l l u -l o se f i b r e s produced under s t a t i c conditions (pw = 57 ym) 121 Figure 31. Scanning electron micrograph of mercerized c e l l u -lose f i b e r s produced under s t a t i c conditions (pw = 254 ym) 121 Figure 32. C e l l u l o s e production and p u r i f i c a t i o n 123 Figure 33. ^. xylinum culture with c e l l u l o s e p e l l i c l e at the surface 125 Figure 34. C e l l u l o s e p e l l i c l e being harvested a f t e r two weeks of incubation 125 Figure 35. Shredded xylinum c e l l u l o s e p r i o r to NaOH-extrac-t i o n 126 Figure 36. A t y p i c a l diffratogram of ^ . xylinum c e l l u l o s e 129 Figure 37. Hydrolysis of 6% NaOH-extracted c e l l u l o s e and de-graded c e l l u l o s e 135 Figure 38. Comparison of y i e l d s observed i n 2.5 N HC1 at 105°C of 6% NaOH-extracted c e l l u l o s e and degraded c e l l u -lose 136 Figure 39. M i c r o c r y s t a l l i n e c e l l u l o s e production 138 Figure 40. Sorption isotherms of _A. xylinum MCC and Av i c e l PH-101 MCC at 25°C 144 Figure 41. Upcurve and downcurve rheograms showing time depen-dent behaviour of 6% dispersions of X. xylinum and Avice l PH-101 MCC at 25° 151 Figure 42. Rheograms for 6, 5, 4 and 3 % A. xylinum MCC d i s -persions at 25°C according to the Power Law flow model 158 Figure 43. Rheograms for 6, 5, 4 and 3 % Avic e l PH-101 MCC dispersions at 25°C according to the Power Law flow model 159 Figure 44. Rheograms for 6 and 3 % Av i c e l and A. xylinum MCC dispersions at 25°C according to the Power Law flow model 161 - xiv -Page Figure 45. E f f e c t of concentration on apparent v i s c o s i t y (y = 100 s" 1) of /\. xylinum MCC dispersions at 25, 35 and 45°C 164 Figure 46. E f f e c t of concentration on apparent v i s c o s i t y (y = 100 s _ 1 ) of Av i c e l PH-101 dispersions at 25, 35 and 45°C 165 Figure 47. E f f e c t of temperature on apparent v i s c o s i t y (shear rate = 100 s" 1) for 3.0 to 6.0 % A. xylinum MCC dispersions 168 Figure 48. E f f e c t of temperature on apparent v i s c o s i t y (shear rate = 100 s" 1) for 3.0 to 6.0 % Av i c e l PH-101 MCC dispersions 169 Figure 49. E f f e c t of pH on apparent v i s c o s i t y of 3% A. xylinum MCC dispersions at 25°C 171 Figure 50. E f f e c t of pH on apparent v i s c o s i t y of 3% A v i c e l PH-101 MCC dispersions 172 Figure 51. E f f e c t of 0.4% NaCl on apparent v i s c o s i t y of 3% A. xylinum or Av i c e l PH-101 MCC dispersions at 25°C .. 175 - XV -ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to Dr. P. M. Townsley for the introduction to t h i s project, for the design and construction of the equipment and his support of t h i s study. Thanks are also extended to Dr. W. D. Powrie, Dr. M. A. Tung and Dr. B. 3. Skura for t h e i r helpful suggestions and review of t h i s t h e s i s . The author i s also very g r a t e f u l to Dr. S. Nakai for his invalu-able advice in the s t a t i s t i c a l analyses and to Dr. L. Paszner for his help f u l guidance i n the c e l l u l o s e c h a r a c t e r i z a t i o n and the suggestion for the preparation of high purity m i c r o c r y s t a l l i n e c e l l u l o s e . The technical advice from S. Yee, A. Speers and L. Robinson i s also grate-f u l l y acknowledged. This thesis i s dedicated to my husband for his support and enthu-siasm throughout t h i s study. - 1 -INTRODUCTION The c e l l u l o s e formed by /A. xylinum i s a well known standard type , s i m i l a r to the h ighly c r y s t a l l i n e homopolymer group which has been represented c l a s s i c a l l y by the alpha c e l l u l o s e or c e l l u l o s e I of co t ton . The c e l l u l o s e i s a l so free of l i g n i n , hemice l lu lose , pec t in or other encrust ing substances. The c e l l u l o s e m i c r o f i b r i l s accumulate e x c l u s i v e l y i n the e x t r a c e l l u l a r phase where they form a h y d r o p h i l i c membranous p e l l i c l e . The g a s - l i q u i d i n t e r f a c e , as the s i t e of p e l l i c l e formation, permits the p o s s i b i l i t y of large sca le c e l l u l o s e membrane product ion . The c e l l u l o s e can be r e a d i l y freed from b a c t e r i a l c e l l s thereby separating product from substrate . A wide va r i e ty of substrates can be u t i l i z e d for c e l l u l o s e synthes i s increas ing the p o t e n t i a l f l e x i b i l i t y of u t i l i z a t i o n of a g r i -c u l t u r a l wastes, such as beet molasses, b lackstrap molasses, coconut m i l k , pineapple pulp trimmings, and s u l f i t e waste l i q u o r . The s i m p l i -c i t y of c e l l u l o s e production suggests that large scale microb ia l c e l l u -lose production plants could be located near sources of convenient and inexpensive substrates . Over the l a s t two decades research into c e l l u l o s e b iosynthes i s by /A. xylinum has increased , s ince i t was through t h i s organism that c e l l u l o s e assembly in l i v i n g systems was f i r s t observed and monitored. U t i l i z a t i o n of ^ . xylinum c e l l u l o s e for i n d u s t r i a l purposes however has been scarce ly explored, i n sp i t e of i t s great po ten t i a l for l a rge- sca le product ion. - 2 -In the l a s t two years increased a t tent ion has been given to indus-t r i a l app l i ca t ions of b a c t e r i a l c e l l u l o s e . In 1982, Mynnat patented a process for f i b e r production from continuous c u l t i v a t i o n of microorgan-isms, per ta in ing to the production of c e l l u l o s e f i b r e s for use in the manufacture of paper. Brown (1983) patented a process for the production of a c e l l u -l o s e - s y n t h e t i c polymer composite f i b r e . The invent ion took advantage of the c e l l u l o s e produced by Acetobacter xylinum in that i t was poss ib le to produce c e l l u l o s e on the surface of a polyester f i b r e , which conferred to the f i b r e many of the phys ica l propert ies of co t ton . The composite polymer produced according to that procedure provided a whole new approach to the manufacture of " c o t t o n - l i k e " goods. The purpose of t h i s the s i s was to develop two app l i ca t ions of A. xylinum c e l l u l o s e . One en ta i l ed the production of a " c o t t o n - l i k e " f ibrous s t rand. This was succes s fu l ly achieved by mechanically d i r e c t -ing A,, xylinum organisms to spin c e l l u l o s e ribbons in to p a r a l l e l or iented s trands . The second a p p l i c a t i o n developed was in the use of A^. xylinum c e l l u l o s e as the raw mater ia l for the production of m i c r o c r y s t a l l i n e c e l l u l o s e (MCC). The m u l t i p l e uses of MCC in the food and pharmaceuti-c a l i n d u s t r i e s , among others , i n d i c a t e the p o t e n t i a l for u t i l i z i n g a highly p u r i f i e d form of A. xylinum c e l l u l o s e as a raw m a t e r i a l . - 3 -LITERATURE REVIEW A. A. Xylinum Cellulose Production As long ago as 1886 Brown described a bacterium which produced a so l id membrane when growing on a carbohydrate r i c h medium. I t was found that these membranes were soluble in ammoniacal copper hydroxide and yielded reducing sugars when hydrolyzed with su l fu r i c a c id . Since i t was known that cotton produced these reactions, the organism was ca l led Acterium xylinum (xy' li«num. Gr. adj . xyl inus of cotton; L. neut. n. xylinum cotton) . The s i m i l a r i t y between /A. xylinum ce l lu lo se and cotton ce l lu lose was confirmed by the production of well oriented x-ray d i f -fractograms of stretched bac ter i a l membranes which were i d e n t i c a l to those of cotton. Electron micrographs showed that these bac ter i a l membranes consist of a th ick network of f ibres and ribbons (Muhlethaler, 1949) s imi la r to that of ce l lu lo se from the c e l l walls of numerous p lants . 1• Description of Acetobacter xylinum The genus Acetobacter refers to a group of bacteria that have the a b i l i t y to oxidize ethanol to acetic ac id . In p a r t i c u l a r , the bacterium Acetobacter xylinum, noted for i t s a b i l i t y to produce vinegar, i s usual-l y found i n wine vats as " . . . a sort of moist s k i n , swollen gelatinous and s l ippery . . . " I t has been referred to in the past as "mother of vinegar" and was the basis of the early vinegar industry (Shramm and Hestr in , 1954). The gelatinous skin i s actual ly a polysaccharide matrix within which the bac ter ia l c e l l s are enmeshed, and i s more commonly - 4 -known as a p e l l i c l e . Because ^ . xylinum i s an ob l iga te aerobe requ i r ing a constant supply of oxygen, i t can be speculated that the funct ion of the polysaccharide p e l l i c l e i s to provide a buoyant environment at the a i r - l i q u i d in ter face (Schramm and H e s t r i n , 1954). ^ . xylinum i s a rod-shaped non-moti le , gram negative bacterium which occurs s ing ly and i n cha ins . The i n d i v i d u a l c e l l s , which are enveloped by a ge lat inous m a t e r i a l , measure 2 ym long and 0.1 ym wide. The polysaccharide p e l l i c l e forms on a l l l i q u i d media i n which growth occurs . The nature of the medium inf luences the thickness of the f i l m which may vary from 2 to 250 mm. The p e l l i c l e becomes car t i lagenous and f a l l s to the bottom i f d i s turbed . The optimum growth temperature of j^. xylinum i s 2 8 ° C (Breed et _al.., 1957). This organism forms acid from glucose , e thanol , propanol and g l y c o l ; but i t does not produce ac id from arabinose, f ruc tose , ga lactose , maltose, l a c to se , r a f f i n o s e , d e x t r i n , s t a r c h , methanol, i sopropanol , butanol , i sobutanol , pentanol , mannitol or acetaldehyde. It produces cata lase ( p o s i t i v e ) ; l i tmus milk i s not changed; indole i s not formed, g e l a t i n i s not l i q u i f i e d ; and i t ox id ize s ace t i c acid to carbon dioxide and water (Lapuz et a l . , 1967). The morphological and p h y s i o l o g i c a l c h a r a c t e r i s t i c s show that t h i s organism belongs to the genus Acetobacter , which according to Breed ^ t a l . (1957), was c l a s s i f i e d as Genus II of the family Pseudomonadacea. The species of Acetobacter may be d i f f e r e n t i a t e d from a l l other Pseudo-monadacea by t h e i r unique a b i l i t y to ox id ize s i g n i f i c a n t quant i t i e s of ethanol under the extremely a c i d i c condi t ions imposed by the presence of from about 2 to more than 11 percent ace t ic a c i d . Acetobacter species - 5 -are aerobic , s t rongly cata lase p o s i t i v e , and ox ida t ive i n t h e i r physio-logy . Within the genus Acetobacter , two groups of species were recog-n i z e d . The f i r s t group cons i s t s of species capable of o x i d i z i n g ace t i c acid to carbon dioxide and water while the second group does not have t h i s a b i l i t y . A. xylinum belongs to the f i r s t group under which f i v e species have been recognized. The d i s t i n c t i o n between the ^ . a c e t i and the other ace t i c ac id o x i d i z i n g species i s based mainly on growth i n Hoyer 's medium, where ethanol i s the sole carbon source. The i n a b i l i t y of / \ . xylinum to u t i l i z e ammonium s a l t s as a so le source of ni trogen (no growth i n Hoyer 's medium) i s due only to the lack of adequate n u t r i e n t s . /A. xylinum w i l l u t i l i z e ammonium s a l t s i f glucose i s supp l i ed . F i n a l l y , a d i s t i n c t i v e character wi th in the c l a s s i f i c a t i o n i s the production of a t h i c k , l ea thery , zoog lea l , c e l l u l o s i c membrane on the surface of l i q u i d s . Buchanan and Gibbons (1975) r e c l a s s i f i e d the genus Acetobacter under the "Genera of Uncertain A f f i l i a t i o n " . 2. Acetobacter xylinum c u l t u r e s The range of u t i l i z a b l e substrates for c e l l u l o s e production by re s t ing c e l l s inc ludes hexoses, t h e i r anhydrides and compounds which presumably y i e l d hexoses as a r e su l t of b a c t e r i a l a c t i o n . When the carbon compound used i s not a free hexose, nor one apparently capable of convers ion in to a hexose, as i n the case of pentoses such as arabinose and xylose , g l y c o l s , p o l y g l y c o l s and e r y t h r i t o l , no membrane formation - 6 -takes p lace . On the other hand mannitol , which i s known to undergo oxidat ion with the formation of fructose under the experimental condi-t i o n s , gives r i s e to high y i e l d s of polysacchar ide . G l y c e r o l behaves s i m i l a r l y , presumably due to a primary ox idat ion to dihydroxyacetone and conversion of t h i s to f ructose . Galactose i s much le s s reac t ive than glucose, while mannose appears to be r e l a t i v e l y i n a c t i v e . The highest y i e l d of the polysaccharide was obtained from f ructose , a r e su l t pre-sumably connected with the recognized fact that t h i s bacterium forms l i t t l e or no ac id from t h i s sugar (Tarr and Hibber t , 1931). Work of t h i s nature was continued by Kaushal and Walker (1951) who studied three other species of Acetobacter . A s t r a i n of j^. acetigenum, i s o l a t e d from East A f r i c a n v inegar , formed c e l l u l o s e from a l l of the eighteen carbohydrates submitted to i t s a c t i o n . By i t s a b i l i t y to convert to c e l l u l o s e <=- and p-methyl-D-glucosides , three pentoses, e r y t h r i t o l and ethylene g l y c o l , r e s p e c t i v e l y , the organism showed i t s e l f to be enzymatical ly more ac t ive than /A. xylinum which proved unable to u t i l i z e these substances for t h i s purpose (Tarr and Hibbert , 1931). As almost a l l e a r l i e r work was done with growing c u l t u r e s , i t d i d not d i s t i n g u i s h between a poss ib le e f fec t of substrate on c e l l growth from that on c e l l u l o s e product ion . This source of ambiguity was e l i m i n -ated by using washed n o n - p r o l i f e r a t i n g c e l l s (Gromet ^ t _ a l . , 1957). Hexoses (glucose and f ruc to se ) , three carbon compounds ( g l y c e r o l and dihydroxyacetone) and hexonates (gluconate, 2- and 5-oxogluconate) have been converted in to c e l l u l o s e by the act ion of washed c e l l s of _A. x y l i n - um. Acetate , pyruvate and c i t r a t e - c y c l e intermediates were ox id ized - 7 -without attendant conversion into c e l l u l o s e . Phosphate esters ( i n c l u d -ing glucose 6-phosphate, ^-glucose 1-phosphate, g-glucose 1-phosphate and ur id ine diphosphoglucose) were presumably not taken up by the c e l l s . Exogenous addi t ion of such esters did not r e s u l t in the formation of c e l l u l o s e . The range of substrates for both c e l l u l o s e and C 0 2 formation depended on the c e l l h i s tory and on the condi t ions of the assay. The work of Dudman (1959b) paid c lo se r a t tent ion to the in f luence of growth condi t ions on c e l l u l o s e y i e l d . Acetate , c i t r a t e and succinate were highly e f f e c t i v e i n s t imula t ing c e l l u l o s e synthesis by \ . a c e t i - genum. Ethanol st imulated growth but d id not increase c e l l u l o s e y i e l d . It was found that appropriate concentrat ions of succinate could cause a l a rge increase in c e l l u l o s e product ion . Thus i t was postulated that succinate increased c e l l u l o s e y i e l d by s p e c i f i c a l l y s t imulat ing c e l l u -lose synthesis rather than growth i n genera l . I t was proposed that succinate was involved in a sparing mechanism where i t i s p r e f e r e n t i a l l y ox id ized i n place of some sugar substrates , which are then freed for c e l l u l o s e synthes i s . The hypothesis was supported by previous observa-t ions of Schramm j?t _al. (1957) i n which acetate, c i t r a t e , succinate and other intermediates of the t r i c a r b o x y l i c a c i d - c y c l e were a l l r e a d i l y ox id ized to C0 2 by washed suspensions of /A. xyl inum. It was also noted that the buffer ing e f fects from the addi t ion of succinate may have been a factor i n the increased c e l l u l o s e y i e l d . An adaptive e f fect of carbohydrate composition of the growth medium on the substrate range was i l l u s t r a t e d by the synthesis of - 8 -c e l l u l o s e from pyruvate by succinate grown c e l l s of xylinum (Benziman and Burger-Rachaminou, 1962). Their observations ind ica ted that the anhydroglucose carbon chain of c e l l u l o s e a r i se s from pyruvate i n xylinum v i a a condensation i n v o l v i n g two molecules of a three carbon compound. Studies by Weinhouse and Benziman (1974) revealed that _A. xylinum c e l l s r e a d i l y ox id ized succ inate , pyruvate and acetate to C O 2 , and synthesized c e l l u l o s e from succinate and pyruvate when they were at r e l a t i v e l y high concentra t ions . Furthermore, any of these t r i c a r b o x y l i c a c i d - c y c l e intermediates promoted c e l l u l o s e synthesis from fructose and gluconate , although re tard ing t h e i r ox ida t ion to C O 2 . Weinhouse and Benziman (1976) further showed that g l y c e r o l and dihydroxyacetone can be u t i l i z e d by ^ . xylinum as a source of carbon and energy for growth and c e l l u l o s e synthes i s . G l y c e r o l u t i l i z a t i o n by c e l l s of _A. xylinum was accompanied by the formation of dihydroxyace-tone, c e l l u l o s e , CO2 and small amounts of acetate . Dudman (1959a) published the f i r s t study concerning the condi t ions which gave maximum c e l l u l o s e y i e l d s with inexpensive and r e a d i l y a v a i l -able substrates , with a view to poss ib le l a rge- sca le production of t h i s po lysacchar ide . D i f f e rent carbohydrate substrates were compared for c e l l u l o s e production by acetigenum and s ix s t r a i n s of ^ . xyl inum. Hydrolyzed molasses was found to give the larges t y i e l d s . C e l l u l o s e y i e l d s var ied over a wide range, equivalent to conversions of 1.9 to 23.5%. C e l l u l o s e synthesis stopped when growth stopped, even when the sugar i n the medium was not exhausted, i n d i c a t i n g that c e l l u l o s e was synthesized only by the a c t i v e l y growing organism. - 9 -In conjunction with the observations previous ly described on c e l l u l o s e production by k. acetigenum i n complex media, a study was conducted on growth and c e l l u l o s e production i n defined media (Dudman, 1959b). Growth and c e l l u l o s e production by A. acetigenum was l i m i t e d by ni trogen concentrat ions below 0.01% (W/V) i n glucose defined medium. Ammonium su l fa te and asparagine + glutamic acid (50% of each) were equivalent as nitrogen sources when compared on a ni trogen weight basis over the range 0.001 - 0.1% N. At higher concentrat ions , ammonium su l f a t e decreased growth and c e l l u l o s e synthes i s , while asparagine + glutamic ac id became s t imula tory . There was no d i r e c t evidence to suggest the mechanism by which the amino ac id mixture became s t imula-to ry , but i t was proposed that i t may have been the r e su l t of deamina-t i o n , by the organism, of some of the asparagine and glutamic ac id to oxa lace t i c and ^ - k e t o g l u t a r i c ac id s , r e s p e c t i v e l y . Because these are intermediates i n the t r i c a r b o x y l i c ac id c y c l e , they would probably act in the same way as a c e t i c , c i t r i c and s u c c i n i c a c id s . Ind i rec t evidence of t h i s was the decreased recovery, i n the amino ac id c u l t u r e s , of u t i l i z e d nitrogen as c e l l - N with the increased concentrat ion of n i t rogen i n the medium. With the goal of u t i l i z i n g coconut m i l k , which i s wasted i n places where copra i s produced, Lapuz et^ _al. (1967) found that supplemented coconut milk i s an exce l l ent growth medium for _A. xyl inum. C u l t u r a l requirements inc luded : a nutr ient medium, preferably one conta ining an ingredient with growth promoting fac tors such as those present i n coconut water, temperature, about 28 °C; pH 5.0 to 5 .5 ; ni trogen source, - 10 -ammonium s a l t s preferably monobasic ammonium phosphate (NHi^POit); carbon source, glucose and sucrose. Among other substances that s t imulate c e l l u l o s e formation, one of the more i n t e r e s t i n g ones i s the c e l l u l o s i c o p t i c a l br ightener Calco-f l u o r White. The use of Ca lco f luor i n the study of Acetobacter species can be traced to Maeda and Ishida (1967), who f i r s t reported the spec i -f i c i t y of i t s binding with c e l l u l o s e . It was because of t h i s s p e c i f i -c i t y that Haig ler et a l . (1980) o r i g i n a l l y u t i l i z e d Ca lco f luor for observing m i c r o f i b r i l assembly i n _A. xyl inum. Haig ler et a l . (1980) reported that Ca lco f luor disrupted the assembly of c r y s t a l l i n e c e l l u l o s e I m i c r o f i b r i l s and t h e i r a s soc ia t ion into composite r ibbons . This d i s rup t ion was traced to a l t e r a t i o n s of hydrogen bonding between glucan chains by the Ca lco f luor which produced a n o n - c r y s t a l l i n e type of c e l l u l o s e . Because t h i s n o n - c r y s t a l l i n e c e l l u l o s e could e a s i l y be made c r y s t a l l i n e by washing out the Ca lco f luor and drying the product, the use of Ca lco f luor demonstrated that i t was pos s ib le to separate the processes of polymerizat ion and c r y s t a l l i z a t i o n in the assembly of c e l l u l o s e m i c r o f i b r i l s . Benziman et a l . (1980) found that the addi t ion of Ca lco f luor to growth medium increased the rate of glucose polymerizat ion into c e l l u l o s e . Since there were not s i g n i f i c a n t increases in general b a c t e r i a l a c t i v i t y , i t was bel ieved that energy which normally was used by c e l l - d i r e c t e d c r y s t a l l i z a t i o n was ava i l ab l e fo r increased glucose po lymer iza t ion . Schramm and Hes t r in (1954) examined the behaviour of A. xylinum i n shaken c u l t u r e s , and reported that although the formation of the - 11 -p e l l i c l e was prevented, growth occurred instead i n the form of s t e l l a t e bodies . C e l l u l o s e production was le s s than ha l f that i n the s t a t i c c u l t u r e s grown under the same c o n d i t i o n s . It was also reported that agi tated condi t ions favoured the growth of mutants unable to synthesize c e l l u l o s e and that prolonged cu l ture or repeated subculture on the shaker led to complete f a i l u r e i n the production of c e l l u l o s e . Dudman (1960) reported that the normally slow rates of growth and c e l l u l o s e synthesis by c e r t a i n s t r a i n s of /A. xylinum i n s t a t i c cu l ture s were accelerated only when growth was c a r r i e d out under condi t ions that prevented the formation of p e l l i c l e s . The dependence of the increased growth rate on obta ining growth i n a f i n e l y d iv ided s ta te , i n which the e f f ec t ive surface area of the s o l i d mass i n contact with the medium was g rea t ly increased, suggested that the slow growth under s t a t i c condi-t ions was caused by the slow rate of penetrat ion of oxygen and nut r i en t s in to the p e l l i c l e . As c e l l u l o s e synthesis i n s t a t i c cu l ture s takes place mainly i n the upper part of the p e l l i c l e , i t was l i k e l y that the l i m i t i n g process i n s t a t i c cu l ture s was the rate of d i f f u s i o n of oxygen into the p e l l i c l e . Accelerated growth was obtained, however, at the expense of decreased c e l l u l o s e y i e l d s . Y i e l d s of c e l l u l o s e i n s t i r r e d , aerated fermenters decreased with increa s ing a i r flow ra te s , although the growth l e v e l remained constant (Dudman, 1960). Results suggested that increased aerat ion decreased the y i e l d of c e l l u l o s e by causing decreased synthesis of c e l l u l o s e per uni t c e l l weight. The inverse r e l a t i o n s h i p between aerat ion and c e l l u l o s e y i e l d s may be in terpre ted to mean that increased aerat ion leads to - 12 -a s h i f t i n the metabolism of the organisms away from c e l l u l o s e synthes i s towards increased ox idat ion of the sugar substrate . I t i s wel l e s t ab l i shed , however, that aerated c u l t u r a l condi t ions favour the se lec-t i o n of c e l l u l o s e l e s s mutants of ce l lu lo se-produc ing s t r a i n s (Schramm and H e s t r i n , 1954). Therefore , both mechanisms were suggested to be involved depending on the s t r a i n and c u l t u r a l c o n d i t i o n s . The s p e c i a l i s t s who c u l t i v a t e A . xylinum under laboratory condi-t ions have wondered for a long time whether the production of c e l l u l o s e i n a tough and leathery p e l l i c l e on a s u i t a b l e l i q u i d medium serves a useful purpose for the organism when i t grows i n nature. General ly two d i f f e r e n t views have been o f fe red . The f i r s t i s that the web of c e l l u -lose m i c r o f i b r i l s affords some support or advantage for the enmeshed c e l l s i n l i q u i d media. The second i s that production of c e l l u l o s e by t h i s bacterium i s e s s e n t i a l l y an accident of evo lut ion without benef i t or detriment to the organism. Cook and C o l v i n (1980) supported the f i r s t view, and reported that cu l ture s of c e l l u l o s e d e f i c i e n t c e l l s of A . xylinum which were i s o l a t e d from s o l i d medium revert from the c e l l u l o s e - d e f i c i e n t cond i t ion to the normal c e l l u l o s e producing form af ter f i v e t rans fer s i n l i q u i d medium. In a d d i t i o n , s e r i a l cu l tures of i n i t i a l mixtures of c e l l u l o s e - d e f i c i e n t c e l l s and normal c e l l s i n the r a t i o of 9 to 1 showed a rap id decrease i n the proport ion of d e f i c i e n t c e l l s , so that a f ter seven t rans fer s i n l i q u i d medium, there were l e s s than 1% of c e l l u l o s e - d e f i c i e n t c e l l s . These r e s u l t s demonstrated that i n l i q u i d medium, c e l l s which were normal i n c e l l u l o s e production overgrew those which were d e f i c i e n t i n - 13 -t h i s capac i ty . This suggests that c e l l u l o s e production i n l i q u i d medium helped t h i s ob l iga te aerobe to obtain a l i m i t e d supply of oxygen by f l o a t i n g the c e l l s c lose to the surface . 