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Proteases production from fermentation of fish solubles Lakhdari, Marc 1978

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PROTEASES PRODUCTION FROM FERMENTATION OF FISH SOLUBLES by MARC LAKHDARI Diplome de l'Ecole Nationale Superieure de Chimie de Paris, 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE IN THE FACULTY OF GRADUATE STUDIES in the Department of CHEMICAL ENGINEERING We accept this thesis as canforrmng to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1978 (^) Marc Lakhdari,1978 In presenting th i s thes i s in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by h is representat ives. It is understood that copying or pub l i cat ion of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of CHEMICAL ENGINEERING The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e MARCH 4th 1978 ABSTRACT The po s s i b i l i t y of using a culture medium derived from herring f i s h solubles to produce proteases was investigated. Following Wah-On's (27) recommandations, the role of the oxygen i n the c e l l and enzyme production was specially considered. A Myxobacteria, Sorangium 495 was used to produce the protease. I t was f i r s t confirmed that the organism was aerobic, and that glucose had a significant effect on growth and protease formation, according to experiments performed i n 250 ml Erlenmeyer flasks and in a 7-liter fermentor. Two different sets of experiments were made i n the latter fermentor, one using culture medium of i n i t i a l protein content of about 4 mg (BSA)/ml, and another between 11 and 16 mg (BSA)/ml. The maximum enzyme activity of 2.47 unit/10 cc was noticed at DO = 100% saturation at the low i n i t i a l protein content. I t was then concluded that oxygen favors protease formation. However, although the importance of oxygen cannot be denied, no consistent pattern to the level of c e l l mass concentration as a function DO level could be found, and this role could not be accurately determined. Furthermore, the kinetic patterns were quite complex, and no simple correlation between the different parameters involved could be deter-mined. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES CHAPTER I 1.1 Intrcduction 1.2 Composition of fish 1.3 Fish processing 1.4 Microbial proteases 1.5 Production of Microbial proteases 1.6 Fish Solubles as culture medium 1.7 Sorangium 495 1.8 Oxygen Transfer CHAPTER II EXPERIMENTAL TECHNIQUES 11.1 Introduction 11.2 Innoculum preparation 11.3 Preparation of Culture Medium 11.4 Apparatus 11.5 Measurement of bacterial growth 11.5.1 Introduction 11.5.2 Experimental procedure 11.6 Enzyme activity determination 11.6.1 Introduction 11.6.2 Measurement of the enzyme activity 11.7 Measurement of the protein content 11.7.1 Introduction 11.7.2 Experimental Analysis 11.8 Measurement of the Glucose concentration 11.8.1 Inrjxduction 11.8.2 The dinitrosalicylic (DNS) method 11.8.3 Experimental procedure CHAPTER III RESULTS AND DISCUSSION III.1 Introduction iv TABLE OF CGS1TENTS (cont'd.) Page 111.2 Deterndnation of concentration of fish solubles 65 111.2.1 Introduction 6 5 111.2.2 Experimental Procedure 66 111.2.3 Results 66 111.3 Preliiruriary experiment: Variation of the tyrosine production with digestion time 69 111.4 Shake Flasks: Salmon Solubles at high protein concentration 69 111.5 Shake Elask Runs: Herring Solubles at different protein concentrations 77 111.5.1 Introduction 77 111.5.2 Shake Flasks: Herring Solubles at i n i t i a l protein concentration of 3.65 mg BSA/ml 77 111.5.3 Shake Flasks: Herring Solubles at i n i t i a l protein concentration of 4.45 mg BSA/ml 78 111.5.4 Shake Flasks: Herring Solubles at i n i t i a l protein concentration of 5.59 mg BSA/ml 79 111.5.5 Shake Flasks: Herring Solubles at i n i t i a l protein concentration of 17.48 mg BSA/ml 79 111.5.6 Shake Flasks: Herring Solubles at i n i t i a l protein concentration of 20 mg BSA/ml 88 111.5.7 Discussion 88 111.5.8 Conclusion 9 8 111.6 7 lit e r Fermentor Runs: Initial protein concentrations around 4 mg BSA/ml 99 111.6.1 Introduction 99 111.6.2 Results and discussion 100 III.6.2.a Cell yield 100 III.6.2.b Protein consumption 115 III.6.2.c Enzyme activity 126 III.6.2.d Glucose consumption 134 111.6.3 Cell yield and Nutrient consumption 139 111.6.4 Conclusion 144 111.7 7 l i t e r Fermentor Runs at high i n i t i a l protein content 152 111.7.1 Introduction 152 111.7.2 Results and discussion 152 111.7.3 Conclusion 17 5 TABLE OF CONTENTS (cont'd.) CHAPTER IV COSTCLUSICN BIBLIOGRAPHY APPENDIX I FLASK RUNS: EXPERIMENTAL DATA Appendix 1.1 Flask Runs: Salmon Solubles at high i n i t i a l protein concentration Appendix 1.2 Herring Solubles APPENDIX II 7- l i t e r Fermentor Runs: I n i t i a l protein concentration at about 4 mg BSA/ml EXPERIMFJNTAL DATA APPENDIX III 7-li t e r Fermentor Runs: High i n i t i a l protein concentration EXPERIMENTAL DATA APPENDIX IV 7-liter Fermentor Runs: High i n i t i a l protein concentration TURBIDITY MEASUREMENTS APPENDIX V 7-liter Fermentor Runs: Runs at low i n i t i a l protein content (around 4 mg BSA/ml) pH CCNTROL vi LIST OF TABLES T a b l e Page 1.1 Parameter of various fish processing plant effluents 2 1.2 Composition of fish 4 1.3 Typical analysis of fish stickwater 8 1.4 Typical analysis of condensed fish solubles 8 1.5 Amino acid analysis of condensed fish solubles 9 1.6 Typical vitamin content of west coast fish solubles 11 1.7 Typical analysis of fish solubles ash constituents 11 1.8 Enzymes production by various microorganisms 15 1.9 Some microbial proteases which have been isolated and characterized 19 1.10 Alternate protein supply sources 21 1.11 Cell Yield of salmon-canning waste-water as a function of cell number per m i l l i l i t e r 23 1.12 Effect on agitation and aeration on ultimate yield 25 2.13 Weight dry cells vs turbidity 44 2.14 Some weight of dry cells: fermentor run #5 46 2.15 Tyrosine calibration data 54 2.16 Protein calibration data 59 2.17 Glucose calibration data 63 3.18 Herring fish solubles solution: protein content in equivalent mg (BSA)/ml function of dilution 67 3.19 Variation of the tyrosine production with digestion time 69 3.20 Shake flasks: Salmon Solubles at high protein concentration. 75 3.21 Shake flasks: Salmon Solubles: Wah-Qn's results 76 v i i LIST OF TABLES (cont'd.) Table Page 3.22 Flask Runs: Results for this work and Wah-On's 94 3.23 Process conditions of the experiments 99 3.24.a Maximum cell yields 116 3.24.b Maximum Protein and Glucose consumption 117 3.24.C Maximum Enzyme Activity 133 3.24.d Maximum Protein and Glucose consumption rates 141 3.25 Yield coefficients based on protein and glucose usage 147 3.26 Process conditions of the Experiments 153 3.27.a Maximum cell yields 161 3.27.b Maximum protein consumption. 164 3.27.C Maximum Enzyme Activity 166 3.28 Yield Coefficients based on Protein usage 168 v i i i LIST OF FIGURES Figure Page 1.1 Fish rendering process 7 1.2 Structure of a pentapeptide 13 1.3 7-liter fermentor CFS Run No. 1 26 1.4 7-liter fermentor CFS Run No. 6 27 1.5 Resistances to oxygen transfer 31 2.6 7-liter fermentor: schematic drawing 41 2.7 7-liter fermentor: head plate penetrations 42 2.8 Weight dry cells vs Turbidity 45 2.9 Weight dry cells vs fermentation time (Fermentor Run #2.5) 47 2.10 Tyrosine, 50 2.11 Calibration curve: Tyrosine concentration ( g/ml) vs absorption time (min.) 55 2.12 Calibration curve; total protein content mg (BSA)/ml vs absorbance at 540 m 60 2.13 Calibration curve: Glucose concentration vs absorbance at 575 m micron 64 3.14 Protein content mg (BSA)/ml vs concentration of Solubles expressed in function of x 68 3.15 Tyrosine concentration (micro mg/ml) vs digestion time (min.) 70 3.16 Flask run: salmon solubles at high i n i t i a l protein concentration weight dry cells (mg/ml) vs fermentation time (hrs.) 73 3.17 Flask run: Salmon solubles. Weight dry cells (mg/ml) vs fermentation time (hrs.) . From Wah-On' s results 74 3.18 Flasks Run #F.l: Weight dry cells (mg/ml) vs fermentation time (hrs.) 80 3.19 Flasks Run #F.l: Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 81 ix LIST OF FIGURES (cx>nt'd.) F i ^ e Page 3.20 Flasks Run #F.l: Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 82 3.21 Flasks Run #F.2: Weight dry cells (mg/ml) vs fermentation time (hrs.) 83 3.22 Flasks Run #F.2: Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 84 3.23 Flasks Run #F.2: Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 85 3.24 Flasks Run #F.3: Weight dry cells (mg/ml) vs fermentation time (hrs.) 86 3.25 Flasks Run #F.3: Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 87 3.26 Flasks Run #F.4: Weight dry cells (mg/ml) vs fermentation time (hrs.) 89 3.27 Flasks Run #F.4: Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 90 3.28 Flasks Run #F.4: Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 91 3.29 Flasks Run #F.5: Weight dry cells (mg/ml) vs fermentation time (hrs.) 92 3.30 Flasks Run #F.5: Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 93 3.31 Flask Run #F.4: Expanded scale showing the two levels of protein consumption 97 3.32 Fermentor Run #1.1 Weight dry cells (mg/ml) vs fermentation time (hrs.) 101 3.33 Fermentor Run #1.2 Weight dry cells (mg/ml) vs fermentation time (hrs.) 102 3.34 Fermentor Run #1.3 Weight dry cells (mg/ml) vs fermentation time (hrs.) 103 3.35 Fermentor Run #1.4 Weight dry cells (mg/ml) vs fermentation time (hrs.) 104 X -LIST OF FIGURES (cont'd.) F i g u r e Page 3.48 Fermentor Run at low i n i t i a l protein concentration: Average protein consumption (mg (BSA)/ml) vs DO (% saturation) 125 3.49 Fermentor Run #1.1 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 127 3.50 Fermentor Run #1.2 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 128 3.51 Fermentor Run #1.4 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 129 3.52 Fermentor Run #1.5 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 130 3.53 Fermentor Run #1.6 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 131 3.54 Fermentor Run#l.l Glucose concentration (mg/ml) vs fermentation time (hrs.) 135 3.55 Fermentor Run #1.2 Glucose concentration (mg/ml) vs fermentation time (hrs.) 136 3.56 Fermentor Run #1.4 Glucose concentration (mg/ml) vs fermentation time (hrs.) 137 3.57 Fermentor Run #1.5 Glucose concentration (mg/ml) vs fermentation time (hrs.) 138 3.58 Fermentor Runs at low i n i t i a l protein concentration. Maximum glucose consumption (mg/ml) vs DO (% saturation).. 142 3.59 Fermentor Runs at low i n i t i a l protein concentration. Average glucose consumption (mg/ml) vs DO (% saturation).. 143 3.60 Fermentor Runs at low i n i t i a l protein concentration. Maximum Enzyme activity (unit/10 cc) vs DO (% saturation). 145 3.61 Fermentor Runs at low i n i t i a l protein concentration. Average Enzyme activity (unit/10 cc) vs DO (% saturation). 146 3.62 Fermentor Runs at low i n i t i a l protein concentration. Cell yield based on protein consumption 148 x i LIST OF FIGURES (cont'd.) P i * u r e Page 3.36 Fermentor Run #1.5 Weight dry cells vs fermentation time (hrs.) 105 3.37 Fermentor Run #1.6 Weight dry cells vs fermentation time (hrs.) 106 3.38. a Dissolved oxygen measurement chart. Run at DO=10% saturation 109 3.39. a Dissolved oxygen measurement chart. Run at DO=30% saturation 110 3.40. a Dissolved oxygen measurement chart. Run at DO=50% saturation-and. at DO=80% saturation I l l 3.38 Fermentor Runs at low i n i t i a l protein concentration: Maximum cell production (mg/ml) vs DO (% saturation) . 112 3.39 Fermentor Runs at low i n i t i a l protein concentration. Average cel l production (mg/ml) vs DO (% saturation) 113 3.40 Fermentor Runs at low i n i t i a l protein concentration. Maximum growth rate (mg/ml x hr) vs DO (% saturation) 114 3.41 Fermentor Run #1.1 Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 118 3.42 Fermentor Run #1.2 Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 119 3.43 Fermentor Run #1.3 Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 120 3.44 Fermentor Run #1.4 Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 121 3.45 Fermentor Run #1.5 Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 122 3.46 Fermentor Run #1.6 Total protein content (mg (BSA)/ml) vs fermentation time (hrs.) 123 3.47 Fermentor Runs at low i n i t i a l protein concentration: Maximum protein consumption (mg (BSA)/ml) vs -DO (% saturation) j _ . 