3. C e l l u l o s e b iosynthes i s a. I n t r a c e l l u l a r processes The bacterium / \ . xylinum has served as a model organism for study-ing c e l l u l o s e b iogenes i s , namely, the b iosynthes i s of l i n e a r p 1,4-glucan chains and t h e i r c r y s t a l l i z a t i o n into c e l l u l o s e f i b r i l s . The polymeric product, formed i n abundance (up to 45% of a l l added g lucose ) , i s deposited e x t r a c e l l u l a r l y and can be r e a d i l y v i s u a l i z e d (Hestr in and Schramm, 1954; Brown et a l . , 1976). Synthesis proceeds without a lag per iod , at a rate that i t i s l i n e a r with respect to c e l l concentra t ion , and i s e s s e n t i a l l y i r r e v e r s i b l e (Hes t r in and Schramm, 1954; Ohad and H e s t r i n , 1962). C e l l u l o s e formation i s not indispensable to the metabo-l i c u t i l i z a t i o n of carbohydrates , e i ther for energy or for carbon. Net pro te in synthesis i s not required for c e l l u l o s e synthesis i n re s t ing c e l l s , s ince the process can proceed normally in the absence of a n i t r o -gen source (Hestr in and Schramm, 1954) and i s not affected by the presence of prote in synthesis i n h i b i t o r s (Webb and C o l v i n , 1967). C e l l u l o s e production i s c o n d i t i o n a l on concurrent ox idat ion processes, and u t i l i z a b l e substrates for c e l l u l o s e synthesis are a l l metabo l i ca l ly associated with the two amphibolic pathways operat ive i n A. xylinum: the pentose cyc le for the ox idat ion of carbohydrates and the c i t r a t e c y c l e for the ox idat ion of organic acids and re la ted compounds (Gromet et a l . , 1957). - n -Two d i f f e r e n t approaches have been offered towards the understand-ing of b iosynthes i s of c e l l u l o s e i n ^ . xyl inum. F i r s t l y , an i n d i r e c t i n  v ivo approach using whole c e l l s included studies on the patterns of i s o t o p i c d i s t r i b u t i o n and l a b e l re tent ion i n the c e l l u l o s e formed by r e s t i n g c e l l s from substrates s p e c i f i c a l l y l a b e l l e d with carbon 14 (Schramm et a l . , 1957; Weinhouse and Benziman, 1976; Cooper and Manley, 1975a; Swissa ^ t j i l . , 1980). A second approach has been to e f fec t the separation of the synthes iz ing enzyme system from the s t ructure of the c e l l (Glasser , 1958; Ben-Hayyim and Ohad, 1965; Swissa _et a l . , 1976; Swissa, 1978; Cooper and Manley, 1975b). The two approaches to the study of c e l l u l o s e b iosynthes i s i n d i -cated that the i n i t i a l steps of the process involved the fo l lowing sequence: « - D - g l u c o s e + ATP % « - D - g l u c o s e - 6 - p h o s p h a t e + ADP a : -D-glucose-6-phosphate £ <=-D-glucose-1-phosphate <*-D-glucose-1-phosphate + UTP t u r i d i n e diphosphoglucose + inorganic pyrophosphate The probable existence of intermediary stages beyond UDP-glucose on the pathway to c e l l u l o s e has a lso been i n d i c a t e d . The synthes i s , in v i v o , of f i b r i l l a r c e l l u l o s e and i n v i t r o of 6 1,4-glucan i s accompanied by formation of g l y c o l i p i d s that appear to contain i n t h e i r carbohydrate port ion e i ther glucose or 8 1 ,4- l inked glucose moieties ( A l o n i and Benziman, 1982). U n t i l a few years ago, i t was genera l ly be l ieved that the synthes i s of c r y s t a l l i n e c e l l u l o s e m i c r o f i b r i l s by ^ . xylinum was an - 15 -e x t r a c e l l u l a r process ( C o l v i n , 1971, 1972). According to these ideas , c e l l u l o s e f i b r i l s were randomly deposited in the surrounding medium and were not appendages of the b a c t e r i a l c e l l (Ben-Hayyim and Ohad, 1965). However, several reports (Brown et _a l . , 1976; Forge and Pres ton , 1977; Zaar, 1977, 1979) s trongly support the concept that each c e l l synthesizes a s ing le ribbon of c e l l u l o s e composed of c r y s t a l l i n e micro-f i b r i l s which elongate cont inuous ly , i n a s soc ia t ion with m u l t i p l e synthes iz ing s i t e s organized i n a row along the l o n g i t u d i n a l axis of the c e l l that , i n t u r n , are associated with corresponding extrusion s i t e s i n the l ipopo lysacchar ide (LPS) layer of the b a c t e r i a . Accord ing ly , the glucan chains comprising the m i c r o f i b r i l s are synthesized i n inmediate contact with the c e l l envelope and must be p h y s i c a l l y attached to the b a c t e r i a l surface (Co lv in and Leppard, 1977; C o l v i n _et _ a l . , 1977). Moreover, i t i s at t h i s end of the growing glucan c h a i n , which i s probably the reducing end, where the incorpora t ion of the new g lucosy l residues occurs (Brown, 1979). Compatible with t h i s concept i s the idea that per iphera l hydro-phobic prote ins are the anchoring s i t e s for the growing glugan cha ins . The formation of B 1 ,4-polyglucan chains would thus require the coopera-t i v e act ion of d i s t i n c t enzyme systems which promote the generation of UDP-glucose, t ransport of ac t ive g lucosy l uni t s to phosphol ipid c a r r i e r s , t rans fer of g lucosy l or c e l l o d e x t r i n to hydrophobic glucopro-t e i n s , and t rans fer of c e l l o d e x t r i n s to "anchor p r o t e i n " i n the outer c e l l wal l ( A l o n i and Benziman, 1982). It has been proposed by Brown and W i l l i s o n (1977) that the enzyme systems which cata lyze the mul t ip l e sequence of react ions leading to the - 16 -synthes i s of c e l l u l o s e are arranged as a multienzyme complex, t r aver s ing the plasma membrane, the per ip lasmic space, and the outer LPS l a y e r . Since a large number of glucan chains cons t i tu te the m i c r o f i b r i l , and each chain i s supposed to have an independent terminus i n the b a c t e r i a l envelope, a mult i subunit enzyme complex would be expected to p a r t i c i p a t e i n the simultaneous synthesis of a number of glucan cha ins . The glucose polymerizing capaci ty wi thin each subunit w i l l , i n t u r n , depend on the a v a i l a b i l i t y of substrates and on the phys ica l i n t e g r i t y and proper s p a t i a l arrangement of i t s d iverse enzymatic and c a r r i e r components. The cooperative ac t ion of the mult i tude of synthes iz ing uni t s thus cons t i tu te s the o v e r a l l c e l l u l o s e - s y n t h e s i z i n g capaci ty of the c e l l . b. E x t r a c e l l u l a r processes In the foregoing , the i n t r a c e l l u l a r transformation of glucose from a free molecule to a part of a polyglucosan has been descr ibed . In the l a s t decade, i t has also become c l e a r how the polymer i s transported to outs ide the c e l l . C o l v i n and Leppard (1977) ind ica ted how the assembly of polymers may form the m i c r o f i b r i l . In /\ . xylinum, c r y s t a l l i n e m i c r o f i b r i l s approximately 3.5 nm i n diameter are assembled i n a s soc ia t ion with rows of p a r t i c l e s (presumed to be groups of multienzyme complexes) that are s i tua ted below extrus ion pores i n the LPS l ayer of the bacterium (Brown ^ t a l . , 1976; C o l v i n and Leppard, 1977; Zaar, 1979). These m i c r o f i b r i l s a s soc ia te , by hydrogen bonding, in to bundles that i n turn form a twisted r ibbon . D a r k - f i e l d l i g h t microscopy has shown that the b a c t e r i a l c e l l turns on i t s axis as i t i s prope l led - 17 -forward by the elongating r ibbon. Ribbons from many bac te r i a interwine into a tough p e l l i c l e of c e l l u l o s e on the surface of the cu l ture medium. The composite ribbon i s analogous to m i c r o f i b r i l s i n walls of other c e l l u l o s e synthes iz ing organisms (Brown et ^ 1 . , 1976). Ribbon extrus ion was f i lmed by using frame by frame a n a l y s i s , and an extrusion rate of 1x10 8 glucose res idues/h was c a l c u l a t e d . In addi-t i o n , i t was observed that the bac te r i a rotate about the l o n g i t u d i n a l axis (Brown, 1981). The important conclus ion that emerged from these s tudies was that m i c r o f i b r i l s which comprise the ribbon were synthesized i n a row p a r a l l e l to the l o n g i t u d i n a l axis of the b a c t e r i a l c e l l (not from the poles of the b a c t e r i a l c e l l as previous ly reported by o t h e r s ) . Haig ler et a l . (1980) showed that d i r e c t dyes and f luorescent br ightening agents a l t e r the _in vivo assembly of c e l l u l o s e r ibbons . ^ . xylinum normally produces a highly c r y s t a l l i n e , e x t r a c e l l u l a r r ibbon of c e l l u l o s e . A v a r i e t y of compounds used as i n d u s t r i a l dyes and b r i g h t -eners for c e l l u l o s i c products and as b i o l o g i c a l s ta ins for c e l l u l o s e bind to the subunits of the ribbon as they are synthesized _in v i v o , prevent t h e i r normal aggregation, and r a d i c a l l y a l t e r the morphology and c r y s t a l l i n i t y of c e l l u l o s e . Most of the dyes and br ighteners found so far to a l t e r c e l l u l o s e assembly are planar de r iva t ive s of t r a n s - s t i l b e n e with subst i tuents capable of hydrogen bonding with the hydroxyl groups groups of l i n e a r 6 (1,4) po lysacchar ides . S i m i l a r l y , carboxy methylce l-l u l o s e (CMC) of varying degrees of polymerizat ion and s u b s t i t u t i o n have been shown to a l t e r c e l l u l o s e assembly by A. xylinum but at a higher - 18 -l e v e l of organizat ion than the dyes and br ighteners . Experimental use of these compounds which in te r rup t c e l l u l o s e assembly at d i f f e r e n t l e v e l s , had allowed the d i r e c t i n v e s t i g a t i o n of the r e l a t i o n s h i p between glucan chain po lymer iza t ion , m i c r o f i b r i l c r y s t a l l i z a t i o n , and f i b r i l assembly i n the biogenesis of c e l l u l o s e I . From these s tud ies , Haig ler e t . a l . (1980) have proposed that polymerizat ion and c r y s t a l l i z a t i o n are coupled processes which can be experimental ly separated _in v i v o , and that biogenesis of c e l l u l o s e I f i b r i l s occurred by a c e l l d i r e c t e d , s e l f assembly process i n / \ . xyl inum. The p o s s i b i l i t y that nat ive c e l l u l o s e can be a l te red provides a mechanism to c o n t r o l c r y s t a l l i t e s i z e . C r y s t a l l i t e s obtained on drying of Ca lcof luor- induced c e l l u l o s e vary i n s i ze depending on the i n i t i a l concentrat ion of Ca lco f luor used to induce the a l t e r a t i o n , a normal 69 A° c r y s t a l l i t e resu l ted when 0.025% Ca lco f luor i s used and a 28 °A c r y s t a l l i t e s re su l ted when 0.1-0.5% Ca lco f luor was used (Haig ler et a l . , 1980; Benziman et a l . , 1980). Brown (1981) reviewed the stages of c e l l u l o s e synthes i s , using the basic model prev ious ly presented by Haig ler et a l . (1980). The process i n general was summarized i n the fo l lowing stages: a. Polymerizat ion to form p-1-4 glucans . b. P a r a l l e l glucan chains produce non-dis soc iable ordered aggre-gates (12 to 15 glucan c h a i n s ) . c . Adjacent glucan ordered aggregates c r y s t a l l i z e in to micro-f i b r i l s (3.5 nm c r y s t a l l i n e elementary m i c r o f i b r i l ) . - 19 -d . M i c r o f i b r i l s from discontinuous segments of extrusion channels aggregate into bundles (6.5 to 7.5 nm). e. Bundles of m i c r o f i b r i l s aggregate to form the s ing le twi s t ing r ibbon . f . D i r e c t i o n of ribbon synthesis i s po la r i zed but can be reversed (the ribbon always forms p a r a l l e l to the l o n g i t u d i n a l ax i s ; however, the p o l a r i t y of i t s synthesis can suddenly be reversed by p h y s i c a l s t r a i n during c e l l d i v i s i o n . g. C e l l - d i r e c t e d c r y s t a l l i z a t i o n i s rate l i m i t i n g . A diagram of the proposed model of c e l l u l o s e assembly i s presented i n F igure 1. B. I n d u s t r i a l Applications of B a c t e r i a l C e l l u l o s e The c e l l u l o s e p e l l i c l e s formed within a sugar- r i ch media by J\. xylinum are used i n the P h i l i p p i n e s as a food product (3esus et a l . , 1971; Lapuz et _ a l . , 1967). A de l i cacy among P h i l i p p i n o s , popular ly known as "na ta " , the c e l l u l o s e p e l l i c l e s are wel l l i k e d for desser t . Lapuz _et _al. (1967) determined the i d e a l condi t ions under which "nata" could be produced i n sugared, coconut-milk medium. This study opened a way of mass-producing "nata" for preservat ion i n heavy syrup and sa le i n the marketplace. The popular i ty of the product has not be confined to the l o c a l community. "Nata" i s gradual ly being introduced in to fore ign markets. It i s ava i l ab l e i n Canada at c e r t a i n Chinese r e t a i l s tore s . Oesus ^ t aL. (1971) reported the i s o l a t i o n and s e l e c t i o n of high-y i e l d i n g A. xylinum s t r a i n s from P h i l i p p i n e m i c r o f l o r a . A. xylinum was - 20 -Figure 1. Proposed model of normal c e l l u l o s e assembly. Glucan chain aggregates from organized multienzyme complexes and extrus ion pores c r y s t a l l i z e into m i c r o f i b r i l s , which then assemble into bundles and the normal ribbon at the c e l l surface (Brown, 1982). - 21 -a l so reported to form "nata" i n tomato j u i c e , c i t r u s j u i c e , coconut milk-sucrose medium and crushed pineapple (Alaban, 1962). Yamanaka j?t j l . (1979) patented a ge l - type dessert which was very s i m i l a r to the "nata" already produced by the P h i l i p p i n o s . B a c t e r i a l c e l l u l o s e produced by A. xylinum has a lso been u t i l i z e d by f i e l d s other than the food indus t ry . Correns et al^. (1972a) patented the production of c e l l u l o s e membranes s u i t a b l e for f i l t e r s from in tac t p e l l i c l e s produced by c u l t i v a t i n g /A. xylinum i n a nutr ient s o l u t i o n . A second patent (Correns ^ t _ a l . , 1972b) involved the production of membranes and sheets from b a c t e r i a l c e l l u l o s e based on conventional papermaking methods. Mynatt (1982) patented an apparatus and process for the product ion of polysaccharide ( ce l lu lo se ) f i b r e s for use i n paper manufacturing. In " F i b e r production from continuous c u l t i v a t i o n of micro-organisms", a method was described i n which f i b r e s were produced by harvest ing the l i b e r a t e d products of continuous micro-organism c u l t i v a t i o n . A s u i t a b l e microorganism such as Sphaerot i lus natans was grown on a p i t t e d m e t a l l i c p la te suppl ied with a f lowing nutr ient substrate . With subs tan t i a l p e l l i c l e growth, the nutr ient flow was hal ted temporari ly while a blade passed over the p late harvest ing the p e l l i c l e growth and depos i t ing the harvest products onto a s l u i c e conveyor. The blade was re t rac ted and the nutr ient flow restored u n t i l the p e l l i c l e growth again became abundant. The harvested products were further processed to remove undesirable n o n - c e l l u l o s i c mater ia l s depending upon the p a r t i c u l a r - 22 -microorganisms used. Thi s process and apparatus thus produces minute c e l l u l o s i c f i b r e s su i t ab le for paper making, thereby e l imina t ing the processing and d iges t ing of wood pulp . As an a l t e r n a t i v e , the apparatus might be modified for the growth of Acetobacter . Brown (1983) patented a method for the production of a c e l l u l o s e -synthet ic polymer composite f i b r e . Hydrophi l i c c h a r a c t e r i s t i c s were imparted to hydrophobic synthet ic substrates , such as polyester f i b r e s , by incubating a cu l ture medium with ^ . xylinum i n the presence of the substrate . Many synthet i c f i b r e s , while having exceedingly useful propert ies such as d u r a b i l i t y , permanent press , e t c . , s t i l l lack some of the phys ica l propert ies des i red i n a cotton f a b r i c . One of the most notable propert ies i s the h y d r o p h i l i c i t y of a cotton f i b r e . Brown's invent ion takes advantage of the c e l l u l o s e produced by Acetobacter i n that i t i s poss ib le to produce t h i s c e l l u l o s e on the surface of polyes ter f i b r e , thereby conferr ing to that f i b r e many of the phys i ca l propert ies of cotton f i b r e . The advantages of the c e l l u l o s e - s y n t h e t i c polymer composite were: a) greater h y d r o p h i l i c i t y and subsequent greater comfort i n wearing; b) greater absorbancy which may be a useful property for d i sposa l bandages, dres s ing , and the l i k e ; c) a na tura l b iosynthet ic react ion being coupled onto the surface of a synthet i c polymer as a substrate ; and d) the a l tered surface proper t ie s of the c e l l u l o s e synthet i c polymer composite might be advantageous for the adsorption of dyes and severa l other agents to the surface . The invent ion also provided a method of enhancing the h y d r o p h i l i c character-i s t i c s of h y d r o p h i l i c mater ia l s ( eg . , cotton or paper) by incubat ing A. - 23 -xylinum i n the presence of a na tura l mater ia l whereby c e l l u l o s e micro-f i b r i l s are produced on and attached to i t s surface . Su i tab le mater ia l s included cotton ( e . g . , to increase the h y d r o p h i l i c nature) and paper ( e . g . , to increase i t s s t rength) . The composite polymer produced according to Brown's invent ion with i t s unique phys i ca l proper t ie s provided a whole new approach to the manufacture of " c o t t o n - l i k e " goods. C. Microcrystalline Cellulose M i c r o c r y s t a l l i n e c e l l u l o s e (MCC) i s one of the few new ingred ient s ava i l ab le to formulators in the l a s t two decades. The f i r s t commercial quant i t i e s were produced i n 1962; however, MCC did not become a f ac tor i n food s t a b i l i z a t i o n u n t i l very l a te i n the 1960's. Although the o r i g i n a l MCC product was designed for use as a bulking agent i n low c a l o r i e foods, these foods have never reached t h e i r expected p o t e n t i a l due to the d i f f i c u l t y i n obta ining a d d i t i o n a l non-ca lo r i c ingredients acceptable for use i n foods. By f a r , the major uses of ed ib le MCC are i n pharmaceutical t a b l e t i n g and i n the s t a b i l i z a t i o n of foods (FMC Corporat ion , B u l l e t i n G-34). M y c r o c r y s t a l l i n e c e l l u l o s e i s a p u r i f i e d , n a t u r a l l y occurr ing ^ - c e l l u l o s e . I t i s a l i n e a r polymer formed by p-1,4 l inked glucose u n i t s , i s completely in so lub le i n water but disperses i n water to form c o l l o i d a l so lu t ions and white opaque g e l s . MCC i s not a chemical d e r i -v a t i v e , but i t i s produced by convert ing f ibrous c e l l u l o s e to c r y s t a l -l i n e c e l l u l o s e or a r e d i s p e r s i b l e g e l . This i s accomplished by a simple ac id h y d r o l y s i s , drying and/or co-process ing with var ious h y d r o p h i l i c - 24 -d i sper s ions . The raw mater ia l for MCC i s " - c e l l u l o s e . Wood contains about 40-50% " - c e l l u l o s e , but the purest natural source of c e l l u l o s e i s cotton f i b e r s or l i n t e r s , which on a dry basis cons i s t of about 98% " - c e l l u l o s e . To date, these two have been the most important commercial sources for raw mater ia l c e l l u l o s e ( B a t t i s t a , 1975). Part of the research of t h i s thes i s points towards the p o t e n t i a l a p p l i c a t i o n of _A. xylinum c e l l u l o s e as a raw mater ia l i n the production of MCC. B a c t e r i a l c e l l u l o s e has been shown to be native c r y s t a l l i n e (type I) c e l l u l o s e , l i k e the " - c e l l u l o s e of co t ton . Furthermore, analy-t i c a l recoveries of D-glucose from hydrolysates of A*, xylinum c e l l u l o s e and cotton are within the same range (nearly 90% of theore t i c value) i n d i c a t i n g the high degree of pur i ty of both mater ia l s (Whi s t l e r , 1963). Although cotton i s the purest form of n a t u r a l l y occurr ing c e l l u -lo se , i t contains severa l impur i t i e s such as wax, pec t ins , and co lour ing matter. Any method of p u r i f i c a t i o n must aim at removing these impuri-t i e s under condi t ions which w i l l not bring about any fundamental change i n the c e l l u l o s e s t ruc ture . I f the condi t ions of p u r i f i c a t i o n are not r i gorous ly c o n t r o l l e d , considerable degradation of the c e l l u l o s e occurs as indica ted by reduced v i s c o s i t y and " - c e l l u l o s e content . This i s p a r t i c u l a r l y true in the case of wood c e l l u l o s e , which must be subjected to a d e l i g n i f i c a t i o n treatment. Degradation of the c e l l u l o s e always occurs during d e l i g n i f i c a t i o n , and i t seems impossible to i s o l a t e a wood c e l l u l o s e with the same degree of polymerizat ion as that of the nat ive wood or plant (Whis t l e r , 1963). The degree of polymerizat ion (DP) of the raw mater ia l i s extremely important s ince i t i s the i n i t i a l DP which w i l l determine the f i n a l - 25 -l e v e l - o f f DP (or LODP) and the c r y s t a l l i n e residue upon h y d r o l y s i s . The LODP value and the c r y s t a l l i n e res idue are higher when the i n t i a l DP value i s higher (Magister ^ t _ a l . , 1975). The commercially ava i l ab l e m i c r o c r y s t a l l i n e c e l l u l o s e comes as a white , f ine powder which i s low i n ash, metals , and so luble organic mater i a l s . I t i s i n s o l u b l e i n water, d i l u t e a c i d , common organic so lvent s , and o i l s . I t i s p a r t i a l l y so lub le , with some swel l ing i n d i l u t e a l k a l i . One of the most important of these products i s a micro-c r y s t a l l i n e c e l l u l o s e sold under the trade name of A v i c e l . I t i s prepared by acid treatment of « - c e l l u l o s e (from wood) under s p e c i a l processing c o n d i t i o n s , as d i s c lo sed by the patent of B a t t i s t a and Smith (1961). By c o n t r o l l e d hydro ly s i s with hydrochlor ic a c i d , ^ - c e l l u l o s e i s converted to two components - an ac id - so lub le f r a c t i o n and an ac id -inso lub le f r a c t i o n . The a c i d - i n s o l u b l e c r y s t a l l i n e residue i s washed and separated. I t i s c a l l e d c e l l u l o s e c r y s t a l l i n e mater ia l or MCC. E s s e n t i a l l y , the amorphous regions of the polymer are hydrolyzed completely , leav ing the c r y s t a l l i n e regions as i s o l a t e d m i c r o c r y s t a l -l i t e s which are defined as the l e v e l - o f f degree of polymerizat ion or LODP c e l l u l o s e ( B a t t i s t a , 1950). In other words, i f the h y d r o l y s i s react ion were cont inued, the degree of polymerizat ion would not change, i n d i c a t i n g that the l e v e l period or l i m i t of r e a c t i v i t y , has been reached. The reported l e v e l - o f f DP cons i s t s of 15-375 anhydroglucose un i t s depending on the i n i t i a l DP of the raw mater i a l ; the const i tuent chains of each aggregate being separate from those of neighboring - 26 -aggregates. These aggregates are character ized by sharp X-ray d i f f r a c -t i o n patterns i n d i c a t i v e of a s u b s t a n t i a l l y c r y s t a l l i n e s t ruc ture ( B a t t i s t a and Smith, 1962, 1965). Acid hydro ly s i s i s followed by mechanical a g i t a t i o n i n a water s l u r r y to free a f r a c t i o n of the unhinged c r y s t a l s . The s l u r r y i s subsequently f i l t e r e d and n e u t r a l i z e d . The r e s u l t i n g f i l t e r cake i s r e s l u r r i e d and the homogeneous aqueous s l u r r y i s spray dr ied to give a white, f ree- f lowing powder cons i s t ing of completely unhinged or d i scon-nected but aggregated m i c r o c r y s t a l s . A dry MCC which i s more conven-i e n t l y redispersed to a s tab le t h i x o t r o p i c ge l can be produced by blending the f i l t e r e d wet cake (40% so l id s ) with a h y d r o p h i l i c b a r r i e r such as CMC. This i s done i n order to homogenize the h y d r o p h i l i c gum throughout the system so that the free microcrys ta l s are coated with a f i l m of the water -d i sper s ib le b a r r i e r . The homogenized mix i s then e i t h e r drum-dried and granulated i n a hammer m i l l or spray d r i e d . These commercial grades of MCC are r e a d i l y d i s p e r s i b l e i n water ( B a t t i s t a , 1975). M u l t i p l e uses of d i f f e r e n t grades of MCC have been l i s t e d by B a t t i s t a (1975). In t h e i r nonfibrous and ge l forms, MCC's have opened up major new app l i ca t ions for pure c e l l u l o s e s never before a v a i l a b l e for commercial use. The various grades of MCC have been engineered to contr ibute unique and useful propert ies to a wide spectrum of commercial products . For example, the regular pharmaceutical grade serves as a pharmaceutical t ab le t b inder . Other grades go into frozen desserts to c o n t r o l i ce c r y s t a l growth. C o l l o i d a l m i c r o c r y s t a l l i n e powders are - 27 -incorporated as rheology c o n t r o l agents i n foods where they may be used as temperature- insens i t ive thickeners for salad dress ings , ho l l anda i se sauces, e tc . One of the f i r s t major commercial uses for MCC was as a non-ca lor i c ingredient for c o n t r o l l i n g the c a l o r i c content of foods, e s p e c i a l l y of fat- loaded foods. I n d u s t r i a l uses of c e l l u l o s e micro-c r y s t a l s as rheology-contro l agents are also being developed, although they have not yet received as much commercial izat ion as well-proven food and pharmaceutical uses. The food grade of m i c r o c r y s t a l l i n e c e l l u l o s e i s "genera l ly recognized as safe" in the Food and Drug Act (FDA) (1958) of the United States and the ADI for man i s "no l i m i t " (WHO, 1974). - 28 -MATERIALS AND METHODS A. Test Organisms The cu l ture s i n t h i s study were obtained i n the freeze dr i ed s tate from the American Type Cul ture C o l l e c t i o n and the Nat ional I n s t i t u t e of Science and Technology, Mani la , P h i l i p p i n e s . Acetobacter xylinum ATCC 10821, ATCC 14851 and the P h i l i p p i n e s t r a i n were resuspended i n the fo l lowing medium (% w/v) : 2% glucose , 0.1% potassium phosphate, 0.05% magnesium s u l f a t e , 0.15% sodium c h l o r i d e , 0.5% peptone and 0.25% yeast ex t rac t , adjusted to pH 5. The cu l ture s were grown i n an incubator at a temperature of 28 to 3 0 ° C . B. Cultivation Method 1. Cul ture propagation and cond i t ions Erlenmeyer f l a sks (250 mL) each conta ining 100 mL of the appropriate medium and equipped with cotton plugs were inocula ted with 10% (v/v) inoculum. Cul tures were l e f t undistrubed on the bench at 28 to 3 0 ° C and subcultured every two weeks. To ensure the maintenance of a pure c u l t u r e , the fo l lowing procedure was used (Cook and C o l v i n , 1980). Cultures were d i l u t e d by a f ac tor of 101*; 0.1 mL of the d i l u t e d c u l t u r e was spread uniformly on agar surfaces i n 10 cm polystyrene P e t r i p la tes (1.5 g agar d i s so lved i n 100 mL of the medium previous ly descr ibed) . Af ter incubat ion of the P e t r i p lates for 4 to 7 days at 28 to 3 0 ° C , two r e p l i c a s of each p la te were made using the same agar medium and incu-bated for 4-7 days at 28 to 3 0 ° C . The incubated plates were sprayed - 29 -with a so lu t ion of Ca lco f luor White M2R New disodium s a l t of 4 , 4 ' - b i s [ 4 - a n i l i n o - 6 - b i s ( 2 - h y d r o x y e t h y l ) a m i n o - s - t r i a z i n - 2 - y l a m i n o ] - 2 - 2 ' - s t i l b e n e d i s u l f o n i c ac id which f luoresces i n the presence of c e l l u l o s e . A stock so lu t ion was prepared with 0 . 1 g of Ca lco f luor in 5 mL of water with 5 mL of 0 . 2 M Na2HP0it added. For spraying, 0 . 1 mL of the stock s o l u t i o n was d i l u t e d into 10 mL of Na2HP0i t . Colonies that produced c e l l u l o s e i n normal quanti ty f luoresced b r i g h t l y when examined with u l t r a v i o l e t l i g h t , wavelength 366 nm, while those co lonies which produced less c e l l u l o s e were markedly darker . C e l l u l o s e producing co lon ie s were picked of f the extra undisturbed r e p l i c a , streaked on agar surfaces to ensure that pure cu l ture s were i s o l a t e d , and then t rans ferred to agar s lopes . Cultures once grown were e i ther stored under r e f r i g e r a t i o n (stock cul tures ) or i n the incubator , but i n e i ther case they were subcultured every 1 to 2 months. 2 . S e l e c t i o n of i n f l u e n c i a l f a c tor s for maximum c e l l u l o s e production i n  defined medium Erlermeyer f l a sks (250 mL) each conta ining 100 mL of n u t r i e n t media and provided with cotton plugs were inoculated with 10% (v/v) of adapted cu l ture ( P h i l i p p i n e s t r a i n ) . To ensure adaptation to each nut r i ent medium mixture the organism was grown through two s e r i a l subcultures i n each mixture before the appropriate f l a sks were inocu-l a t e d . Dupl ica te cu l tures were incubated at 2 8 - 3 0 ° C for 1 week. The F r a c t i o n a l F a c t o r i a l design L 2 7 ( 3 1 3 ) of Taguchi ( 1957) was used to se lec t the f ac tor s which may s i g n i f i c a n t l y af fect c e l l u l o s e product ion, namely, sucrose and peptone concentrat ions and pH, as wel l as poss ib le - 30 -i n t e r a c t i o n s . The nutr ient media composition (Lapuz jst _a l . , 1967) was as fo l lows : sucrose (at 3 l e v e l s , 4, 6 and 10%), peptone (at 3 l e v e l s , 0 .30, 0.50 and 1.23%), 0.1% potassium phosphate, 0.05% magnesium s u l f a t e , 0.15% sodium c h l o r i d e , and 0.25% yeast e x t r a c t . The pH was adjusted with ace t i c ac id at 3 d i f f e r e n t l e v e l s , 4 .0 , 4.5 and 5 .5 . Af ter one week of incubat ion , the c u l t u r e f l u i d was decanted and the p e l l i c l e s were l e f t i n the f l a s k s . P e l l i c l e s were thoroughly washed with repeated changes of d i s t i l l e d water over an 8 h p e r i o d . Mercuric c h l o r i d e so lu t ion (1 mL, 10% w/v) was added to the f i r s t changes of water to prevent the growth of contaminants (Dudman, 1959a). At t h i s stage i t was assumed that the p e l l i c l e s contained only c e l l u l o s e plus organisms. Next, 100 mL of 8% (w/v) NaOH were added to the f l a sks and shaken overnight to ensure complete d i s s o l u t i o n of the c e l l u l a r mater ia l from the p e l l i c l e . To determine c e l l u l o s e content , the a l k a l i - e x t r a c t e d p e l l i c l e s were washed f i r s t with d i l u t e ace t i c ac id (1% v/v) u n t i l they remained acid for severa l hours, as ind ica ted by the add i t ion of methyl red , and then with repeated changes of d i s t i l l e d water to remove the a c i d . The c e l l u l o s e p e l l i c l e s were hung on glass rods , allowed to dra in fo r 8 h , and then dr ied to constant weight at 1 0 5 ° C i n tared c r u c i b l e s (Dudman, 1959a). Data c o l l e c t e d from the 27 nutr ient media were analyzed using a F a c t o r i a l Ana ly s i s of Variance Taguchi ' s L27 ( 3 ) 1 3 program wri t ten for a Monroe c a l c u l a t o r (Model 1880, Monroe, Orange, NO). - 31 -C. C u l t u r e S t a b i l i t y C e l l u l o s e producing co lon ie s of ATCC 14851, ATCC 10821 and the P h i l i p p i n e s t r a i n were propagated i n the fo l lowing defined medium: 10% sucrose, 0.5% peptone, 0.1% potassium phosphate, 0.05% magnesium s u l f a t e , 0.15% sodium c h l o r i d e , 0.5% peptone and 0.25% yeast ex t rac t , adjusted to pH 4 .5 . S t a t i c and swir led cu l ture s were incubated at 2 8 - 3 0 ° C . Swirled c u l t u r e s i n 250 mL Erlenmeyer f l a sks were incubated i n a New Brunswick Psychrotherm incubator at 150 rpm. After 48 h incuba t ion , cu l ture s were t rans fer red to fresh media at a 10% v/v inoculum l e v e l . This t rans fer was designated t rans fer 1. Subsequent t rans fer s were done every 48 h . At each t r a n s f e r , the cu l ture s were d i l u t e d and spread on agar surfaces i n 10 cm P e t r i p lates (1.5 g agar d i s so lved i n 100 mL of the medium descr ibed above). Af ter 6-7 days growth, they were sprayed with Ca lco f luor and the numbers of c e l l u l o s e d e f i c i e n t (mutants) and normal (wild) co lonies were determined. The percentage of the co lonies found to be d e f i c i e n t was c a l c u l a t e d . Colonies were observed with respect to co lour , opac i ty , form, e leva t ion and margin under a Steromicroscope (Model Wild M3, Wild Heerbrugg, Swi tzer land) . C e l l u l o s e d e f i c i e n t co lonies were i s o l a t e d from r e p l i c a s and maintained on s lopes . C e l l u l o s e d e f i c i e n t s t r a i n s as w e l l as the wi ld type were t rans ferred to l i q u i d medium and allowed to grow for 2 weeks at 2 8 - 3 0 ° C under s t a t i c c o n d i t i o n s . Cultures of c e l l u -lose d e f i c i e n t co lonies were tested for p e l l i c l e formation by v i s u a l comparison with cu l tures of the wi ld type . - 32 -0 . Growth Curves Cul tures were grown i n 100 mL of defined medium i n 150 mL f l a s k s , under s t a t i c condi t ions at 2 8 - 3 0 ° C . Dupl icates were removed from the incubator and analyzed at growth i n t e r v a l s of 0, 4, 8, 12, 16, 20, 30 and 40 days. At the time of a n a l y s i s , the cu l ture f l u i d was decanted for pH measurement and r e s i d u a l sugar determinat ion. The p e l l i c l e s were l e f t i n the f l a sks and washed with repeated changes of d i s t i l l e d water. Mercuric c h l o r i d e so lu t ion (1 mL, 10% w/v) was added to prevent the growth of contaminants. 