124 x i i LIST OF FIGURES (cont'd.) E i ^ u r e Page 3.63 Fermentor Runs at low i n i t i a l protein concentration. Cell yield based on glucose consumption 149 3.64 Fermentor Runs at low i n i t i a l protein concentration. Enzyme production based on glucose consumption 150 3.65 Fermentor Runs at low i n i t i a l protein concentration. Enzyme production based on protein consumption 151 3.66 Fermentor Run #2.1 Weight dry cells (mg/ml) vs fermentation time (hrs.) 155 3.67 Fermentor Run #2.2 Weight dry cells (mg/ml) vs fermentation time (hrs.) 156 3.68 Fermentor Run #2.3 Weight dry cells (mg/ml) vs fermentation time (hrs.) 157 3.69 Fermentor Run #2.4 Weight dry cells (mg/ml) vs fermentation time (hrs.) 158 3.70 Fermentor Run #.25 Weight dry cells (mg/ml) vs fermentation time (hrs.) 159 3.71 Fermentor Run #2.6 Weight dry cells (mg/ml) vs fermentation time (hrs.) 160 3.72 Maximum cell yield (mg/ml) vs DO (I saturation) 162 3.73 Maximum protein consumption (mg BSA/ml) vs DO (% saturation) 163 3.74 Fermentor Runs at high i n i t i a l protein concentration. Mass cells/Protein used 167 3.75 Fermentor Run #2.1 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 169 3.76 Fermentor Run #2.2 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 170 3.77 Fermentor Run #2.3 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 171 3.78 Fermentor Run #2.4 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 172 x i i i LIST OF FIGURES (cont'd.) E i ^ u r e Page 3.79 Fermentor Run #2.5 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 173 3.80 Fermentor Run #2.6 Enzyme activity (unit/10 cc) vs fermentation time (hrs.) 174 3.81 Fermentor Runs at high i n i t i a l protein concentration. Enzyme activity protein used 176 3.82 Fermentor Run #2.1 Protein concentration (mg BSA/ml) vs fermentation time (hrs.) 177 3.83 Fermentor Run #2.2 Protein concentration (mg BSA/ml) vs fermentation time (hrs.) 178 3.84 Fermentor Run #2.3 Protein concentration (mg BS/A/ml) vs fermentation time (hrs.) 179 3.85 Fermentor Run #2.4 Protein concentration (mg BSA/ml) vs fermentation time (hrs.) 180 3.86 Fermentor Run #2.5 Protein concentration (mg BSA/ml) vs fermentation time (hrs.) 181 3.87 Fermentor Run #2.6 Protein concentration (mg BSA/ml) vs fermentation time (hrs.) 182 AC^ OWLEDGET^ IENTS I wish to thank Dr. Richard Branion, under whose direction this work was undertaken, for his patience and guidance throughout this work. I also wish to thank Dr. George Strasdine of the Fisheries Research Board of Canada for providing reference materials and stock culture. Furthermore, I wish to thank Mr. D.K. Ferguson for his help and advice for conducting the experiments and in the use of some of the equipment. Appreciation is also extended to the personnel of the Chemical Engineering Department for their technical support. I wish to thank Ms. Maria Lowe for typing this thesis. Finally, I wish to thank Environment Canada, Fisheries and Marine Service, the National Council of Canada and the Canada Council for providing financial support. 1 CHAPTER ONE T . l INTRODUCTION Fish canning is an important industry in British Columbia. Salmon canning and herring processing operations, either for reduction to meal and o i l , or for food purposes, are the only two types of operations which carry major problems of liquid waste disposal. (1) For most plants, waste water flows are of the order of 200 to 1,000 USGPM. The major pollutants in these effluents are flesh particles, scales, blood, slime and soluble proteins. Table 1.1 adapted from Clagget (1) shows some typical parameters of such waste waters. Relatively recently, a variety of methods has been developed to dispose of these waste waters. Previously, they were discharged to waterways; there, the proteinaceous matter decomposed rapidly creating pollution problems. Treatment of these waste waters should include systems for recovering as much proteinaceous and oily materials as possible (1, 2, 3, 4, 5). One way of reducing the polluting effects of fish plant waste waters while producing a marketable byproduct would be to use them as a culture medium for the production of microbial proteases. The high protein content of the wastes make them suitable for this purpose. There is a small, but significant demand for enzymes which hydro-lyze proteins: these are called proteolytic enzymes or proteases. Industrial production of these could be economically profitable. Uses of such enzymes are discussed in section 1.4. Table 1.1 'PARAMETERS OF VARIOUS FISH PROCESSING PLANT EFFLUENTS (1) SPECIES PROCESSED BOD SUSPENDED SOLIDS TOTAL SOLIDS mg/1 #/1000# fish mg/1 #/l'000# fish mg/1 #/1000# fish Halibut 200 4 t 350 7.2 500 10.3 Grey Cod 435 2.2 300 1.5 600 3.0 Lingcod 460 4.1 235 2.2 550 5.2 Sole 200 1.4 125 0.8 300 1.9 Ocean Perch 75 0.7 50 1.3 85 2.2 Salmon Canning 3500 28 1500 12 3000 24 Food Herring 3850 22 3000 21 6000 42 3 1.2 COMPOSITION OF FISH Fish consists of skin, flesh and bones. Skin consists mainly of water (about 80%) and about 16% proteins. Bones contain much mineral matter, principally calcium and phosphate, which amounts to about 14% of the bone material. Water (about 75%) and proteins (9%) constitute the other major components of fish bone. Flesh is the main component of fish. It is predominately made up of numerous cells, especially muscle fibres, held together by connective tissues, and surrounded by extracellular fluids. Blood vessels and nerve fibres are less impor-tant on a weight basis. Fish flesh also contains a large amount of fat and water. The quantities present vary according to the diet of the fish, the season and its environment. Fat is present in muscle tissue as microscopic globules both within the fibres and in the extracellular fluids. Summer herring is expected to contain about 20% fat, autumn herring about 10 to 15%, winter herring about 5 to 10%, and spring herring less than 5%. In the fish, fat is closely related to water, and the total of the two constitutes 80% of the composition of the fish as shown in Table 1.2. adapted from Burgers (6). Myosin and actin are the main proteins of the fish; these are com-bined in the muscle as actomyosin. The protein content of healthy fish flesh is about 16 to 18% (wet basis). In their natural state, these proteins are associated with a high proportion of water. Any processing or cooking of fish will tend to denature the proteins, which results in a change of reactivity and a loss of solubility. TABLE 1.2 COMPOSITION OF FISH (6) % Water % Protein % Fat Well fed herring 60 20 20 Starved herring 75 20 5 N.B.: The amount of protein remains the same in both cases, but the fat content of the well fed fish increases at the expense of water. The total water fat content is constant in both cases. 5 Apart from these major constituents, fish contain a variety of minor components. "The extractive consists of a group of otherwise unrelated substances that share the properties of solubility in water and of usually possessing some kind of taste. Volatiles overlap with extractives. They are those compound that are released in small quantities in gaseous form from foods, thereby,contributing their characteristic odors as well as taste. They consist mainly of nitro-genous bases. (6) The principal free sugar of fresh fish is glucose. Ribose is also present attached to a complex nitrogenous substance and to phos-phate. Fish flesh contains a variety of different minerals. Among them are: potassium, sodium, calcium, magnesium, iron, copper, manganese, zinc and cobalt. The fish contains some vitamins as well, especially vitamin A and D. However, vitamins occur in too small amounts to have any effects on flavor or texture. T.3 FISH PROCESSING Fish solubles are made from fish stickwater which is a by-product from the rendering processes. In the rendering of fish, the fish and/ or fish waste are steam cooked, and then separated under pressure into an aquaeous extract and fish pulp. The water is extracted from the fish pulp leaving a fish meal of about 8% moisture. This meal can be sold as fertilizer or as animal feed. The press water is screened to 6 remove any solids and then passed through a centrifuge. The sludge from this process i s returned to the press cake, and the c l a r i f i e d press liquor i s passed to an o i l centrifuge. This o i l centrifuge yields two products: the f i s h o i l , and the stickwater. Since good quality condensed fi s h solubles cannot be made from stickwater with a high o i l content, the stickwater i s treated with acid i n holding tanks. This process removes the last amount of fatty matter by hydrolysis, as well as suspended proteinaceous matter. Addition of acid eventually produces more stable condensed f i s h solubles having lower viscosity and a pH of about 4.5. This stickwater i s then eva-porated which reduces i t s water content from 95% to about 50% (7). The process i s outlined in Figure 1.1 taken from Soderquist (8). The evaporative concentration of stickwater produces a brown, viscous, f i s h smelling liq u i d called "solubles". Solubles contain about 50% of solids, including ash, fat and crude proteins. Its speci-f i c gravity i s around 1.20 at 20° C, and i t s pH i s about 4.5. The approximate (composition of stickwater and condensed fi s h solubles are given i n Tables 1.3 and 1.4 respectively (7). The fish solubles obtained by this process are rich i n amino acid as shown i n Table 1.5 (7). Vitamin analysis reveals, i n part, the reason why condensed fi s h solubles are used so intensively for animal nutrition (Table 1.6 (7) ). The ash content of f i s h solubles i s quite considerable, from 8.5% to 9.5% (dry basis). Several minerals are present: potassium and sodium 7 'Figure 1.1 FISH RENDERING PROCESS (8) PROCESS WASTES DISPOSAL RAW PRODUCT LIQUOR CENTRIFUGE OIL SLIME, WATER^ --( OIL, WATER)--( STI<IKWAT_R^  EVAPORATOR SOLUBLES WATER, SOLIDS)J RECEIVING WATER 8 Table 1.3 TYPICAL ANALYSIS OF FISH STICKWATER (7) PARAMETER VALUE "Total solids Ash Fatty substances Crude protein (N x 6.25) Table 1.4 TYPICAL ANALYSIS OF CONDENSED FISH SOLUBLES (7) ' PARAMETER 'VALUE .Total solids 50.43% Ash 8.86% Fat 4.8% Crude protein (N x 6.25) 33.85% Sp. gr. at 20° C 1.20 pH 4.5 5.6% 0.95% 0.60% 3.5% * Table 1.5 i AMINO ACID ANALYSIS OF CONDENSED FISH SOLUBLES (7) % CRUDE PROTEIN AMINO ACID (N X 6.25) ASSAY METHOD Arginine 4.84 Mb.a Histidine 5.79 Chem.k Lysine 4.87 Mb. Leucine 4.67 . Mb. Isoleucine 2.73 Mb. Phenylalanine 2.33 Chem. Tryptophan 0.35 Mb. Methionine 1.51 Mb. Threonine 2.55 Chem. Cystine 0.58 Chem. Glutamic acid 8.44 Chem. Proline 6.70 Chem. Mb. = microbiological. Chem. = chemical. 10 are the main ones (Table 1.7 (7) ). From the above, i t can be determined that f i s h solubles contain significant amounts of amino acids, vitamins, minerals and sugars. Thus, they should be a rich source of nutrients and energy for the support of the growth of microorganisms and hence should be an excel-lent culture medium, either i n themselves or i n combination with other nutrients. T.4 MICROBIAL PROTEASES The primary structure of proteins i s defined as the covalent backbone structure of polypeptide chain. The peptide bond i s the sole covalent linkage between amino acids i n the linear backbone structure of proteins (9). Figure 1.2 shows the structure of a pentapeptide i l l u s t r a t i n g the role of the peptide bonds. (from Lehninger (9) ) A l l proteolytic enzymes, or proteases, are characterized by their a b i l i t y to catalyse hydrolytic cleavage of peptide linkages between amino acids (9) (10). The result of the overall reaction can be described by: C = 0 2 Table 1.6 TYPICAL VITAMIN CONTENT OF WEST COAST FISH SOLUBLES VITAMIN ug/g VITAMIN pg/g Riboflavin 22 Pyridoxin 12.5 Pantothenic Acid 84 Choline 1100 Thiamine 7.5 Folic Acid 0.23 Niacin 390 Vitamin B^2 0.47 Ref. (7) Table 1.7 TYPICAL ANALYSIS OF FISH SOLUBLES ASH (XNSTITUENTS CONSTITUENT % CONSTITUENT o, "5 Potassium (K) 1.93 Iron (Fe) 0.0249 Sodium (Na) 1.87 Magnesium (Mg) 0.016 Phosphorous (P) 0.85 Copper (Cu) 0.007 Calcium (Ca) 0.0869 Iodine (I) 0.007 Manganese (Mn) 0.0869 Muminum (Al) 0.005 TOTAL ASH 8.86% 12 The action of proteases can be very specific. For example, trypsin, a digestive enzyme secreated by the pancreas catalyses the hydrolysis of only those peptide bonds i n a polypeptide chain whose carbonyl function i s donated by either a lysine or an arginine re-sidue, regardless of the length or amino acid sequence of the chain. (9) These enzymes are classified into two distinct groups: the endo-peptidases and the exopeptidases. (10) The endopeptidases hydro lyze the internal peptide bonds i n high molecular weight protein molecules. The results of specific cleavage of peptide bonds located inside long chains of polypeptids are peptides such as proteoses and peptones. Endopeptidases do not carry the disruption of the peptide chains further than the poly- or dipeptide stage. The exopeptidases can disrupt the poly- and dipeptides to the stage of amino acids. Terminal peptide bonds adjacent to free polar groups are attacked and free amino acids are liberated. (11) A l l micro-organisms feeding upon proteins produce proteolytic en-zymes. They hydrolyse the proteins into the amino acids necessary to their metabolism for the production of their own cellular and enzyme proteins. Table 1.8 adapted from Keay (12) shows seme enzyme production by various microorganisms. Generally, these are extracellular enzymes and are endopeptidases. They differ from each other i n the pH optima-at which they work. They are divided into three main groups, acid, neutral and alkaline, depending upon the pH range i n which their activity i s greatest. (13) I t seems that the majority of microbial enzymes studied to date have been produced extracelluTarly, and then isolated i n active form 13 Fig. 1.2 STRUCTURE OF A PENTAPEPTLDE 14 from the culture f i l t r a t e s of the appropriate organism. Enzymes are frequently found as mixtures of proteases. In addition, other enzymes such as lipases, amylases, or cellulases may be present. Such mixture of enzyme are called "crude enzyme mixtures". Commercial application i s usually only feasible with these crude enzyme mixtures because the isolation of a pure enzyme i s a complex and costly procedure (12). The enzymatic activity of these crude mixtures can be very complicated morever quantitative measurement of their activity becomes d i f f i c u l t . Although these crude enzyme mixtures are impure, they are widely used