1. pH measurement The pH of the c u l t u r e f l u i d was determined immediately a f ter sampling using a pH meter (Accumet Model 230, F i sher S c i e n t i f i c C o . , P i t t s b u r g h , PA) . 2. Nitrogen determination ( c e l l - n i t r o g e n ) Washed p e l l i c l e s were weighed i n the f l a sks and an equal volume of 2N NaOH was added. The f l a sks were stoppered and shaken overnight . Nitrogen determinations were c a r r i e d out on 1 mL samples of the a l k a l i n e ext rac t s ; from these r e s u l t s , the nitrogen content of the p e l l i c l e s was ca l cu l a ted (Dudman, 1959a). Nitrogen was determined i n dup l i ca te according to the rapid micro-Kje ldahl method of Concon and Sol tes s (1973). The amount of ni trogen i n each digested sample was determined using an Auto Analyser II (Technicon Instruments C o r p . , Tarrytown, NY). A l l ni trogen analyses were ca l cu l a ted on a dry ba s i s . - 33 -3. C e l l u l o s e determination To determine c e l l u l o s e content , the a l k a l i - e x t r a c t e d p e l l i c l e s were washed f i r s t with d i l u t e a c e t i c ac id (1% v/v) u n t i l they remained acid as demonstrated by the addi t ion of methyl red i n d i c a t o r , and then with repeated changes of d i s t i l l e d water to remove the a c i d . The c e l l u l o s e p e l l i c l e s were allowed to dra in for 8 h and then dr ied to constant weight at 1 0 5 ° C (Dudman, 1959a). 4 . Sugar u t i l i z a t i o n and convers ion At the time the c u l t u r e f l u i d was decanted for r e s i d u a l sugar determination for the d i f f e r e n t growth i n t e r v a l s , samples were f i l t e r -s t e r i l i z e d with a M i l l i p o r e membrane (0.22 \im) to remove organisms. T o t a l carbohydrate analyses of the organism-free cu l ture f l u i d s were determined by the p h e n o l - s u l f u r i c acid method of Dubois et _al. (1956). Absorbance was determined at 490 nm on a Beckman Spectrophotometer (Model DB, Beckman Instruments I n c . , F u l l e r t o n , C A . ) . T r i p l i c a t e analyses were c a r r i e d out on dup l i ca te c u l t u r e s . T o t a l carbohydrate content was estimated from a standard curve for sucrose. The sugar u t i l i z e d by the cu l ture s was c a l c u l a t e d by d i f ference between the re s idua l sugar found and at i n i t i a l l y present i n the medium (day 0 ) . Percent t o t a l sugar conversion to c e l l u l o s e ("conversion of t o t a l sugar") and percent of the sugar u t i l i z e d by the cu l ture ("conversion of u t i l i z e d sugar") to c e l l u l o s e was ca l cu la ted by comparing the a c t u a l , corrected c e l l u l o s e y i e l d to the number of grams of t o t a l or u t i l i z e d sugar i n 100 mL sample and expressing t h i s as a percentage. - 34 -E. Average Degree of Polymerization (DP) vs. Incubation Time Stock cu l ture s of ATCC 14851 and the P h i l i p p i n e s t r a i n were propa-gated in defined medium for 48 h at 2 8 - 3 0 ° C . After 48 h of incuba t ion , cu l ture s were t rans ferred to fresh media at a 10% (v/v) inoculum l e v e l , and l e f t undisturbed. Dupl icate cu l tures were removed at growth i n t e r -va l s of 5, 8, 11, 13, 15, 20, and 32 days. At the time of harvest , the cu l ture f l u i d was decanted, the p e l l i c l e s were washed with d i s t i l l e d water and shredded i n a Waring Blendor regulated with a Var i ab le Autotransformer (600 rpm). The shredded f ib re s were passed through severa l l ayers of cheesec loth , the f r a c t i o n on the c l o t h was scraped o f f , resuspended i n water, and c o l l e c t e d by cent r i fuga t ion (9000 x g for 15 min) . Thi s process was repeated severa l times u n t i l centr i fugates were solute f r e e . Then, the f i b r e s were immersed i n 2% (v/v) NaOH s o l u t i o n for 3 h to remove a l k a l i - s o l u b l e components and neut ra l i zed with 1% (v/v) ace t i c acid (Takai et _a l . , 1975). F i n a l l y , they were washed exhaust ively with large volumes of d i s t i l l e d water and freeze d r i e d . The degree of polymerizat ion (DP) for the p u r i f i e d c e l l u l o s e was measured by the Cupriethylenediamine disperse v i s c o s i t y pipet method (Cannon-Fenske Viscometer) for pulp (Tappi , 1966). The Cupriethylendiamine disperse v i s c o s i t y method made use of two cupriethylenediamine s o l u t i o n s : 0.167 M in copper and 1.0 M in copper. An amount of freeze dr ied sample equivalent to 0.125 g of moisture-free c e l l u l o s e was weighed and placed i n the d i s s o l v i n g b o t t l e ( t h i s was c a l c u l a t e d by determining the moisture on a separate p o r t i o n ) . The bot t l e s conta ining the two Cu ( E n ) 2 so lut ions were connected to 25 mL - 35 -side-arm Mohr buret tes . The burettes were f i l l e d by maintaining a 2 p s i nitrogen pressure on the b o t t l e s . 15 mL of 0.167 M so lu t ion were added to the d i s s o l v i n g b o t t l e , taking care that the c e l l u l o s e was thoroughly wetted. Then, 10 mL of 1.0 M so lu t ion were added, the b o t t l e was f lushed with a stream of ni trogen for at l ea s t 15 s, and quick ly capped. The bo t t l e was shaken for 15 min to disperse the c e l l u l o s e completely. At the end of the shaking, the so lu t ion was poured in to the v i s c o s i t y tube and placed i n a water bath maintained at 2 5 ° C . The e f f lux time was determined i n d u p l i c a t e s . V i s c o s i t y ( in m P a « s ) was ca l cu la ted with the formula: V=Ctd, in which C i s the viscometer constant (C2oo =0-1 a r , d C3oo=0.25), t i s the time i n seconds and d i s the density of the c e l l u l o s e so lu t ion (d=1.052 g/mL). Once the v i s c o s i t y measurement was obtained, the approximate DP was deduced from nomogram tables (Rydholm, 1965). F. Cellulosic Fibre Production and Characterization 1. Desc r ip t ion of the F i b r e Production Apparatus Acetobacter xylinum cu l ture s were d i rec ted to grow within a confined u n i d i r e c t i o n a l flow of s t e r i l e medium. This was accomplished i n pre l iminary experiments by growing the cu l ture in the apparatus depicted i n Figure 2. As shown i n t h i s f i g u r e , the apparatus included two 2 L Erlenmeyer f l a sks stoppered with cotton plugs connected with a s lop ing glass tube (29.5 cm long and 4.5 cm ins ide diameter) provided with ground glass 24/40 j o i n t s at both ends. The s loping glass tube provided a growing surface along a s t r a ight l i n e path where the - 36 -P U M P Figure 2. F i b r e product ion apparatus (pre l iminary de s i gn ) . - 37 -c e l l u l o s e f i b r e s were deposi ted. The flowing inoculated medium was c i r c u l a t e d with a flow rate of 10 L/min by a March O r b i t a l Magnetic Dr ive pump (Model MDX-3, March Mfg. I n c . , IL) regulated with a v a r i a b l e autotransformer. The f l u i d i n the Erlenmeyer f l a sks was s t i r r e d with magnetic s t i r r i n g bars to prevent p e l l i c l e formation i n the f l a s k s . The apparatus was placed i n an incubator room at 2 8 - 3 0 ° C . Based on the same p r i n c i p l e of u n i d i r e c t i o n a l flow (Townsley, 1981; personal communication), a second apparatus was designed which provided a l a rger growing surface along a s t r a i g h t - l i n e path. A side view of the apparatus diagram i s shown i n Figure 3. In t h i s apparatus, the s t e r i l e chamber was provided by a tank (58 cm long , 9.5 cm high and 35 cm wide) constructed of polycarbonate sheet (12.7, 9.2 and 6.3 mm t h i c k for bottom, s ide wal ls and top , r e s p e c t i v e l y ) . The bottom part of the tank was provided with a rubber gasket f i t t e d in s ide a groove carved along the edges of the s ide w a l l s , which allowed a t i g h t sea l for the cover . The growing surface was a ramp (50 cm long and 35 cm wide) pro-vided with a number of V-shaped grooves or channels with s ides approxi-mately 3 mm long (Figures 4 and 5 ) . The ramp was i n c l i n e d s l i g h t l y ( 7 ° ) so that growing medium placed at the top of the ramp adjacent the grooves flowed slowly down (flow rate of 12.2 L/min) the grooves to the bottom. The growing medium was then c o l l e c t e d and r e c i r c u l a t e d to the top of the ramp by a March O r b i t a l Magnetic Drive Pump (Model MDX-3, March Mfg. I n c . , IL) regulated with a v a r i a b l e autotransformer. The pH of the growing medium was c o n t r o l l e d at 4.5 by a pH c o n t r o l system Speedomax H (Model 300, Leed and Northrup C o . , North Wales, PA) provided with a pH e lectrode which was placed i n a glass j a r through which - 38 -Figure 3 . F i b r e production apparatus ( s ide view) . - 39 -Figure Grooves or channels i n the f i b r e production apparatus. - 40 F i g u r e 5. Ramp with carved grooves or channels prov id ing a s t r a i g h t l i n e flow path. - 41 -growing medium c i r c u l a t e d i n and out (Figure 6 ) . 2N NaOH was fed in to the tank by a p e r i s t a l t i c pump (Model 1201, Harvard Apparatus Co. I n c . , M i l l i s , MA) at an average flow rate of 0.42 mL/min (Figure 7 ) . Inter-mittent flow was accomplished by means of a Paragon timer (15 min s e t t i n g c a p a b i l i t y up to 48 h o n / o f f , AMF Paragon E l e c t r i c C o . , Guelph, ON). In a d d i t i o n , f i l t e r s t e r i l i z e d a i r was sparged through the tank at a rate of 0.04 - 0.28 L a i r / L medium/min (Figure 8 ) . A i r was suppl ied by an a i r compressor (Webster C o . , London, ON) connected to a flow meter (model No. 3, Gilmont Instruments I n c . , Great Neck, NY). The flow meter was c a l i b r a t e d by Gilmont and a graph was supplied to obtain the proper readings by i n t e r p o l a t i o n . The apparatus was located i n an incubator room maintained at 2 8 - 3 0 ° C . The polycarbonate apparatus with i t s tubing connections and a i r f i l t e r were s t e r i l i z e d i n an autoclave at 1 2 0 ° C and 15 ps i g for 45 min. The pH e lectrode was s t e r i l i z e d by f lu sh ing i t with Cry-Oxide ethylene oxide (11% ac t ive ingred ient , AMSC0) overn ight . I t was then a s e p t i c a l l y attached to the tubing connected to the po lycar-bonate apparatus. 2 . Process for the production of f i b r e s S t e r i l i z e d defined medium (4.5 L) inoculated with 10% (v/v) inoculum were dispensed in to the s t e r i l e polycarbonate tank. The growing medium was c i r c u l a t e d through the ramp for 4 d with a flow rate of 12.2 L/min e i t h e r i n t e r m i t t e n t l y or cont inuous ly . In pre l iminary experiments, a f l a t wooden ramp was used as a growing surface for the product ion of f i b r e s by two s t r a i n s of / \ . xylinum ATCC 14851 and the P h i l i p p i n e s t r a i n . Both s t r a i n s were tested for t h e i r a b i l i t y to - 42 -F i g u r e 6. pH c o n t r o l l e r Speedomax provided with a pH e l e c t r o d e . - 43 -Figure 7. Polycarbonate chamber provided with a i r f i l t e r , cotton plug ( a i r vent ) , and NaOH feeder. F i g u r e 8. Polycarbonate cover with a i r f i l t e r a t tached. - 45 -produce c e l l u l o s e under these p a r t i c u l a r c o n d i t i o n s . C e l l u l o s i c f i b r e s were observed for uniformity of growth over the wooden ramp for the two organisms. Since i t was observed that continuous or in termi t tent flow determined the r i g i d i t y of the f i b r e s at harvest , p a r t i c u l a r l y for one of the s t r a i n s ( P h i l i p p i n e s t r a i n ) , i t was necessary to te s t the t e n s i l e strength of the f i b r e s produced under the fo l lowing c o n d i t i o n s : c o n t i n -uous flow and in termi t tent flow (1 h on/1 h o f f ) . Four t r i a l s were required for t h i s t e s t , incubat ion of ATCC 14851 and the P h i l i p p i n e s t r a i n with continuous flow and with one hour flow at one hour i n t e r -v a l s . A i r was sparged through the apparatus at a rate of 0.04 L a i r / L medium/min. The cu l ture s were incubated for four days at 2 8 - 3 0 ° C for each t r i a l . C e l l u l o s e was harvested, washed with several changes of water to remove remaining c u l t u r e f l u i d , and dipped into a 10% (v/v) g l y c e r o l so lu t ion for 1 h . This procedure was adopted i n attempts to e l iminate b r i t t l e n e s s upon drying of the c e l l u l o s e f i b r e s . C e l l u l o s e was cut into bone shaped pieces 5 cm long , 3 cm wide (widest parts) and 2 cm wide (narrowest center part) and d r i e d i n a vacuum oven at 6 0 ° C for 2.5 h (40% moisture wet b a s i s ) . T r i p l i c a t e samples from each treatment were subjected to t e n s i l e s trength determinat ion. Fol lowing t h i s experiment, in te rmi t tent flow (1 h on/1 h off) was adopted for fur ther t r i a l s . In a d d i t i o n , ATCC 14851 s t r a i n was se lected for further s tud ie s . The e f fect of rate of aerat ion on growth and c e l l u l o s e y i e l d s was examined with cu l ture s grown as described above. Cultures were a l l grown under the same condi t ions with the exception that they were aerated at d i f f e r e n t rates (0.040, 0.102 and 0.282 L - 46 -a i r / L media/min). Sugar u t i l i z a t i o n , sugar convers ion, c e l l u l o s e and pH were determined fo l lowing the methods previous ly described for the s t a t i c c u l t u r e s . Samples (50 mL) were a s e p t i c a l l y removed d a i l y for sugar analyses and pH determinat ion. At the end of the incubat ion per iod , 25 mL samples were placed in to a vesse l cup used to measure the d i s so lved oxygen. This vesse l cup was threaded so that i t could be screwed onto an oxygen probe (YSI model 57 oxygen meter and YSI model 5739 oxygen probe, Yellow Springs Instruments C o . , I n c . , Yellow Spr ings , OH) excluding the a i r . A small t e f l o n magnetic bar was used to achieve c i r c u l a t i o n . C e l l u l o s e and ni trogen i n the p e l l i c l e were determined only at the end of the incubat ion per iod . In add i t ion to these 3 t r i a l s at d i f f e r e n t aerat ion ra tes , a fourth t r i a l was performed fo l lowing the same parameters but t h i s time the growth medium was pH c o n t r o l l e d at 4.5 and a i r was sparged at a rate of 0.04 L a i r / L medium/min; a l l t r i a l s were c a r r i e d out i n d u p l i c a t e . At t h i s stage, a f i n a l set of condi t ions was adopted for further f i b r e product ion . pH c o n t r o l l e d defined medium (pH 4.5) flowed down a ramp at a rate of 12.2 L/min. Flow was c i r c u l a -ted for 1 h at 1 h i n t e r v a l s . The apparatus was sparged with a i r at a flow rate of 0.04 L a i r / L medium/min and incubated for 4 days at 28-3 0 ° C . 3. T e n s i l e proper t ie s determination The standard load-e longat ion te s t conducted to specimen f a i l u r e , was performed using an Instron Univer sa l Test ing Instrument (Model 1122, Instron C o r p . , Canton, MA) equipped with a tens i le /compress ive load c e l l with a f u l l sca le range of 10 g to 500 kg and a s t r i p chart recorder . - 47 -T e n s i l e s t rength , modulus of e l a s t i c i t y and percent elongation were ca lcu la ted for each sample from i t s load-elongat ion curve . T e n s i l e strength or s t ress at f a i l u r e was defined as the r a t i o between the force applied and the cross s ec t iona l area . The rupture force was determined from the peak height i n the load-elongat ion curve and converted into uni t s of force (Newtons). The cross s ec t iona l area, expressed in c m 2 , was determined for each i n d i v i d u a l sample. Gust p r i o r to the t e s t , measurements of the samples were taken with the aid of a micrometer and a r u l e r . Cross s e c t i o n a l area at the narrowest part of the "bone-shaped" s t r i p s and strands were ca lcu la ted by m u l t i p l y i n g width by th icknes s . The s t r i p s were wider at the ends to be gripped by the pneumatic ac t ion clamps so that the specimens would not f a i l at the clamping s i t e s . In the case of threads, the diameter was measured and the cross s e c t i o n a l area was c a l c u l a t e d . Modulus of e l a s t i c i t y i s defined as the res i s tance of the f i b r e extension to the appl ied force , or a measure of the force required to produce an extension. I t was estimated from the slope of the i n i t i a l part of the curve or the tangent of the angle between the i n i t i a l part of the curve and the h o r i z o n t a l ax i s , expressed i n uni t s of N/cm. F i n a l l y , percent elongation or breaking extension i s expressed by the r a t i o between the extension at the breaking point and the i n i t i a l length (gauge length) x 100. The extension at the breaking point was estimated from the distance (on the h o r i z o n t a l axis) between the point of f i r s t tension and the breaking point d iv ided by the magni f icat ion r a t i o 10 (chart speed 50 mm/min/crosshead speed 5 mm/min). - 48 -Pretes t setup procedures were required i n order to e s t a b l i s h some factors a f fec t ing the t e n s i l e propert ies of the f i b r e s . To ensure uniform t e s t i n g , the same condi t ions were used for a l l s i m i l a r t e s t s . Factors considered were the f o l l o w i n g : test specimen length (gauge l ength) , t e s t ing speed (cross head speed), moisture i n the specimen and shape of the test specimen. The gauge length was 2 cm for a l l t e s t s , t h i s test specimen length was found to minimize the e f fect known as the "weak l i n k " e f fect (Booth, 1964). A su i t ab le crosshead speed for a l l the te s t s was 5 mm/min and a chart speed of 50 mm/min. Thus, the magni-f i c a t i o n r a t i o (Chart speed/Cross head speed) was 10. I f c e l l u l o s e samples were produced i n sheets, s t r i p s 5 cm long and 3 cm wide were cut and shaped into a bone form (2 cm wide at the c e n t e r ) . At the time of t e s t i n g , the samples were mounted c e n t r a l l y , securely gripped along the f u l l width to prevent s l i p p i n g , and the jaws were al igned i n a p a r a l l e l fashion so that the load was appl ied uniform-ly across the f u l l specimen center width. C e l l u l o s e f i b r e s produced i n the channels or grooves were already uniform strands (0.2 cm wide and 50 cm long) which were tested with t h e i r o r i g i n a l shape unless otherwise s p e c i f i e d . The ef fect of moisture content on the t e n s i l e strength of f i b r e s was determined by e q u i l i b r a t i n g the samples under d i f f e ren t r e l a t i v e humid i t i e s . Six bone-shaped samples were placed i n each of the des icca-tors conta ining the fo l lowing saturated s o l u t i o n s : KOH (9% ERH), potas-sium acetate (22% ERH), NaN02 (65% ERH), NaCl (76% ERH), and K2SQK (97% ERH). Samples were allowed to e q u i l i b r a t e for 48 h; ha l f of the samples were subjected to t e n s i l e strength te s t ing and the other ha l f of the - 49 -samples were analyzed for moisture content. Previous to e q u i l i b r a t i o n at the d i f f e ren t r e l a t i v e humidi t ie s , the samples were dr ied i n a vacuum oven for 15 h at 6 0 ° C . Thus, dr ied samples would e q u i l i b r a t e by water adsorpt ion . Pre l iminary experiments showed that desorption took from 7 to 10 days due to the high water content of the samples i n the swollen s t a te . At the time of t e n s i l e t e s t i n g , sample thickness was measured by means of a micrometer. The Instron was operated with a crosshead speed of 5 mm/min, a chart speed of 50 mm/min, a 5 kg load and a 2 cm gauge length . Tens i l e strength was p lo t ted against moisture content . Tens i l e t e s t i n g of f i b r e s produced by two d i f f e ren t s t r a i n s under d i f f e ren t flow modes ( in termi t tent and continuous) was c a r r i e d out using the same set of condi t ions as above. 4 . Mapping Super Simplex Opt imizat ion of mercer izat ion treatment Mercer iza t ion i s perhaps the most commonly employed chemical treatment of cotton f i b r e s and yarns . In t h i s process the swel l ing and modifying act ion of sodium hydroxide upon the c e l l u l o s e f i b r e s of cotton ha i r s i s u t i l i z e d to advantage to impart c e r t a i n des i rab le propert ies such as l u s t e r , improved strength and increased d y e a b i l i t y . A great deal of work has been done on various aspects of mercer iza t ion , and studies have been c a r r i e d out to determine the optimum condi t ions under which c e l l u l o s e should be mercerized i n order to impart the desired improved p r o p e r t i e s . As a r e s u l t , information i s a v a i l a b l e on the extent of changes i n the s t r u c t u r a l and morphological propert ies of cotton af ter swel l ing i n NaOH under various condi t ions of treatment - 50 -(Warwicker et a l . , 1966; Peters , 1967; Warwicker and Hallam, 1975; Rajagopalan et ^ 1 . , 1975). Since i t was shown that the ef fect of any treatment intended to modify the propert ies of the f i b r e s was grea t ly inf luenced by the o r i g i n a l c h a r a c t e r i s t i c s of the f i b r e s , i t was neces-sary to f ind the optimum condi t ions under which ^ . xylinum c e l l u l o s e f ib re s would achieve maximum t e n s i l e s t rength . Mapping Super Simplex Opt imizat ion (MSO) was c a r r i e d out to optimize the treatment condi t ions using a modified super-simplex opt imi-za t ion program (MSS) (Nakai, 1982), wri t ten for a Monroe 1880-88 programmable c a l c u l a t o r , a grouping and matching program (Nakai et a l . , 1984) wri t ten for the UBC Amdahl 470 V/8 computer, and a simultaneous factor s h i f t program (Nakai et a L . , 1984) wri t ten for a Monroe 1880-88 programmable c a l c u l a t o r . Three fac tors were optimized for maximum t e n s i l e s t rength , namely, temperature, NaOH concentrat ion and t ime. Based on pre l iminary experiments, the boundary values of each factor were e s t ab l i shed . Thus, sodium hydroxide concentrat ions ranged between 6 and 22% (w/v); temperature ranged between 2 and 8 0 ° C and time ranged between 10 and 60 min. T r i p l i c a t e c e l l u l o s e strands were treated under var ious c o n d i t i o n s , neu t ra l i zed with 1% (v/v) ace t i c a c i d , washed several times with d i s t i l l e d water and dipped into a 10% (v/v) g l y c e r o l s o l u t i o n for 1 h . During treatment, strands were securely gripped with two clamps to prevent entanglement. The treated strands were dr ied under vacuum at 6 0 ° C for 2.5 h to reach a f i n a l moisture content of 40% (wet basis) and immediately subjected to t e n s i l e t e s t i n g . I n d i v i d u a l strands were securely gripped along the f u l l width with the upper gr ip and twisted by r o t a t i o n of a - 51 -clamp attached to the free end to introduce 10 turns per cm, a t o t a l of 20 turns in the 2 cm gauge length for a l l tested samples. Twist was introduced to the f i b r e s i n order to develop strength due to cohesive forces , as i s the common prac t i ce in t e x t i l e s (Booth, 1964). Diameter of the threads was measured with a micrometer once they were secure. The standard load-elongat ion tes t conducted to specimen f a i l u r e was c a r r i e d out with a 5 mm/min crosshead speed, a 50 mm/min chart speed, a 2 kg f u l l scale load range and a 2 cm gauge length . The mean t e n s i l e strength ca l cu la ted with each treatment represented the response value entered into the program. 5. S t a t i s t i c a l analyses a) Ef fect of A . xylinum s t r a i n and flow mode on t e n s i l e s trength The purpose of the ana lys i s was to determine i f the s t r a i n and the flow mode had a s i g n i f i c a n t e f fect on t e n s i l e s t rength . Data c o l l e c t e d were coded for " s t r a i n " (2 l eve l s ) and "flow mode" (2 l eve l s ) and analyzed using a two-way ana lys i s of variance (ANOVA) program package (*MFAV) (Le, 1980) ava i l ab l e for use on the UBC computer (Amdahl 470 V/8) system. b) E f fec t of batch on t e n s i l e proper t ie s Tens i l e s t rength , modulus of e l a s t i c i t y and percent e longat ion or breaking extension were tested i n c e l l u l o s e f i b r e s produced from two d i f f e r e n t batches. The standard load-elongat ion te s t s conducted to specimen f a i l u r e were c a r r i e d out with the Instron operated with a 5 mm/min crosshead speed, a 50 mm/min chart speed, a 2 kg f u l l scale load - 52 -range and a 2 cm gauge l ength . Eight r e p l i c a t e s were evaluated from each batch. A one-way ana lys i s of variance (ANOVA) with "Batch" as the s i n g l e factor (2 l eve l s ) was performed using the *MFAV program. c) Ef fect of Mercer iza t ion on t e n s i l e proper t ie s Tens i l e s t rength, modulus of e l a s t i c i t y and percent elongation or breaking extension were tested i n non-treated and mercerized f i b r e s . The load-elongat ion te s t s were conducted with the Instron operated with the same condi t ions previous ly s ta ted . Eight r e p l i c a t e s were evaluated from each treatment. A one-way ana lys i s of variance with "treatment" as the s ing le factor (2 l eve l s ) was performed using the *MFAV program. 6. F ibre microstructure a) L ight microscopy C e l l u l o s i c f i b r e s were observed under a Wild M20 Microcope (Wi ld , Heerbrugg, Switzerland) equipped with a Pentax ME 35 mm camera. C e l l u -l o s i c s t r i p s were d i a lyzed with d i s t i l l e d water immediately a f ter harvesting and stained with a 1% aqueous so lu t ion of Chlorazo l Black E (Paszner, 1982; personal communication). Phase contrast was used and photographic images were recorded on Kodak Ektachrome colour s l i d e f i l m . Stained f i b r e s were also observed using po la r i zed l i g h t . Phase po la r i zed l i g h t was obtained by i n s e r t i n g a Nico l prism in the micro-scope substage. - 53 -b) Scanning e l ec t ron microscopy S t ruc tura l examination of c e l l u l o s i c f ib re s produced e i ther wi th in a confined u n i d i r e c t i o n a l f lowing medium or under s t a t i c c o n d i t i o n s , non-treated and mercerized samples, was accomplished using scanning e l ec t ron microscopy techniques . Samples, approximately 3x3x1 mm cubes, were f ixed with 4% v/v glutaraldehyde i n 0.066 M phosphate pH 7.0 for 15 h at 4 ° C . Three 5 min r inses i n 0.066 M phosphate buffer were followed by secondary f i x a t i o n in 1% osmium te t rox ide i n phosphate buffer for 60 min. Rinses of phosphate buffer (3x5 min) preceeded a lcohol dehydration through a ser ie s of increas ing ethanol concentrat ions (5 min each i n 50, 60, 70, 80; 2x10 min i n 90; and 3x20 min i n 100%). Replacement of ethanol with amyl acetate was accomplished through 10 min changes of 25, 50, and 75% amyl acetate i n absolute e thanol , followed by 1 h i n 100% amyl acetate . The samples were then dr ied i n a Parr 5770 C r i t i c a l Point Drying Bomb (Parr Instrument C o . , Mol ine , IL) using carbon dioxide as the t r a n s i t i o n a l f l u i d ( c r i t i c a l temperature and pressure: 3 0 ° C ; 7468 kPa). The dr ied samples were mounted with aluminum paste on aluminum stubs and coated with gold by vacuum evaporat ion. A Cambridge Steroscan 250 Scanning E lec t ron Microscope (Cambridge C o . , Cambridge, England) equipped with a Po laro id 545 land f i l m camera was used to observe the s t r u c t u r a l d e t a i l of the samples and photographic images were recorded on pos i t i ve /nega t ive f i l m (type 55). An operating voltage of 20 kV was employed. - 54 -7. C r y s t a l l i n i t y index, c r y s t a l s i z e and degree of po lymer izat ion  determination C r y s t a l l i n i t y index and c r y s t a l s i ze of mercerized f i b r e s produced under confined u n i d i r e c t i o n a l flowing media were determined i n d u p l i -cate using X-ray d i f f r a c t i o n methods (de ta i l ed d e s c r i p t i o n of the method i s given i n sect ion G2). The degree of polymerizat ion was determined by the Cupriethylenediamine disperse v i s c o s i t y method (TAPPI, 1966; d e t a i l e d d e s c r i p t i o n of the method was given i n sect ion E) i n d u p l i c a t e . G. Microcrystalline Cellulose (MCC) Production 1. C e l l u l o s e production C e l l u l o s e production s tarted with the incubat ion of s t a t i c c u l -t u r e s . Defined medium was inoculated with 10% inoculum ( P h i l i p p i n e s t ra in) and l e f t undisturbed for two weeks at 2 8 - 3 0 ° C . C e l l u l o s e p e l l i -c l e s were harvested af ter two weeks incubat ion and d ia lyzed overnight with tap water. The d ia lyzed c e l l u l o s e was then shredded into small pieces with a Waring Blendor regulated with a va r i ab le autotransformer (600 rpm). The shredded c e l l u l o s e was drained through several l ayers of cheesecloth to remove excess water and subjected to NaOH e x t r a c t i o n . 2 . C e l l u l o s e p u r i f i c a t i o n (NaOH extrac t ion) An ex t rac t ion treatment which resul ted in a highly p u r i f i e d c e l l u -lose (_> 98% c e l l u l o s e ) without any s t r u c t u r a l damage was i n v e s t i g a t e d . C e l l u l o s e , in the swollen s tate (98% moisture wb) and freeze dr ied (2% - 55 -moisture, wb) were treated with increas ing NaOH concentrat ions 1, 3, 6 and 8% (w/v). Weighed, swollen samples were mixed with NaOH so lu t ions i n a r a t i o of 1 to 2; thus the f i n a l NaOH concentrat ion was approximately 2/3 of the i n t i a l concentrat ion assuming the samples were 100% water. Mixtures were shaken for 3 h at 2 5 ° C , neut ra l i zed with 1% (v/v) ace t ic acid and r insed with d i s t i l l e d water to remove the a c i d . Treated samples were freeze dr ied and ground with a Wiley m i l l equipped with a 40 mesh screen. Dupl icate samples were subjected to nitrogen determination according to the method of Concon and Soltess (1973) and the digested samples were analyzed using an Auto-Analyser III (Technicon Instruments C o . , Tarrytown, NY). The degree of polymerizat ion (DP) was measured by the Cupriethylenediamine disperse v i s c o s i t y p ipet te method (TAPPI, 1966) i n d u p l i c a t e . C r y s t a l l i n i t y and c r y s t a l s ize were determined by using a X-ray d i f f r a c t i o n method. Approximately 1 g of ground sample was shaped in to a th in disk by compression with a hydraul ic press (750 psig for 1 min) . The samples were c a r e f u l l y placed i n p o s i t i o n i n a Dutch P h i l i p s 1009 X-ray Di f fTactometer ( P h i l i p s E l e c t r o n i c , Mahwah, N3) and scanned between 9 ° to 3 0 ° (20) angle. The x-rays were generated from a water-cooled copper target at 30 kV and 15 mA current , monochromatized by a n i c k e l f i l t e r and passed through a 0 . 5 ° divergence s l i t . The goniometer consis ted of a propor t iona l counter through a 0 . 1 ° rece iv ing and 0 . 5 ° s ca t ter s l i t , ampl i f ied by the preampl i f i e r c i r c u i t and the r e s u l t i n g s igna l s were fed into a recorder . The three maxima (peaks) obtained corresponded to 002, 101 and 10T planes of the c e l l u l o s e c r y s t a l l a t -t i c e . Percent c r y s t a l l i n i t y was derived by comparing the i n t e n s i t y - 56 -of x-rays d i f f r a c t e d by the c r y s t a l l i n e port ion with that d i f f r a c t e d by the n o n - c r y s t a l l i n e (amorphous) port ion in the sample (Browning, 1967). The method uses the i n t e n s i t y maxima of the 002 peak d o 0 2 ) a n a " inten-s i t y minima at 18 ° 20 ( I a m ) . The c r y s t a l l i n i t y index was ca lcu la ted with the fo l lowing formula: CrI = I o ° 2 " I « " x 100 1Q02 The c r y s t a l l i t e s i zes of c e l l u l o s e were obtained from the l i n e breath of 002 peak. The usual Sherrer formula was used, D = ° - 9 X 8 cos 0 where: D i s the c r y s t a l l i t e s i ze i n A " ; X i s the wavelength i n A° of the x-ray used (XCu = 1.54 ° A ) ; 0 i s the Bragg angle in degrees ( 2 2 . 6 ° ) ; and 8 i s the ha l f breadths of the peaks, i n rad . Data c o l l e c t e d for a l l the measured var i ab le s were coded for " c o n d i t i o n " (2 l e v e l s , swollen and freeze dried) and "treatment" (5 l e v e l s ; 1, 3, 6 and 8% NaOH e x t r a c t i o n ) ; an ana lys i s of variance (ANOVA) program package (*MFAV) ava i l ab l e for use on the UBC computer was used to analyze the data . Dupl ica te values were ava i l ab l e for a l l v a r i a b l e s . Data were subjected to further s t a t i s t i c a l ana lys i s using Duncan's mul t ip l e range test ava i l ab le with the MFAV program. Duncan's mul t ip l e range tes t ( L i , 1964) i s used to compare the means of the main e f f ec t s . 3. Bleaching Freeze dr ied NaOH-extracted c e l l u l o s e samples s t i l l contain some co lour ing matter and have a ye l lowish c o l o u r a t i o n . Thus, a mild - 57 -bleaching process was necessary to obtain a good white co lour . The treatment was c a r r i e d out with sodium c h l o r i t e as the bleaching agent. The method represents further refinements on the Gayme and Wise c h l o r i t e c e l l u l o s e technique (Browning, 1967) i n that i t was adjusted to s u i t t h i s type of c e l l u l o s i c mate r i a l , s ince the sever i ty of the treatment required depends on the amount of n o n - c e l l u l o s i c mater ia l present in the sample. Water (200 mL) was added to a 500 mL Erlenmeyer f l a s k , heated to 7 0 ° C , and added to 5 g of sodium c h l o r i t e , NaOCl2• The equivalent of 5 g (dry) of unbleached samples were added and shaken to mix. G l a c i a l ace t i c acid (2 mL) was added and mixed again. The mixture was held in a water bath at 7 0 ° C for 3 h . The d ige s t ion was stopped and the mixture was washed with 1% ace t i c a c i d . F i n a l l y , the c e l l u l o s e was r insed very thoroughly with water, drained through cheesecloth, and freeze d r i e d . k. Hydro lys i s A study was c a r r i e d out on the ef fect of time on the weight l o s s and the degree of polymerizat ion of p u r i f i e d c e l l u l o s e samples using d r a s t i c condi t ions of h y d r o l y s i s , i . e . 2.5 N hydrochlor ic acid at b o i l i n g temperatures ( B a t t i s t a , 1950). C e l l u l o s e samples of known moisture content were weighed and added to b o i l i n g 2.5 N HC1 i n a r a t i o 1 (g) : 150 (mL). The hydroch lor i c acid so lu t ion was heated by means of a G l a s s - c o l mantle connected with a powerstat. A steady stream of ni trogen gas (100 cc per min) was administered through one opening of a three neck f l a sk for the purpose of keeping the temperature of the acid uniform, e l iminat ing bumping, and excluding oxygen from the hydrolyzing - 58 -medium. The temperature was kept at 105 ± 0 . 5 ° C . The samples were l e f t in the b o i l i n g acid for p r e c i s e l y the times s p e c i f i e d , 15, 30, 60 and 120 min. The apparatus was dismantled and the contents were rap id ly t rans ferred to a tared f r i t t e d glass f i l t e r of M p o r o s i t y . The sample was then washed repeatedly with d i s t i l l e d water, d i l u t e ammonium hydroxide (5%), and more d i s t i l l e d water u n t i l a c i d - f r e e , a f ter which i t was dr ied i n a vacuum oven to constant weight at 1 0 5 ° C . Changes i n the degree of polymerizat ion with hydro ly s i s time were recorded and p l o t t e d . C e l l u l o s e y i e l d s r e s u l t i n g from acid hydro ly s i s (based on weight of residue) were determined at each time i n t e r v a l ; DP and y i e l d s were determined i n d u p l i c a t e . The aim of t h i s study was to f ind the time i n t e r v a l needed to reach the " l e v e l - o f f degree of po lymer iza t ion" (LODP) or when a r e l a -t i v e l y constant degree of polymerizat ion was reached. Once the hydrolyz ing condi t ions were e s t ab l i shed , they were adopted for further work. 5. Spray Drying The r e s u l t i n g n e u t r a l i z e d , f i l t e r e d c e l l u l o s e c r y s t a l s were r e s l u r r i e d to about 3% s o l i d s and subjected to the act ion of a Waring Blendor for 30 min to free a f r a c t i o n of the unhinged c r y s t a l s . The homogeneous aqueous s l u r r y was spray dr ied with a Niro Atomizer (Copenhagen, Denmark) at a 9 6 - 1 0 0 ° C i n l e t temperature and 3 6 ° out le t temperature. The s l u r r y was fed into the Atomizer with a flow rate of - 59 -20 mL/min. After spray drying, the product was stored i n bottles placed in a desiccator u n t i l used for the various t e s t s . H. Microcrystalline Cellulose (MCC) Characterization I . Physical property t e s t s a) C r y s t a l l i n i t y and c r y s t a l l s i z e Estimation of c r y s t a l l i n i t y and c r y s t a l s i z e was performed on t r i p l i c a t e samples of the MCC prepared i n t h i s study, as well as on commercial MCC A v i c e l PH-101 by methods previously described. b) Average degree of polymerization and p a r t i c l e s i z e The degree of polymerization was determined in t r i p l i c a t e by the Cupriethylenediamine disperse v i s c o s i t y method as previously described (TAPPI, 1966). Average p a r t i c l e s i z e was determined by measuring 20 p a r t i c l e s i n each sample dispersion using a Wild M40 Microscope (Wild Heerbrugg, Switzerland) provided with an ocular and stage micrometer. Both te s t s were performed on commercial MCC Avic e l PH-101 and prepared MCC. c) Moisture adsorption T r i p l i c a t e MCC samples were allowed to e q u i l i b r a t e for 48 h at 25°C with the following saturated solutions: potassium acetate (22 % ERH), sodium ch l o r i d e (76% ERH) and potassium s u l f a t e (97% ERH). Water a c t i v i t i e s were determined by placing the samples in a Rotronic Hygroskop DT DMS 100 water a c t i v i t y measuring s t a t i o n (Rotronic - 60 -Ag, Z u r i c h , Swi tzer land) , and allowed to e q u i l i b r a t e for 1 h . The Hygroskop was c a l i b r a t e d with a saturated barium c h l o r i d e s o l u t i o n . Moisture content was determined by drying i n a vacuum oven at 105 kPa vacuum and 8 0 ° C for 15 h . Moisture contents were ca l cu l a ted as a percentage (dry basis) and r e s u l t s presented as the average of t r i p l i -cate analyses . d) Zeta p o t e n t i a l MCC d i spers ions (0.1%) were prepared i n .01 M phosphate buffer (pH=6.95) by using a Polytron high speed b lender / son ica tor . Zeta po tent i a l s of dup l i ca te d i spers ions were determined with a Laser Zee^M (Model 500, Pen Kern, I n c . , Bedford H i l l s , NY) and the values were corrected for temperature. e) Colour determination Colour of the MCC samples was measured with a Hunterlab Colour Dif ference Meter (Model D 25 D, Hunter Associates Laboratory, I n c . , F a i r f a x , VA) with the L sca le and the standard No. W 272. 2. Chemical composition a) C e l l u l o s e determination T r i p l i c a t e MCC samples were subjected to a primary and a secondary hydro lys i s as described by Whist ler (1963). Tota l carbohydrate content of the digested samples was determined by the phenol-su lphur ic acid method of Dubois et a l . (1956) using a glucose standard curve . - 61 -Primary hydrolys i s : Cel lulose (0.3 g) in 3.0 mL ^50,+ (72%) was maintained at 30°C for 1 h . The samples were then d i luted with 84 mL of d i s t i l l e d water i n a tared 250 mL Erlenmeyer f l a sk . Secondary Hydrolysis : The f lasks were covered with an inverted beaker and placed in an auto-clave for 1 h (15 psig ; 120°C). Neut ra l i za t ion : The hydrolysates were cooled and neutral ized to pH 5.5 by addition of a saturated solut ion of barium hydroxide during vigorous ag i ta t ion with a mechanical s t i r r e r . Solutions were d i luted to the weight of 261.7 g exactly equivalent to 250 mL of so lut ion plus suspended BaSO^ (11.7 g) . The neutral ized hydrolysates were centrifuged and al iquots (200 mL) were taken from the clear supernatant. Aliquots were stored under re f r igera-t i o n u n t i l t o t a l carbohydrate analyses were performed. b) Nitrogen determination Samples (60 mg) of spray dried MCC, experimental and commercial, were placed i n a 30 mL micro-Kjeldahl f lask and digested using the method of Concon and Soltess (1973). Digested samples were analyzed for nitrogen content by means of a Technicon Autoanalyzer (Technicon Instruments, Co. , Tarrytown, NY). Samples were also analyzed for ammonia nitrogen using a Kjeldahl D i s t i l l i n g Uni t , a saturated boric solut ion as the trapping agent and - 62 -NaOH-Na2S2n3 as the re leas ing agent. D i s t i l l a t e s were t i t r a t e d with 0.02 N HC1 (AOAC, 1980). Analyses were c a r r i e d out in t r i p l i c a t e . c) Ash determination Samples (1 g) were heated at 550 ± 5 0 ° C u n t i l completely charred i n a muffle furnace, then i gn i t ed at 800 ± 2 5 ° C u n t i l free from carbon, cooled i n a des iccator and weighed (FCC I I I , 1981). Analyses were ca r r i ed out i n t r i p l i c a t e . 3. Rheologica l proper t ie s Samples of MCC, prepared from _A. xylinum c e l l u l o s e and commercial A v i c e l PH-101 were dispersed i n d i s t i l l e d deionized water to make d u p l i -cate d i spers ions of 6, 5, 4 and 3% (w/v). So l id s were dispersed by using a Polytron high speed b lender / sonicator for 2 min. Dupl ica te samples were then subjected to v i scometr ic evaluat ions using a c o a x i a l c y l i n d e r Fann Viscometer (Model 35, Fann Instrument C o . , Houston, TX) . Tests were c a r r i e d out over a range of temperatures (25, 35 and 4 5 ° C ) , shear rates (3-600 rpm) and pH (4.0 , 5.5 and 7 .0 ) . The ef fect of 0.4% (w/v) NaCl on the flow behaviour of 3% d i spers ions was also s tud ied . Each sample was loaded, allowed to e q u i l i b r a t e and sheared over a shear rate range of 0 to 600 rpm at 10 s i n t e r v a l s fol lowed by a decrease in shear rate to 0, again at 10 s i n t e r v a l s . The sample was then sheared with the same increase/decrease c y c l e . The d i a l readings (0) were recorded from the meter for each speed (rpm). Using a - 63 -T h e o l o g i c a l ana lys i s program (Computer-Aided Rheologica l Ana ly s i s of D r i l l i n g F l u i d s ; Speers, 1982), ava i l ab l e through an Apple II microcom-puter , data from a l l experiments were analyzed. By using l i n e a r regres-s ion techniques, t h i s program ca l cu l a t e s Bingham, Power law and Casson model parameters. The Bingham model was expressed by the equation n = rip^ + — , where r i ^ was the p l a s t i c v i s c o s i t y , was the y i e l d p o i n t , Y was the shear rate and r\ was the apparent v i s c o s i t y . The Casson model was represented by the equation / n = k x + k 0—1- , where k 0 was a~11 and llo / Y Y . N - 1 k\ was n The Power Law model was expressed by the equation n = my where m was the consistency c o e f f i c i e n t and n was the flow behaviour index. These techniques also i n d i c a t e the degree to which the model f i t s the data . The program also provides a g raphica l p o r t r a i t of a versus y (or transforms) , and n versus y. In t h i s program the 0 ( d i a l readings) values obtained at severa l rpms were treated assuming that Newtonian shear rates occur i n the annular gap. The shear rate and s t res s values were ca l cu l a ted from the fo l lowing two equations: 2 4Itrpm r c . ^ c G Y = — r, rp- and a - ,— 60 ( r c z - r t / ) 2 n r b Z h e where r^ was the radius of the bob (1.725 cm), r c was the radius of the cup (1.842 cm), S c was the spr ing constant of the viscometer (387 dyne/cm degree), and h e was the e f f e c t i v e height of bob (corrected for end e f f e c t , 4.05 cm). These two equations for shear rate and shear s t ress were modified by s u b s t i t u t i o n of the values to equations Y =1.703 rpm in s - 1 and o=0.511 0 in Pa. Therefore , to c a l c u l a t e the Power law index n and consistency c o e f f i c i e n t s m from data, n was ca l cu l a ted using - 64 -regress ion of log a on log y according to the above equations. Once the n was c a l c u l a t e d , a true m was determined by using a corrected shear 2In rate ( y c ) given by the expression y c = ^ ^ P " 1 —J^Q _ — . The corrected 60 r c / n _ r b / n shear rate values were then used i n further computations. Dispers ions were also tested for time dependence. P l o t s of the upcurve and downcurve at 2 5 ° C at each concentrat ion were subjected to a l i n e comparison program t i t l e d "NLIN" , ava i l ab l e on the UBC Food Science Department computing ID, to test the presence of a s i g n i f i c a n t d i f f e r -ence between two regress ion l i n e s . This program compares pa i r s of l i n e s to test for homogeneity of re s idua l var iances , slopes and l e v e l s . The s i g n i f i c a n c e of these comparisons was based on a F-value generated i n each case. In t h i s comparison of Power Law rheograms the ri and y data were transformed to logarithms to provide a l i n e a r funct ion so that a d i f f e rence i n slopes would i n d i c a t e that n values d i f f e r e d , and a d i f ference in l e v e l s would i n d i c a t e that m values d i f f e r e d . To tes t i f the d i spers ions were t h i x o t r o p i c , that i s , i f the f l u i d possesses a r e v e r s i b l e decrease i n v i s c o s i t y to some equ i l ib r ium value with time at a constant shear ra te ; ge l strengths (10 sec and 10 min) were recorded at 3 rpm by shearing the d i sper s ion at 600 rpm for 10 sec , then the shear rate was brought to zero for a rest period of 10 sec or 10 min (Gray and Dar ley , 1981). In addi t ion a tes t was c a r r i e d out to check i f the two MCC samples te s ted , commercial A v i c e l PH-101 and MCC prepared from A. xylinum c e l l u l o s e , presented s i g n i f i c a n t l y d i f f e r e n t v i s c o s i t i e s over a range of concentrat ions (3, 4, 5 and 6%) and shear rates (3-600 rpm) at 2 5 ° C . P lo t s of the equ i l ib r ium curves of both MCC - 65 -at 2 5 ° C at each concentrat ion were subjected to the l i n e comparison program. In t h i s comparison of Power law rheograms, a d i f ference i n s lopes would ind ica te that (n-1) values d i f f e r e d , and a d i f f e rence i n l e v e l s would ind ica te that m values d i f f e r e d . Using the Power law models apparent v i s c o s i t i e s were ca l cu l a ted at 100 s " 1 . These apparent values were then used to examine v i s c o s i t y - c o n c e n t r a t i o n r e l a t i o n s h i p . The v i s c o s i t y - c o n c e n t r a t i o n model used was a Power-type r e l a t i o n s h i p expressed by the equation ri = ac*3 or log r\ = log a + b log C, where a and b were constant parameters (Harper and EL S a h r i g i , 1965). The ef fect of temperature on apparent v i s c o s i t i e s was s tudied by using an Arrhenius- type r e l a t i o n s h i p expressed by the equation where A i s the frequency f a c t o r , AE i s the a c t i v a t i o n energy, T i s the absolute temperature, R i s the un iver sa l gas constant and e i s natura l logarithm base (Harper and EL S a h r i g i , 1965). n - Ae AE/RT or log n = log A + AE ^ 2.303R T - 66 -RESULTS AND DISCUSSION A. Selection of Influential Factors in Defined Medium Data c o l l e c t e d for the 27 nutr ient media tes ted , fo l lowing a Frac-t i o n a l F a c t o r i a l Experimental Design L 2 7 ( 3 ) 1 3 of Taguchi (1957) were analyzed by ana lys i s of variance to determine the s i g n i f i c a n c e of pH, sucrose and peptone concentrat ion and poss ib le i n t e r a c t i o n s on c e l l u l o s e product ion . The r e s u l t s are presented i n Table 1. The fac tors of pH and sucrose concentrat ion were computed to be highly s i g n i f i c a n t sources of v a r i a t i o n (P < 0 .01) . Peptone concentrat ion was also found to be s i g n i f i c a n t (P < 0 .05) . The ef fect curve i n Figure 9a i l l u s t r a t e s a remarkable increase i n c e l l u l o s e production towards a c i d i c pH, as i t i s expected for bac te r i a of the genus Acetobacter , reaching an optimum at pH 4 . 5 . These r e s u l t s agreed very c l o s e l y with those reported by Lapuz et a l . (1967), who ind ica ted that A. xylinum i s d i s t i n c t l y a c i d - t o l e r a n t , t h r i v i n g even as low as at pH 3.5 , and that t h i c k e r p e l l i c l e formation was always observed i n nutr ient media with an i n i t i a l pH of 4.5 to 6 .0 . In coconut water medium, p e l l i c l e formation was observed to be optimum at pH 5.0 to 5 .5 . This study agreed with t h e i r f ind ings even though t h e i r method of assessing c e l l u l o s e production was based on volume displacement by the ge lat inous mater ia l ( c e l l u l o s e p e l l i c l e ) , whereas i n t h i s study, dry c e l l u l o s e weights were determined. In pre l iminary experiments severa l sugar substrates were t e s ted , namely, glucose, f ructose , sucrose and supplemented coconut water. A l l of them sustained good c e l l u l o s e product ion, however, the aim in t h i s - 67 -Table 1 . Ana lys i s of variance (Taguchi ' s L 2 7 3 ) for c e l l u l o s e y i e l d s obtained from 27 nutr ient media. Source of v a r i a t i o n DF Mean square F - r a t i o PH 2 159.28 x 10 3 38 .90* * Sucrose concentrat ion 2 139.32 x 10 3 34 .03* * Peptone concentrat ion 2 22.64 x 10 3 5.53* pH x sucrose concentrat ion 4 37.30 x 10 3 5.93* pH x peptone concentrat ion 4 37.08 x 10 3 9 . 0 5 * * Peptone concentrat ion x sucrose concentrat ion 4 2.02 x 10 3 0.49 E r r o r 8 4.09 x 10 3 T o t a l 26 * s i g n i f i c a n t at p < 0.05 * * s i g n i f i c a n t at p < 0.01 4 0 0 3 0 0 r -E X (3 UJ 5 o O 2001-0\ Co iooh F i g u r e 9. 4 6 p H SUCROSE CONCENTRATION (a ) ( b ) Effect Curves re su l t ing from the s e l e c t i o n factors i n defined media. 10 0,2 0,4 0,6 0.8 1.0 1,2 1,4 PEPTONE C O N C E N T R A T I O N , % (c ) of i n f l u e n t i a l - 69 -study was to f ind a s u i t a b l e defined medium for the s t r a i n s under inves-t i g a t i o n that would allow large y i e l d s of c e l l u l o s e from an inexpensive and r e a d i l y ava i l ab l e substrate . Therefore , sucrose was chosen as the sugar substrate for t h i s experiment and for further work. The e f fect of sucrose concentrat ion on c e l l u l o s e production revealed that i f the amount of sucrose i s l i m i t e d , the amount of c e l l mater ia l and thus the c e l l u l o s e would also be reduced. The ef fect of low nutr ient concentrat ions on growth rate was suggested by Brock (1979). I t was proposed that at low nutr ient concentra t ions , the nutr ient cannot be transported into the c e l l at s u f f i c i e n t l y rapid rates to s a t i s fy a l l the metabolic demands for the n u t r i e n t . As the nutr ient concentrat ion i s increased , a concentrat ion w i l l be reached that i s no longer l i m i t i n g , and further increases then no longer lead to increases i n t o t a l crop . As i t i s shown i n Figure 9b, c e l l u l o s e y i e l d s were grea t ly increased as sucrose concentrat ion increased; however, the optimum concentrat ion was not determined s ince the e f fect curve d id not show a maximum or a p la teau . In determining the optimum sucrose concentrat ion further experimentation would be requ i red , searching that region beyond 10%. F a c t o r i a l designs present t h i s l i m i t a t i o n , where the r e s u l t s are h ighly dependent upon the choice of l e v e l s tested (Nakai, 1982). These re su l t s agreed well with those reported by Lapuz et a l . ' (1967) , who reported maximum c e l l u l o s e production with 10% glucose or sucrose when 2, 6 and 10% sugar concentrat ions were t e s ted . C e l l u l o s e y i e l d s however, as reported by Dudman (1960), were reduced when sugar - 70 -concentrat ions i n hydrolyzed molasses were above 3.7% for one of t h e i r s t r a i n s , and were unaffected by concentrat ions of up to 10.7% with a d i f f e r e n t s t r a i n . These disagreements may have a r i s e n , f i r s t l y , from using d i f f e r e n t s t r a i n s from those used i n t h i s study. Secondly, t h e i r c u l t u r e s were shaken thus increas ing aerat ion which may have lead to a s h i f t in the metabolism of the organism away from c e l l u l o s e towards increased ox idat ion of the sugar substrate (Dudman, 1960). Dudman (1959b) reported that the inf luence of glucose concentra-t i o n i n defined medium on c e l l u l o s e production was r e l a t i v e l y s l i g h t unless succinate was present as an adjunct . The addi t ion of succinate to the medium caused the c e l l u l o s e y i e l d s to show a marked response to glucose concentrat ion reaching a maximum at 5% concentra t ion . Again , t h i s v a r i a t i o n i n reponse to sugar concentrat ion may be a t t r i b u t e d to using d i f f e r e n t s t r a i n s . Peptone proved to be i n h i b i t o r y at the maximum l e v e l tested (1.23%) which i s equivalent to 0.2% n i t rogen . The e f fect curve (Figure 9c) shows a s l i g h t plateau at low peptone concentra t ions , 0.3 and 0.5% equivalent to .05 and .08% nitrogen concentrat ions , r e s p e c t i v e l y . The i n h i b i t o r y e f fect of peptone at 1.23% (0.2%N) agrees wel l with f igures reported by Dudman (1959b). It was reported that cu l ture s conta ining nitrogen concentrat ions ranging from 0.001 up to 0.1% as ammonium sulphate or as asparagine + glutamic acid gave s i m i l a r r e s u l t s when c e l l u l o s e y i e l d s were determined, but a d i f ference was found when nitrogen concentrat ions were increased . Ammonium su l fa te at concentra-t ions equivalent to 0.2% N caused decreased growth and c e l l u l o s e y i e l d s , - 71 -while the equivalent concentrat ion of asparagine + glutamic acid gave r i s e to increased growth and c e l l u l o s e y i e l d s . There was no d i r e c t evidence reported to suggest the mechanism by which the amino acid mixture became s t imula tory . Gaudy and Wolfe (1961) reported that peptone concentrat ions higher than 0.25% s i g n i f i c a n t l y i n h i b i t e d c e l l u l o s e formation in cu l ture s of Sphacre t i lus natans. The r a t i o of c e l l u l o s e formation to t o t a l growth decreased with increa s ing peptone concentra t ion , although t o t a l weight continued to increase . I t was suggested that the disturbance i n the metabolism due to high concentrat ions of peptone may have been, as observed by others , due to growth i n h i b i t i o n by tryptophan, c y s t i n e , and methionine i n concentrat ions as low as 0.2 mg/L. However, other researchers found no i n h i b i t i o n with tryptophan at 100 mg/L; tyros ine and cys t ine retarded growth at t h i s concentra t ion . Others reported delayed growth at concentrat ions of 0.1% of l e u c i n e , threonine , cy s t ine and t y r o s i n e . A poss ib le explanation for the i n h i b i t o r y ef fect of high peptone concentrat ions on c e l l u l o s e formation by A^ xylinum, might be that increased ox ida t ion of the substrate occurred rather than c e l l u l o s e formation; as suggested by the lower f i n a l pH and c e l l u l o s e y i e l d recorded for those cu l ture s conta ining 1.23% peptone (0.2% nitrogen) at pH 4 .0 , whereas t h i s behaviour was not observed at higher pH va lues . Thi s hypothesis i s c l e a r l y supported by the highly s i g n i f i c a n t in terac-t i o n showed between pH and peptone concentrat ion (p < 0 .01) . A s i g n i f i c a n t i n t e r a c t i o n was also observed between pH and sucrose concentrat ion (p < 0 .05) . This i n t e r a c t i o n ind ica ted a d i f f e r e n t - 72 -response to increas ing sugar concentrat ion at the d i f f e r e n t pH te s ted . The aim of t h i s study was to develop a c a r e f u l l y defined formula-t i o n for determining the s p e c i f i c requirements of c e l l u l o s e production which would be adopted for further work. Defined medium was preferred s ince i t permits the determination of those s p e c i f i c requirements for growth and c e l l u l o s e formation, and simultaneous v a r i a t i o n of the two most e s s e n t i a l n u t r i t i o n a l f ac tor s to mic rob ia l a c t i v i t y 1) a source of energy for c e l l metabolic processes, a carbon source, and 2) a source of mater ia l s from which c e l l u l a r matter can by synthes ized, a nitrogen source. It appears that by simultaneously varying nutr ient concentra-t i o n s and medium pH, much more valuable information can be obtained than when only one fac tor i s studied at a time as was the case i n a l l a v a i l a b l e reports on nut r i ent requirements of A . xylinum which t o t a l l y ignore i n t e r a c t i o n s among f a c t o r s . Other advantages of defined medium inc lude i t s r e p r o d u c i b i l i t y , t rans lucency, and the r e l a t i v e ease of c e l l u l o s e recovery and p u r i f i c a -t i o n . However, t h i s phase should be followed by a t r a n s i t i o n to a natura l medium i n order to scale-up the formulation to a commercially v i a b l e process . Natural medium might, for example, be beet molasses which i s a by-product of the r e f i n i n g of table sugar from the sugar beet; b lackstrap molasses which i s the mother l i q u o r remaining a f ter the c r y s t a l l i z a t i o n of brown sugar from the crude sugar j u i c e extracted from sugar cane; r e f i n e r ' s cane molasses which i s the residue remaining af ter white sugar has been r e c r y s t a l l i z e d from brown sugar. In genera l , beet and cane molasses have comparable amounts of fermentable sugar (48-58 t o t a l sugar) , potassium, t race minera l s , n i a c i n , pyr idoxine and - 73 -i n o s i t o l . Beet molasses can have a f i v e - f o l d higher organic nitrogen content , but ha l f of i t i s betaine which i s not a s s imi lab le by some spec ie s . Cane molasses i s s u b s t a n t i a l l y r i c h e r i n b i o t i n , pantothenic a c i d , thiamin, magnesium and ca lc ium. Molasses appears to be an e s p e c i a l l y useful medium component, s ince i t i s a good source of n i t rogen , inorganic cons t i tuent s , and vitamins as wel l as carbohydrates . These n u t r i t i o n a l c h a r a c t e r i s t i c s combined with i t s economy make molasses one of the most widely u t i l i z e d raw mater ia l s i n i n d u s t r i a l fermentation media ( Z a b r i s k i e , 1980). In f ac t , experiments had been described determining the inf luence of some n u t r i t i o n a l factors on c e l l u l o s e production i n media based on blackstrap molasses. Di f fe rent carbohydrate sources were compared and were found to be i n the fo l lowing order : hydrolyzed molasses>molasses>glucose>-sucrose (Dudman, 1959b). An a g r i c u l t u r a l waste mater ia l which i s an exce l lent growth medium for A. xylinum may be supplemented coconut milk (Lapuz et _a l . , 1967). The accelerated growth of t h i s organism when coconut milk i s added to a basal medium i s due to the presence of growth promoting factors which were reported to be present i n i t by Shantz and Steward (1952; 1955). Another source of carbohydrate i s found as a waste mater ia l from the s u l f i t e paper-pulping indus t ry . S u l f i t e waste l i q u o r i s the spent f l u i d remaining af ter wood for paper manufacturing i s digested to form a c e l l u l o s e f i b e r pulp using calcium b i s u l f i t e . The l i q u o r contains l i g n i n , s u l f i t e , and hexoses derived from the hydro ly s i s of hemice l lu-l o s e s . Treated s u l f i t e waste l i q u o r has been used i n the production of - 74 -s i n g l e c e l l pro te in (SCP) and ethanol ( Z a b r i s k i e , 1980). The primary ob jec t ive of those processes had been to upgrade t h i s waste stream into a useful product . Pineapple pulp derived from trimmings and cores i n the pineapple-canning industry i s another waste mater ia l high i n carbohydrates able to support growth of A .