i n industry. (13) (14) Food processing and brewing, the two industries that f i r s t ex-ploited enzymes, are s t i l l the major users. I t has been estimated that fungal enzyme preparations are used i n the manufacture of 75% of a l l bread baked i n the U.S.A. (13) The proteolytic enzymes have many applications i n the processing of food products, such as meat and f i s h which have a high protein content. Hydrolysed protein, prepared enzy-mically, i s an ingredient of a wide variety of freeze-dried products such as soup powder and baby foods. Enzymes are often used as meat tenderizers. Papain extracted from papaya, and bromelain from pine-apples are probably the most widely used enzymes for this purpose. Many fi s h are processed on a large scale primarily for their o i l content. In that case, the use of enzymes to break down the fi s h pro-tein greatly f a c i l i t a t e s extraction of the o i l . Similarly, the use of proteolytic enzymes to f a c i l i t a t e o i l extraction from olives has been suggested. Use of enzymes can improve o i l removal i n sauces as well (Worchester, soya etc.) 15 ' Table 1.8 Enzyme production by various microorganisms Organism Enzymes B. subtilis NRRL B3411 B. subtilis Marburg B. subtilis IAM 1523 B. subtilis N' B. amyloliquefaciens Neutral protease, subtilisin Novo, liquefying <* -amylase B. subtilis var. amylosacchariticus Neutral protease, subtilisin Novo, saccharifying c<-amylase B. subtilis B. pumilis B. licheniformis Subtilisin Carlsberg B. thermoproteolyticus Thermolysin B. megaterium MA. B. cereus NCTC 945 Aeromonas proteolytica Streptomyces naraensis Neutral proteases Streptomyces rectus Streptomyces fradiae Thermostable alkaline protease Streptomyces griseus K-l Aspergillus saitoi Aspergillus oryzae Neutral and alkaline proteases and peptidases Acid protease Acid, neutral, and alkaline proteases, amylase 16 The cheese, making industry uses rennet which i s a complex mixture of enzymes obtained from the stomachs of milk fed calves. I t seems that supplies of animal rennet are beconing short. Microbial rennet can be produced and used as a substitute. For example, a suitable enzyme i s produced from "Mucor" and "Rizophus" and i s now used i n Japan for cheese making. (14) Outside the food industries, enzymes are used i n various areas, such as the manufacturing of spot removal agents, dehaifing of hides and i n pharmaceutical products. The pharmaceutical industries require more purified enzymes; among them, proteolytic enzymes can be used as digestive aids. The use of proteolytic enzymes as additives for waste treatment has been developed to some extent. This i s not unexpected since the breakdown of large proteins i s their function. Domestic sewage i s a rich biological medium and, not surprisingly can support the growth of a large number of different microorganisms. I t has been suggested (15) that enzymes could be used i n restarting a microbiological waste treatment system which has been stopped due to poisoning of the biomass. I t has been reported that enzymes are useful i n clearing small size biological treatment process which have been over loaded. (15) Mthough said to be quite efficient (15) the widespread use of enzyme preparations i n waste treatment w i l l remain too expensive considering the present cost of enzyme production and the high quantity of material to be treated. 17 1.5 Production of Microbial proteases Enzyme production seems to be a profitable industry because over 4 million dollars worth of proteolytic enzymes were marketed in 1969. (14) The usual industrial fermentation for the growth of micro-organisms has been applied, as well as the classical microbiological technicjaes. For large scale growth, batchwise or continuous fermen-tation can be used. Tray culture or submerged culture techniques are employed. If the enzyme i s extracellular, the culture medium can be par-ti a l l y purified and/or concentrated, and the product may be directly ready for sale. If the enzyme is intracellular, the organisms are harvested, often by centrifugation. The cells then must be broken down by various means to release the enzymes. It is important that the product be uni-form since standardization is often required. (14) A wide variety of enzymes are presently available in commercial quantities. Moreover, a wide variety of processes for enzyme production have been considered. Thus in proposing any new process i t is important that the right strain of micro-organisms is selected to give the best yield of the required enzyme. Considerable exploratory investigations are then required to establish optional culture conditions for tempera-r •  ture, pH, oxygen and substrate requirements. The wide range of protease producing organisms together with the number of potential process options suggests infinite possible production sequences. However, for 18 economical reasons, only those organisms which produce very high yields of enzymes from very cheap substrates should be considered. Additional restrictions arise from the fact that only enzymes which are regarded as safe can be produced. This became an important factor, especially after the use of much advertized enzyme containing detergents which caused allergic reactions in many users. The danger of such detergents became widely publicized and they had to be subsequently withdrawn from use. In Japan, Tomanaga and Yanayita (15) (16) developed a method to produce continuously proteases from aspergillus Niger on a medium composed of amino acids, glucose, salts and Na2 So^. Satisfactory yields were achieved. Jonsson and Martin were able to produce protease from Aspergillus fumigatus also at very high yield. (18) Keay produced proteases from the genus 'bacillus in large numbers as shown in Table 1.9. (19) Industrial waste material has been used as culture media to pro-duce proteinases. Kline, McDonnel and Linewater used waste asparagus butts to grow B. subtilis and lightly active proteases were produced. (20) Waste matter and by-products derived from the fish canning indus-tries have also been used as culture medium to produce proteases. (21) T.6 FISH SOLUBLES AS OTLTUEE_MEDIUM Fish solubles are used in animal nutrition. However, i t may be 19 Table 1.9 Seme Microbial Proteases Which Have Been Isolated and Characterized Acid proteases Neutral proteases Alkaline proteases Asp. candidus Asp. saitoi Asp. niger Asp. awamori Asp. oryzae Asp. fumigatus Pen. janthinellum Pen. notatum Phiz, chinensis Phiz, bligosporus Paecilcmyces varioti Mucor pusillus Mucor hiemalis Trametes sanguinea Alternaria tenuissima Candida albicans Endothia parasitica Byssochlamys fulva Bacillus cereus Bacillus subtilis Bacillus megaterium Bacillus cereus Bacillus thermoproteolyticus Streptomyces naraensis Streptomyces griseus Asp. oryzae Asp. flaws Asp. ochraceus Asp. parasiticus Aeromonas proteolytica Aeromonas hydrophila Pseudomonas aeruginosa Clostridium histolyticum Proteus mirabilis Micrococcus caseolyticus Trychophyton granulosum Halobacterium salinarum Micromonosporium sp. Bacillus subtilis Bacillus licheniformis Bacillus pumilis Streptomyces griseus Streptomyces fradiae Streptomyces rectus Streptomyces moderatus Asp. oryzae Asp. sydowi Asp. flavus Asp. sojae Asp. melleus Asp. fumigatus Thermomonospora fusca Thermoactinomyces vulgaris Penicillium cyaneofulvum Alternaria tenuissima Bactericides amylophilus Cliocladium roseum Arthrobacter sp. 20 that fermentation of this material could produce a more valuable product. This would have been true in the past, but at the moment, the price of protein sources is rapidly increasing. It has been suggested that a liquid high in protein should serve as an excellent medium for the growth of micro-organisms producing extracellular pro-teases. Compared to other protein sources, its cost is comparatively low. (Table 1.10) Fish based culture media have been used in a variety of situations. For example, fish peptone solutions were used to grow a fish pathogen called hemophilus piscium. (22). This pathogen is responsible for an ulcer disease of trout, i t was found to grow very well on fish peptone solutions. Production of such micro-organisms allowed further studies on fish diseases. Several other cultures were grown on fish peptones media. These peptones were incorporated into agar medium, and compared to other commercially available peptones (23). In most cases, the fish peptones were comparable or better in supporting bacterial growth especially for fastidious lactobacilli, suggesting a response to the wide variety of available amino acids and known vitamins. Furthermore, in many species a l l colonies on the test media appeared to be about the same size, shape, and color as those on the standard medium. These experi-ments have demonstrated that fish peptone can, in most cases, sustain good growth and recovery of micro-organisms, from either mixed popu-lations, or specific species. The fish peptones have sufficient protein content and variety of amino acids, and are low enough in fat content to be considered for this purpose. Table 1.10 ALTERNATE PROTEIN SUPPLY SOURCES (14, 15) MATERIAL PRICE/POUND* (cents) PRICE/POUND PROTEIN (cents) Soy Meal and Flour Soy Protein Cone. Fish Solubles Fish Meal (feed grade) Fish Protein Cone. Cottonseed Flour Wheat Flour Dry Skim Milk 3.5-6.5 21.5 2.50 6.3-8.5 10-16 11 6.6 14.4-21.0 Yeast-Torula (sulfite waste) 15-16 8-14.8 26.5-35 7.8 10.5-14.2 13-20 20 60 40-60 27-29 * Based on 1969 market. 22 Settle screening studies have been made to see what kind of organisms would grow on f i s h waste (24 (25). In the majority of these cases, the bacteria grown on f i s h waste were protein consuming organisms. This was to be expected since the medium used i s rich i n protein. The Fisheries Research Board of Canada, at the Vancouver Laboratory, studied the s u i t a b i l i t y of salmon canning waste water as a microbial growth medium (26). Studies using six different species of bacteria chosen as being representative of those types which might be of commercial value showed that Sorangium 495 gave the maximum yield of c e l l s . (Table 1.11) Based on these results, Wah-Cn (27) used Sorangium 495 to study the production of proteolytic enzymes by fermentation of f i s h plant waste. I t was found that the f i l t e r e d f i s h solubles media gave better protease yields that the unfiltered media. Maximum enzyme production was obtained i n a medium containing 1% glucose at a protein concentration equivalent to 3.85 mg BSA/ml, when the i n i t i a l pH was fixed at 7.0. An i n i t i a l pH of 8.0 was observed to be best for c e l l production. The addition of glucose increased the growth of the organism. This glucose containing medium gave a biphasic growth curve i n some situations. Studies i n a 7- £ fermentor indicated that the inflexion i n the growth curve observed at the lower agitation and aeration rates might have been a complex result of i n i t i a l amino acid u t i l i z a t i o n on the one hand and enzymatic digestion of larger poly-peptide units plus enzyme secretion and oxygen limitation on the other. Wah-On's results showed that growth and protease synthesis were inde-pendent. Enzyme production was affected more by the rate of agitation 23 Table 1 . 1 1 (26) CELL YIELD OF SAIMDN-CANNING WASTE WATER AS A FUNCTION OF CELL NUMBER PER MILLILITER CELLS/ML % OF MAXIMUM MEDIA GIVING (log ) FROM TABLE 1.4 MAXIMUM GROWTH Sorangium sp. A. aerogenes Bacillus sp. P. putrefaciens St. faecalis 9.34 8.60 7.20 6.83 6.76 19.1 9.3 3.6 0.3 1.2 SCE - S polypeptone - S trypticase - S SCE - S polypeptone - S L. plantarum 6.74 0.3 peptone - S S : salts 24 than by a i r flew rate. Table 1.12 taken from Wah-On summarize the. . effect of agitation and aeration on ultimate yield. Fig. 1.3 shows some typical results of his obtained i n a 7-litre fermentor. Enzyme activity and glucose consumption increase with time. The growth curve can be divided up into 4 phases: (1) I n i t i a l growth (2) f i r s t stationary phase (3) Secondary growth (4) f i n a l deceleration and stationary phase. The duration of the second phase was greatly decreased, from 40 to 20 hours, as the agitator speed increased from 300 rpm to 400 rpm. At s t i l l higher agitation rates, 500 and 600 rpm, the f i r s t stationary phase of the second portion was completely eliminated. Increasing the aeration rate from 1 1/mn to 2 1/mn also eliminated the second phase as shown by f i g . 