^ xylinum (Alaban, 1962; V i l l a n u e v a , 1937). There-fore , a great va r i e ty of substrates might be u t i l i z e d for c e l l u l o s e synthesis increas ing the p o t e n t i a l f l e x i b i l i t y of u t i l i z a t i o n of a g r i -c u l t u r a l wastes. B. Culture S t a b i l i t y There i s ample evidence i n the l i t e r a t u r e of the high mutab i l i ty of c e l l s within species of the ace t i c acid bac te r i a (Shimwell and Car r , 1958; Schramm and H e s t r i n , 1954; Cook and C o l v i n , 1980). Therefore , the s t a b i l i t y of the three s t r a i n s ava i l ab l e i n t h i s study was assessed before any of them was chosen for further work. Cul tures were grown under s t a t i c and swir led c o n d i t i o n s , s ince both s i t u a t i o n s were of i n t e r e s t for the a p p l i c a t i o n s intended. Table 2 shows the percentage of c e l l u l o s e - d e f i c i e n t co lonies from s t a t i c and swir led l i q u i d cu l ture s on agar surfaces as a funct ion of the number of t r a n s f e r s . There was a rapid increase in the proport ion (and therefore of t o t a l numbers) of c e l l u l o s e d e f i c i e n t co lonies i n the swir led c u l t u r e s ; 100% of the co lonies were c e l l u l o s e d e f i c i e n t between the f i f t h (10 days) and s ix th (12 days) s e r i a l t rans fer for the three s t r a i n s . The swir led cu l tures d id not develop the c h a r a c t e r i s t i c p e l l i c l e formed i n s t a t i c c u l t u r e s , ins tead , c e l l u l o s e i n the swir led Table 2. Mean values of percentage of c e l l u l o s e " d e f i c i e n t " CFU i n l i q u i d c u l t u r e s , s w i r l e d and s t a t i c , and a f u n c t i o n of number of t r a n s f e r s (n=3). C e l l u l o s e - d e f i c i e n t CFU (%) No. of ATCC 14851 ATCC 10821 P h i l i p p i n e s t r a i n Transfers Swirled S t a t i c Swirled S t a t i c S w i r l e d S t a t i c 1 0 0 0 0 0 0 2 35.4 ( 1 . 2 ) 1 0 8.1 (7.0) 9.1 (3.1) 1.0 (0.2) 0 3 82.6 (5.7) 0 32.6 (0.6) 9.0 (0.7) 28.4 (3.0) 0 4 96.1 (0.9) 0 50.0 (5.0) 10.7 (4.1) 97.0 (2.1) 13.8 (6.0) 5 97.6 (0.7) 0 100.0 (0.0) 16.0 (3.1) 97.0 (3.2) 25.0 (2.1) 6 100.0 (0.0) 0 100.0 (0.0) 20.0 (3.0) 100.0 (0.0) 75.9 (10.4) 7 100.0 (0.0) 0 100.0 (0.0) 20.7 (2.6) 100.0 (0.0) 85.5 (1.0) 8 100.0 (0.0) 0 100.0 (0.0) 14.6 (0.6) 100.0 (0.0) 87.0 (11.2) 9 100.0 (0.0) 9.2 (9.1) 100.0 (0.0) 18.1 (3.4) 100.0 (0.0) 100.0 (0.0) 10 100.0 (0.0) 10.3 (2.8) 100.0 (0.0) 18.8 (1.9) 100.0 (0.0) 100.0 (0.0) Standard d e v i a t i o n . - 76 -c u l t u r e s occurred i n the form of spher i ca l bodies . I t appears that the c e l l s growing i n swir led medium gradual ly l o s t the a b i l i t y to form c e l l u l o s e and assumed a d i f fuse growth hab i t a t . On the contrary , cu l tures grown under s t a t i c condi t ions were remarkably more s t ab le , as shown by the low percentage of c e l l u l o s e - d e f i c i e n t c o l o n i e s . In a d d i t i o n , a l l the s t r a i n s developed the c h a r a c t e r i s t i c leathery c e l l u l o s e p e l l i c l e at the surface of the l i q u i d medium. These re su l t s seem to agree wel l with the hypothesis of Schramm and Hes t r in (1954). They proposed that i n s t a t i c l i q u i d medium a f l o a t -ing p e l l i c l e insures an abundant oxygen supply to the c e l l s , and i n swir led l i q u i d medium, on the other hand, the c e l l s need not be f loated by the p e l l i c l e i n order to obtain oxygen and a c t i v e l y p r o l i f e r a t i n g forms which are d e f i c i e n t i n c e l l u l o s e forming a b i l i t y might thus gain ascendancy. Observations concerning the slow p r o l i f e r a t i o n of c e l l u l o s e d e f i c i e n t co lonies of the P h i l i p p i n e s t r a i n i n s t a t i c cu l tures i n s e r i a l t rans fers do not agree with r e s u l t s reported by Cook and Co lv in (1980). These researchers demonstrated that i n l i q u i d medium, c e l l s which are normal i n c e l l u l o s e production overgrow those which are d e f i c i e n t i n t h i s capac i ty . The reason for t h i s negative s e l e c t i o n i n s t a t i c l i q u i d medium was specu la t ive , but i t was proposed that c e l l s which produce l e s s c e l l u l o s e are le s s able to form a p e l l i c l e that tends to keep them on the surface of the medium. Because, _A. xylinum i s an ob l iga te aerobe, c e l l u l o s e - d e f i c i e n t c e l l s would thus be less able to obtain the oxygen which they need and therefore would be at a d e f i n i t e disadvantge to normal c e l l s under those c o n d i t i o n s . - 77 -One i n t e r e s t i n g feature of s t a t i c cu l tures of the P h i l i p p i n e s t r a i n which was completely d i f f e r e n t from that of the other two s t r a i n s was p e l l i c l e thickness and p o r o s i t y . The former produced highly hydrated porous p e l l i c l e s approximately 1 to 2 cm t h i c k , whereas the other two form very compact tough p e l l i c l e s 0.3 to 0.5 mm t h i c k . I t appears that cu l ture s with th ick porous p e l l i c l e s would have many more enmeshed c e l l s within the p e l l i c l e matrix which do not need to produce c e l l u l o s e to be able to obtain the oxygen necessary for s u r v i v a l . With the other two t h i n p e l l i c l e producing s t r a i n s , however, c e l l s have to s truggle to reach the surface to obtain oxygen. On the basis of the types of co lonies grown on agar two d i s t i n c t types were observed. C e l l u l o s e producing co lonies were round with umbonate e l e v a t i o n , undulate margin, tough, opaque and cream i n pigmen-t a t i o n , while c e l l u l o s e d e f i c i e n t co lonies were round, convex, e n t i r e margin, soft and t rans lucent for s t r a i n s ATCC 14851 and 10821. Colony morphology of the P h i l i p p i n e s t r a i n was s l i g h t l y d i f f e r e n t . C e l l u l o s e producing co lonies were round, of pu lv inate e l e v a t i o n , e n t i r e margin, tough, opaque and cream in pigmentation whereas the c e l l u l o s e d e f i c i e n t co lon ie s had the same features except that they were f l a t t e r and extremely mucoid. When c e l l u l o s e d e f i c i e n t co lonies as wel l as the wi ld type ( i s o l a t e d from rep l i ca s ) were t rans ferred to l i q u i d s t a t i c c u l t u r e s , a l l of them formed the c h a r a c t e r i s t i c zoogleal f i l m at var ious rates of product ion . As expected, the wi ld type formed the charac te r i s -t i c tough c e l l u l o s e p e l l i c l e i n 3 to 4 days whereas the c e l l u l o s e d e f i -c i en t co lonies formed f r a g i l e p e l l i c l e s a f ter 1 to 2 weeks incubat ion . - 78 -Therefore , i n the present i n v e s t i g a t i o n , i t was concluded that a l l the s t r a i n s tested were unstable under swir led cond i t ions , while ATCC 14851 was the most s table of the three when grown under s t a t i c condi-t i o n s . C. Growth Curves C e l l u l o s e y i e l d s a f ter 40 days of incubat ion var ied from 6500 mg (64% conversion of t o t a l sugar) with the P h i l i p p i n e s t r a i n , to 600 mg (6.05% conversion of t o t a l sugar) produced by ATCC 10821, which may be considered a low-y ie ld ing s t r a i n . ATCC 14851 produced much le s s c e l l u -lose than the P h i l i p p i n e s t r a i n , however i t y ie lded almost as much c e l l u l o s e as ATCC 10821 (Figure 10). The nitrogen content i n dry c e l l u l o s e af ter 40 days of incubat ion , which i s an i n d i c a t i o n of c e l l growth, var ied between narrower l i m i t s (1.81-0.47%) than the c e l l u l o s e y i e l d s , showing that the degree of growth was r e l a t i v e l y s i m i l a r between the three s t r a in s at the end of incubat ion (Figure 11). Thus, i t appears that the P h i l i p p i n e s t r a i n i s a high c e l l u l o s e -y i e l d i n g s t r a i n , whereas the other two s t r a i n s appear to s h i f t t h e i r metabolism away from c e l l u l o s e synthesis towards other metabolic path-ways. The pH drop (to approximately 3.0) for the two l o w - y i e l d i n g s t r a i n s during the exponential phase, suggests that an ox ida t ive mechanism predominated in these organisms. The pH for the P h i l i p p i n e s t r a i n remained constant during the exponential phase and s tar ted to decrease a f ter 14 days of incubat ion (Figure 10). The two low-y ie ld ing s t r a i n s were only able to u t i l i z e approximately 25 to 30% of the sugar, Figure 10. C e l l u l o s e p r o d u c t i o n ( — - ) and pH changes ( — ) d u r i n g g r o w t h o f t h r e e A. x y l i n u m s t r a i n s I n 100 mL o f d e f i n e d medium. 9.0 8.0 M 2. 7.0 3 > •5 6.0 o o> E o 2 5.0 5 4.0 ul % 3.0 EC 2.0 1.0 -A ATCC 14 8 5 1 • A T C C 1 0 8 2 1 • P H I L I P P I N E STRAIN 9 0 8 0 • 8 12 16 2 0 3 0 40 , D A Y S Figure 1 1 . Sugar conversion of t o t a l sugar ( ) and ni trogen content per 100 g of c e l l u l o s e (---) of three A . xylinum s t r a i n s i n 100 mL of defined medium. DC < a co - i < r-o u. O CO c > z o o oc < o 3 CO oo o - 81 -from which only about 45% was converted to c e l l u l o s e by ATCC 14851 and about 20% by ATCC 10821. On the contrary , the P h i l i p p i n e s t r a i n was able to u t i l i z e up to 73%, of which 87% was converted to c e l l u l o s e (Figure 12). The conversion of t o t a l sugar in the medium reached a maximum of 64% with the P h i l i p p i n e s t r a i n (Figure 11). Contrary to these r e s u l t s , Dudman (1959a) reported a maximum of 23.5% conversion of t o t a l sugar, when A. xylinum s t r a i n Hes t r in was grown on hydrolyzed molasses for 30 days eventhough 94% of the sugar was u t i l i z e d . Comparison of the growth curves of the three ^ . xylinum s t r a i n s suggested that t h i s organism var ie s widely i n i t s e f f i c i e n c y to convert sugar to c e l l u l o s e . Thus, i t appears that the P h i l i p p i n e s t r a i n i s the only one whose i n d u s t r i a l a p p l i c a t i o n may be economically f e a s i b l e . However, i t should be emphasized that no attempt was made to further improve sugar conversion by the use of a d d i t i o n a l adjuncts (Dudman, 1959b). D. Degree of Polymerization vs. Incubation Time As shown i n Table 3, the degree of polymerizat ion (DP) of the P h i l i p p i n e s t r a i n s l i g h t l y decreases with incubat ion time to a mimimum of 2000 DP uni t s a f ter 20 days of incuba t ion . The same behaviour appears evident for ATCC 14851 as ind ica ted by the decreasing v i s c o s i t y a f ter 5 days of incubat ion (Table 4 ) . Takai et a l . (1975) reported a maximum DP of 3500 at ta ined a f ter 250 h incubat ion of s t r a i n AHV-1595 of ^ . xylinum which then also decreased. This group of researchers in terpre ted the decreasing DP Figure 12. Sugar u t i l i z a t i o n ( ) and sugar conversion ( ) of three A . xylinum s t ra ins i n 100 mL of defined medium. - 83 -Table 3. The v i scometr ic average degree of polymerizat ion of b a c t e r i a l c e l l u l o s e produced by incubat ion of the P h i l i p p i n e s t r a i n of A. xyl inum. Incubation I n t r i n s i c v i s c o s i t y 3 time (days) ( m P a » s ) D.P . 5 96.30 + 1.13 2300 8 84.98 + 0.67 2200 11 86.59 + 1.62 2200 13 70.99 + 0.24 2050 15 73.49 + 1.23 2075 20 66.60 + 0.60 2000 32 63.14 + 0.70 2000 aMean i n t r i n s i c v i s c o s i t y ± S .D. (n=4). - 84 -Table 4. The average degree of polymerizat ion of b a c t e r i a l c e l l u l o s e produced by incubat ion of ATCC 14851 of _A. xylinum (n=4). Incubation I n t r i n s i c v i s c o s i t y 3 time (days) ( m P a « s ) D.P . 5 251.02 ± 11.05 >2350 8 126.24 ± 20.0 >2350 11 131.02 ± 12.3 >2350 13 144.02 ± 18.68 >2350 15 109.50 ± 2.12 >2350 20 109.00 ± 5.01 >2350 32 105.00 ± 2.03 >2350 aMean i n t r i n s i c v i s c o s i t y ± S .D. - 85 -to be the r e s u l t of the act ion of c e l l u l a s e released in to the medium at the same time as c e l l u l o s e m i c r o f i b r i l s were being formed. The ac t ion of a c e l l u l a s e i n cu l tures of _A. xylinum, however, could not be confirmed by other authors . A more prec i se i n t e r p r e t a t i o n of t h i s DP drop was given by Marx-F ig in i (1982) cons ider ing the polymerizat ion mechanism and taking into account a c e r t a i n synchronizat ion of the b a c t e r i a . The r e l a t i o n s h i p between molecular weight, r eac t ion ra te , time of synthes i s , and generation time of the bac te r i a i n b a c t e r i a l polymerizat ion processes has been t h e o r e t i c a l l y der ived assuming a Poisson l i k e polymerizat ion mechanism and taking in to account an exponential increase of chain i n i t i a t i o n and a l i n e a r chain growth. The t h e o r e t i c a l approach ( F i g i n i , 1974) confirmed the assumption of a Poisson l i k e mechanism and revealed furthermore that the experimental ly observed decrease of DP i s a consequence of a c e r t a i n synchronizat ion of the bac te r i a and the n o n s t a t i s t i c a l polymerizat ion mechanism. A decreasing DP could also be in terpre ted to be the r e su l t of the formation of mutants which possess d i f f e r e n t degrees of c e l l u l o s e d e f i -c i ency , but s t i l l able to produce shorter f i b r i l s . This would r e s u l t i n higher p o l y d i s p e r s i t y of the molecular weight, and consequently, a lower average DP would be determined. E. C e l l u l o s i c F i b r e s P r o d u c t i o n 1. F i b r e production apparatus The pre l iminary design of t h i s apparatus resul ted i n the success-f u l production of c e l l u l o s e f i b r e s which became interwined to form a - 86 -f ibrous strand or iented p a r a l l e l to the medium flow. U n i d i r e c t i o n a l o r i e n t a t i o n of the f i b r e s was f i r s t confirmed by observing the sta ined f i b r e s by l i g h t microscopy. Photographs showing wel l defined areas of b i re f r i gence are shown and discussed i n the f i b r e microstructure s e c t i o n . This f i b r e production apparatus provided a completely s t e r i l e system which could be a s e p t i c a l l y inoculated with cu l ture s of xyl inum. A second apparatus was designed based on the same p r i n c i p l e of u n i d i r e c t i o n a l flow (Townsley, 1981; personal communication) of s t e r i l e medium, which would provide a l a rger growing surface . Further improve-ments were the i n c l u s i o n of an a i r f i l t e r through which a i r was sparged in to the chamber, a growing surface provided with channels where i n d i -v idua l strands were synthes ized , the adaption of a pH c o n t r o l system which maintained an optimum pH and a timer which provided the means to i n t e r m i t t e n t l y c o n t r o l the f low. 2. Process for the production of f i b r e s Two v a r i a b l e s , the xylinum s t r a i n and the flow mode (continuous or i n t e r m i t t e n t , which appeared to determine the t e n s i l e strength of f i b r e s were cons idered. Flow rate was f ixed at 12.2 L/min s ince i t was the lowest flow rate which s t i l l provided a smooth evenly d i s t r i b u t e d flowing media. a) E f fec t of A . xylinum s t r a i n and flow mode on t e n s i l e s trength Values for t e n s i l e strength obtained through load-elongat ion t e s t s of c e l l u l o s e f i b r e s produced by incubat ion of two s t r a i n s of / \ . xylinum under two flow modes are presented i n Table 5. Results of a two-way - 87 -Table 5 . Tens i l e strength of c e l l u l o s e f i b r e s produced by two s t r a i n s of ^ . xylinum c u l t i v a t e d under continuous or in te rmi t tent flow (Mean ± S . D . ) . Tens i l e s t rength, N/cm (n=3) ATCC 14851 P h i l i p p i n e s t r a i n Intermit tent Continuous Intermit tent Continuous 8340 ± 1200 5760 ± 920 4000 ± 4 2 0 N . D . 1 1 Not detected. - 88 -ana ly s i s of variance are reported i n Table 6. The fac tors of s t r a i n of /\. xylinum and flow mode were computed to be h ighly s i g n i f i c a n t sources of v a r i a t i o n (P<.01). Tens i l e strength of high polymers had been reported to be inf luenced by the t o t a l amount and o r i e n t a t i o n of c r y s t a l l i n e mater ia l i n a preferred d i r e c t i o n ( B a t t i s t a , 1958). Several researchers had reported on the r e l a t i o n between c r y s t a l l i n e o r i e n t a t i o n and rupture propert ies such as t e n s i l e strength and extension at break of cotton f i b r e s ; these observations ind ica ted that the s t r u c t u r a l alignment of c e l l u l o s e had a major in f luence on the t e n s i l e propert ies of the f i b r e s (Ooshi , et _ a l . , 1967; Shelat et j i l . , 1960; Egle and Grant, 1970). This i n t e r n a l f i b r e arrangement i s determined by extensive hydrogen bonding; molecules of c e l l u l o s e associate through OH bonding, i n t r a and i n t e r -chain (Nissan, 1977). Thus, the s i g n i f i c a n c e of water in the formation of b a c t e r i a l c e l l u l o s e m i c r o f i b r i l s i s obvious. Wet c e l l u l o s e i s surrounded by layers of water molecules; one of the e f fects of drying i s the removal of these mul t ip l e l a y e r s , permitt ing a c lose a s soc ia t ion of the chains and the i r r e v e r s i b l e formation of hydrogen bonds between c l o s e l y opposed hydroxyl groups. Sometimes these a s soc ia t ions are extensive and strong enough to form the c r y s t a l l i t e s ( C o l v i n , 1977). The mechanism by which f i b r e s were produced by in te rmi t tent flow i n t h i s study, involved the combination of these aggregated and des iccated stages as explained by C o l v i n (1977). Intermit tent flow allowed des i cca t ion to take place with the concurrent c lo se r a s soc ia t ion of c e l l u l o s e cha ins . Strong a s soc ia t ions resu l ted i n the formation of or iented c r y s t a l l i t e s consequently increas ing the t e n s i l e strength of - 89 -Table 6. Ana lys i s of variance of mean t e n s i l e strength for c e l l u l o s e f i b r e s produced by two s t r a i n s of _A. xylinum c u l t i v a t e d under continuous or in termi t tent f low. Source of v a r i a t i o n DF Mean square F - r a t i o S t r a i n of A. xylinum 1 0.7774 X 10 8 138.82** Flow mode 1 0.3178 X 10 8 56.75* * S t r a i n x flow mode 1 0.0136 X 10 8 2.42 Er ror 8 0.0056 X 10 8 T o t a l 11 * * S i g n i f i c a n t at p < 0 .01 . - 90 -the f i b r e s . On the contrary , c e l l u l o s e chains of f i b r e s produced under continuous flow were constant ly surrounded by water molecules which were competing with adjacent chains for those OH groups involved i n hydrogen bonding, consequently weakening a s soc ia t ions among c e l l u l o s e cha ins . S i g n i f i c a n t d i f ferences i n the t e n s i l e strength of f i b r e s produced by two d i f f e r e n t s t r a in s also suggest d i f ferences i n c r y s t a l l i n e o r i e n -t a t i o n . V i s u a l comparison of c e l l u l o s e f i b r e s produced by the two s t r a in s ind ica ted that the P h i l i p p i n e s t r a i n produced a f i b r e network much more highly hydrated than that produced by ATCC 14851. Again , c lose aggregation of c e l l u l o s e chains appeared to play an important r o l e i n t e n s i l e s t rength . The i n t e r a c t i o n between s t r a i n and flow mode was computed to be a nons ign i f i cant source of v a r i a t i o n (P > 0 .05) . Fol lowing t h i s experiment, in te rmi t tent flow (1 h on/1 h off) and s t r a i n ATCC 14851 were adopted for fur ther t r i a l s . b) Influence of aerat ion rate on growth and c e l l u l o s e y i e l d The inf luence of aerat ion rate on growth and c e l l u l o s e y i e l d s was examined i n cu l ture s grown i n defined medium. Cul tures were a l l grown under the same c o n d i t i o n s , but aerated at d i f f e r e n t rates (0.04, 0.102, and 0.282 L a i r / L medium/min). The rate of sugar u t i l i z a t i o n reached a maximum of 15% at the end of incubat ion when a i r flow was sparged at a rate of 0.10 L a i r / L medium/min, whereas lower or higher a i r flow rates resul ted i n approximately 6.8 and 7.5% sugar u t i l i z a t i o n (Figure 13). In a l l t r i a l s , pH f e l l to about 3.2 to 3.6. Consequently, i t appears - 91 -O 0 . 0 4 U a i r / L med ium/min A 0 . 1 0 " II 0 1 2 3 4 I N C U B A T I O N T I M E , D A Y S Figure 13. Influence of a i r flow rate on sugar u t i l i z a t i o n ( ) and ( ) of A. xylinum ATCC 14851 grown in defined medium. - 92 -appears that c e l l u l o s e y i e l d s could be increased i f the cu l tures were neutra l i zed to keep the pH i n a more favorable range. Thus, a fourth t r i a l was examined fo l lowing the same parameters as for the other three t r i a l s , except that the pH was c o n t r o l l e d at 4.5 and a i r was sparged at .04 L a i r / L medium/min. Sugar u t i l i z a t i o n i n the pH c o n t r o l l e d t r i a l followed the same trend as that in the non c o n t r o l l e d t r i a l , reaching a f i n a l sugar u t i l i z a t i o n of 6.8%. However, c e l l u l o s e y i e l d i n the pH c o n t r o l l e d t r i a l represented an increase of approximately 23% over the non c o n t r o l l e d t r i a l . Furthermore, the most favourable conversion of u t i l i z e d sugar to c e l l u l o s e of 10.24% was i n the pH c o n t r o l l e d t r i a l (Figure 14). C e l l u l o s e y i e l d s and sugar conversion were af fected by the a i r flow rates used; c e l l u l o s e y i e l d s reached a maximum of 3100 mg with an a i r flow rate of 0.10 L a i r / L medium/min), with lower y i e l d s observed at lower or higher flow rates (Figure 14). However, the most favourable conversion of u t i l i z e d sugar to c e l l u l o s e of 8.34% was with the lowest a i r flow rate (0.04 L a i r / L medium/min). Increased aerat ion decreased the c e l l u l o s e to nitrogen r a t i o C/N from 74.6 (0.04 L a i r / L medium/min) to 15.6 and 14.7. Thus, i t appears that c e l l u l o s e synthes i s /un i t of growth was reduced by increased aera-t i o n . This inverse r e l a t i o n s h i p may be interpre ted to mean that increased aerat ion lead to a s h i f t i n the metabolism of the organisms away from c e l l u l o s e towards increased ox idat ion of the sugar substrate (Dudman, 1960) . The C/N for the pH c o n t r o l l e d t r i a l was within the highest va lues , 60.6, as i t would be expected for a process with the highest sugar conversion (10.24%). - 93 -A I R F L O W R A T E I L a i r / L m e d i u m / min) F i g u r e 14. Inf luence of a i r flow rate on c e l l u l o s e y i e l d ( o o ) , sugar convers ion ( A A ) , n i t rogen content ( • • ) and d i s s o l v e d oxygen ( • • ) of A. xylinum ATCC 14851 grown i n def ined medium. Symbols in brackets represent pH c o n t r o l l e d t r i a l s . - 94 -Disso lved oxygen (DO) was measured at the end of the t r i a l s to ensure anaerobic condi t ions did not occur , p a r t i c u l a r l y with lower a i r flow ra te s . DO values ranged from 3.3 to 3.5 ppm (Figure 14). Thus, i t appears that eventhough A,, xylinum i s a h ighly aerobic organism, a l i m i t e d supply of oxygen i s a l l that was required for maximum sugar conversion to c e l l u l o s e , s ince accelerated growth occurred with increased aerat ion at the expense of decreased c e l l u l o s e y i e l d s . The fo l lowing c u l t i v a t i o n condi t ions were adopted for fur ther f i b r e product ion: pH c o n t r o l l e d defined medium was used with i n t e r m i t -tent flow at a rate of 12.2 L medium/min; a i r was sparged in to the system at a rate of 0.04 L a i r / L medium/min, and the inoculated appara-tus was incubated for 4 days at 2 8 - 3 0 ° C . F. Cellulosic Fibre Characterization 1. Tens i l e proper t ie s a) E f f ec t of moisture content on t e n s i l e s trength The e f fect of moisture content on t e n s i l e strength of c e l l u l o s i c f i b r e s i s depicted i n Figure 15. Each bar represents the mean of t r i p -l i c a t e analyses of samples which were e q u i l i b r a t e d over d i f f e r e n t saturated s a l t s o l u t i o n s . The mechanical behaviour of the f i b r e s t ructure appeared to be great ly inf luenced by the moisture content i n the specimen. I t has long been recognized that the moisture r e l a t i o n s h i p s of various f i b r e types d i f f e r and the degree to which the f i b r e propert ies are modified w i l l vary . The load-e longat ion curve for a hydrophobic mater ia l when tested - 95 -7000 -5000 -6000 -• \ Z x" C3 z UJ CC I- 40001-U i - J (/) z Ul h- 3000 2000 — 1000 — N a N 0 2 E R H 6 5 °/o C H 3 C O O K E R H 2 2 % Na C I E R H 7 3 °/o 2 0 40 6 5 9 5 M O I S T U R E , °/o C w b ) Figure 15. Mean t e n s i l e strength of _A. xylinum c e l l u l o s e f i b r e s e q u i l i -brated over f i v e saturated s a l t solutions (n=3) . - 96 -i n the dry s tate w i l l be s i m i l a r to the curve obtained from a wet s tate ; on the other hand, the curves obtained when te s t ing h y d r o p h i l i c mater-i a l s dry and wet w i l l exh ib i t s i g n i f i c a n t d i f ferences (Booth, 1964). Since c e l l u l o s e f i b r e s are highly h y d r o p h i l i c , t e n s i l e strength was expected to be highly inf luenced by the moisture content i n the sample. The mechanical behavior of the f ib re s in t h i s study could be explained i n terms of chain mob i l i ty i n the c e l l u l o s e f i b r e s t r u c t u r e . Maximum t e n s i l e strength i n a polymeric s t ructure i s at ta ined when the load i s appl ied unformly to a l l chain segments, i . e . , a pe r f ec t ly or iented s t r u c t u r e . With imperfect o r i e n t a t i o n , strength can be improved i f more chains are u t i l i z e d i n r e s i s t i n g the load . Or ien ta t ion i s p o s s i b l e , however, only i f there i s chain segment mobi l i ty under the tes t cond i -t i o n s . Chain mob i l i ty i s useful i n delaying f a i l u r e of b r i t t l e mater-i a l s because s t r a i n energy can be d i s s ipa ted i n viscous flow and/or t rans ferred away from a region of l o c a l i z e d s t ress concentra t ion , e . g . , the t i p of a propagating crack (Warburton, 1970). C e l l u l o s e i s composed p r i n c i p a l l y of s t i f f , r i g i d c e l l u l o s e cha ins . The greater part of each molecule i s constrained to a c r y s t a l l i n e l a t t i c e . There i s l i t t l e opportunity for chain mob i l i ty i n such a s t ruc ture ; however, the l i t t l e that ex i s t s i s of cons iderable u t i l i t y . It appears that approximately 40% moisture provided the c e l l u l o s e s t ructure with the required chain mobi l i ty needed to achieve maximum o r i e n t a t i o n when subjected to s t r a i n , consequently, maximum t e n s i l e strength was a t t a ined . As moisture content increased beyond 40% t e n s i l e strength decreased. This r e s u l t may be in terpre ted i n terms of excessive hydrogen bonding with water - 97 -rather than between c e l l u l o s e cha ins , thus when a load was app l i ed , c e l l u l o s e chains possessed excessive mob i l i ty cont r ibu t ing to d i s o r i e n t a t i o n . On the other hand, as the moisture content approximated 0% the mater ia l became extremely b r i t t l e and t e n s i l e strength decreased. For subsequent t e s t i n g , there fore , i t was e s s e n t i a l that a standard moisture content in the sample was maintained. b) Mapping super simplex Opt imizat ion (MSO) of mercer iza t ion  treatment The ob jec t ive of t h i s study was to f ind the condi t ions under which the t e n s i l e strength of ^ . xylinum c e l l u l o s e f i b r e s was maximized. After 18 experiments using a modified super simplex opt imiza t ion (MSS) (Nakai , 1982) the i t e r a t i o n was stopped s ince the l a s t three v e r t i c e s to replace the worst ( v e r t i c e s 15, 16 and 18) had approximately the recommended 10% di f ference i n the response s i ze (Nakai ^ t _ a l . , 1984). Af ter car ry ing out eighteen experiments, the response values were p lo t ted against the fac tor l e v e l s on separate graphs for each f a c t o r . The large and small l i m i t s of each factor were determined and entered in to the grouping-matching program to obtain a se r ie s of matched data . The matched data points were jo ined i n each graph and the most probable l i n e s were drawn. Figures 16a, b, and c show the r e s u l t of Mapping with the 18 data from MSS; the condi t ions for each treatment are summarized i n Table 7. From these r e s u l t s , a target value for each fac tor was set as fol lows (the present best value i s shown i n parentheses) : NaOH 8.31 - 98 -(8.31)%, temperature 70 ( 5 9 . 0 9 ) ° C , and time 20 (24.62) min. Af ter two experiments using simultaneous s h i f t , the search was terminated s ince the response value became worse. The MSO showed the presence of an optimum at 8.3% NaOH, 5 9 ° C and 24 min, with a maximum t e n s i l e strength of 83,300 ± 1 8 0 0 N/cm which was an increase over the untreated f i b r e s of 281%. The mean t e n s i l e strength of the untreated f i b r e s was 29,600 ± 4 2 0 0 N/cm . The re su l t s i n t h i s study showed the usefulness of mapping super simplex opt imiza t ion i n f ind ing the best condi t ions for the mercer izat ion treatment of / \ . xylinum c e l l u l o s e . Furthermore, MSO could be extremely he lp fu l in t e x t i l e research s ince numerous s tudies have been c a r r i e d out to determine the optimum condi t ions under which c e l l u l o s i c mater i a l s , i . e . , cotton should be mercerized (Warwicker and Hallam, 1975; Peters , 1963). However, t h e i r r e s u l t s were far from being the true optimum; the treatments var ied only one factor at a t ime, thus e l imina t ing the strong i n t e r a c t i o n s among the factors which take place during swel l ing of c e l l u l o s e by the act ion of sodium hydroxide. On the contrary , super simplex opt imizat ion allowed the simultaneous v a r i a t i o n of the three factors involved i n mercer i za t ion . Furthermore, a f ter 18 experiments, the mapping procedure provided a useful overview of the response surface around the optimum. The e f fec t s of the mercer izat ion treatments on the mechanical proper t ie s of c e l l u l o s i c f i b r e s were explained by Warwicker jet a l . (1966) on the basis of changes i n f i b r e o r i e n t a t i o n and improvements i n the uni formity of the strength along the f i b r e . This was a t t r ibu ted to - 99 -F igure 16a. Mapping responses of experiments to determine condi t ions to maximize t e n s i l e strength of ^ . xylinum c e l l u l o s e f i b r e s . Response value plotted against NaOH, %. - 101 -H E A T I NG T I M E , min F i g u r e 16c. Response values p l o t t e d against heating t ime, min. - 102 -Table 7 . M e r c e r i z a t i o n treatments and mean t e n s i l e strength values obtained with Mapping Super Simplex Optimization (n=3). NaOH Temperature Time T e n s i l e Strength Vertex No. (%) C O (min) (N/cm2) 1 6.00 2.0 10.0 32,500 + 4200 a 2 21.00 20.3 21.7 5,000 + 400 3 9.77 75.5 21.7 63,200 + 2800 4 9.77 20.3 57.1 61,600 + 3200 5 4.00 44.8 37.5 51,600 + 2500 6 9.69 80.0 60.0 8,300 + 400 7 7.53 39.8 34.2 63,000 + 1300 8 14.06 45.6 37.9 11,100 + 900 9 7.39 45.1 37.6 56,200 + 800 10 11.06 50.6 41.1 44,800 + 2500 11 8.55 43.9 36.9 60,100 + 2200 12 9.51 46.5 38.5 69,400 + 1300 13 8.11 80.0 10.0 82,000 + 1900 14 10.71 80.0 12.6 15,100 + 900 15 8.54 57.2 27.4 82,400 + 1800 16 7.66 46.9 28.8 71,900 + 1500 17 6.70 76.2 5.6 61,100 + 3200 18 8.31 59.0 24.6 83,300 + 1800 19 8.31 70.0 20.0 72,400 + 1300 20 8.50 72.0 15.0 79,700 + 1500 untreated sample 29,600 + 4200 aStandard d e v i a t i o n . - 103 -an increase i n strength at the weakest points i n the f i b r e . Thus, i t was suggested that mercer izat ion increased the t e n s i l e strength of f i b r e s by e l imina t ing the weak po int s ; however, very l i t t l e i s known about the fac tors in f luenc ing uniformity and these deserve further i n v e s t i g a t i o n . Modi f i ca t ion of the c e l l u l o s e s t ructure takes place due to the high a f f i n i t y of NaOH. The a l k a l i i s able to penetrate not only the amorphous regions but also the c r y s t a l l i n e ones. During t h i s process , the i n t e r c h a i n forces are weakened and the strength of the mater ia l decreases, but i s recovered when the f i b r e i s deswollen and d r i e d . The process of mercer izat ion i s i r r e v e r s i b l e because of the d i s t o r t i o n of the polymer network and of the changes i n c r y s t a l l i n e s t ruc ture (Peters , 1967). Several authors reported other s t r u c t u r a l changes taking place ; i . e . the transformation of the o r i g i n a l c e l l u l o s e I to c e l l u l o s e II when the f i b r e i s completely mercerized as revealed by X-ray examination of nat ive and mercerized cotton f i b r e s (Peters , 1963), and the reduct ion of the amount of c r y s t a l l i n e mater ia l and e f fects on the c r y s t a l l i t e s i ze as ind ica ted by the l e v e l i n g of f degree of polymerizat ion dependent on a l k a l i concentrat ion (Warwicker j^t _ a l . , 1966). Although severa l theor ie s of how swel l ing occurs have been proposed, i t appears that no theory accounts for a l l the known f a c t s . The most popular theory (Peters , 1963) postulated that the hydroxyl groups of the glucose uni t s behave as weak acids which d i s s o c i a t e - 104 -independently of each other . In an a l k a l i n e medium, these w i l l become d i s soc ia ted to an extent dependent on caus t ic soda concentra t ion , and the presence of ions i n the c e l l u l o s e gives r i s e to an osmotic pressure which w i l l cause water to enter u n t i l counter balanced by the e l a s t i c forces of the swollen polymer. When the a l k a l i n e so lu t ion i s replaced by water, undissociated hydroxyl groups are reformed; the osmotic pressure therefore f a l l s , l eav ing the c e l l u l o s e i n i t s o r i g i n a l chemical s t a te . I f the osmotic pressure becomes high enough, however, the polymer w i l l be permanently d i s t o r t e d . Thus i t appears that the response values ( t e n s i l e strength) obtained in t h i s study, when the three i n t e r a c t i n g fac tors were v a r i e d , were d i r e c t l y re la ted to the degree of swel l ing taking place i n the c e l l u l o s e s t r u c t u r e . The extent of swel l ing i s determined by the osmotic pressure created between the c e l l u l o s e and the external s o l u t i o n . As i t can be seen i n Figure 16, excessive swel l ing appeared to occur with increas ing a l k a l i concentra-t ion as shown by ver t i ce s 2 (21% NaOH, 2 0 . 3 8 ° C , 21.78 min) and 8 (14.06% NaOH, 4 5 . 6 ° C , 37.9 min) , or by increas ing temperature and/or time as shown by v e r t i c e s 6 (9.69% NaOH, 8 0 . 0 ° C , 60 min) and 14 (10.71% NaOH, 8 0 ° C , 12.6 min). Extensive swel l ing resu l ted i n mechanically damaged f i b r e s , as ind ica ted by the lower t e n s i l e strength values of these treated f i b r e s than those of untreated f i b r e s . Mean t e n s i l e strength values for v e r t i c e s 2, 8, 6, 14 and for untreated f i b r e s were 5000 ± 4 0 0 , 11,100 ± 9 0 0 , 8300 ± 4 0 0 , 15,100 ± 9 0 0 , and 29,600 ± 4 2 0 0 N/cm 2 , r e s p e c t i v e l y . - 105 -c) E f fec t of mercer izat ion on t e n s i l e proper t ie s A t y p i c a l load-elongat ion curve conducted to specimen f a i l u r e i s depicted i n Figure 17. This curve d e t a i l s features such as the e l a s t i c and flow reg ions , the e x t e n s i b i l i t y of the mater ia l and the ul t imate t e n s i l e strength and rupture c h a r a c t e r i s t i c s . The shape of the curve i s one t y p i c a l l y obtained when f i b r e s are subjected to an external fo rce ; i t i s balanced by i n t e r n a l forces developed i n the molecular s t ructure of the m a t e r i a l . In the ear ly stages of s t re tch ing the m a t e r i a l , the e longat ion i s mainly concerned with the deformation of the amorphous regions i n which bonds are s tretched and sheared. I f the s t ress were removed at t h i s stage most of the extension would be recovered and the mater ia l would e x h i b i t e l a s t i c p r o p e r t i e s . By increas ing the s t res s fu r ther , the curve bends s l i g h t l y and large extensions are produced by small increases i n s t r e s s . A sort of p l a s t i c " f low" of the mater i a l occurs . The long-chain molecules rearrange themselves with further breaking of secondary bonds; the curve begins to bend towards the force ax i s u n t i l the breaking point i s reached. Such t y p i c a l behaviour was described by Booth (1964) for d i f f e r e n t f i b r e s ; showing s l i g h t l y d i f f e r -ent shapes for f i b r e s with d i f f e r e n t molecular s t ruc ture . Since phys i -c a l and chemical treatments a f fect the load-elongat ion proper t ie s of f i b r e s (Booth, 1964), the e f fect of mercer izat ion on the t e n s i l e proper-t i e s of A. xylinum c e l l u l o s e f i b r e s was s tud ied . Values for t e n s i l e s t rength , modulus of e l a s t i c i t y and percent elongation obtained through load-elongat ion ana lys i s of mercerized and unmercerized f i b r e s are presented i n Table 8. Results of a one-way ana lys i s of variance with treatment as the s ing le factor are reported i n Tables 9, 10 and 11. - 106 -S P E C I M E N R U P T U R E E X T E N S I O N , c m Figure 17. T y p i c a l load-elongat ion curve of A. >c^inum c e l l u l o s e f i b r e s conducted to specimen f a i l u r e . - 107 -Table 8 . Mean values of t e n s i l e propert ies of mercerized and unmercer-i zed f i b r e s (n=8). Treatment Tens i l e Strength (N/cm 2) Elongat ion Modulus of E l a s t i c i t y (N/cm) Mercer iza t ion 22,100 ± 3900 3 6.56 ± 2.29 22.18 ± 9.74 Contro l 4,700 ± 400 10.21 ± 3.98 4.07 ± 0.83 a Standard d e v i a t i o n . Table 9 . Ana lys i s of variance for t e n s i l e strength of mercerized and unmercerized f i b r e s . Source of v a r i a t i o n DF Mean square F - r a t i o Treatment 1 0.1217 x 1 0 1 0 158.49** Er ror 14 0.7680 x 10 7 Tota l 15 • • S i g n i f i c a n t at p < 0 .01 . - 108 -Table 10. Ana lys i s of variance for e longat ion of mercerized and unmercerized f i b r e s . Source of v a r i a t i o n DF Mean square F - r a t i o Treatment Error T o t a l 1 14 15 53.29 10.52 5.06 Table 11. Ana ly s i s of variance for modulus of e l a s t i c i t y of mercerized and unmercerized f i b r e s . Source of v a r i a t i o n DF Mean square F - r a t i o Treatment Er ror Tota l 1 14 15 1311.40 47.85 27 .40 * * * * S i g n i f i c a n t at p < 0 .01. - 109 -T e n s i l e strength (N/cm ) and modulus of e l a s t i c i t y (N/cm) were s i g n i f i c a n t l y greater (p < 0.01) for the mercerized f i b r e s , whereas e longat ion (%) was not s i g n i f i c a n t l y d i f f e r e n t for the mercerized and unmercerized f i b r e s . The increased t e n s i l e strength of mercerized c e l l u l o s i c f i b r e s has already been explained i n terms of f i b r e o r i e n t a -t i o n and improved uniformity along the f i b r e (Warwicker ^ t a l . , 1966). The modulus of e l a s t i c i t y (N/cm) gives a measure of the force required to produce an extension or r e f l e c t s the s t i f f n e s s of the f i b r e s ; a higher modulus for mercerized f i b r e s ind ica te s i n e x t e n s i v i l i t y , whereas a lower modulus ind ica te s greater e x t e n s i b i l i t y (Booth, 1964). I t appears that such behaviour could be a t t r i b u t e d to increased o r i e n t a t i o n of the f i b r e s during mercer i za t ion ; there fore , the t y p i c a l chain rearrangement taking place when f i b r e s are subjected to an external force i s cons t ra ined . d) E f f ec t of batch on t e n s i l e proper t ie s Values for t e n s i l e s t rength , modulus of e l a s t i c i t y and percent elongation obtained through load-elongat ion analyses of f i b r e s produced from two batches are presented i n Table 12. Results of a one-way analy-s i s of variance with batch as the s i n g l e factor are reported i n Tables 13, 14 and 15. Tens i l e propert ies of f i b r e s in the two batches were not s i g n i f i -cant ly d i f f e r e n t . These analyses support the conclus ion that there was no v a r i a t i o n of f i b r e s t r u c t u r e , and consequently t e n s i l e proper t ie s when f i b r e s were produced i n two t r i a l s . However, t h i s was true - 110 -Table 12. Mean values of t e n s i l e proper t ie s of c e l l u l o s e f i b r e s pro-duced from two batches (n=8). T e n s i l e Strength Elongat ion Modulus of E l a s t i c i t y Batch (N/cm 2) (%) (N/cm) 1 4700 ± 400 3 10.21 ± 3.98 4.07 ± 0.83 2 5300 ± 1 1 0 0 9.43 ± 6.03 5.81 ± 1.92 a Standard d e v i a t i o n . Table 13. Ana lys i s of variance for t e n s i l e strength of f i b r e s produced i n two d i f f e r e n t batches. Source of v a r i a t i o n DF Mean square F - r a t i o Batch Er ror T o t a l 1 14 15 0.14652 x 10 7 0.66208 x 10 6 2.21 - 111 -Table 14. Ana lys i s of variance for elongation of f ib re s produced i n two d i f f e r e n t batches. Source of v a r i a t i o n DF Mean square F - r a t i o Batch Er ror Tota l 1 14 15 2.48 26.11 0.095 Table 15. Ana lys i s of variance for modulus of e l a s t i c i t y of f i b r e s produced i n two d i f f e r e n t batches. Source of v a r i a t i o n DF Mean square F - r a t i o Batch Error T o t a l 1 14 15 12.02 2.19 5.48 - 112 -provided that the inoculum was a pure cu l ture of a c e l l u l o s e producting s t r a i n . Therefore , pe r iod i c i s o l a t i o n of c e l l u l o s e producing organisms was necessary i n maintaining a pure cu l ture which would provide uniform-i t y throughout the t r i a l s . 2. F i b r e Micros t ruc ture a) L ight microscopy Micrographs dep ic t ing stained c e l l u l o s e f i b r e s matrix embedded with bac te r i a are presented i n F igure 18 and 19. Observation of F igure 18 c l e a r l y reveal s bac te r i a which are genera l ly u n i d i r e c t i o n a l l y o r i e n t e d . Such pattern i s the r e s u l t of c e l l u l o s e f i b r e s being produced by incubating A. xylinum within a confined u n i d i r e c t i o n a l flow of medium. I n i t i a l l y , some organisms are deposited on the growing surface as medium i s c i r c u l a t e d over i t ; therea f te r , more of them become attached to the c e l l u l o s e ribbons already synthesized and attached to the growing surface . Consequently, ribbons from many bac te r i a associate with each other i n a p a r a l l e l f a sh ion . C e l l u l o s e ribbon assembly by _A. xylinum has already been observed by several workers (Brown et a l . , 1976; C o l v i n and Leppard, 1977; Zaar, 1979); however, i n a l l o f these studies ^ . xylinum was grown in s t a t i c cu l ture s where c e l l u l o s e ribbons were interwined i n a d i sorganized fa sh ion . Therefore , t h i s study was the f i r s t report on the c h a r a c t e r i z a t i o n of the s t ructure of c e l l u l o s e r ibbons which were produced i n an organized p a r a l l e l fashion (see Figure 19). A micrograph dep ic t ing the c e l l u l o s e f i b r e s under po la r i zed l i g h t i s presented in Figure 20. Well defined u n i d i r e c t i o n a l luminous areas - 113 -F i g u r e 18. Light micrograph of stained c e l l u l o s e f i b r e matrix embedded with bacteria (pw=130um), pw = photo width. F i g u r e 19. Light micrograph of c e l l u l o s e f i b r e s in an organized paral-l e l fashion (pw=130ym). - 114 -F igure 20. U n i d i r e c t i o n a l L y oriented c e l l u l o s e f ib re s under po la r i zed l i g h t (pw-130ym). - 115 -can be observed which i n d i c a t e a high degree of u n i d i r e c t i o n a l o r i e n t a -t i o n of t h i s an i so t rop ic m a t e r i a l . O p t i c a l l y an i so t rop ic or b i r e f r i n -gent mater ia l s rotate the plane of po la r i zed l i g h t and appear luminous, whereas o p t i c a l l y i s o t r o p i c mater ia l s cannot rotate the plane of po l a r i zed l i g h t and remain dark on r o t a t i o n between crossed polars (Whis t l e r , 1963). In genera l , b i r e f r i g e n c e i s used as a r e l a t i v e measure of o r i e n t a t i o n and i s defined as the d i f ference of r e f r a c t i v e indexes in two p r i n c i p a l d i r e c t i o n s ; most natural and synthet i c f i b r e s are an i so t rop ic or b i r e f r i n g e n t and can e a s i l y be observed through the p o l a r i z i n g microscope (Happey, 1978). b) Scanning e l ec t ron microscopy Scanning e lec t ron micrographs of c e l l u l o s e f i b r e s produced under u n i d i r e c t i o n a l flow are presented i n Figures 21, 22 and 23. F ibres i n F igures 22 and 23 were subjected to mercer i za t ion , whereas f i b r e s i n Figure 21 were unmercerized. Examination of the mercerized samples (Figures 22 and 23) revealed that a high degree of swel l ing occurred when c e l l u l o s e f i b r e s were exposed to NaOH; i n d i v i d u a l f i b r e s appeared glued to each other adopting a very compact s t ruc ture . On the contrary , a f i n e r and looser s t ructure was observed i n unmercerized f i b r e s (Figure 21) . Width of i n d i v i d u a l c e l l u l o s e ribbons ranged between 0.1-0.2 ym. A common feature observed i n Figures 21, 22 and 23 was a high degree of p a r a l l e l o r i e n t a t i o n of the f i b r e s . Scanning e lec t ron micrographs of c e l l u l o s e f i b r e s produced under s t a t i c condi t ions are presented i n Figures 24 to 31. Observation of Figure 21. Scanning e l e c t r o n micrograph of unmercerized c e l l u l o s e f i b r e s produced under u n i d i r e c t i o n a l flow showing a loose f i b r e s t r u c t u r e (pw=10pm). Figure 22. Scanning e l e c t r o n micrograph of mercerized c e l l u l o s e f i b r e s produced under u n i d i r e c t i o n a l flow (pw=247i.im). - 117 -Figure 23. Scanning electron micrograph of mercerized c e l l u l o s e f i b r e s produced under u n i d i r e c t i o n a l flow showing a compact f i b r e structure (pw=11ijm). Figure 24. Scanning electron micrograph of unmercerized c e l l u l o s e f i b r e s produced under s t a t i c conditions showing a network of entangled loose disoriented c e l l u l o s e f i b r e s (pw=11ym)". F igure 25. Scanning electron micrograph of unmercerized c e l l u l o s e f i b r e s produced under s t a t i c conditions showing a bacterium entrapped within the matrix (pw=12ym). - 119 -F igure 26. Scanning electron micrograph of unmercerized c e l l u l o s e f i b r e s produced under s t a t i c conditions (pw=28ym). Figure 27. Scanning electron micrograph of unmercerized c e l l u l o s e f i b r e s produced under s t a t i c conditions (pw=208ym). - 120 -F i g u r e 29. Scanning electron micrograph of mercerized c e l l u l o s e f i b r e s produced under s t a t i c conditions showing a compact structure (pw-22|im). - 121 -Figure 30. Scanning e l e c t r o n micrograph of mercerized c e l l u l o s e f i b r e s produced under s t a t i c c o n d i t i o n s (pw=57]jm). Figure 31. Scanning e l e c t r o n micrograph of mercerized c e l l u l o s e f i b r e s produced under s t a t i c c o n d i t i o n s (pw=254ym). - 122 -these f igures c l e a r l y revealed a network of entangled d i so r i en ted c e l l u l o s e f i b r e s . Figure 25 shows a bacterium entrapped within the c e l l u l o s e matr ix . It i s approximately 2 ym long and 0.6 tim wide, which i s i n c lose agreement with dimensions reported previous ly (Breed, et a l . , 1957). Again , unmercerized f i b r e s (Figures 24 to 27) show a f ine and loose s t ruc ture ; whereas mercerized f i b r e s show a swollen and compact s t ructure (Figures 28 to 31). Such compact s t ructures also appeared in the e lec t ron micrographs of Peters (1967). I t was descr ibed that the wel l or iented f i b r e s of cotton became comparatively d i s o r i e n t e d , and assumed a crimped appearance after mercer i za t ion . 3. C r y s t a l l i n i t y index, c r y s t a l s i z e and degree of polymerizat ion Mercerized strands presented the t y p i c a l X-ray di f fractogram of c e l l u l o s e I (Figure 36). According to Peters (1967), c e l l u l o s e I i s transformed to c e l l u l o s e II when f i b r e s are completely mercer ized. Therefore , i t appears that the mercer izat ion treatment given to the f i b r e s i n t h i s study, did not cause extensive swel l ing which would bring about a change i n the d i f f r a c t i o n pattern of c e l l u l o s e . C r y s t a l l i n i t y index and c r y s t a l s i ze ca l cu l a ted for the mercerized strands were 95.0 ± 1 . 4 1 % and 61.60 ± 0 . 7 0 ° A , r e s p e c t i v e l y . The degree of polymerizat ion of the mercerized strands was 2000 ± 2 0 0 . G. Microcrystalline Cellulose (MCC) Production 1. C e l l u l o s e production and p u r i f i c a t i o n C e l l u l o s e was produced and p u r i f i e d according to the procedure o u t l i n e d i n Figure 32. Production s tar ted with the incubat ion of ^ . - 123 -Culture medium (10% sucrose defined medium) Inoculum (Philippine s t r a i n / of _A. xylinum) Incubation for 2 weeks at 28°C V Cellulose harvest V D i a l y s i s with tap water V Shredding in a Waring blendor draining through cheese cloth V NaOH Extraction Neutralization draining through cheese cloth V Bleaching V Rinsing with water draining through cheese cloth V Freeze drying Figure 32. Cellulose production and p u r i f i c a t i o n . - 124 -xylinum i n 10% sucrose defined medium, where ce lu lose p e l l i c l e s were grown adjacent to the surface of s t a t i c growing medium (Figure 33). Af te r 2 weeks of incubat ion , c e l l u l o s e p e l l i c l e s were harvested by decanting the cu l ture f l u i d and removing c e l l u l o s e (Figure 34) . Intact p e l l i c l e s were d i a lyzed overnight with tap water to remove remaining growing medium and then shredded to f a c i l i t a t e NaOH ext rac t ion (Figure 35) . The use of NaOH so lut ions to remove b a c t e r i a l c e l l s from c e l l u l o s e p e l l i c l e s has been wel l documented i n the l i t e r a t u r e (Schramm and H e s t r i n , 1954; Dudman, 1959a; Kaushal and Walker, 1954). However, due to the nature of t h e i r work, no a t tent ion was given to the pos s ib le s t r u c t u r a l damage taking place i n the c e l l u l o s e matrix . Studies by various researchers (Warwicker, £ t a l . , 1966; Ranby, 1952; Peter s , 1967) had shown the detr imental ac t ion of wel l ing agents, such as NaOH, on the phys i ca l s t ructure of var ious c e l l u l o s e s , namely, decreased c r y s t a l l i n i t y and degree of po lymer iza t ion . Furthermore, i n pre l iminary experiments of t h i s study, i t was observed that c e l l u l o s e p e l l i c l e s which were soaked i n 8% (w/v) NaOH for 3 to 4 days at room temperature had a promounced decrease in the degree of polymerizat ion (approx. 700 DP u n i t s ) . Consequently, a very low y i e l d was recovered a f ter ac id hydro ly s i s (30-40%). Taking these facts into cons idera t ion , c e l l u l o s e was subjected to various ex t rac t ion treatments to inves t i ga te whether s t r u c t u r a l damage was taking place under the condi t ions te s ted . C e l l u l o s e , whether i n the swollen wet state or freeze dr ied s ta te , was subjected to increas ing NaOH concentrat ions ranging from 1 to 8% (w/v) for 3 hours. Degree of - 125 -Figure 3 4 . C e l l u l o s e p e l l i c l e being harvested a f t e r two weeks of incu-bation. - 126 -Figure 35. Shredded ^. xylinum cellulose prior to NaOH extraction. - 127 -po lymer iza t ion , c r y s t a l l i n i t y index, c r y s t a l s i ze and nitrogen content of the var ious samples are reported i n Table 16. A t y p i c a l di f fractogram for / \ . xylinum c e l l u l o s e i s presented i n Figure 36, showing the main d i f f r a c t i o n maxima of the s t ructure corres-ponding to the 101, 10T and 002 planes from which the c r y s t a l l i n i t y index and c r y s t a l s i ze were der ived . Results of a two way ana lys i s of variance for each of the four var i ab le s tested are presented i n Tables 17, 18, 19 and 20. The fac tor "treatment" was computed to be a h ighly s i g n i f i c a n t source of v a r i a t i o n (p < 0.01) for a l l determined v a r i a b l e s , except for c r y s t a l s i z e . The fac tor " c o n d i t i o n " was found to be a highly s i g n i f i c a n t source of v a r i a t i o n (p < 0.01) for a l l var i ab le s tested except for the degree of po lymer iza t ion . The r e s u l t s of a Duncan's mul t ip l e range te s t on nitrogen content revealed that each treatment was s i g n i f i c a n t l y d i f f e r -ent (p < 0.05) from the o thers , except for 6 and 8% NaOH concentra t ion . Increasing NaOH concentrat ion s i g n i f i c a n t l y (p < 0.05) reduced the ni trogen content of the c e l l u l o s e matrix , u n t i l i t appeared to approach equ i l ib r ium at concentrat ions between 6 and 8%. Sodium hydroxide ex t rac t ion was e f f e c t i v e i n removing entrapped b a c t e r i a . Ex t r ac t ion of nitrogenous impur i t i e s was s i g n i f i c a n t l y (p < 0.01) more e f f i c i e n t i n freeze dr i ed treated samples than i n the swollen samples. These r e s u l t s were somewhat expected, s ince the swollen samples had a d i l u t i o n fac tor of approximately 2/3. However, i t i s poss ib le that not only the f i n a l NaOH concentrat ion during ex t rac t ion accounted for the decreased nitrogen i n the freeze dr ied samples, but - 128 -Table 16. E f f e c t of NaOH e x t r a c t i o n of swollen and freeze d r i e d c e l l u -l o s e on Degree of Po l y m e r i z a t i o n (DP), C r y s t a l l i n i t y Index ( C r I ) , c r y s t a l s i z e and nitrogen content (Mean values ± SD) 1. Condition of NaOH CrI C r y s t a l s i z e Nitrogen c e l l u l o s e (%) D.P. («) (°A) (%) Swollen 0 300 + 70.7 86.45 + 0.14 66.35 + 1.62 0.90 + 0.14 1 1200 + 141.4 94.85 + 1.69 61.60 + 0.70 0.71 + 0.07 3 1400 + 35.3 95.00 + 1.14 61.60 + 0.70 0.53 + 0.02 6 2050 + 162.6 95.00 + 1.41 61.60 + 1.13 0.31 + 0.01 8 1900 + 106.0 94.80 + 0.70 61.60 + 4.24 0.15 + 0.02 Freeze-dried 0 300 + 70.7 86.40 + 0.14 66.35 + 1.62 0.90 + 0.14 1 1200 + 141.4 89.20 + 2.12 66.35 + 1.69 0.48 + 0.02 3 1600 + 106.0 87.50 + 0.28 66.30 + 2.54 0.21 + 0.01 6 1800 + 141.4 89.60 + 1.41 66.30 + 0.7 0.09 + 0.01 8 1700 + 70.7 92.20 + 1.69 66.30 + 3.53 0.10 + 0.02 - 129 -D I F F R A C T I O N A N G L E 20, d e g r e e s Figure 36. A t y p i c a l d i f f ratogram of A. xylinum c e l l u l o s e . - 130 -Table 17. Ana lys i s of variance of nitrogen content of NaOH-extracted c e l l u l o s e samples 3 . Source of v a r i a t i o n DF Mean square F - r a t i o Condi t ion 1 0.134 33 .52* * Treatment 4 0.397 99 .32* * C x T 4 0.017 4.42 n . s . E r r o r 10 0.004 a These data are shown i n Table 16. • • S i g n i f i c a n t at p < 0 .01. Table 18. Ana lys i s of variance of c r y s t a l l i n i t y index of NaOH-extracted c e l l u l o s e samples 3 . Source of v a r i a t i o n DF Mean square F - r a t i o Condi t ion 1 90.40 82 .18* * Treatment 4 30.40 2 7 . 6 3 ^ C x T 4 8.00 7.27 n . s . E r r o r 10 1.1 3 These data are shown i n Table 16. • • S i g n i f i c a n t at p < 0.01. - 131 -Table 19. Ana lys i s of variance of degree of polymerizat ion of NaOH-extracted samples 3 . Source of v a r i a t i o n DF Mean square F - r a t i o Condit ions 1 0.0125 x 10 6 1.08 n . s . Treatment 4 1.6805 x 10 6 146.13** C x T 4 0.0325 x 10 6 2.82 n . s . E r r o r 10 0.0115 x 10 6 a These data are shown i n • ^ S i g n i f i c a n t at p < 0.01 Table 16. • Table 20. Ana lys i s of samples 3 . var iance of c r y s t a l s i ze of NaOH-extracted Source of v a r i a t i o n DF Mean square F - r a t i o Condi t ions 1 71.10 16.15** Treatment 4 4.50 1.02 n . s . C x T 4 4.40 1.00 n . s . E r r o r 10 4.40 3 These data are shown i n Table 16. * * S i g n i f i c a n t at p < 0 .01. - 132 -a l so an increased NaOH adsorption rate determined by changes i n the f i b r e morphology. S i g n i f i c a n t l y (p < 0.01) lower c r y s t a l l i n i t y indexes i n the freeze dr ied samples supports the assumption of an increased NaOH adsorption ra te . Magister et _al. (1975) reported a s i m i l a r phenomenon when c e l l u l o s e was hydrolyzed; higher reac t ion rates for dr ied c e l l u l o s e were reported ( a i r d r i e d , freeze d r i e d , a i r dr ied and wetted again) than for moist c e l l u l o s e s . I t was suggested that one of the e s s e n t i a l factors was the development of mechanical s t resses , contrac t ions during the primary shrinkage stage of the drying process , r e s u l t i n g i n microtears which then ass i s ted i n the h y d r o l y t i c degradation. The c r y s t a l l i n i t y indexes of treated samples were s i g n i f i c a n t l y (p < 0.05) higher than those of untreated samples. These r e s u l t s do not i n d i c a t e that c r y s t a l l i n i t y per se was increased by the treatment, but rather that a subs tant i a l amount of impur i t i e s and amorphous c e l l u l o s e extracted by NaOH accounted for the percentage of amorphous mater ia l i n the untreated samples thus, decreasing the r a t i o Ioo2-Iam/Io02« A c r y s t a l l i n i t y index of approximately 95% ca l cu l a ted i n t h i s study agrees wel l with a C r . I . of 96% reported by Correns and Purz (1975). It appears that some d e c r y s t a l l i z a t i o n took place i n the freeze d r i e d samples, and these r e s u l t s agree wel l with those reported by P a t i l et a l . (1965). It was reported that sodium hydroxide acts as a decrys-t a l l i z i n g agent on cotton c e l l u l o s e ; NaOH concentrat ions ranging from 0 to 50% resu l ted i n C r . I . ranging from 70 to 45%. - 133 -The degree of polymerizat ion was s i g n i f i c a n t l y (p < 0.05) higher for the treated samples than for the untreated ones, and the D.P. of 6 and 8% treated samples were not s i g n i f i c a n t l y (p > 0.05) d i f f e r e n t . Since the D.P. i s not an absolute measurement, but only an average, impur i t i e s and short f i b r i l s i n the untreated samples may have caused low D.P. averages. Treated samples freed from impur i t i e s r e s u l t i n true est imation of c e l l u l o s e chain s i z e s . Increasing NaOH concentra-t i o n s i g n i f i c a n t l y (p < 0.05) reduced impur i t i e s as indica ted by the ni trogen content . Consequently, the average D.P. also increased u n t i l i t appeared to l e v e l o f f at 6 and 8% NaOH. In conc lus ion , an ex t rac t ion treatment with a NaOH concentrat ion ranging between 6 and 8% on swollen c e l l u l o s e e f f i c i e n t l y removed embedded bac ter i a without any detr imental e f fec t on f i b r e s t ruc ture . The y i e l d of p u r i f i e d c e l l u l o s e after treatment with 6% NaOH was 43 ± 1 . 4 4 % . NaOH ex t rac t ion of swollen c e l l u l o s e not only e l iminated degradation, but also e l iminated the freeze drying s tep. Thus, the process of c e l l u l o s e p u r i f i c a t i o n could be continuous. Since at t h i s stage, c e l l u l o s e was highly p u r i f i e d (approximately 98% c e l l u l o s e ) , a very mild bleaching treatment was necessary to obtain a good white c o l o u r . This mild treatment involved only one sequence of c h l o r i t e and ac id a d d i t i o n . On the contrary , several sequences are usua l ly involved during d e l i g n i f i c a t i o n of wood pulp by the c h l o r i t e method r e s u l t i n g i n cons iderable depolymerizat ion (Browning, 1967). Bleaching resu l ted i n 88% y i e l d thus, a 12% loss in c e l l u l o s e and co lour ing matter. The DP value of the bleached sample was 1500, - 134 -there fore , some depolymerizat ion had taken place during c h l o r i t e treatment. Modi f i ca t ions of the c h l o r i t e method are concerned with the time and sequence of the c h l o r i t e and acid add i t ions , the c o n t r o l of pH during the c h l o r i t e treatments, and the temperature at which the treatment i s c a r r i e d out . For example, Browning (1967) reported that c h l o r i t e markedly reduced the DP of cotton at pH 2 ( 7 0 ° C ) ; but at pH 5 ( 7 0 ° C ) the drop in pH was n e g l i g i b l e , although at 9 0 ° C the DP markedly decreased. Sodium c h l o r i t e at pH 2.9 and 2 0 ° C ox id izes reducing groups to carboxyl groups, but s ide react ions occur and a d d i t i o n a l ox ida t ion react ions take place i n cotton which leads to lower molecular weight. Thus, i t i s suggested that i f further work were to be done to avoid depolymerizat ion of A. xylinum c e l l u l o s e during b leaching , c o n t r o l of pH during the c h l o r i t e treatment should be cons idered. 