1.4. The effect was therefore thought to be related to some mass transfer limitation on disolved oxygen. The nature of the nitrogen source may have contributed to' the overall effect as well. The f i r s t portion of the growth curve might be ^interpreted^ as representing the depletion of free amino acids and nitrogeneous bases present i n the medium. After this i n i t i a l depletion of the simple nitrogenous materials, bacteria metabolism could be limited by the rate at which the proteolytic enzyme could convert the larger polypeptides to simple assimilable forms. This depends on the rate of enzyme production which seems to be determined by the level of agitation. Table 1.12 (27) EFFECT OF AGITATION AND AERATION ON ULTIMATE YIELDS MEDIA AIR FLOW RATE ( /nvin) AGITATOR SPEED rpm DRY WEIGHT MAX TIME TO MAX (g/ ) (hrs) PROTEOLYTIC ACTIVITY MAX TIME TO MAX (units/10cc) (hrs) SUGAR UTILIZATION MAX TIME TO MAX (%initial) '(hrs) FILTERED 2% v/v 300 400 1.00* 0.85* 113§ 94 8.84 9.95 134 116± 96.3 95.0 .134 116| CONDENSED 1 500 1.29 52^ 3 Z4 10.52 68| 95.4 77 FISH SOLUBLES 600 1.62 35 7.82 52^ 3 Z4 94.4 '4 + 1% GLUCOSE 750 2.05 2°i 6.94 45 92.4 45 2 400 1.55 4 7774 47 94.3 56 500 1.55 16 9.86 45 94.1 ABOVE MEDIUM minus 1% GLUCOSE SHAKER INCUBATOR 1.00 32 2.85 45 — — NB plus 1% GLUCOSE 1 400 0.83 15 2.28 45 16.0 34 Global maximum Figure 1.3 (27) 7-LITRE FERMENTOR CFS RUN NO. 1 (Agitator speed: 300 rpm; Air flow rate: 1 /min) 26 o o o • a n C > > O 8 < m Q LU (7) Z) LU CO O O ID -J O ©-•o-A -•• -pH CELLS ACTIVITY GLUCOSE >-© J Q. [1.2 c 1.8 ^ I LU . o 0 80 TI M E (hrs.) 27 Figure 1.4 (27) 7-LITRE FERMENTOR CFS RUN NO. 6 (agitator speed: 400 rpm; Mr flow rate: 2 /min) 28 As no oxygen measuring probe was available at the time, the exact role of the oxygen could not be studied. Moreover, during the fermentation, no attempt was made to regulate the pH by the automatic addition of acid or base. It was recommended that additional studies on the effect of dissolved oxygen concentration should be carried out. Better pH and foam control during fermentation were recommended as well. Based on these recommendations, the objective of the present work is to study the effect of dissolved oxygen level on the yield of pro-duction of proteolytic enzymes from Sorangium 495 grown on a fish solubles medium. 1.7 Sorangium 495 Sorangium 495 is a soil bacteria isolated by Gillespie and Cook in 1964. (28) It was f i r s t obtained for this study from G. A. Strasdine (Vancouver Laboratory, Fisheries Research Board of Canada), and later on from F. D. Cook of the Alberta Institute of Pedology. The genus Sorangium is a member of the Sorangiaceae which belongs to the order Myxobacterales. Those bacteria are characterized by their delicate cell walls, which give them a great flexibility, and the gliding, non flagellar movement they have when they are in contact with solid surfaces. The vegetative cells of the myxobacteria are gram ne-gative rods which during vegetative growth remain perfectly indepently of one another. Cooperative action of many thousands of vegetative cells can form structure known as fruiting bodies. This is why direct photometric methods of measuring their growth, such as turbidity, are impossible. 29 The vegetative cells of the family Sorangiaceae are short, rigid cells with blunt end. This makes them different from the members of the other families such as Myxococcaceae, Archangiaceae, and Polyangiacea, which possess vegetative cells which are long, flexible and tapered (29). Many isolates from the Sorangium species are strongly cellulolytic. It was shown by Gillepsie and Cook (28) that the Sorangium 495 was able to produce at least two extracellular enzymes: a protease and a lysin. The protease hydrolyzes casein and haemoglobin and is inactive against bacterial cell walls while the lysin hydrolyzes bac-terial cell walls but is inactive on proteins. The protease activity differs for the two substrates with changing pH. The casein hydrolysis shows a sharp maximum at pH 8.5 and haemoglobin breakdown occurs over a wide pH range (7.0 - 9.5) with a broad maximum centered at near pH 8.5. Those differences reflect the differences in the two substrate molecules. The lysin hydrolyzing bacterial cell walls was most active at pH 9.0, the activity dropping off rapidly on either side of this value. The specific site of action of lysin on ce l l walls could not yet be determined. Separation of the two different extracellular enzymes elaborated by the Sorangium 495 was achieved by means of stepwise elution of protein from hydroxylapatite columns. 1.8 Oxygen Transfer Sorangium growing on fish solubles and producing protease is an aerobic organism, that is i t requires oxygen for the metabolic processes whereby i t derives its energy for the ce l l growth and reproduction. 30 This oxygen must be available to the organism in the liquid medium in which i t is growing. Oxygen is only slightly soluble in water based media so the store of dissolved oxygen will be -rapidly depleted during periods of rapid organism growth unless i t is replenished. This replenishment is carried out in large scale fermentation by sparging air bubbles into the liquid medium. (30). Wah-On (27) noted some effects of agitation and aeration rate on microbial growth rate and enzyme production rate which he attributed to limitations in the rate of oxygen transfer. In the case of shake flask culture, oxygen transfers from air to the liquid medium through the free surface of the liquid. Shaking of these flasks causes the liquid contents to swirl resulting in the more rapid mass transfer. The simplest way to picture the transfer of oxygen from a gas phase (air) to a solid phase (bacteria) suspended in a liquid phase (fermentation medium which is mostly water) is to consider a number of resistances to mass transfer which are in series. (31) . A sketch of this system appears as Fig. 1.5 the resistances proposed are: (1) a diffusional resistance between the bulk of the liquid and the gas liquid interface. (2) a resistance to movement through the interface. (3) a diffusional resistance through the film of liquid that is postulated to surround the interface. (4) a resistance to transport of dissolved oxygen from the liquid film at the-gas liquid interface to another 32 liquid film proposed as surrounding the bacterial cell. (5) a diffusional resistance to transfer through this film surrounding the bacteria cell or cells. (6) a diffusional resistance to transfer through the cell wall. (7) a resistance to reaction in the metabolic scheme of reactions within the c e l l . If the cells grow in clumps rather than as individuals there will be a further resistance to diffusion of oxygen towards the cells in the interior of the floe. Other possibilities are discussed by Bailey and Ollis (31). It has been shown by Calderbank (32) that the resistance to trans-fer of DO across the gas film is much less than that across the liquid film for typical fermentation systems. If mixing is sufficiently good to ensure a uniform concentration of DO in the bulk of the liquid we can express the mass transfer rate as: N = a (0* - CL) (1) where C* is the concentration of DO in equilibrium with the supply air. is the concentration of DO in the fermentation medium, a is the interface area is a mass transfer coefficient. N is the rate of transfer of oxygen from the gas to the liquid phase. 33 The rate of the mass transfer of DO from the liquid to the bacterial cells can be expressed by: N' = E£ a' (CL - C q*) (2) where: N' is the rate of transfer of oxygen from the liquid to the cells. K' is a mass transfer coefficient a' is the area of cell surface Co* is the concentration of DO in equilibrium with the cells. Under steady state conditions the two rates of oxygen transfer must be equal that is the rate of oxygen consumption by the organisms equals the rate of oxygen transferred into the solution. However conditions in a batch fermentation are not steady state. The number of cells is constantly increasing (at least during the growth stages) hence a' is constantly increasing. So in order to maintain a constant value of DO concentration N would have to be increased. Since C* is fixed for a particular fermentation medium and since C is to' be held at some constant value a must be raised. Calderbank (32) has shown that for gas to liquid transfer for bubbles that is dependent only on the physical properties of the gas and the liquid. Hence for a particular gas and liquid k^ is constant. So a must be varied. Various workers ( (34) (35) (36) ) have shown that a depends upon the level of agitation and the rate of air supplied to the fermenter. For example, Calderbank (33) found that: where: a = interfacial area per unit volume of dispersion P = power input to the dispersion via the agitator. V - volume of fluid. Pc = continuous phase density, cr = interfacial tension. Vs = superficial gas velocity. Vt = terminal velocity of single bubble. In equation (3) the superficial gas velocity appears directly. Thus effects of changes in. the rate of air supply on a can be esti-mated. The amount of power put into the medium depends on the size of the agitator and its speed. Hence agitator speed can be used to bring about changes in a. In the 7-1 fermentor used in some of the experiments described in this thesis, DO concentration could be controlled automatically. DO level was sensed by a DO probe and compared to a set point value. To increase (or decrease) the rate of oxygen transfer the speed of the agitator was increased (decreased) and/or the gas flow rate was increased (decreased) although the oxygen transfer rate could be con-trolled by using either agitator rpm control or gas flow rate control individually, in these experiments both modes were used simultaneously. One of the aims of this study was to see i f there was some critic a l DO concentration above which cell growth and proteins production were not affected by DO concentration and below which they were. 35 CHAPTER II  EXPERIMENTAL TECHNIQUES IX. 1 Introduction The bacterium Sorangium 495 was grown on a fish solubles based medium. (Fish solubles are discussed in Section 1.4). This section describes the way the Sorangium cultures were grown, and maintained. The preparation of the culture used to grow the bacteria is also des-cribed as well as the equipment involved. In order to follow the fermentation process, cell growth, total protein content of the medium, enzyme activity, and sometimes glucose content of the medium were measured as functions of time. Since the dissolved oxygen (DO) level was a parameter of particular interest, i t was controlled and measured using a DO probe and controller. Enzyme activity was of particular interest as well since the aim of the study was to find a proper way to produce proteolytic enzymes. II.2 Innoculum preparation Sorangium 495 was obtained from the Alberta Institute of Pedology, at the University of Alberta in Edmonton. Cultures were transplanted regularly using heart infusion agar slants. About 6 gms of brain heart infusion agar (Difco) and 4 grams of agar were dissolved in 200 ml of distilled water. This was usually enough to cover 5 culture plates and to f i l l 4 test tubes. The cultures were transferred using the 36 usual techniques (37). The cultures were then incubated at 30w C. These cultures were never let grow more than a week on the same medium. Such frequent transplants of course increase the chances for contamination, but were necessary in order to keep active organisms always available. Once, new cultures had to be obtained since i t appeared that the old cultures had lost their activity after being transplanted too many times. A loop of Sorangium in the exponential phase of growth was taken from the slants, and innoculated into 100 mis of nutrient broth. This broth was a standard one consisting of 0.5%'peptone and 0.3% beef ex-tract. 250 ml shake flasks containing 100 mis of this nutrient broth were incubated in a NBS shaker (controlled environment incubator shaker New Brunswick Scientific Co.) at a constant temperature of 30° C and shaken at about 200 rpm. The culture reached its exponential phase after about 2.3 days; then 10 mis were innoculated into 250 ml/;flasks containing 100 mis of fish solubles solutions. For the large fermentor studies, where 3 litres of fish solubles solution were used as culture medium, at least 200 mis of fast growing innoculum solution were poured in. Larger amounts of innoculum were used whenever the lag phase had to be reduced. Extra care was taken to avoid contamination since<even i f the Sorangium used here can produce antibacterial and antifungal substances (38) i t seems to be a poor competitor, and can therefore be overgrown by more rapidly growing eubacteria when both are placed in such a rich medium (39). 37 II.3 Preparation of Culture Medium The condensed fish solubles was obtained from B.C. Packers, Richmond, B.C. It is a thick, brownish, smelly liquid, at pH = 4 which has to be kept refrigerated at about 6° C to prevent spoilage. Some frozen fish solubles left by Wah-On were used in some preliminary experiments which were largely unsatisfactory. The fermentation medium was obtained by diluting a given amount of fish solubles into a fixed volume of distilled water. It was well stirred, and any solids were removed by filtration through Whatman fi l t e r paper #1 to provide a stock solution. From this concentrated solution, other dilutions were made. In some prelinuinary work, some old salmon solubles were used. These were diluted to various concentrations. Then, the solutions were filtered. This filtrate constitutes a protein medium corresponding to the appropriate amount of proteins from the original solubles volume used. The pH was adjusted from 4.0 to 7.0 by neutralization with NaOH. Further dilutions were then made. It was found that the best way to check concentration was to measure the actual protein content by the biuret method (see Section II.7). This frozen salmon solubles left from previous years turned out to be unfit for production of proteolyticly active solution, although some growth was noticed. It is quite probable that the long storage time and/or the freezing process had denaturated and/or hydrolyzed the proteins, so fresh waste was obtained from B.C. Packers. At the time, the herring season was just terminating, so fresh, unsalted, fish solubles concentrate was available. In order to make 38 any colorimetric measurements more accurate, only filtered solutions were used. Wah-On (27) found an increase in enzyme activity with addition of glucose, so glucose was added in sane cases to make the solution 1% in glucose by weight. The herring solubles was usually diluted enough to make a solution with a protein concentration equivalent to about 4 mg BSA/ml (see Section II.7). That was the optimal condition found by Wah-On (27) for protease formation rather than for maximum growth. Some tests were also done at higher i n i t i a l protein contents. Unlike Wah-On, I used distilled water in preference to tap water, even though a l l the media were later sterilized. pH adjustment of 300 mis of concentrated solubles from 4 to 7 was made using a small amount (about 20 mis) of very concentrated NaOtt. The solutions were sterilized for 20 to 30 minutes in an autoclave under a steam pressure of 15 psig at a temperature of approximately 135° C. A slight drop in the pH was noticed after sterilization of the medium. No attempt was made to readjust the pH before innoculation. II.4 Apparatus The preliminary experiments studying growth were carried out in standard 250 ml: j Erlenmeyer flasks stoppered with non absorbent cotton. Cultivation was done in an incubator shaker working at about 200 rpm, providing a controlled environment. Temperature' was fixed at 30° C; the shaking rate of the apparatus to seme extent controlled the degree of agitation and aeration. pH could not be monitored here. 39 Another series of experiments was conducted in a large 7 l i t r e fermentor (Modular Bench Top Fermentor New Brunswick Scientific Co. NBS). The pH was maintained between 6.8 and 7.2 (7.0+0.2) by a NBS automatic pH controller, and temperature was controlled at 31 + 1° C. During growth, a large amount of foam was formed due to the nature of the culture medium, and to the aeration and agitation. A diluted antifoam solution (10 mis. of antifoam B emulsion (10% silicone defoamer Dow Corning) in 500 ml water) was automically introduced by means of an automatic antifoam addition system (New Brunswick Scientific Co.). Aeration and agitation were maintained by an automatic dissolved oxygen (DO) controller. The DO could be measured during the entire growth period (and recorded on a chart paper) using an electrochemical membrane type probe' of the type described by Johnson, Borkowski and Engblom (40). This probe produces a millivolt signal which is directly proportional to the rate of oxygen diffusion through the membrane. This rate in turn is proportional to the oxygen partial pressure in the liquid in which the probe is immersed. During the experiments, a direct reading meter with a scale of 0-100% provided a continuous in-dication of the percent saturation of the liquid with oxygen. The complete regulating system contains a DO servo module with servo elements for changing the agitation speed, air flow rate or both, in order to maintain a selected DO level during growth. Samples could be taken from the fermenting culture without any risk of contamination by using a NBS saripling/innoculator device. This consists of a removal glass collecting tube and a built in air f i l t e r chamber. Inlet and outlet lines with transfer tubing, pinch clamps and 40 a sterilizable plastic syringe pump are also provided in this device for forcing liquid into and out of the fermentor. The agitation was provided i n the fermentor by a shaft with two impellers, centrally mounted and driven through the head plate. The impellers had six 3/4 x 3/4 cm stainless steel blades set 60° apart, having diameters of 5 cm, with their flat surfaces perpendicular to the bottom of the vessel. The lower one was located 7 cm from the . bottom of the fermentor. The upper, one was 20 cm above the lower one. The fermentor itself consists of a 7 l i t r e , cylindrical, glass, growth vessel, 15 cm in diameter and 45 cm high. Four stainless steel baffle plates, each 42 x 2 cm are fixed to the head plate. These baffles were placed with their surfaces perpendicular to the fermentor walls, and their lower units 3 cm above the bottom of the vessel. The baffle assembly was also used for the introduction of the air, and for the control of temperature as water circulates through them. Figure 2.6 and 2.7 adapted from Ferguson (41) is a schematic drawing of the fer-mentor described above. II.5 Measurement of bacterial growth II•5.1. Introduction A standard turbidity method is very often used to measure the level of bacterial growth (42) . However, at the end of the growth period of this organism, a variation of color occurs, and slimy material appears; this works against the use of direct turbidimetric measurements as in-dicators of growth. Dilution, or plate counts are quite precise, but 41 Fiqure 2.6 1-Z Fermentor: schematic drawing 4 baffles, 2 cm. wide on a 8 cm. sq. pH electrodes Mr inlet line Stirrers, 3/4 cm. sq. on a 3 cm. x 5 cm. diam. annulus Thermowell Sample outlet 15 cm. ID fermentor i pH electrodes Mr inlet. Baffle 42 Figure 2.7 Head Plate Penetrations U.V. or Quartz Lamp Thermistor Well-Foam Probe Water Out-Inlet Line Inlet Line" Level Probe pH Additions— and Antifoam Inlet Grounding Stud Air in pH Penetration Plugs Water In Inoculation Port Thermometer Well Air Out Sample Line Addition Line Harvest Line (Continuous Fermentor only) 7.5 LITER Exhaust Air Condenser Air Out Thermistor Well Foam Probe Water Out Inlet Line Inlet Line Level Probe pH Additions and Antifoam Inlet Grounding Stud pH Penetration Plugs Water In Inoculation Port Thermometer Well Air In Sample Line Addition Line Harvest Line (Continuous Fermentor only) / 43 too tedious to perform. So weighing of the dry cells was the method chosen to measure cel l growth. This is a direct measurement, and although i t is not totally equivalent to the cell number per unit volume according to Monod (43), i t constitutes a direct index of pro-duction of cells, and is a simple way to follow growth. Some turbidity measurements were also made to compare with the weighing method (Fig. 2.7 data from Appendix III A, Table 2.13.) II.5.2 Experimental procedure A set of four 20 ml. samples of the same broth was centrifuged (IEC International Centrifuge Universal Model UV) for 30 minutes at about 4,000 rpm. The liquid phase was kept for further analysis, whereas the solids were washed in distilled water and resuspended using a Vortex mixer. After a second centrifugation, the washed solids were collected again, and resuspended in about 10 mis. of distilled water. The four solutions were then poured into four different preweighed aluminium dishes and let dry for at least 12 hours (overnight). After that time, the dishes were weighed again, and the dry cell weights were calculated. The dry cell weight corresponding to the particular fer-mentation time is reported as the main of these four observations. The precision on the weights was quite good, since a fine precision balance was used. Table 2.14 shows an example of such measurements. Figure 2.8 shows a typical variation of weight of dry cells with fermen-tation time. 1 Table 2.13 Weight dry cells vs direct turbidity weight dry direct turbidity fermentor cells mg/ml run # Yi Xi 0.83 0.58 1 0.43 0.54 1 . 0.45 0.56 1 4.59 3.00 2 3.51 4.86 2 4.64 5.22 2 5.44 6.12 2 5.09 6.12 2 4.95 6.12 2 3.54 2.85 3 4.41 3.00 3 1.70 1.47 4 2.44 2.25 4 2.72 2.85 4 0.93 1.05 5 1.64 1.80 5 2.35 2.40 5 2.49 2.50 5 2.84 2.65 5 1.73 1.80 6 1.14 1.86 6 1.72 2.40 6 2.07 2.40 6 2.32 2.40 6 Regression line: Yi = 0.82 x + 0.38 Yi = 0.82 Xi + 0.38 r 2 = 0.84 45 Figure 2.8 Weight of dry cells vs direct turbidity o to' in a >—*' t— i — i a •—i o a a 4^* (D * A A 0 0 4 ^ + + O'o 1.0 2.0 3.0 4.0 5.0 6.0 WEIGHT DRY CELLS MG/ML 46 Table 2.14 SOME WEIGHT OF DRY CELLS Example FERMENTOR RUN # 2.5 INITIAL DISSOLVED OXYGEN = 70% SATURATION innoculation weight dry mean standard Time Hrs. cells mg/ml mg/ml deviation 2.0 0.900 0.9263 0.0225 0.915 0.945 0.945 4.0 2.2601 2.3076 0.0465 2.3701 2.3100 2.2900 8.0 2.4800 2.4963 0.0221 2.5100 2.4750 2.5200 10.0 2.1350 2.1775 0.0323 2.1700 2.2050 2,2000 47 LP* ' a in' 21 CO _ J _ J > — CC a f_D a. — i r V UJ 2* FIG. 2.9 Fermentor Run # 2.5 Dissolved Oxygen fixed at DO=20 % Saturation Initial protein concentration CQ=15.63 MG (BSA) / ML Weight Dry Cells MG/ML vs Fermentation Time (Hrs.) CD CD CD CD CD CD Q CD CD CD ^ , 1 1 1 1 1 0.0 B.O 16.0 24.0 32.0 40.0 48.0 T I M E H R S £3 48 Turbidimetric measurements (44) were made directly. The optical density of the sample (percent transmittance 915 mjj) was measured against a blank composed of the broth solution before inno-culation. The suspensions were diluted so that the measurements were in the reliable part of the scale of the spectrophotometer, i.e. between 20 and 90 percent transmittance. II.6 Enzyme activity determination  II.6.1 Introduction Solubles are rich mixtures of protein molecules, peptides and amino acids. Sorangium 495 is able to utilize the amino acids in this solution and to,produce a protease which enables i t to break down the longer chain proteins so that they too can be used. As the enzyme is a protein itself, and its structure is generally not well known, a direct determination of the amount of enzyme present during the growth is not feasible. However, since the objective of this project is to produce a protease, i t is necessary to have some method of detecting the presence of such a protease and to determined how much of i t there is. This is why the amount of enzyme present in the culture broth during the growth period is characterized by the result i t produces, that i s , by its activity. Enzyme activity can be expressed in arbitrary units, depending on the conditions of the experiments. However, i t seems reasonable to try to follow the recoitmendations of the Enzyme Commission of the Inter-national Union of Biochemistry (45): 49 "A unit of any enzyme is defined as the amount which catalyses the transformation of one micromole of substance per ntinute, or when more than one bond of a more complex substrate (e.g. protein, poly-saccharide, etc..) is attacked, one micro equivalent of the group concerned by minute under well defined conditions. The temperature should be stated, and i t is suggested that, where practical, be optimal". Here, the activity of a proteolytic enzyme, i.e. a protein hydro-lyzing enzyme, had to be measured. One cannot easily measure a micro-mole of a protein, let alone the mixed bag of protenaceous materials present in fish solubles. One way of measuring the activity of a proteolytic enzyme is Anson's colorimetric method (46). The activity of the enzyme is measured by its ability to break down long chained protein molecules into smaller fragments. The measurements of the concentration of a characteristic end product after the enzyme solution has digested the protein based substrate constitutes a standard measure-ment of the enzyme's activity. Anson measured the activity of pepsin, trypsin, papain, and cathepin using hemoglobin as a substrate. The end product chosen for measurement was tyrosine, a simple amino acid containing an aromatic ring. The presence of delocalized electrons on the benzene ring is probably responsible for the colorimetric reaction (Figure 2.10.). After removing the undigested proteins ''by precipitation with trichloro-acetic acid (TCA), a blue color is produced by the reaction of the tyrosine present in the supernatant digest with Folin phenol. „ . Figure 2.10 TYROSINE lyDLEXZULAR WEIGHT _'18l uncharged polar group J l Tyrosine (9) Tyrosine is a basic amino acid which is characterized by the presence of a benzene ring linked to a OH group, (Phenolic group) Although none of the 20 amino acids found in protein absorbs light in the visible range, tyrosine is one among three amino acids, (the two others are tryptophan and phenylalanine) which absorb significantly in the ultraviolet. 