2 . E f fec t of ac id h y d r o l y s i s on weight l o s s and degree of  polymerizat ion Changes i n the degree of polymerizat ion and y i e l d with the time of hydro ly s i s by b o i l i n g with 2.5 N HC1 are shown in Figures 37 and 38 for 6% NaOH-extracted bleached samples. A highly degraded c e l l u l o s e (extracted with 8% NaOH for 3-4 days) i s also included to i l l u s t r a t e the dependence of the degree of polymerizat ion and the f i n a l y i e l d on the extent of preswel l ing of c e l l u l o s e samples by sodium hydroxide so lu-t i o n s . B a t t i s t a (1950) had reported the hydro ly s i s of severa l c e l l u -loses ( p u r i f i e d co t ton , bleached cotton and wood pulp) by using 2.5 and 5N HC1 at 5, 18, 40 and 1 0 5 ° C . In t h i s work, the use of b o i l i n g 2.5 N - 135 -a. a < N oc UJ 2 >O 0. UJ Ul CC C3 Ul Q UJ C3 < CC Ul > < 1500 1400 1200|-10004-8 0 0 H 600h 400|-200r-120 T I M E , m i n F i g u r e 3 7 . Hydrolyses of 6% NaOH-extracted ce l lu lose ( • ) and degraded ce l lu lo se ( • ) . - 136 -T I M E , m i n Figure 38. Comparison of y i e l d s observed in 2.5 N HCi at 1 0 5 ° C of 6% NaOH-extracted c e l l u l o s e ( • ) and degraded ceLLulose ( • ) . - 137 -HC1 was recommended, s ince 5N H Q gave r i s e to the formation of humic substances and at the lower temperatures the degree of polymerizat ion tended to l e v e l - o f f at higher va lues . Therefore these condi t ions were adopted in t h i s study. In Figure 37, i t i s shown how quick ly the basic degree of polymer-i z a t i o n reaches what appears to be a f a i r l y constant value and that t h i s l e v e l i n g of f i s higher for the 6% NaOH-extracted (D.P. 200-220) than for a degraded c e l l u l o s e (D.P. 150-175). Furthermore, the 6% NaOH-extracted c e l l u l o s e l o s t weight with time of hydro ly s i s at a much slower rate than did degraded c e l l u l o s e (see Figure 38). These same trends were observed by B a t t i s t a (1950) when p u r i f i e d cotton (nat ive c e l l u l o s e ) and regener-ated c e l l u l o s e (viscose t i r e yarn) were subjected to acid hydro ly s i s with b o i l i n g 2.5 N HC1 for 15 min. P u r i f i e d cotton (D.P.=3200) y ie lded a LODP of 242, whereas regenerated c e l l u l o s e (D.P.=470) y ie lded a LODP of 43; y i e l d s upon hydro ly s i s were 91 and 70%, r e s p e c t i v e l y . On the basis of the data shown in Figures 37 and 38, a time of 15 minutes i n b o i l i n g 2.5 N HC1 was adopted as being the optimum point at which the LODP (220) was reached with minimum weight losses (5%). At t h i s stage of the process , c e l l u l o s e was been converted to m i c r o c r y s t a l l i n e c e l l u l o s e (MCC) s ince the amorphous mater ia l was completely removed. The c r y s t a l l i n e mater ia l was then removed by f i l t r a t i o n or c e n t r i f u g a t i o n , neut ra l i zed with 5% NH^OH, and washed with severa l changes of d i s t i l l e d water (see Figure 39). The r e s u l t i n g neutra l i zed f i l t e r e d c e l l u l o s e c r y s t a l s were r e s l u r r i e d to 3% s o l i d s and subjected to mechanical d i s i n t e g r a t i o n to free the unhinged c r y s t a l s and to form a homogeneous aqueous s l u r r y which could be e a s i l y spray d r i e d . - 138 Freeze dr ied c e l l u l o s e Hydro lys i s with 2.5 N HC1 N e u t r a l i z a t i o n with 5% NH^OH F i l t r a t i o n or c e n t r i f u g a t i o n Washing with d i s t i l l e d water Reconst i tu t ion to 3% s o l i d s Mechanical d i s i n t e g r a t i o n with a Waring Blendor Spray drying F igure 39. M i c r o c r y s t a l l i n e c e l l u l o s e product ion. - 139 -MCC production was terminated by spray drying the homogeneous s l u r r y at a temperature of 96 to 1 0 0 ° C ( i n l e t temperature). MCC in the powder form was stored i n bo t t l e s placed i n a des icca tor u n t i l required for the c h a r a c t e r i z a t i o n t e s t s . H. Microcrystalline Cellulose Characterization I . Chemical composi t ion, c e l l u l o s e , n i trogen and ash content C e l l u l o s e determination was c a r r i e d out by f i r s t subject ing the c e l l u l o s e to a t o t a l h y d r o l y s i s . The procedure required a primary hydro ly s i s with strong mineral acid followed by a secondary hydro ly s i s i n d i l u t e a c i d . The primary hydro ly s i s resul ted i n the formation of a mixture of o l i go sacchar ide s . I t was the funct ion of the secondary h y d r o l y s i s to complete the conversion to monomeric sugars. The i n i t i a l weights of c e l l u l o s e samples were corrected dry weights (moisture content of spray dr i ed sample was 4.0 ± 0 . 7 % dry b a s i s ) . The t h e o r e t i c a l glucose y i e l d i s equal to the c e l l u l o s e weight m u l t i p l i e d by the fac tor (1.111); t h i s f ac tor i s the r a t i o 180/162, 162 i s the anhydroglucose M.W. and 180 i s the monohydrate M.W. Recovered glucose was determined by the phenol s u l f u r i c ac id method of Dubois et _al. (1951). C e l l u l o s e content i n the sample was c a l c u l a t e d by the r a t i o recovered g l u c o s e / t h e o r e t i c a l glucose y i e l d X 100. The r e s u l t s are shown i n Table 21. C e l l u l o s e content ind ica ted that ^ . xylinum MCC was p u r i f i e d to 98.5%, wi thin the requirements of MCC as i s l i s t e d in the FCC III (1981) which s tates that i t should not be less than 97.0% and not more than the - 140 -Table 21. Chemical composition of m i c r o c r y s t a l l i n e c e l l u l o s e s (Mean ± S . D . ) . 3 3 3 3 Sample C e l l u l o s e Moisture Nitrogen (% d .b . ) Ash (MCC) (% d .b . ) (% w.b.) T o t a l Ammonia (% d .b . ) A . xylinum 98.5 ± 1.4 4.05 ± 0.7 1.1 ± 0.4 1.01 ± 0.1 1.15 ± 0.1 A v i c e l PH-101 99.2 ± 0.3 2.64 ± 0.2 N.D. N.D. 0.85 ± 0.1 an=3. b Not detected. - 141 -equivalent of 102.0% of carbohydrate as c e l l u l o s e , ca l cu la ted on a dr ied ba s i s . Values for t o t a l ni trogen content were r e l a t i v e l y high for / \ . xylinum MCC. It appears that most of i t was in the ammonium form as ind ica ted by the nitrogen determined as ammonia. These r e s u l t s r e f l e c t the need for exhaustive washing with water a f ter n e u t r a l i z a t i o n with NHi + O H to e l iminate the NH^Cl being formed. An ash content of 1.15% for A. xylinum MCC might be the r e su l t of using t e c h n i c a l grade reagent for the c h l o r i t e treatment; sodium c h l o r i t e which was 80% NaOCl 2 was used for b leach ing . 2. P h y s i c a l proper t i e s a) C r y s t a l l i n i t y , c r y s t a l s i z e , average degree of po lymer iza t ion ,  p a r t i c l e s i z e determination Values for / \ . xylinum MCC and A v i c e l PH-101 are summarized i n Table 22. C r y s t a l l i n i t y index, c r y s t a l s i ze and D.P. were s i g n i f i c a n t l y (p < 0.01) l a rger for A. xylinum MCC than for commercial MCC ( A v i c e l PH -101) as ind ica ted by a t - t e s t . These r e s u l t s c l e a r l y i n d i c a t e that the preparat ion of MCC from non-degraded high D.P . _A. xylinum c e l l u l o s e resul ted i n a product which was h ighly c r y s t a l l i n e with a high LODP va lue . Commercial MCC A v i c e l PH-101, however, i s prepared from highly degraded c e l l u l o s e from wood pulp . Degradation of c e l l u l o s e always occurs during d e l i g n i f i c a t i o n and i t seems impossible to i s o l a t e a wood c e l l u l o s e with the same D.P. as that i n the o r i g i n a l p l a n t . Consequently, c r y s t a l l i n i t y i s pa r t ly destroyed and the LODP was s i g n i f i c a n t l y lower (p < 0 .01) . - 142 -Table 22. Mean values of c r y s t a l l i n i t y index, c r y s t a l s i z e , average degree of polymerizat ion and p a r t i c l e s i ze for A. xylinum MCC and A v i c e l PH-101 MCC. Sample (MCC) C r y s t a l l i n i t y Index 3 (*) C r y s t a l S i z e 3 ( ° A ) a D.P. P a r t i c l e 0 s i ze (pm) A. xylinum 9 8 . 1 5 x i (0 .91) c 57 .25* (1.76) 220.0X (14.1) 1.05 x X 0 .70 x (0.60) (0.06) A v i c e l PH=101 84.90y (1.41) 45.47Y (3.43) ioo.oy (0.0) 3.50y X 1.75y (0.30) (0.20) 3n=3. bn=20. c Standard d e v i a t i o n . V a l u e s sharing a common s u p e r s c r i p t w i t h i n a column are not s i g n i f i -c a n t l y d i f f e r e n t (p > 0.05). - 143 -The c r y s t a l l i n i t y index ca l cu l a ted i n t h i s study for A v i c e l PH-101, agrees wel l with a c r y s t a l l i n i t y index of 82% reported by Paquot (1982). Mean p a r t i c l e s i ze values reported i n Table 22 were obtained a f t e r d i sper s ing 0.1% MCC so lu t ions by a Polytron high speed b lender / soni -ca tor . Large aggregates, approximately ten times the p a r t i c l e s i ze were observed before d i spers ing with a P o l y t r o n . Thus, i t i s poss ib le that the values presented i n Table 22 represent dimensions of aggregates rather than i n d i v i d u a l p a r t i c l e s . Values reported i n the l i t e r a t u r e ( B a t t i s t a , 1975) for mechanically d i s in tegra ted MCC are wi thin the range of 150A-5pm. The s i ze and the shape of the microcrys ta l s depend on the h i s tory of the precursor c e l l u l o s e f i b r e s ( B a t t i s t a , 1975). b) Moisture adsorption Moisture adsorption isotherms of / \ . xylinum MCC and A v i c e l PH-101 MCC are depicted i n Figure 40. I t appears that A v i c e l PH-101 reta ined l e s s moisture than /jk. xylinum MCC. The reason for t h i s behaviour could be due to the fact that A v i c e l PH-101 was more highly p u r i f i e d than _A. xylinum MCC, as i t can be observed from t h e i r respect ive chemical compositions (see Table 21) . Another important fac tor which may determine t h i s sorpt ion behav-i o u r , however, i s the t o t a l surface area. Since A. xylinum MCC had a s i g n i f i c a n t l y (p < 0.05) smaller p a r t i c l e s i ze than A v i c e l PH-101 (Table 22) , the t o t a l surface area ava i l ab l e for water adsorption i n A . xylinum - 144 -Figure 40. Sorption isotherms of _A. xylinum MCC and A v i c e l PH-101 MCC at 2 5 ° C - 145 -MCC was greater . Water adsorption of A v i c e l PH-101 determined i n t h i s study i s i n c lose agreement with those r e s u l t s reported by Paquot (1982). A moisture content of 16% (d.b) at 93% RH ( 2 0 ° C ) was recorded for A v i c e l PH-101. Browning (1967) has pointed out that as a f i r s t approximation, there i s a d i r e c t r e l a t i o n s h i p between the capaci ty of a f i b e r for water adsorption and the quanti ty of amorphous mater ia l i n the f i b r e . Sorp-t ion occurs mainly, i f not e n t i r e l y , i n the n o n c r y s t a l l i n e reg ions , and depends on the a v a i l a b i l i t y of free hydroxyl groups. The surfaces of the c r y s t a l l i t e s also p a r t i c i p a t e in the s o r p t i o n . A . xylinum MCC was s i g n i f i c a n t l y (p < 0.01) more c r y s t a l l i n e than A v i c e l PH-101, that i s , i t had le s s amorphous regions than A v i c e l PH-101 (Table 22). Consequently, i t would be expected to r e t a i n le s s water than A v i c e l PH-101; however, the opposite occurred . Therefore , i t appears that a combination of f ac tors determines the sorpt ion behaviour of m i c r o c r y s t a l l i n e c e l l u l o s e . c) Zeta p o t e n t i a l determination The mean corrected zeta p o t e n t i a l values for 0.1% d i spers ions of A. xylinum MCC and A v i c e l PH-101 i n 0.01 M phosphate buffer (PH=6.95) were -3.2 ± 0 . 4 5 mV and -6.05 ± 2 . 1 9 mV. The magnitude of the e l e c t r o s t a t i c charge (zeta po tent i a l ) of small s o l i d p a r t i c l e s dispersed i n water or c o l l o i d s determines the s t a b i l i t y of the d i s p e r s i o n . A s table c o l l o i d i s one i n which the c o l l o i d p a r t i c l e s remain separate and d i s t i n c t (d i spersed) . An unstable - 146 -c o l l o i d i s charac ter ized by p a r t i c l e s that gradual ly agglomerate. I f the charge of the p a r t i c l e s i s h igh , the p a r t i c l e s repel one other and the c o l l o i d i s s t ab le . I f the charge i s near zero, the random motion (Brownian motion) of the p a r t i c l e s cause them to c o l l i d e and to become attached to one another (Sennett and O l i v i e r , 1965). Thus, i t appears that strong agglomeration or i n c i p i e n t i n s t a b i l i t y i s expected for d i sper s ions of ^ . xylinum MCC and A v i c e l PH-101. The zeta p o t e n t i a l obtained for A v i c e l PH-101 i n t h i s study, t o t a l l y disagrees with r e s u l t s reported i n the l i t e r a t u r e . Paquot (1982) reported zeta p o t e n t i a l values ranging from -35 to -40 mV i n a pH range from 5 to 9 for A v i c e l PH-101. d) Colour determination A. xylinum MCC was s l i g h t l y le s s white than A v i c e l PH-101 as r e f l e c t e d i n i t s lower " L " value (Table 23). A v i c e l was more ye l lowish than A .^ xylinum MCC as ind ica ted by i t s higher "b " value; however, A,, xylinum MCC appeared more green as ind ica ted by the " a " va lue . It i s suggested that the greenish shade i s the r e su l t of n e u t r a l i z a t i o n with NH40H during MCC preparat ion (Paszner, 1982; personal communication). 3. Rheologica l proper t ie s Data from a l l experiments were analysed by using the r h e o l o g i c a l ana ly s i s program for the purpose of s e l ec t ing the best model that f i t the data; c o e f f i c i e n t s of determination (r ) and p lo t s of o versus y (or - 147 -Table 23. Colour of A. xylinum MCC and A v i c e l PH-101 MCC. Hunterlab sca le Sample L 1 a 2 b 3 Standard No. W272 93.10 -0.60 0.40 A . xylinum MCC 90.23 -0.18 1.41 A v i c e l PH-101 91.91 0.02 3.69 lL white 2a=0 grey a<0 green a>0 red 3 b=0 grey b>0 yellow b<0 blue - 148 -transforms) were examined. The Power Law accurately described the flow behaviour at the concentrat ions and temperatures tested for both MCC, A . xylinum and A v i c e l PH-101. Consistency c o e f f i c i e n t s , flow behaviour i n d i c e s , c o e f f i c i e n t s of determination (r ) and the number of points used i n each regress ion for the upcurves and downcurves of ^ . xylinum or A v i c e l PH-101 MCC are shown i n Tables 24 and 25. S t a t i s t i c a l analyses of the slopes (n) and l e v e l s (m) of A. xylinum MCC curves revealed s i g n i f i c a n t d i f ferences (p < 0 .05) ; there fore , i t appears that the flow proper t ie s of / \ . xylinum MCC are time dependent. S t a t i s t i c a l analyses of the slopes (n) and l e v e l s (m) of A v i c e l PH-101 MCC curves , however, revealed no s i g n i f i c a n t d i f f e rence (p < 0 .05) ; therefore i t appears that the flow proper t ie s of A v i c e l PH-101 MCC were not time dependent. Rheograms of the upcurves and downcurves for 6% d i sper s ions of /A. xylinum and A v i c e l PH-101 MCC are depicted i n Figure 41. For a time dependent f l u i d , the increas ing and decreasing rheograms w i l l not c o i n c i d e , that i s , they w i l l show hys teres i s (Holdsworth, 1971). Thus, the wel l defined hys teres i s observed for A. xylinum MCC further confirms i t s time dependence. The concavi ty of the l i n e s i d e n t i f i e s these curves as c h a r a c t e r i s t i c of time dependent t h i n n i n g . Gel strength r a t i o s of _A. xylinum MCC and A v i c e l PH-101 are shown i n Tables 26 and 27. The ge l strength r a t i o G 0 / G i o r e f l e c t s the progress ive nature of s t r u c t u r a l bui ldup i n the d i spers ions at r e s t , i f the ge l s trength i s measured immediately a f ter shearing and a f ter i n c r e a s i n g l y longer periods of r e s t , 10 minutes i n t h i s s tudy. The values obtained are found to increase at a decreasing rate u n t i l a - 149 -Table 24. Consistency c o e f f i c i e n t s and flow behaviour ind ice s for the upcurve and downcurve of ^ . xylinum MCC at 2 5 ° C and various concentra t ions . Concentrat ion (%) Curve d i r e c t i o n m (Pas n ) n r 2 N 6 upward 1 .037 a i 0.395 x 0.983 12 downward 0 .379 b 0.540 y 0.983 12 5 upward 1.067 3 0.347 X 0.970 12 downward 0 . 2 5 7 ° 0 .542 y 0.995 12 4 upward 0 .679 3 0.335 X 0.941 12 downward 0 . 3 0 8 ° 0 .447 X 0.964 12 3 upward 0 .309 3 0.417 X 0.972 12 downward 0 . 2 3 5 ° 0 .422 X 0.060 12 1m and n values sharing a common superscr ipt l e t t e r within a column for each upcurve and downcurve are not s i g n i f i c a n t l y d i f f e r e n t (p > 0 .05) . - 150 -Table 25. Consistency c o e f f i c i e n t s and flow behaviour ind ice s for the upcurve and downcurve of A v i c e l PH-101 MCC at 2 5 ° C and v a r i -ous concentra t ions . Concentrat ion Curve m (%) d i r e c t i o n (Pas n ) n r 2 6 upward 0 . 4 6 5 a l 0 .407 x 0.971 12 downward 0 .344 a 0 .437 x 0.975 12 5 upward 0 .245 a 0.422* 0.987 12 downward 0 .256 3 0 .399 x 0.982 12 4 upward 0 .275 a 0 .375 x 0.992 12 downward 0 .282 a 0 .339 x 0.955 12 3 upward 0 .309 a 0 .308 x 0.975 12 downward 0 .296 a 0 .256 x 0.817 12 1m and n values sharing a common superscr ip t l e t t e r within a column for each upcurve and downcurve are not s i g n i f i c a n t l y d i f f e r e n t (p > 0 .05 ) . Figure 41. Upcurve and downcurve rheograms showing time dependent behaviour of 6% d i spers ions of A. xylinum MCC and A v i c e l PH-101 MCC at 2 5 ° C . - 152 -Table 26. Gel strength r a t i o ( G o / G 1 0 ) of 6, 5, 4 and 3 % d i spers ions of A,, xylinum MCC at d i f f e r e n t temperatures. Concentrat ion Temperature (56) ( ° C ) Gel s t rength 1 ( G o / G 1 0 ) 6 25 1/4 35 2/4 45 1/2 5 25 2/3 35 1/2 45 1/2 4 25 1/1 35 1/1 45 1/1 3 25 1/1 35 1/1 45 1/1 - 153 -Table 27. Gel strength r a t i o ( G o / G 1 0 ) of 6, 5, 4 and 3 % d i sper s ions of A v i c e l PH-101 MCC at d i f f e r e n t temperatures. Concentrat ion Temperature (%) ( ° C ) Gel s t rength 1 ( G o / G 1 0 ) 25 1.5/3.0 35 1.5/3.0 45 1.5/3.0 25 1.0/2.0 35 1.0/2.0 45 1.0/1.0 25 1.0/1.0 35 1.0/1.0 45 1.0/1.0 25 1.0/1.0 35 1.0/1.0 45 1.0/1.0 - 154 -maximum value i s reached, then t h i s behaviour i s a manifestat ion of the phenomenon of th ixotropy (Gray and Dar ley , 1981). Thus, i t appears that 6 and 5% di spers ions of both MCC present such behaviour, whereas no ge l s trength was detected (G 0 /Gio=D a t lower concentrat ions . Thi s may be due to the fact that the ge l s trength increased very r a p i d l y immediately a f ter cessat ion of shear ing, so that the i n i t i a l gel strength was very s e n s i t i v e to t ime. In conc lus ion , i t appears that both MCC presented time dependent Theological behaviour of the t h i x o t r o p i c type, that i s , there i s a r e v e r s i b l e isothermal change i n v i s c o s i t y with time at a constant rate of shear. M i c r o c r y s t a l l i n e c e l l u l o s e aqueous gels had been reported to present t h i x o t r o p i c behaviour; the flow proper t ie s of these gels were in terpre ted i n terms of a p a r t i c l e network model which i s r e v e r s i b l y d i srupted by the a p p l i c a t i o n of an outs ide mechanical force (Hermans, 1963). A second approach to evaluat ing time e f fec t s on v i s c o s i t y of MCC di spers ions i s suggested for future work, by recording the shear s t res s decay curve at a constant shear rate by means of a s t r i p - c h a r t at pre-determined time i n t e r v a l s . Consistency c o e f f i c i e n t s , flow behaviour i n d i c e s , c o e f f i c i e n t s of detemination (r ) and the number of points used i n each regress ion for the equ i l ib r ium curves of / \ . xylinum and A v i c e l MCC are shown i n Tables 28 and 29. Since the n values are < 1, that i s , v i s c o s i t y decreases with shear rate the d i spers ions were non-Newtonian pseudoplas t ic . Suspensions - 155 -Table 28. Consistency c o e f f i c i e n t s and flow behaviour ind ice s of ^ . xylinum MCC d i sper s ions at various temperatures and concen-t r a t i o n s . Concentrat ion Temperature m ( « ) ( ° C ) (Pas n ) n r 2 N 6 25 0.150 0.634 0.981 12 35 0.150 0.610 0.989 12 45 0.160 0.590 0.988 12 5 25 0.173 0.575 0.987 12 35 0.160 0.570 0.983 12 45 0.160 0.570 0.989 12 4 25 0.200 0.502 0.979 12 35 0.170 0.510 0.942 12 45 0.190 0.470 0.954 12 3 25 0.243 0.406 0.981 12 35 0.210 0.410 0.966 12 45 0.210 0.410 0.966 12 - 156 -Table 29. Consistency c o e f f i c i e n t s and flow behaviour ind ice s of A v i c e l PH-101 MCC d i sper s ions at var ious temperatures and concentra-t i o n s . Concentrat ion Temperature m (%) ( ° C ) (Pas n ) n r 2 N 6 25 0.336 0.437 0.987 12 35 0.300 0.410 0.965 12 45 0.310 0.390 0.974 12 5 25 0.260 0.373 0.981 12 35 0.230 0.380 0.985 12 45 0.220 0.400 0.980 12 4 25 0.294 0.325 0.991 12 35 0.240 0.330 0.934 12 45 0.260 0.310 0.952 12 3 25 0.342 0.268 0.998 12 35 0.290 0.250 0.928 12 45 0.310 0.180 0.694 12 - 157 -of long-chain polymers are known to be t y p i c a l pseudoplas t lcs . At r e s t , the chains are randomly entangled, but they do not set up a s t ructure because the e l e c t r o s t a t i c forces are predominantly r e p u l s i v e . When the f l u i d i s i n motion, the chains tend to a l i gn themselves p a r a l l e l to the d i r e c t i o n of flow; t h i s tendency increases with increase i n shear r a te , so that the e f f e c t i v e v i s c o s i t y decreases (Gray and Dar ley , 1981). At the concentrat ions and temperatures te s ted , no t y p i c a l trends were observed for the consis tency c o e f f i c i e n t s and flow behaviour i n d i c e s . Rheograms for d i f f e r e n t concentrat ions of ^ . xylinum and A v i c e l PH-101 MCC are depicted i n Figures 42 and 43, r e s p e c t i v e l y . These f igures demonstrate t h i s decrease i n v i s c o s i t y with increas ing shear ra te s . Table 30 summarizes the Power Law parameters for d i f f e r e n t concen-t r a t i o n s of A. xylinum and A v i c e l PH-101 MCC d i sper s ions at 2 5 ° C . S t a t i s t i c a l analyses of the slopes (n-1) and l e v e l s (m) at each concentra t ion , revealed s i g n i f i c a n t d i f ferences (p < 0 .05) ; there fore , i t appears that apparent v i s c o s i t i e s for the two types of MCC were s i g n i f i c a n t l y d i f f e r e n t over the e n t i r e range of shear r a te s . By examining t h e i r rheograms depicted i n Figure 44, i t appears that A v i c e l PH-101 i s more pseudoplas t ic , that i s i t presents more shear th inning than ^ . xylinum MCC. Consequently, apparent v i s c o s i t i e s at high shear rates were greater for A. xylinum MCC than for A v i c e l PH-101, while apparent v i s c o s i t i e s at low shear rates (Y=1S~1) were greater for A v i c e l PH-101 than for A. xylinum MCC. Greater pe sudop la s t i c i ty of A v i c e l PH-101 MCC may be explained i n terms of i n t e r a c t i o n s between p a r t i c l e s . A v i c e l PH-101 appears to be - 158 -316.2 1 0 1 0 0 1 0 0 0 S H E A R R A T E , S~ 1 Figure 42. Rheogram for 6, 5 4 and 3 % A xylinum MCC di spers ions 2 5 ° C according to the Power law flow model. - 159 -10 1 0 0 1 0 0 0 S H E A R R A T E , s"' Figure 43. Rheogram for 6, 5, 4 and 3 % A v i c e l PH-101 MCC dispers ions at 2 5 ° C acording to the Power Law flow model. - 160 -Table 30. Consistency c o e f f i c i e n t s and flow behaviour ind ice s for bac-t e r i a l and A v i c e l PH-101 MCC di spers ions at 2 5 ° C and various concentra t ions . Concentrat ion (%) MCC m (Pas n) n r 2 N 6 A. xylinum 0 . 1 5 0 a l 0.634 x 0.981 12 A v i c e l 0 .336 a 0.437y 0.937 12 5 A. xylinum 0 .173 a 0.575 x 0.987 12 A v i c e l 0 . 2 6 0 ° 0.373y 0.981 12 4 A . xylinum 0 .200 a 0.502 x 0.979 12 A v i c e l 0 . 2 9 4 ° 0.325y 0.991 12 3 A . xylinum 0 .243 3 0.406 x 0.981 12 A v i c e l 0 . 3 4 2 ° 0.268y 0.998 12 m and n values sharing a common superscr ipt l e t t e r within a column for ^ . xylinum and A v i c e l MCC at each concentrat ion are not s i g n i f i c a n t l y d i f f e r e n t (p < 0 .05) . - 161 -1 10 1 0 0 1 0 0 0 S H E A R R A T E . S _ 1 F igure 4 4 . Rheogram for 6 and 3 % A v i c e l PH-101 MCC (A) and A. xylinum MCC (B) d i spers ions at 2 5 ° C according to the Power law flow model. - 162 -more aggregated than / \ . xylinum MCC as ind ica ted by the s i g n i f i c a n t l y l a rger average p a r t i c l e s i ze of A v i c e l PH-101 (Table 22) . Consequently, there was more breakdown or rearrangement of the s t ructure r e s u l t i n g i n a f l u i d which was more shear s e n s i t i v e . Shear dependency reveal s the nature of s t ructures e x i s t i n g i n the system. Rha (1978) a t t r i b u t e d the presence of pseudoplast ic behaviour to the presence of severa l f a c t o r s : 1) high-molecular weight compounds or large p a r t i c l e s at s u f f i c i e n t concentra t ion , and 2) high i n t e r a c t i o n between the p a r t i c l e s , causing aggregation, or a s soc ia t ion by secondary bonding. Thus, i t appears that the d i f ferences i n v i s c o s i t y of A v i c e l PH-101 and /A. xylinum MCC were determined by mechanical forces ; that i s , s o l i d - s o l i d , s o l i d - l i q u i d and l i q u i d - l i q u i d i n t e r a c t i o n s . However, the in f luence of e lectrochemica l forces should not be ignored, eventhough zeta p o t e n t i a l values for A v i c e l PH-101 and A. xylinum MCC (-6.05 ± 2 . 1 9 and -3.2 ± 0 . 4 5 mV, re spec t ive ly ) ind ica ted very small e l e c t r o s t a t i c charge. Using the Power law parameters shown i n Tables 28 and 29, apparent v i s c o s i t i e s were ca l cu l a ted at 100 s - 1 . These apparent v i s c o s i t i e s (at Y=100 s - 1 ) were used to examine v i s c o s i t y - c o n c e n t r a t i o n r e l a t i o n s h i p s . The v i s c o s i t y - c o n c e n t r a t i o n model f i t the data accurate ly over the concentrat ion range tested as i s ind ica ted by the r in Tables 31 and 32. Rheograms of these data are depicted i n Figures 45 and 46, where the logarithm of apparent v i s c o s i t y vs the logarithm of concentrat ion were regressed. - 163 -Table 31. Apparent v i s c o s i t y (at Y-100 S ~ 1 ) - c o n c e n t r a t i o n r e l a t i o n s h i p for /A. xylinum MCC di spers ions from 25 to 4 5 ° C . Temperature a b r ( ° C ) (m Pa s) (of log r ^ = 1 0 0 s _ i to log C) 25 6.397 0.825 0.999 35 5.420 0.858 0.998 45 5.3662 0.849 0.986 Table 32. Apparent v i s c o s i t y (at y=100 s " 1 ) - c o n c e n t r a t i o n r e l a t i o n s h i p for A v i c e l PH-101 MCC di spers ions from 25 to 4 5 ° C . Temperature a b r C O (m Pa s) (of log n « _ 1 0 0 s _ i to log C) 25 3.630 0.983 0.870 35 2.704 1.053 0.953 45 1.591 1.366 0.998 - 164 -100 3 6 CONCENTRATION, % Figure 45, E f fect of concentrat ion on apparent v i s c o s i t y (y=100 s~1) of A . xylinum MCC di spers ions at 25, 35 and 4 5 ° C . Figure 46. Ef fect of concentrat ion on apparent v i s c o s i t y (y=100 s" 1 ) of A v i c e l PH-101 MCC di spers ions at 25, 35 and 4 5 ° C . - 166 -Apparent v i s c o s i t i e s (at y=100 s - 1 ) were a lso analysed with respect to the r e c i p r o c a l of absolute temperature. An Arrhenius type model was used to f i t the data . This model f i t the data accura te ly , except for the 5% d i sper s ion of A v i c e l PH-101 as i n d i c a t e d by t h e i r 9 c o e f f i c i e n t s of determination (r ) shown i n Tables 33 and 34. The a c t i v a t i o n energies and frequency fac tors were a l so inc luded i n these t ab l e s . The data were p lo t ted on semilogarthmic coordinates with v i s c o s i t y on the logar i thmic ordinate and the r e c i p r o c a l of the absolute temperature on the ar i thmet ic absc i s sa . These rheograms are depicted i n F igures 47 and 48. It appears that very small changes i n apparent v i s c o s i t y took place at the temperatures t e s ted , with a general trend towards greater changes at lower concentra t ions . Tables 35 and 36 show the consis tency c o e f f i c i e n t s and flow behaviour ind ice s of 3% di spers ions of A v i c e l PH-101 and A . xylinum MCC at d i f f e r e n t pH va lues . These data were also portrayed i n a graphic form i n Figures 49 and 50. k. xylinum MCC d id not appear to be g rea t ly affected by pH, whereas A v i c e l PH-101 showed greater v i s c o s i t i e s at lower pH. A v i c e l PH-101 response to pH was somehwat expected s ince the system acquired a f l o c c u l a t e d appearance as the pH was adjusted to 4 . 0 . The term f l o c c u l a t i o n i s l i m i t e d to the loose a s soc i a t ion of p a r t i c l e s which form f loes or gel s t ructures (Gray and Dar ley , 1981). Such behaviour was not as c l e a r l y observed with _A. xylinum MCC, s ince i t s e l e c t r o s t a t i c charge, as represented by i t s zeta p o t e n t i a l va lue , was lower than for A v i c e l PH-101. F l o c c u l a t i o n i s known to be the r e s u l t of compression of the double layers when an e l e c t r o l y t e i s added (Gray and - 167 -Table 33. Apparent v i s c o s i t y (at shear rate=100 s )-temperature r e l a -t i o n s h i p for xylinum MCC d i sper s ions at d i f f e r e n t concentra t ions . Temperature AE A r C O (k c a l mole) (m Pa s) (of log T I - = 1 0 0 S _ I to 1 0 0 0 / T ) 6 1.304 3.025 0.950 5 0.960 4.745 0.875 4 1.871 0.845 0.991 3 1.211 1.990 0.875 Table 34. Apparent v i s c o s i t y (at shear rate=100 s )-temperature r e l a -t i o n s h i p for A v i c e l PH-101 MCC d i sper s ions at d i f f e r e n t con-c e n t r a t i o n s . Temperature AE A r C O (k c a l mole) (m Pa s) (of log rr = 1 0 0 s - i to 1 0 0 0 / T ) 6 2.824 0.206 0.950 5 0.4602 6.55 0.582 4 1.8352 0.575 0.901 3 4.7469 0.003 0.999 - 168 -1 2 . 