51 reagent. Conversion of the color value to enzyme activity is readily made from a standard calibration curve. Similar methods have been widely used to measure the activity of known proteolytic enzymes. For example, studies on trypsin (47), hymotrypsin (48), pepsin (49) and more generally various proteases (50) can be cited. The substance to be digested by the enzyme can be casein instead of hemoglobin. Wah-On (27) chose the method used by Petrova and Vintsyanaite (51) to measure the activity of the enzyme produced by Sorangium 495 growing on fish solubles. Considering the equipment available, and in order to be able to compare the results with Wah-On's, the same method was decided upon. II.6.2 Measurement of the enzyme activity: experimental part The measurement of the proteolytic activity was done following the method used by Petrova and Vintsyunaite (51). The reagents were: - 5% solution of trichloroacetic acid (CCl^ COOH) - 6% solution of sodium carbonate (Na2 CO^). - Folin reagent obtained in prepared from Fisher Scientific Co. N.S., U.S.A. This reagent is a 2N solution of Folin Ciocalteau reagent to be diluted five fold. - 0.025 M tris HC1 buffer Ph - 8.5 (British Drug House Ltd.) - 2% Casein solution: 2 gm of casein "Hammarsten" was suspended in about 45 ml of the 0.025 M tris buffer and was vigorously stirred with a magnetic stirrer. After dissolution of the 52 casein, the solution was made up to 100 mis. - Tyrosine solution: 10 mg of tyrosine was dissolved in 'in 5 ml of 0.1 N NaOH, transferred to a 25 ml measuring flask, and brought to the mark with water. This solution containing 400 pg of tyrosine per ml was used to prepare solutions of lower concentration containing 5 to 100 pg of tyrosine per ml. A calibration curve was made. One ml of samples of tyrosine solutions of known concentration were taken and poured into 4 ml of 6% sodium carbonate and 1 ml of five fold diluted Folin's reagent. These solutions were immediately mixed, and left at room temperature (20 to 25° C) for 30 minutes in order to let the color develop. The depth of color was determined In a photometer at wavelength 660 mp in colorimeter tubes. A calibration straight line linking the absorption at 660 mp to the tyrosine concentration was drawn through those points using a least square f i t regression method. A control was simultaneously set up for the reagents using 1 ml of water instead of the tyrosine solution, and keeping the same reagents in the same amount. (See Fig. 2.11 and Table 2.15). Enzyme activity was determined by letting 1 ml of the enzyme active solution to react with 2 ml of casein solution at a constant temperature of 30° C for 10 mn. Good mixing was assured by using a Vortex mixer; the time was accurately measured with a stop watch. When the 10 min incubation time was over, 5 ml of trichloroacetic acid were poured in to stop the reaction. At such an acid pH, the long chain proteins 53 present i n the casein solution precipitated. The solution was mixed again and l e f t for about 2 to 5 min i n the 30° C bath. Then, the white precipitate was f i l t e r e d out, and the liquid phase was retained for measurement of i t s tyrosine content. The amount of tyrosine was measured using the standard method outlined above, except that 1 ml of f i l t r a t e was used instead of 1 ml of tyrosine solution. A control was simultaneously set up by adding f i r s t 5 ml of trichloroacetic acid to the enzyme solution and then 2 ml of casein solution. This solution was mixed, f i l t e r e d , and 1 ml of the f i l t r a t e was used instead of 1 ml of d i s t i l l e d water as the blank i n the tyrosine. The enzyme unit i n this method i s defined as the amount which i n one minute forms proteolysis products which are not precipitated with trichloroacetic acid and contain one micro-equivalent of tyrosine. Therefore, the number of enzyme units per ml of the investigated solution w i l l be: E = (a x 8) / (181 x 10) = a x 0.00442 E i s the enzyme activity unit (micro-e.g. tyrosine/ml min) a number of g of tyrosine found from the calibration curve. 8 i s the dilution factor after precipitation with trichloroacetic acid. 181 i s the microgram molecular weight of tyrosine. 10 i s the proteolysis period i n minutes. Table 2.15 TYROSINE CALIBRATION concentration absorption Tyrosine jj g/ml at 660 mp Xi Yi 0.0 0.008 2.5 0.032 5.0 0.063 10.0 0.13 15.0 0.24 20.0 0.27 regression line: Yi = 0.014 Xi x 0.0002 r 2 = 0.98. 55 Figure 2.11 Calibration Curve Tyrosine Concentration V (Micro Gr/Ml) vs Absorption at 660 m Micron 8 • 0-0 4.0 B.O 12.0 16.0 20.0 TYR C0NC. MICRO GR/ML 56 II.7 Measurement of the protein content  II.7.1 Introduction Fish solubles solution has a high protein content. Sorangium uses these proteins as a nitrogen source and probably also as a carbon source. This is why the protein content was chosen as a parameter to measure in following the growth of the bacteria. Proteins in solutions such as this one are macromolecules of i l l defined molecular weight. There are various methods available for the estimation of the protein content of a solution. As tryptophan and tyrosine residues are present in protein solutions, protein content could be estimated directly by light absorption at 280 my. However, this method does not seem to be extremely specific. The protein solution could be exceptio-nally poor or rich in those amino acids, and thus make this method not reliable. (52) Turbidity could also decrease the accuracy of such measurements The main disadvantage.though, would seem to be the interference of nucleic acids which are apt to be present. These absorb more strongly at 260 mp than at 280 my. Colorimetric methods at present are widely used to determine the protein content of a solution. The color is obtained by adding Folin-Ciocalteau reagent to a protein solution. It reacts with'phenols and phenolic amino acids, to form a reduced phosphomolybdic-phosphotun-gstic acid complex which is blue in color (53). However, as i t is closely related to the presence of tyrosine and other phenolic amino-acids, this method is subject to the same objections as the direct 57 absorption method. Following Wah-On, i t was decided to use the biruet reaction method. Biruet is the simplest compound possessing in its molecular structure pairs of carbonyl groups linked through nitrogen or carbon: CONH2 NH Biruet i CONH2 When biruet is treated with cupric ions in an alkaline sodium potassium tartrate solution, two biruet molecules are joined to form a violet colored compound (52). OH OH / I CCNH„ Cu NHoC0 2 2| NH NH C0-NH--K K-NHoC0 1 2 i I OH OH Since proteins contain these pairs of linkage, they react to give the characteristic color which is directly proportional to the amount of proteins present. For most proteins, peptide bonds occur with appro-ximately the same frequency per gram of material. This is the reason why deviation are encountered less frequently than with the Folin re-agent method. The color obtained is quite dense, and i t is believed that the chromophore (light absorbing centre) is a complex between the peptide backbone, and cupric ions. 58 II.7.2 Experimental Analysis The biruet reagent was obtained commercially (British Drug House, Toronto). It is a blue colored liquid, and seems quite stable when kept at cool temperatures. It should not be used i f any black or reddish precipitates are present. As a calibration curve must be drawn relating color intensity to protein concentration, solutions of known protein concentrations were prepared. Standard solutions of Bovine Serum Albumine (BSA) were made by dissolving 0.25 gm of crystalline BSA in 25 mis of distilled water. This solution contains 10 mg/ml of BSA, and is used to prepare solutions of lower concentrations containing 1 to 10 mg BSA/ml. To 4 ml of biruet reagent, 1 ml of the solutions of known concen-tration of BSA were added and after mixing by swirling using the Vortex mixer, the solutions were let stand for 30 minutes at room temperature Table 2.16 PROTEIN CALIBRATION DATA Concentration Absorption at BSA mg/ml 540 mp Yi Xi mean 0.0 0.0 1.0 0.05 2.0 0.115 3.0 0.18 4.0 0.22 5.0 0.27 6.0 0.32 7.0 0.37 8.0 0.44 9.0 0.50 10.0 0.54 Regression analysis Y = 18.44 X - 0.04 r 2 = 0.998 Figure 2.12 CALIBRATION CURVE: TOTAL PROTEIN CONTENT MG (BSA) ML VS ABSORBANCE AT 540 nt|J PRDT BSfl MG/ML 61 (20 to 25° C) . The per cent transmission was measured using a Spectronic 20 at 540 mp against a blank consisting of 4 ml of biruet reagent plus 1 ml of water. Each measurement was made 3 times, and the mean value taken. Ten different concentrations were tested, covering the maximum reliable part of the spectrophotometer. The results are shown in Table 2.16 and Figure 2.11. Protein concentrations in the fermentation medium were expressed as equivalents of BSA. II.8 Measurement of the Glucose Concentration 11.8.1 Introduction Wah-On noted that the addition of glucose to the fermentation medium he used had a significant effect on the results. Therefore, glucose was added to the medium in the majority of the present experi-ments. However, at one time, a run was made forgetting to add glucose. The growth was surprisingly modest for a dissolved oxygen level of 80% saturation. A second run at the same DO level was made, and a much higher level of growth was observed. Thus, i t was decided to measure the consumption of glucose by the bacteria during growth. In previous work (27) the DNS method had been chosen to measure glucose so i t was used in this study too. 11.8.2 The dinitrosalicylic (DNS) method Wah-On used the DNS method used by Summer (54) to determine the amount of glucose in his samples. In this method, the sugar solution 62 is allowed to react with dinitrosalicylic acid reagent. The reagent contains sodium hydroxide, dinitrosalicylic acid, Rochelle salt, phenol and sodium bisulfite. As dissolved oxygen can destroy part of the sugar present in the sample, the presence of the Rochelle salt is necessary. Addition of sodium bisulfite helps to stabilize the color produced when the DNS is heated with glucose. This test was designed for the measure-ment of glucose in urine. The fish medium is different in chemical composition than urine, although its smell is as bad. The addition of phenol which was probably added as a preservative (54) does not seem to be necessary. A modification of the Summer method described by Snell (55) was chosen. II.8.3 Experimental Procedure A reagent containing 1% of dinitrosalicylic acid, 0.05% of sodium sulfite and 1% of sodium hydroxide was prepared. Three mis of sample was poured into 3 mis of this reagent, and left for 15 minutes at 100°C in a boiling water bath; then 1 ml of 40% Rochelle Salt solution was added. Next the sample was cooled to room temperature and the absorbance of the solution measured with the spectrophotometer at 575 m . A calibration curve was made using solutions containing between 0.00 and 1.30 mg of glucose. The absorption values at 575 m versus the different concentrations of glucose are shown in Table 2.17 and Figure 2.12. 63 Table 2.17 Glucose Calibration Data Glucose Absorption at 575 mp concentration mg/ml Xi #1 #2 #3 mean Yi 0.00 0.00 0.00 0.00 0.00 0.32 0.08 0.08 0.09 0.08 0.64 0.18 0.18 0.18 0.18 0.96 0.34 0.33 0.32 0.33 1.28 0.39 0.39 0.40 0.39 Regression line Yi = 0.32 Xi - 0.01 r 2 = 0.98 64 Figure 2.13 CALIBRATION CURVE GLUCOSE COTCENTRATLON (mg/ml) VS ABSORBANCE AT 575 m micron 0 . 4 T T GLUCOSE CONC MG/ML 1.6 1 2.0 65 CHAPTER III  RESULTS AND DISCUSSION 111.1 Introduction Preliminary experiments were made in order to develop a labo-ratory procedure for preparing fish solubles solutions at i n i t i a l concentrations of about 4 mg BSA/ml. A correlation between dilutions of filtered fish solubles and their protein content was made. Since some problems arose during the deterinination of enzyme activity, an experiment was made to check the variation of tyrosine production with enzyme digestion time using a casein solution. Salmon solubles solutions as well as herring solubles solutions at different i n i t i a l protein contents in shake flasks were innoculated with Sorangium 495. Following these tests, the 7.5 liter^fermentor was used to grow Sorangium 495 in 31 of solution of herring fish solubles at different dissolved oxygen (DO) levels. There were two distinct sets of experiments: one at high i n i t i a l protein concentrations of between 15 mg BSA/ml and 20 mg BSA/ml, and an other one at i n i t i a l protein contents of between 4 mg BSA/ml and 6 mg BSA/ml. 111.2 Determination of concentration of fish solubles III.2.1 Introduction The concentration of the culture medium plays an important role in the growth of Sorangium 495 (27). The i n i t i a l culture medium con-66 centration before innoculation has to be known as well as its con-centration during the fermentation. The major ingredient of the medium is the protein content. This was measured by the biruet reaction method discussed in Section II.7. This expresses the values of concentration in equivalent Bovine Serum Albumine ing (BSA)/ml units. An experiment was designed in order to determine approximately the amount of protein, which was soluble in 250 mis of distilled water, from 50 mis of concentrated fresh herring fish solubles obtained from B.C. Packers Richmond, B.C. This kind of solubles was mostly used in this work. The salmon solubles left by Wah-On in frozen form turned out to be unfit for the growth of Sorangium 495. 111.2.2 Experimental Procedure 50 mis of fish solubles were poured into 250 mis of distilled water and stirred for at least 5 hours at room temperature. The sus-pension was quite turbid and had a dark brown color. It was filtered through Whatman paper #4, and a clear, lighter brown solution was obtained. Its unknown protein concentration was called x in mg BSA/ml and different dilutions of x were made as indicated in Table 3.18. 111.2.3 Results The results are shown in Table 3.18 and are plotted in Figure 3.14. As expected, the protein content of the fish solubles varied linearly as a function of the dilution. This leads to the conclusion that the proteins are well distributed in the solution of solubles and that their TABLE 3.18 Herring fish solubles solution/ Protein content in equivalent mg BSA/ml function of dilution Protein content Absobance JProtein content function of at 540 mu BSA mg/ml mean mean 0.03 x 0.06 0.06 0.05 0.056 0.49 0.49 0.40 0.460 0.04 x 0.08 0.07 0.04 0.063 0.67 0.58 0.75 0.667 0.08 x 0.19 0.19 0.17 0.183 1.64 1.64 1.46 1.580 0.10 x 0.22 0.23 0.22 0.223 1.90 1.99 1.90 1.930 0.20 x 0.48 0.48 0.46 0.473 4.19 4.19 4.02 4.133 0.25 x >> 1.00 68 Figure 3.14 PROTEIN CONTENT MG (BSA)/ML vs CONCFJNTRATION OF SOLUBLES EXPRESSED AS A FUNCTION OF X. 0.0 0.04 i r 0.0B 0.12 PRDT FCT X 69 solubility is not concentration dependent over the range of interest to this work. To obtain a solution containing about 4 mg BSA/ml, 50 mis of raw fish solubles were poured into 250 mis of distilled water and after filtration (through Whatman paper #4) and pH adjustment (to 7.0), the liquid phase was diluted to 4 litres. 111.3 Prelirninary experiment: Variation of the tyrosine production  with digestion time Tyrosine concentration which is indicative of enzyme activity was determined by the method of Petrova and Vintsyunaite outlined in Section II.6. Some difficulty was experienced in the early stages of the work in obtaining reproducible values of tyrosine content. Therefore a test was carried out to study the effect of the contact time between enzyme (protease) and substate (casein) on the results. The outcome is shown in Table 3.19 and Figure 3.15. Any time greater than 10 minutes should produce maximum release of tyrosine. From the results, i t was decided to let the enzyme solution digest the casein substrate for 10 minutes for the measurement of the enzyme activity. (See Section II.6) 111.4 Shake flasks: Salmon solubles at high protein concentration A set of flasks containing salmon solubles at an i n i t i a l protein concentration equivalent to about 30 mg BSA/ml was innoculated with Sorangium 495. The salmon solubles were taken from a frozen liquid TABLE 3.19 Variation of the tyrosine production with digestion time. digestion time seconds mn absorption 660 mp mean Tyrosine cone, yg/ml mean 360 6 0.30 0.30 0.30 0.30 16.74 16.74 16.74 16.74 480 8 0.35 0.35 0.35 0.35 18.94 18.94 18.94. 18.94 600 10 0.38 0.38 0.38 0.38 20.26 20.26 20.26 20.26 720 12 0.32 0.32 0.32 0.32 18.50 18.50 18.50 18.50 900 15 0.35 0.32 0.32 0.33 18.94 18.50 18.50 18.65 1200 20 0.35 0.35 0.35 0.35 18.94 18.94 18.94 18.94 FIGURE 3.15 TYROSINE CONCENTRATION MICRO G/ML vs DIGESTION TIME (Mir) . 72 left by Wah-On. Growth was noticed (fig. 3.16) and maximum of 4.15 mg/ml of cell mass concentration was obtained after 40 hours which is higher than the 2.54 mg/ml obtained by Wah-On for a similar run after 125.5 hours innoculation. Initial c e l l production rate was quite high too, although later i t became rather inconsistent. Eventually a decrease in cell mass concentration was observed. The growth rate was not steady (see Table 3.20), and cells were not produced regularly. A plot of the weight of the dry cells produced against time (Fig. 3.16) showed an overall level of growth which was relatively important com-pared to Wah-On's results (Fig. 3.17 and Table 3.21). However, the irregularity of cell production makes these results less credible. The measurement of the protein concentration gave very erratic results, showing an increase in protein content with time which is hard to explain. It was thought that perhaps the solubles solution used had deteriorated over its long storage period. Measurements of the tyrosine levels showed that the amount of tyrosine present in the samples was not increasing regularly. As the medium was quite concentrated, there was a' large amount of tyrosine already present at the start of the fermentation. The enzyme activity was not significant enough to create a detectable increase in tyrosine content. In fact, i t seemed as i f the bacteria were feeding on the existing amino-acids, tyrosine included in solution. In some cases, the amount of tyrosine used by the bacteria seemed to be larger than any amount produced by enzyme activity which could explain why the tyrosine content of those samples was lower than that i n i t i a l l y present. 73 -©-FIGURE 3.16 • FLASKS RUN: SALMON SOLUBLES WEIGHT DRY CELLS (MG/ML) vs FERMENTATION TIME (HRS.) CD CD CD CD CD CD ° ° ° 3 0 0 4 1 : 0 T I M | ° - H D R S B 0 - ° "">•» 120-0 74 FIGURE 3.17 FLASKS RUN: SALMON SOLUBLES WEIGHT DRY CELLS (MG/ML) vs FERT'ENTATION TIME (HRS.) *FROM WAH-ON'S RESULTS tn in C3 cn" CD CO C J -_| A A CD irj A m • A A a a A . 0.0 20.0 -1 1 40.0 60.0 TIME HRS 80.0 ~1 100.0 120.0 'TABLE 3.20 Shake Flasks: Salmon Solubles at High Protein Concentration innoculation time T hrs. weight dry cells X mg/ml Cells production AX mg/ml AT ' hrs. Growth rate AX AT Total Proteins P mg BSA/ml -AP mg BSA/ml Tyrosine cone, g/ml 5 0.00 2.48 5 0.50 31.30 0.00 56.40 15 2.48 0.19 10 0.02 31.30 3.68 77.56 20 2.67 -0.95 5 -0.19 27.62 -3.68 85.49 25 1.72 -0.07 4 -0.02 31.30 0.00 51.55 29 1.65 1.25 6 0.21 31.30 -3.69 56.40 35 2.90 1.25 5 0.25 34.99 0.74 102.68 40 4.15 -2.10 10 -0.21 34.25 -3.31 89.90 50 2.05 0.22 10 0.02 43.20 -3.31 95.18 60 2.27 -0.10 25 -0.004 46.51 17.83 56.40 85 2.17 0.13 5 0.03 38.68 -5.53 74.03 90 2.30 44.21 71.09 TABLE 3.21 .Shake Flasks: Salmon Solubles: Wah-On's Results innoculation weight dry cells cell production time Growth rate Proteolytic activity time x AX AT AX hrs. mg/ml mg/ml hrs. AT unit/10 cc 0.00 0.00 -0.06 7.00 0.01 7.00 0.06 -0.88 12.00 0.07 19.00 0.94 0.146 0.12 11.00 0.01 30.00 1.06 0.199 0.28 13.00 0.02 43.00 1.34 0.345 0.30 12.00 0.03 55.00 1.64 0.345 0.16 12.00 0.01 67.00 1.80 0.663 0.08 12.25 0.01 79.25 1.88 0.875 0.30 11.00 0.03 90.25 2.18 0.862 0.10 12.00 0.01 102.25 2.28 0.924 0.24 12.50 0.02 114.75 2.52 -0.02 10.75 0.001 125.50 2.54 1.286 0.00 12.50 0.00 138.00 2.54 1.255 -0.12 12.00 -0.01 150.00 2.42 1.295 maximum yield Time to max Xmax max. cell Xmax Tmax Tmax production mg/ml hrs. Xmax This work 4.15 40 0.10 2.48 Wah-On's 2.54 125.5 0.02 0.88 77 This happened after 25 hours as well as after 29 hours and 60 hours (Table 3.20). It was concluded from those results that the enzyme activity produced was negligible. No new salmon solution was available, so as fresh herring fish solubles could be easily obtained, i t was decided to conduct further experiments using this,medium. Basically, the nature of the protein molecules in this medium would be similar to the salmon solubles. III.5 Shake Flasks Runs: Herring Solubles at different Protein  Concentrations 111.5.1 Introduction Several sets of flasks containing 100 mis of herring solubles of different i n i t i a l protein concentrations were innoculated with Soran-^ gium 495. The different i n i t i a l protein concentrations of the solutions were 3.65 mg BSA/ml, 4.45 mg BSA/ml, 5.59 mg BSA/ml, 17.48 mg BSA/ml and 20.00 mg BSA/ml. The experiments were designed so that both high and low i n i t i a l protein content solutions could be tested. Weight of dry cells, enzyme activity and total protein content were measured. The results obtained were compared with Wan-On's and are discussed in Section III.5.7. 111.5.2 Shake Flasks: Herring Solubles at i n i t i a l Protein Concentra- tion of 3.65 mg BSA/ml. Ten samples were taken over a period of 125 hours of fermentation 78 time. The results are shown in Appendix I.2.a. A high level of cell mass concentration was observed (Fig. 3.18) reaching a maximum of 4.82 mg/ml after 36 hours. However, the cell population started to decline after 48 hours of fermentation time. This was probably due to a lack of nutrients after such a long fer-mentation process, the main nutrients having been already consumed by the bacteria. Measurement of the protein content (Fig. 3.19) confirmed this. Enzyme activity was detectable. Activity i n i t i a l l y rose, reaching a maximum at around 30 hours then drecreased. The maximum obtained was 0.44 unit/10 cc at that time (Figure 3.20). The enzyme activity followed the same pattern as the cell growth although maximum activity was noted prior to maximum growth. III.5.3 Shake Flasks: Herring Solubles at i n i t i a l Protein Concen- tration of 4.45 mg BSA/ml. Eleven samples were taken over a period of 81 hours of fermentation time. The results are shown in Appendix I.2.b. Here too, a large degree of growth was observed with cell production reaching 2.82 mg/ml after 25.5 hours of fermentation time. Cell popu-lation declined after 30 hours of fermentation (Figure 3.21). Measurement of the protein content showed that after a rapid consumption of the proteins by the organism, the amount of protein present in solution increased slightly. This is hard to explain (Figure 3.22) . 79 Enzyme activity was found to increase slowly during the growth period. /After 30 hours fermentation, i t reached its inaximum level of 0.77 units/10 cc (Figure 3.23). 111.5.4 Shake Flasks: Herring Solubles at i n i t i a l Protein Concen- tration of 5.59 mg BSA/ml Eleven samples were taken over a period of 109 hours of fermentation time. The results are shown in Appendix I.2.c. Growth occurred with cell yields reaching 4.21 mg/ml after 53 hours of fermentation. After this time, cell population started to decrease (Figure 3.240. Protein molecules were slowly consumed (Figure 3.25). In this experiment however, the measurement of tyrosine content during growth gave no indication of enzyme activity (Appendix I.2.c) since the values did not increase with time. 111.5.5 Shake Flasks: Herring at i n i t i a l Protein Concentration of 17.48 mg BSA/ml Eleven samples were taken over a period of 28 hours of fermentation time. Results are shown in Appendix I.2.d. Growth was observed. After 28 hours of fermentation time, a yield of 3.66 mg/ml of dry cells was obtained. However, the observations were then terminated so maximum yield may not have been reached. The growth was slow and regular (Figure 3.26). The protein concentration decreased as expected, but not regularly, 80 FIGURE 3.18 FLASKS RUN # F.l HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN . CONCENTRATIONS .65 MG (BSA) /ML WEIGHT DRY CELLS (MG/ML) V S FERMENTATION TIME (HRS)r. CD CD CD 0 CD CD CD r 1 1 r 0.0 26.667 53.333 - r j ^ 0 ^ l D 6 ' 6 7 1 3 3 - 3 3 i 6 0 - 0 81 FIGURE 3.19 FLASKS RUN # F.l HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATION3.65 MG (BSA)/ML TOTAL PROTEIN CONTENT(MG (BSA)/ML)vs FERMENTATION TIME(HRS.) X X X x x w X x X o n I 1 1 1 1 1 0.0 2B.667 53.333 80.0 106.67 133.33 160.0 TIME HRS 82 FIGURE 3.20 FLASKS RUN # F.l HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN (XNCENTRATION=3.65 MG (BSA) /ML ENZYME ACTIVITY(UNIT/10 CC)vs FERMENTATION TIME (HRS.) o <!> O 1 1 1 1 1 1 0.0 26.667 53.333 80.0 106.67 133.33 160.0 TIME HRS 83 FIGURE 3.21 FLASKS RUN # F.2 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CCNCT3SITRATION=4.45 MG(BSA)/ML WEIGHT DRY OILS (MG/ML )vs FF^ MENTATION TIMEl(HRS.) CD CD CD CD CD CD CD CD CD o.o - i 1 1 1 1 1 26.667 53.333 60.0 106.67 133.33 160.0 TIME HRS 84 FIGURE 3.22 FLASKS RUN # F. 2 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATIONS.45 MG(BSA) /ML TOTAL PROTEIN CONTENT (MG (BSA)/ML)Vs FERMENTATION TIME(HRS.) a a — i ID LO C O R C Q a _ I— O cn a. cc X X X X X X X X X a 0.0 26.667 53.333 80.0 106.67 133.33 160.0 TIME HRS FIGURE 3.23 FLASKS RUN # F. 2 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATION=4.45 MG (BSA)/ML ENZYME ACTIVITY(UNIT/10 CC)vs FERMENTATION TIME(HRS.) 09 <!> c_> cc <!> <!> 0.0 26.667 53.333 TIME HRS 80.0 106.67 86 LD I D z : CO ro >— CC a ' 1-4 22 03 FIGURE 3.24 FLASKS RUN # F.3 FLASKS SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATI0N=5.59 MG (BSA)/ML WEIGHT DRY CELLS'(MG/ML)vs FERMENTATION TIME (HR.) CD 0 CD 0 0 O 0 0 O 5^ 1 1 1 1 — i 1 0 0 2B.667 53.333 80.0 106.67 133.33 160.0 TIME HRS 87 FIGURE 3.25 FLASKS RUN # F. 3 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATION=5.59 MG (BSA) /ML TOTAL PROTEIN CONTENT(MG (BSA)/ML)vs FERMENTATION TIME(HRS.) x x x X x X x x X X O.D 26.667 53.333 80.0 106.67 133.33 160.0 TIME HRS 88 and two steps were observed (Figure 3.27). This could result from i n i t i a l utilization of the short chain protein material in the solubles followed by generation of an extracellular protease capable of attacking the longer chain proteins and making them available as a substrate for the organism. The graphical representation of the measured enzyme activity during the fermentation process was not very informative (Figure 3.28). The concentration of tyrosine decreased or did not increase substantially, This seemed to indicate that the bug was using more tyrosine than the amount of tyrosine produced by the enzymes. Hence the activity of the enzymes i f any, was low. 111.5.6 Shake Flasks: Herring Solubles at i n i t i a l Concentration of  20 mg BSA/ml Twelve samples were taken over a period of 150 hours of fermentation time. Results are showed in Appendix I.2.e. Growth was slow (Figure 3.29) and i t took 85.5 hours to produce a maximum of 2.66 mg/ml cells. The protein was consumed quite slowly (see Figure 3.30). The 2 stages of protein consumption noted in ~ Figure 3.27 were not seen. No enzyme activity could be observed. 111.5.7 Discussion The f i r s t three experiments used medium with low i n i t i a l protein concentrations between 3 mg BSA/ml and 6 mg BSA/ml. The growth patterns-obtained were consistent. At the beginning 89 FIGURE 3.26 FLASKS RUN # F.4 HERRING SOLUBLES 1% GLUCOSE ADDED INTIAL PROTEIN CONCTHTRATI0N=17.48 MG (BSA)/ML WEIGHT DRY CELLS(MG/ML)vs FERMENTATION TIME(HRS.) (_) • J >— CC a 0.0 CD CD "1 1 1 26.667 53.333 80.0 TIME HRS 106.67 133.33 160.0 90 FIGURE 3.27 FLASKS RUN # F.4 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATIONS7.48 MG (BSA)/ML TOTAL PROTEIN CONTENT(MG (BSA)/ML)vs FERMENTATION TIME(HRS.) CM U 3 \u to /N U 3 - J 8 I ~K CQo. O CC CL IU3 Q - U D I—(D O m cn m cn' o i 1 1 1 1 r 0.0 26.667 53.333 80.0 106.67 133.33 160.0 TIME HRS 919 FIGURE 3.28 FLASKS RUN # F.4 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATION=17.48 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 CC)vs FF,RMENTATION TIME (HRS.) 4> <s> <!