581 , 3.1 3.2 3.3 3.4 1 0 0 0 / T , ° K I I I I I 4 5 3 5 25 T E M P E R A T U R E . ° C Figure 47. Ef fect of temperature on apparent v i s c o s i t y (shear rate=100 s" ) for 6.0 to 3.0 % _A. xylinum MCC d i s p e r s i o n s . - 169 -31.62 3.1 3.2 3.3 3.4 1 0 0 0 / T , °K~' 4 5 3 5 25 T E M P E R A T U R E, ° C F igure 48. E f fec t of temperature on apparent v i s c o s i t y (shear rate=100 s" 1 ) for 6.0 to 3.0% A v i c e l PH-101 MCC d i s p e r s i o n s . - 170 -Table 35. Consistency c o e f f i c i e n t s and flow behaviour ind ice s for a 3% d i s p e r s i o n of / \ . xylinum MCC at 2 5 ° C and at d i f f e r e n t pH. pH m n r 2 4.0 0.270 0.459 0.984 5.5 0.241 0.473 0.990 7.0 0.304 0.411 0.991 Table 36. Consistency c o e f f i c i e n t s and flow behaviour i n d i c e s for a 3% d i s p e r s i o n of A v i c e l PH-101 MCC at 2 5 ° C and at d i f f e r e n t pH. pH m n r 2 ( P a « s n ) 4.0 5.5 7.0 0.427 0.387 0.376 0.405 0.269 0.288 0.991 0.991 0.988 - 171 -1 0 0 0 SHEAR RATE, S"' F igure 49. E f f ec t of pH on apparent v i s c o s i t y of 3% A. xylinum MCC d i s -pers ions at 2 5 ° C . — ure 50. E f f e c t of pH on apparent v i s c o s i t y at ?_5°C f o r 3% A v i c e l PH-101 MCC d i s p e r s i o n s . - 173 -Dar ley , 1981), and so the higher the e l e c t r o s t a t i c charge, the higher the s e n s i t i v i t y to e l e c t r o l y t e s and the greater the chances for f l o c c u -l a t i o n . Table 37 presents the Power law parameters for 3% d i sper s ions o f e i t h e r A v i c e l PH-101 or ^ . xylinum MCC and e i ther of the two d i spers ions + 0.4% NaCl . These data are also shown i n Figure 51. E i t h e r of the 3% d i sper s ions appeared f loccu la ted when prepared with 0.4% NaCl . Thi s f l o c c u l a t i o n was more c l e a r l y observed at high shear rates where apparent v i s c o s i t i e s were higher for the d i spers ions prepared with 0.4% NaCl than for e i ther of the 3% MCC d i s p e r s i o n s . A study was made by Edelson and Hermans (1963) on the ef fect of adding e l e c t r o l y t e to m i c r o c r y s t a l l i n e c e l l u l o s e ge ls ; i t was reported that provided the ge l and the e l e c t r o l y t e so lu t ion were properly mixed, only a small increase in the shear s t ress at any shear rate was measured. It was concluded that the negative charges present on the c e l l u l o s e microcrys t a l s do not appreciably contr ibute to the i n t e r p a r t i c l e forces respons ib le for the ge l p roper t i e s . - 174 -Table 37. Consistency c o e f f i c i e n t s and flow behaviour i n d i c e s for a 3% d i s p e r s i o n of A v i c e l PH-101 MCC or A. xylinum MCC and A v i c e l or A. xylinum MCC + 0.4% NaCl at 25*0. Sample m ( P a « s n ) _A. xylinum MCC A v i c e l MCC /A. xylinum MCC or A v i c e l MCC +0.4% NaCl 0.243 0.342 0.286 0.406 0.981 12 0.268 0.998 12 0.413 0.972 12 - 175 -S H E A R R A T E , S Figure 51. E f f e c t of 0.4% NaCl on apparent v i s c o s i t y of 3% A. x y l i and A v i c e l PH-101 MCC d i s p e r s i o n s at 25°c! ~ - 176 -CONCLUSIONS I n d u s t r i a l app l i ca t ions of A. xylinum c e l l u l o s e have been scarce ly explored, i n s p i t e of i t s great po ten t i a l for large sca le product ion . This study was conducted to develop two a p p l i c a t i o n s of A. xylinum c e l l u l o s e . The f i r s t part of the study was aimed at i n v e s t i g a t i n g n u t r i e n t requirements of Acetobacter xylinum and to compare three s t r a i n s i n terms of c e l l u l o s e production and cu l ture s t a b i l i t y . Previous reports on carbohydrate and nitrogen requirements and pH of the c u l t i v a t i o n media of A. xylinum (Lapuz et j a l . , 1967; Dudman, 1959) were confirmed. A defined medium which consis ted of 10% sucrose and 0.5% peptone among other ingredients at pH 4 .5 , was formulated. This formulat ion was adopted for further work i n t h i s study; however, i t i s suggested that future work should fol low t h i s phase by a t r a n s i t i o n to natura l media or a g r i c u l t u r a l waste i n order to scale-up the formulat ion to a commercially v i a b l e process . High m u t a b i l i t y of c e l l s wi th in species of the ace t i c ac id bac te r i a was also confirmed (Shimwell and C a r r , 1958; Schramm and H e s t r i n , 1954; Cook and C o l v i n , 1980). The three s t r a i n s tested were unstable under swir led cond i t ions , 100% of co lon ie s were c e l l u l o s e -d e f i c i e n t a f ter the f i f t h and s ix th s e r i a l t r ans fer s (10 to 12 days) . On the contrary , a l l the s t r a in s were much more s table under s t a t i c c o n d i t i o n s ; ATCC 14851 being the most s table with only 9 to 10% c e l l u -lose d e f i c i e n t co lonies af ter the ninth to tenth s e r i a l t rans fer (18 to 20 days) . - 177 -C e l l u l o s e y i e l d s a f ter 40 days of incubat ion var ied from 6.5 g/100 mL media (64% conversion of t o t a l sugar) with the P h i l i p p i n e s t r a i n to 0.6 g/100 mL media (6.05% conversion of t o t a l sugar) produced by ATCC 10821. The h i g h - y i e l d i n g s t r a i n was able to u t i l i z e up to 73% of the sugar, from which up to 87% was converted to c e l l u l o s e . On the other hand, the two low-y ie ld ing s t ra ins were only able to u t i l i z e 25 to 30% of the sugar, from which only 45% was converted to c e l l u l o s e . Thus i t appears that the P h i l i p p i n e s t r a i n i s a high c e l l u l o s e y i e l d i n g s t r a i n whereas the other two s t r a i n s appear to s h i f t t h e i r metabolism away from c e l l u l o s e synthesis towards other metabolic pathways. Therefore , i t was concluded that these organisms vary widely in t h e i r e f f i c i e n c y to convert sugar to c e l l u l o s e . The second part of t h i s study involved the cont inuat ion of the development of a process which integrated c e l l u l o s e I f i b r i l s into a " s y n t h e t i c " f i b r e o r i g i n a l l y developed i n t h i s laboratory (Townsley, 1974; unpublished r e s u l t s ) . A method was devised to force A. xylinum c e l l s to sp in c e l l u l o s e ribbons into p a r a l l e l s tab le f i laments by c u l t i v a t i o n of the organisms in a s t r a i g h t - l i n e flow path . E l ec t ron micrographs c l e a r l y revealed p a r a l l e l o r i e n t a t i o n i n the f ine f i b r e s t ruc ture . This i n v e s t i g a t i o n confirmed an e lec t ron microscopic study c a r r i e d out in t h i s laboratory (Townsley and Tung, 1974; unpublished r e s u l t s ) . Brown (1982) suggested the in tegra t ion of c e l l u l o s e I f i b r i l s produced by A. xylinum into a synthet ic f i b r e . According to him, a novel system requires that c e l l u l o s e I s t ructures be reta ined throughout - 178 -the l a t e r stages of synthet ic f i lament formation. Thereby, an integrated process for developing a c e l l u l o s e f i b r i l would incorporate a natura l polymer i n the ear ly stages of i t s aggregation, thus a l lowing a greater amount of synthet ic engineer ing . Therefore , i t appears that the synthet ic f i b r e developed i n t h i s study w i l l encourage more integrated research in t h i s f i e l d which may benefi t i n d u s t r y . T e n s i l e strength of the f ib re s was improved by using the mercer i-za t ion treatment commonly used for c o t t o n . Optimum condi t ions for the treatment as determined by using Mapping Super Simplex Opt imizat ion were 8.3% NaOH, 5 9 ° C and 24 minutes. A t e n s i l e s trength of 83,300 ± 1 8 0 0 N/cm was achieved, an increase over the untreated f i b r e s of 281%. The re su l t s of t h i s study thus indica ted that the wel l known mercer iza t ion treatment used i n t e x t i l e s could succes s fu l ly be appl ied to c e l l u l o s i c f i b r e s produced by bac te r i a only i f the treatment cond i t ions were optimized to t h i s p a r t i c u l a r c e l l u l o s e f i b r e s t r u c t u r e . F i n a l l y , the t h i r d part of t h i s study cons i s ted of the development of a process for the p u r i f i c a t i o n and hydro ly s i s of j^. xylinum c e l l u l o s e for the manufacture of mycrocrys ta l l ine c e l l u l o s e (MCC). MCC from ^ . xylinum c e l l u l o s e was produced by a simple procedure. The method consis ted of only one ex t rac t ion procedure with 6% NaOH for 3 hours, one bleaching sequence with NaOCl 2 for 2 hours and h y d r o l y s i s with 2.5 N HC1 for 15 minutes. Nevertheless , a h ighly p u r i f i e d (98.5 ± 1 . 4 % c e l l u l o s e ) and highly c r y s t a l l i n e (98.15 ± 0 . 9 1 % c r y s t a l l i n i t y index) m i c r o c r y s t a l l i n e c e l l u l o s e was obtained without causing any detr imenta l e f fect on the f i b r e s t ruc ture . On the cont ra ry , commercial - 179 -MCC i s prepared from highly degraded c e l l u l o s e from wood pulp as revealed by a low c r y s t a l l i n i t y index of 84.90 ± 1 . 4 1 % . Degradation of the c e l l u l o s e always occurs during the severe p u r i f i c a t i o n treatments to which wood pulp i s subjected t o . The flow behaviour of A. xylinum MCC d i sper s ions was s tudied and compared to commercial A v i c e l PH-101. Both MCC di sp layed non-Newtonian pseudoplast ic behaviour. _A. xylinum MCC d i sper s ions were found to be t h i x o t r o p i c . The m u l t i p l e uses of MCC in the food and pharmaceutical i n d u s t r i e s ind ica te the great p o t e n t i a l that A^ xylinum c e l l u l o s e may have as a raw m a t e r i a l . Results of t h i s study indicated that the two a p p l i c a t i o n s o f A' xylinum c e l l u l o s e developed are t e c h n i c a l l y f e a s i b l e . What remains i s the need to determine economic f e a s i b i l i t y . - 180 -LITERATURE CITED Alaban, C. A. 1962. Studies on the optimum condi t ions for "nata de coco" bacterium or "nata" formation i n coconut water. P h i l i p p . A g r i c . 45: 491-516. A l o n i , Y. and M. Benziman. 1982. Intermediates of c e l l u l o s e synthes i s i n Acetobacter i n C e l l u l o s e and other natura l polymer systems bio-genesis , s t ructure and degradation. Ed. Brown, R. M . , Plenum Press , London, pp. 341-359. AOAC (Assoc ia t ion of O f f i c i a l A n a l y t i c a l Chemists ) . 1980. O f f i c i a l Methods of A n a l y s i s , 13th E d i t i o n , ed. Horowitz, W., Washington, DC. B a t t i s t a , 0. A. 1950. Hydrolys i s and c r y s t a l l i z a t i o n of c e l l u l o s e . Ind. Eng. Chem. 42: 502-507. B a t t i s t a , 0. A. 1958. Fundamentals of High Polymers. Reinhold Pub-l i s h i n g C o . , New York, NY, pp. 97-117. B a t t i s t a , 0. A. 1975. Mic rocry s t a l Polymer Sc ience . McGraw-Hill C o . , New York, NY. pp. 1-106. B a t t i s t a , 0. A. and P. A. Smith. 1961. L e v e l - o f f Dr. P. C e l l u l o s e Products . U .S . Patent 2,978,446 (to American Viscose Corpora t ion ) . B a t t i s t a , 0. A. and P. A. Smith. 1962. M i c r o c r y s t a l l i n e c e l l u l o s e . Ind. Eng. Chem. 54: 20-29. B a t t i s t a , 0. A. and P. A. Smith. 1965. C o l l o i d a l macromolecular pheno-mena. Am. S c i . 53: 151-173. Ben-Hayyim, G. and I . Ohad. 1965. Synthesis of c e l l u l o s e by Acetobac- ter xylinum V I I I . On the formation and o r i e n t a t i o n of b a c t e r i a l c e l l u l o s e f i b r i l s in the presence of a c i d i c po lysacchar ides . 3. C e l l B i o l . 25: 191-107. Benziman, M. and H. Burger-Rachamimou. 1962. Synthesis of c e l l u l o s e from pyruvate by succinate grown c e l l s of Acetobacter xyl inum. 0. B a c t e r i o l . 84: 625-630. Benziman, M . , C. H. H a i g l e r , R. M. Brown, A. R. White and K. M. Cooper. 1980. C e l l u l o s e b iogenes i s : Polymerizat ion and c r y s t a l l i z a t i o n are coupled processes i n Acetobacter xyl inum. Proc . N a t l . Acad. S c i . USA 77: 6678. Booth, 3. E . 1964. P r i n c i p l e s of t e x t i l e t e s t i n g . Temple Press Books L t d . , London, pp. 212-213. Breed, R. S . , G. D. Murray and N. R. Smith. 1957. Bergey's Manual of Determinative Bac te r io logy . 7th E d i t i o n . The Wil l iams and Wi lk ins C o . , Ba l t imore , MD, pp. 1094. - 181 -Brown, A. 3. 1886. Quoted i n Muhlethaler , K. (1949). The s t ruc ture of b a c t e r i a l c e l l u l o s e . Biochem. Biophys. Acta . 3: 527-535. Brock, T . D. 1979. Biology of microorganisms. 3rd E d i t i o n . P r e n t i c e - H a l l I n c . , Englewood C l i f f s , NO, pp. 217-218. Brown, R. M. 1979. Quoted in A l o n i Y. and Benziman, M. (1982). Intermediates of ce lu lose synthesis in C e l l u l o s e and other natural polymer systems biogenes i s , s t ructure and degradat ion. Ed. Brown, R. H . , Plenum Press , London, pp. 357. Brown, R. M. 1981. Integrat ion of biochemical and v i s u a l approaches to the study of c e l l u l o s e b iosynthes i s and degradation _in The Ekman-Days 1981 b iosynthes i s and biodegradation of wood components, V. 3, Inter-na t iona l Symposium on Wood and Pulping Chemistry, Stockholm, June 9-12. Brown, R. M. 1982. C e l l u l o s e and other natura l polymer systems b i o -genesis , s t ructure and degradation b iogenes i s , s t ruc ture and degrada-t i o n . Plenum Press , London, pp. 442-447. Brown, R. M. 1983. Production of a c e l l u l o s e synthet ic polymer compo-s i t e f i b e r . U .S . Patent 4,378,431. Brown, R. M . , 3. H. M. W i l l i son and C. L . Richardson. 1976. C e l l u l o s e b iosynthes i s i n Acetobacter xylinum: V i s u a l i z a t i o n of the s i t e of synthesis and d i r e c t measurement of the in vivo process . Proc . N a t l . Acad. S c i . , USA 73: 4565-4569. Brown, R. M. and 3. H. M. W i l l i s o n . 1977. Quoted in A l o n i , Y. and M. Benziman. 1982. Intermediates of c e l l u l o s e synthes i s in C e l l u l o s e and other natural polymer systems biogenes is , s t ructure and degrada-t i o n . Ed. Brown, R. M . , Plenum Press , London, pp. 358. Browning, B. L . 1967. Methods of wood chemistry. V. 2, In ter sc ience Pub l i sher s , New York, pp. 499-502. Buchanan, R. E. and N. E . Gibbons. 1975. Bergey's manual of determina-t i v e bac te r io logy . 8th E d i t i o n . The Wil l iams and Wilk ins C o . , Bal t imore, MD, pp. 276. C o l v i n , 3. R. 1971. Quoted in A l o n i , Y. and M. Benziman (1982). Intermediates of c e l l u l o s e synthesis _in C e l l u l o s e and other natura l polymer systems b iogens i s , s t ructure and degradat ion. Ed. Brown, R. M . , Plenum Press , London, pp. 342. C o l v i n , 3. R. 1972. Quoted in A l o n i , Y. and M. Benziman (1982). Intermediates of c e l l u l o s e synthesis in_ C e l l u l o s e and other natura l polymer systems biogenes i s , s t ructure , and degradat ion, R. M. Brown ( ed . ) , Plenum Press , London, pp. 432. C o l v i n , 3. R. 1977. A new look at c e l l u l o s e b iosynthes i s i n r e l a t i o n to s tructure and i n d u s t r i a l use. Tappi 60: 59-61. - 182 -C o l v i n , 3. R. and G. G. Leppard. 1977. The b iosynthes i s of c e l l u l o s e by Acetobacter xylinum and Acetobacter acet igenus. Can. 3. Micro-b i o l . 23: 709-717. C o l v i n , 3. R . , L . C. Sowden and G. G. Leppard. 1977. The s t ructure of c e l l u l o s e producing b a c t e r i a , Acetobacter xylinum and Acetobacter  acetigenus. Can. 3. M i c r o b i o l . 23: 790-797. Concon, 3. M. and D. So l te s s . 1973. Rapid microk je ldahl d ige s t ion of cereal grains and other b i o l o g i c a l mater i a l s . A n a l . Biochem. 53: 35-41. Cook, K. E . and 3. R. C o l v i n . 1980. Evidence for a b e n e f i c i a l i n f l u -ence of c e l l u l o s e production on growth of Acetobacter xylinum i n l i q u i d medium. Current M i c r o b i o l . 3: 203-205. Cooper, D. and 3. Manley. 1975a. C e l l u l o s e synthes i s by Acetobacter  xyl inum. I . Low molecular weight compounds present i n the region of synthes i s . Biochim. Biophys. Acta . 381: 78-96. Cooper, D. and 3. Manley. 1975b. C e l l u l o s e synthesis by Acetobacter  xylinum. I I . Inves t iga t ion into the r e l a t i o n between c e l l u l o s e synthesis and c e l l envelope components. Biochim. Biophys. Acta . 381: 97-108. Cooper, K. M. 1980. C e l l u l o s e b iogenes i s : Po lymer iza t ion and c r y s t a l -l i z a t i o n are coupled processes in Acetobacter xyl inum. Proc . N a t l . Acad. S c i . USA 77(11): 6678-6682. Correns, E . and H. 3. Purz . 1975. Proper t ie s of b a c t e r i a l c e l l u l o s e . C e l l u l o s e Chem. Technol . 9: 449-469. Correns , E . , A. Groebe and H. 3. Purz . 1972a. Membranes and f l a t shaped a r t i c l e s based on c e l l u l o s e used for separat ion processes . Ger. Patent 80,307. Correns , E . , A. Groebey, H. 3. Purz , H. H. Scharz and I. Hagen. 1972b. manufacture of membranes and sheets from b a c t e r i a l c e l l u l o s e . Ger . Patent 92,136. Dubois, M . , K. A. G i l l e s , 3. K. Hamilton, P. A. Rebers and F . Smith. 1956. C o l o r i m e t r i c method for determination of sugars and re l a ted substances. A n a l . Chem. 28: 350-356. Dudrnan, W. F . 1959a. C e l l u l o s e production by Acetobacter acetigenum and other Acetobacter spp. 3. Gen. M i c r o b i o l . 21: 312-326. Dudman, W. F . 1959b. C e l l u l o s e production by Acetobacter s t r a i n s i n submerged c u l t u r e . 3. Gen. M i c r o b i o l . 22: 25-39. Dudman, W. F . 1960. C e l l u l o s e production by Acetobacter s t r a i n s i n submerged c u l t u r e . 3. Gen. M i c r o b i o l . 22: 25-34. - 183 -Dudman, W. F . 1959b. C e l l u l o s e production by Acetobacter s t r a i n s i n submerged c u l t u r e . 3. Gen. M i c r o b i o l . 22: 25-39. Edelson, M. R. and 3. Hermans. 1963. Flow of gels of c e l l u l o s e m i c r o c r y s t a l s . I I . E f fect of added e l e c t r o l y t e . 3. Polym. S c i . , Part C, 2: 145-152. Eg le , C. 3. and 3. N. Grant . 1970. I n t e r r e l a t i o n s of s t r u c t u r a l and phys ica l proper t ie s of untreated cot tons . T e x t i l e . Res. 3. 40: 158-168. F i g i n i , R. V. 1974. Quoted in M a r x - F i g i n i , M. (1982). The c o n t r o l of molecular weight and molecular-weight d i s t r i b u t i o n i n the biogenesis of ce lu lose in C e l l u l o s e and other natural polymer systems. Ed . Brown, R. H . , Plenum Press , London, pp. 260. FCC I I I . 1981. Food Chemicals Codex I I I . General te s t s and apparatus. 3rd E d i t i o n , pp. 80. FDA. 1958. Food and Drugs Act and Regulat ions . Quoted i n FMC Corporat ion, Food and Pharmaceutical Products D i v i s i o n . B u l l e t i n A v i c e l PH m i c r o c r y s t a l l i n e c e l l u l o s e . FMC Corporat ion , Food and Pharmaceutical Products d i v i s i o n . B u l l e t i n G-34. A v i c e l MCC product d e s c r i p t i o n . Forge, A. and R. D. Pres ton. 1977. An e lec t ron microscope examination of Acetobacter xylinum showing the u l t r a s t r u c t u r e of the c e l l s and the a s soc ia t ion of c e l l u l o s e m i c r o f i b r i l s . Ann. Bot. 41: 437-446. Gaudy, E . and R. S. Wolfe. 1961. Factors a f f ec t ing f i lamentous growth of Sphaerot i lus Natans. App l . M i c r o b i o l . 9: 580-584. Glas ser , L . 1958. The synthesis of c e l l u l o s e i n c e l l - f r e e extracts o f Acetobacter xyl inum. 3. B i o l . Chem. 232: 627-636. Gray, G. R. and H. C. H. Dar ley . 1981. Composition and proper t ie s of o i l wel l d r i l l i n g f l u i d s . 4th E d i t i o n , Gulf Pub. C o . , Book D i v i s i o n , Houston, pp. 160-213. Grornet, Z . , M. Schramm and S. H e s t r i n . 1957. Synthesis of c e l l u l o s e by Acetobacter xylinum enzyme systems present i n a crude extract of glucose-grown c e l l s . Biochem. 3. 67: 679-689. H a i g l e r , C. H . , R. M. Brown and M. Benziman. 1980. C e l c o f l u o r White ST a l t e r s the iv^ vivo assembly of c e l l u l o s e m i c r o f i b r i l s . Science 210: 903-906. H a i g l e r , C. H. and M. Benziman. 1982. Biogenesis of c e l l u l o s e I micro-f i b r i l s occurs by c e l l - d i r e c t e d self-assembly i n Acetobacter xylinum  in C e l l u l o s e and other natura l polymer systems b iogenes i s , s t ructure and degradation, Ed. Brown, R. M . , Plenum Press , London, pp. 273-297. - 184 -Happey, F . 1978. Appl ied f i b e r sc ience . Academic Press , I n c . , London, pp. 114. Harper, 3. C. and A. F . E l S a h r i g i . 1965. Vi scometr ic behaviour o f tomato concentrates . 3. Food S c i . 30: 470-476. Hermans, 3. 1963. Flow of gels of c e l l u l o s e m i c r o c r y s t a l s . I . Random and L iqu id c r y s t a l l i n e ge l s . 3. Polym. S c i . , Part C, 2: 129-144. H e s t r i n , S. and M. Schramm. 1954. Synthesis of ce lu lo se by Acetobacter  xylinum. I I . Preparat ion of f reeze-dr ied c e l l s capable of polymer-i z i n g glucose to c e l l u l o s e . Biochem. 3. 58: 345-352. Holdsworth, S. D. 1971. A p p l i c a b i l i t y of r h e o l o g i c a l models to the i n t e r p r e t a t i o n of flow and processing behaviour of f l u i d food products . 3. Texture Studies 2: 393-418. 3esus, E . G . , R. M. Andres and E . T . Magno. 1971. A study on the i s o l a t i o n and screening of microorganisms for production of d iver se-textured nata. P h i l i p . 3. S c i . 100: 41-49. 3osh i , V. S . , B. R. Shelat and T. Radhakrishnan. 1967. Some mechanical propert ies of swollen and stretched cotton using d i f f e r e n t swel l ing agents. T e x t i l e Res. 3. 37: 989-994. Kaushal , R. and T. K. Walker. 1951. Formation of c e l l u l o s e by c e r t a i n species of Acetobacter . Biochem. 3. 48: 618-621. L e , C. D. 1980. *MFAV (Analys i s of variance - covar i ance ) , Computing Centre L i b r a r y , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B . C . Lapuz, M. M . , E . G. Gal lardo and M. A. Pa lo . 1967. The nata organism -c u l t u r a l requirements, c h a r a c t e r i s t i c s , and i d e n t i t y . P h i l i p . 3. S c i . 96: 91-109. L i , 3. C. R. 1964. S t a t i s t i c a l Inference, I I . Edward Brothers , I n c . , Ann Arbor , Mich . pp. 270-273. Maeda, H. and N. I sh ida . 1967. S p e c i f i c i t y of binding of hexopyronsyl polysaccharide with f luorescent br ightener . 3. Biochem. 62(2) : 276-278. Magis ter , G . , F . Loth and B. P h i l i p p . 1975. Studies on the in f luence of f i b r e morphology on the h y d r o l y t i c degradation of c e l l u l o s e . C e l l u l o s e Chem. Technol . 9: 471-476. M a r x - F i n g i n i , M. 1982. The c o n t r o l of molecular weight and molecular-weight d i s t r i b u t i o n i n the biogenesis of c e l l u l o s e i n C e l l u l o s e and other natura l polymer systems b iogenes i s , s t ructure and degradat ion. Ed . Brown, R. M . , Plenum Press , London, pp. 243-271. Muhlethaler , K. 1949. The s tructure of b a c t e r i a l c e l l u l o s e . Biochim. Biophys. A c t a . 3: 527-535. - 185 -Mynatt, R. L . 1982. F iber production from continuous c u l t i v a t i o n of microorganisms. U.S . Patent 4,320,198. Nakai, S. 1982. Comparison of opt imizat ion techniques for a p p l i c a t i o n to food product and process development. 3. Food S c i . 47: 144-152. Nakai , S . , K. Koide, K. Eugster. 1984. A new mapping super-simplex opt imizat ion for food product and process development. 3. Food S c i . 49(4) : 1143-1148. Nissan, A. H. 1977. Lecturs on Fiber Science. Ed. Walker, W. C , New York, The 3oint Textbook Committee of the Paper Industry , pp. 7-17. Ohad, D. D. and S. H e s t r i n . 1962. Synthesis of c e l l u l o s e by Acetobacter xyl inum. V. U l t r a s t r u c t u r e of polymer. 3. C e l l B i o l . 12: 31-46. Paquot, M. 1982. Charac ter iza t ion of two m i c r o c r y s t a l l i n e c e l l u l o s e s and study of t h e i r e l e c t r o k i n e t i c behaviour. Lebensn. Wiss. u . Technol . 15: 148-152. Paszner, L . 1982. Personal communication. Facul ty of Fore s t ry , U n i -v e r s i t y of B r i t i s h Columbia, Vancouver, B . C . P a t i l , N. B . , N. E . Dweltz and T. Radhadrishnan. 1965. Studies on d e c r y s t a l l i z a t i o n of co t ton . T e x t i l e Res. 3. 35: 517-523. Peters , R. H. 1963. Quoted i n Peters , R. H. (1967). Mercer iza t ion of cotton in T e x t i l e Chemistry, V. I I . , E l s e v i e r , Amsterdam, pp. 347. Pe ter s , R. H. 1967. Mercer iza t ion of cotton _in T e x t i l e Chemistry, V. I I . E l s e v i e r , Amsterdam, pp. 328-365. Rajagopalan, A . , G. M. Venkatesh and N. E . Dweltz. 1975. D i f f e r e n t i a l response of cottons to s lack mercer iza t ion . T e x t i l e Res. 3. 45: 409-413. Ranby, B. G. 1952. Physico-chemical i n v e s t i g a t i o n s on b a c t e r i a l c e l l u l o s e . Arkiv for Kemi 4: 249-255. Rha, C. 1978. Rheology of f l u i d foods. Food Technol . 39(7) : 77-82. Rydholm, S. A. 1965. Pulping Processes. In ter sc ience P u b l i c a t i o n s . 3ohn Wiley and Sons, L t d . , New York, pp. 1120. Schramm, M. and S. H e s t r i n . 1954. Factors a f f ec t ing production of c e l l u l o s e at the a i r / l i q u i d inter face of a c u l t u r e of a c u l t u r e of Acetobacter xyl inum. 3. Gen. M i c r o b i o l . 11: 123-129. Schramm, M . , Z. Gromet and S. H e s t r i n . 1957. Synthesis of c e l l u l o s e by Acetobacter xyl inum. I I I . Substrates and I n h i b i t o r s . Biochem. 3. 67: 669-679. - 186 -Shantz, E . M. and F . C. Steward. 1952. Coconut milk f ac to r : the growth promoting substance in coconut m i l k . Amer. Chem. Soc. 74: 6133. Shantz, E . M. and F . C. Steward. 1955. The i d e n t i f i c a t i o n of compound A from coconut milk as 1 .3-diphenylurea. Amer. Chem. Soc. 77: 6351. Shelat , B. R. , T . Radkahrishnan and B. V. I y e r . 1960. The r e l a t i o n between c r y s t a l l i t e o r i e n t a t i o n and mechanical proper t i e s of mercerized cot tons . T e x t i l e Res. 3. 30: 836-842. Shimwell, 3. L . and 3. G. Car r . 1958. Old and new ce l lu lo se -kproduc ing Acetobacter spec ie s . 3. Ins t . Brew. 64: 477-484. Sennet, P. and 3. P. O l i v i e r . 1965. The Inter face Symposium. Ind. Eng. Chem. 57: 33-50. Speers, R. A. 1984. Computer-aided rheo log i ca l ana lys i s of d r i l l i n g f l u i d s . O i l and Gas 3. ( in press ) . S t e e l , R. and T . K. Walker. 1957. C e l l u l o s e l e s s mutants of c e r t a i n Acetobacter spec ie s . 3. Gen. M i c r o b i o l . 17: 12-18. Swissa, M . , H. Weinhouse and M. Benziman. 1976. A c t i v i t i e s of c i t r a t e synthase and other enzymes of Acetobacter xylinum in s i t u and in  v i t r o . Biochem. 3. 153: 499-501. Swissa, M. 1978. Quoted i n A l o n i , Y. and M. Benziman (1982). In ter -mediates of c e l l u l o s e synthesis _in C e l l u l o s e and other natura l polymer systems biogenes i s , s t ructure and degradat ion. Ed. Brown, R. M . , Plenum Press , London, pp. 343. Swissa, M . , Y. A l o n i , H. Weinhouse and M. Benziman. 1980. Intermediary steps in Acetobacter xylinum c e l l u l o s e synthes i s : s tudies with whole c e l l s and c e l l - f r e e preparations of the wi ld type and a c e l l u l o s e l e s s mutant. 3. B a c t e r i o l . 143: 1142-1150. Taguchi, G. 1957. Experimental Designs. Maruzen P u b l i s h i n g , Tokyo. Taka i , M . , Y. Tasuta and S. Watan. 1975. Biosynthes i s of c e l l u l o s e by Acetobacter xyl inum. Polymer. 3. 7: 137-155. TAPPI (Technica l As soc ia t ion of the Pulp and Paper Indus t ry ) . 1966. Method T230-M50. T a r r , H. L . and H. Hibber t . 1931. Studies on react ions r e l a t i n g to carbohydrates and polysacchar ides . XXXV. Polysacchar ide synthes i s by the act ion of Acetobacter xy l inus on carbohydrates and re la ted compounds. Can. 3. Research. 4: 372-388. Townsley, P. M. 1981. Personal communication. Department of Food Science, Facul ty of A g r i c u l t u r a l Sciences , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B . C . - 187 -Townsley, P. M. and M. A. Tung. 1974. Unpublished r e s u l t s . Department of Food Science, Faculty of A g r i c u l t u r a l Sciences, Univers i ty of B r i t i s h Columbia, Vancouver, B.C. Vi l l anueva , L. 3. 1937. The effect of varying amounts of sugar added to pineapple pulp mash on ac id i ty and y i e l d of "nata de p i n a " . P h i l i p p . A g r i c . 26: 508-514. Warburton, C. E. 1970. Variance in breaking-strength d i s t r i b u t i o n of yarns raveled from chemically treated cotton f a b r i c s . Tex t i l e Res. 3. 40: 75-80. Warwicker, 3. 0. and P. Hallam. 1975. The effect of a lka l ine and acid swelling agents on the mechanical properties of cotton f i b e r s . 3. Text. Ins t . 66: 61-76. Warwicker, 3. 0 . , R. 3e f f r i e s , R. L. Colbran and R. N. Robinson. 1966. A review of the l i t e r a t u r e on the effect of caust ic soda and other swell ing agents on the f ine structure of cotton. Sh i r ley I n s t i t u t e Pamphlet No. 93. St Ann's Press, Altrincham, England. Webb, T. E. and 3. R. C o l v i n . 1967. The e x t r a c e l l u l a r proteins of Acetobacter xylinum and t h e i r r e l a t ion to ce l lu lo se synthesis . Can. 3. Biochem. 45: 465. Weinhouse, H. and M. Benziman. 1974. Quoted in A l o n i , Y. and M. Benziman (1982). Intermediates of ce l lu lo se synthesis _in Cel lu lose and other natural polymer systems biogenesis, structure and degradation. Ed. Brown, R. M . , Plenum Press, London, pp. 342. Weinhouse, H. and M. Benziman. 1976. Phosphorylation of g lycero l and dihydroxyacetone i n Acetobacter xylinum and i t s possible regulatory r o l e . 3. B a c t e r i o l . 127: 747-754. Whis t ler , R. L. 1963. Methods in Carbohydrate Chemistry. V. 3: Ce l lu lo se , Academic Press, New York, pp 3, 55-57 and 344-345. WHO (World Health Organization). Food Addit ives Ser ies . 1974. No. 5 . Yamanaka, S. , T. Tanaka and K. Takinami. 1979. Polysaccharide. 3apan. Patent 7,937,889. Zaar, K. 1977. The biogenesis of ce l lu lo se by Acetobacter xylinum. Cytobiologie . 1: 1-15. Zaar, K. 1979. V i s u a l i z a t i o n of pores (export s i tes) correlated with ce l lu lo se production in the envelope of the gram-negative bacterium Acetobacter xylinum. 3. C e l l B i o l . 80: 773-777. Zabr i sk ie , D. W. 1980. Traders' Guide to Fermentation Media Formula-t i o n . Traders Protein D i v i s i o n , Traders O i l M i l l Co . , Fort Worth, Texas. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096177/manifest

Comment

Related Items