> I I 1 1 1 1 0.0 26.667 53.333 80.0 106.67 133.33 160 0 TIME HRS 92 FIGURE 3.29 FLASKS RUN # F.5 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL "PROTEIN CONCENTRATIONS MG (BSA) /ML WEIGHT DRY CELLS(MG/ML)vs FERMENTATION TIME(HRS.) 1 = $ 21 <n CD CO 01 CD >— CC a O 0 U J 23 0 0 O 0 0 0 0 o 0 a a 0.0 1 I 1 26.667 53.333 80.0 TIME HRS 106.67 "I 133.33 160.0 93 FIGURE 3.30 FLASKS RUN # F.5 HERRING SOLUBLES 1% GLUCOSE ADDED INITIAL PROTEIN CONCENTRATIONS MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA)/ML)vs FFJRMENTATION TIME (HRS,.) °~| X xX XX x r- I X U 3 U3 U3-J X X cn ,rn —!cn \.cn_| CE 03 o . 63 CC CL r— _ J t o fc-to" cn cn tn tn X X o~i 1 1 1 1 r 0.0 26.667 53.333 80.0 106.67 133.33 160.0 TIME HRS TABLE 3.22 Flasks Runs: Results for this work and Wah-On's in i t i a l Protein Maximum Time to Maximum enzyme concentration yield max activity X max Co X max T max T max E max mg BSA/ml mg/ml hrs. mg/ml hr. unit/10 cc 3.65 4.45 5.59 17.48 20.00 4.87 2.82 4.21 3.47 2.66 36 21 53 25 85.5 0.14 0.13 0.08 0.14 0.04 0.44 0.77 0.25 3 low i n i t i a l protein content 2.52 125.5 0.02 12.0 w -P r H 2:100 v/v g u W Si high i n i t i a l „ • _ n n n 9 protein content 2 ' 5 4 1 2 5 " 5 °- 0 2 !- 2 9 £ 6:100 v/v 95 rapid growth occurred. Then the cell population s t i l l increased, but the rate of production of cells decreased. Finally the cell population itself decreased corresponding to the death of bacteria. This was thought to be due to the lack of nutrient. As shown in Table 3.21 the maximum cell yield obtained (4.87 mg/ml) was for the series at i n i t i a l protein concentration of 3.65 mg BSA/ml. This maximum cell yield is higher than the maximum of 2.52 mg/ml obtained by Wah-On for similar runs (Table 3.22). This maximum was even above those obtained during fermentation at the higher i n i t i a l protein con-tents (20 mg BSA/ml and 17.48 mg BSA/ml). However, during those last two experiments, no rapid decline in the cell population occurred. This was expected since at high i n i t i a l protein concentrations, the nutrients were more available and hence the cell population having more nutrients could survive longer. For a. medium of higher i n i t i a l protein content, Wah-On found a smaller maximum yield of 2.54 gm/ml compared to the maximum yield of 3.47 gm/ml obtained for the medium of i n i t i a l protein content of 17.48 mg BSA/ml. Decline of cell population at the end of the fermentation process was also noted by Wah-On. Growth rates were calculated (Appendix 1.2) as well as specific growth rates but no relationship between i n i t i a l protein concentration and growth rate is apparent. A l l the maximum growth rates occurred at the beginning of the exponential growth phase and their values are a l l around 0.30 mg/ml per hr. Usually, proteins were slowly and regularly consumed as expected, although inconsistent results were obtained for the run starting at an in i t i a l protein content of 20 mg BSA/ml. This was probably because 96 this rich medium contained so much amino acid and low molecular weight peptide that the culture could grow on those without having to use the large protein molecules. This could also explain why Wah-On found that growth at the high protein concentration was as good as, or better than, that obtained at the optimum concentration for protease formation. This utilization of short chained easily digestable material could also explain the two steps observed during the protein consumption measurements for the medium of i n i t i a l protein concentration of 17.48 mg BSA/ml (Figure 3.31). Enzyme activity was found for the f i r s t two runs, but in the other cases, the tyrosine measurements were not very informative. It seemed that high concentration media were not suited for enzyme production. This could be explained by noting that i f the bacteria could consume relatively more short chain proteins which are present in rich protein medium, i t would not need to produce enzymes to break down the larger protein molecules into easily digestable short chained peptides. These results agreed with Wah-On's. However, the enzyme activity found was usually lower than the values obtained by Wah-On as showed in Table 3.19. Wah-On could obtain an activity of 12.0 unit/10 cc whereas the maximum activity found was 0.77. Although some activity was obtained for the run corresponding to an i n i t i a l protein concentration of 17.48 mg BSA/ml, the values were not very high. Only the f i r s t two runs at 3.65 and 4.45 mg BSA/ml gave regular production of enzyme, which followed a pattern compatible with the growth obtained and the protein consumption. The maximum levels were 0.77 unit/10 cc and 0.44 unit/10 cc respectively. Those values are 97 FIGURE 3.31= FLASKS RUN # F.4 HERRING SOLUBLES AT INITIAL PROTEIN CONCENTRATION CQ=17.48 MG (BSA) /ML TOTAL PROTEIN CONTENT(MG(BSA)/ML)vs FERMENTATION TIME (HRS.) (1% GLUCOSE ADDED) 98 well below the 12.0 unit/10 cc found by Wah-On. Ill.5.8 Conclusions These shake flasks experiments showed that Sorangium 495 could grow on a herring solubles medium. This was not entirely unexpected since Wah-On (27) was able to grow this organism on salmon solubles. These tests also demonstrate that the data obtained frequently behaved in a very erratic fashion leading to the conclusion that kinetic analysis of the data which involves graphical differentiation would be difficult i f not impossible. The results were disappointing in terms of production of enzyme activity. Measured activities were low and sometimes were not even detectable. Best results in terms of this activity were obtained at low levels of i n i t i a l protein concentration. At higher levels i t is postulated that there is sufficient amino acid and peptide content in the medium that the organism is not required to produce an extracellular protease to provide itself with a suitable amount of nutrient. III.6 l-l Fermentor Runs Initial protein concentration around 4 mg (BSA)/ml. III.6.1 Introduction Five runs were made in the l-l fermentor using 3 £ of medium Table 3.23 outlines the process conditions used. In addition to these, a fermentation was done at 80% DO saturation without glucose. The failure to add glucose was an error, but the results were in-cluded so a comparison could be made between the two runs at 80% DO saturation with and without glucose. TABLE 3.23 Process conditions of the experiments Dissolved oxygen Initial Protein Number of Duration of . level concentration samples Fermentation analyzed % Saturation mg BSA/ml hrs. 0 5.74 9 61 30 5.49 15 134 50 4.88 12 95 80 5.31 11 93 100 4.14 12 96 80 * 4.08 13 106 Without glucose 100 The weight of dry cells, protein content and enzyme activity were measured for each run. In addition, the glucose content was determined. III.6.2 Results and discussion The results of the fermentation runs are plotted in Figure 3.32 to Figure 3.57. Tabulation of these data appears in Appendix II. III.6.2.a Cell yield Figure 3.32 to 3.37 are plots of cell mass concentration against fermentation time with DO as a parameter. The obvious and immediate conclusion i s that Sorangium 495 is an aeorbic organism because at 0% DO, there is very l i t t l e growth (Figure 3.37) whereas at 30% DO or greater level of DO a greater growth is observed. Figure 3.37 shows the variation of the weight of dry cells produced with fermentation time at DO=0% saturation. The amount of cells produced is quite low compared to the other runs, and a maximum of 0.42 mg/ml is obtained after 38 hours of fermentation. The maximum values obtained at higher DO levels can reach 3 mg/ml (see Figures 3.32 and 3.36). The second conclusion is that growth seems to require glucose, because the fermentation at 80% DO with no glucose resulted in more or less the same amount of cell mass as the fermentation at 0% DO (Figure 3.34). 101 o cn cn rn cn' to .to FIGURE #3.32 FERMENTOR RUN #1.1 DISSOLVED OXYGEN FIXED AT DO=100% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4.14 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FERMENTATION TIME (HRS.) CD CD 0 0 O CD a C D ™ 2 E CO I u j a I CD CN >— CC a 5*1 I CD LU CO CD , 1 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 102 FIGURE #3.33 FERMENTOR RUN #1.2 DISSOLVED OXYGEN FIXED AT DO=80% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.31 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FFJ3MENTATION TIME (HRS.) O ° 0 ° 0 0 ; ^ 1 1 1 1 — — 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 103 FIGURE #3.34 FFJWENTOR RUN #1.3 DISSOLVED OXYGEN FIXED AT DO=80% SATURATION (NO GLUCOSE) INITIAL PROTEIN CONCENTRATION CQ=4.08 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FERMENTATION TIME (HRS.) ° o ° 0 ° o o ° m m ° © o $ 1 — — - — I 1 1 , , °-0 20.0 40.0 y j ^ ' ° H p 5 8°'° 1 0 0 ' ° i 2 0 ' ° 104 FIGURE #3.35 FFJRMENTOR RUN #1.4 DISSOLVED OXYGEN FIXED AT DO=50% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4.88 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FERMENTATION TIME (HRS.) CD CD Q ° ° m 0 CD CD CD U 0 0 1 1 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 105 FIGURE #3.36 FERMENTOR RUN #1.5 DISSOLVED OXYGEN FIXED AT DO=30% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.49 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FERMENTATION TIME (HRS.) o © © o u © © © © © © 1 1 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 106 FIGURE #3.37 FERMENTOR RUN #1.6 DISSOLVED OXYGEN FIXED AT DC=0% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.74 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) V S FERMENTATION TIME (HRS.) on rn rn oi" it-=iuo 21 to CM CD CO C J •. CM >— a C D 0 ! •—i —•" L U CD to 0.0 CD CD CD CD CD CD CD 20.0 40.0 60.0 80.0 TIME HRS 100.0 120.0 107 The data also show that cell lysis seems to require a reasonably high level of dissolved oxygen because at 30% DO or greater, the cell mass concentration rises to a innaximum and then drops off. This could not be observed for the 0% DO run. There is no consistent pattern to the level of cell mass con-centration as a function of DO level for DO levels above 30%. So there must be some critical DO levels between 0% DO and 30% DO above which the growth of the organism in terms of cell mass concentration is independent of DO level. Studies have been made to determine the critical values of oxygen concentration below which various organisms cannot survive (35). It was noted that these values were about the same for many different microorganisms (between 2% and 10% DO). However, many of the numbers cited (35) were approximate because of the difficulty in measuring accurately such low concentrations of oxygen (56). In this work, the oxygen control for DO levels below 20% DO was very difficult to perform. For example, a run was tried at 10% DO. To keep the DO level at 10% DO, the control system allowed air to en-ter the fermentor and the agitation rate increased. This caused the DO level to increase sharply, and although small quantity of air was provided, this was sufficient to reach a DO level above 10% DO saturation. Figuref-3.38a shows a typical part of the chart of the measurements of the DO during this run. Under such conditions, i t was thought that no measurement was worth being taken. DO control was more stable at 30% DO (Figure 3.39a)although same difficulties 108 were encountered during the exponential growth phase. At higher DO levels, a relatively good control could be obtained (Figure 3.40a). Calculation of growth rates (Appendix II) show for a l l 'runs that the cell production was highest near the beginning of the fermentation process, and then the rate of production started to decline. Subsequently the cell population decreased resulting in negative values of growth rate. In Table 3.24.a rnaximum cell yields (X mg/ml) obtained at m different DO levels and the time T.. i t took to reach them are re-ported. These values are plotted in Figure 3.38. The average cell Xm yields (— mg/ml x hr) could then be calculated and plotted versus the DO levels for each run (Figure 3.39). No definite pattern linking maximum or average cel l production to DO level is apparent which i s consistent with the previous results obtained from the global growth curves. The maximum growth rates are recorded too in this table and plotted in Figure 3.40. An i n i t i a l growth rate was observed for the run at DO=0%, but this has no consequence since the growth stopped very rapidly without oxygen. Higher maximum growth rates were ob-served at medium DO levels (around 50% saturation) and then decreases when oxygen was more abundant. The time i t took to obtain maximum cell production was shorter than at other DO levels (13 hrs at 50% DO whereas the T for a l l other runs were between 30 and 50 hrs.) However, m this did not correspond to a higher amount of cell produced. This suggests that above the criti c a l DO level, the vitality of the culture broth is greater at mid DO but that the overall cell production cannot 109 Figure 3.38a Dissolved Oxygen Measurement Chart. Run at DO=10% Saturation DO levels: 110 Figure 3.39a Dissolved Oxygen Measurement Chart Run at DO=30% Saturation DO levels Run at DO=30% Saturation Perturbation during the Exponential Growth Phase. DO levels I l l Figure 3.40a Dissolved Oxygen Measurement Chart Run at DO=50% Saturation Run at DO=80% Saturation 112 cn m"' r-L U - . " C J X 5 1 ui Figure # 3.38 FFJ3MENT0R RUNS AT LOW INITIAL PROTEIN CONCENTRATION. X(M) MAXIMUM CELL PRODUCTION (MG/ML) vs DO (% SATURATION) CP a a CD 0 O CD 1 1 1 1— 1 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION 113 Figure #3.39 FERMENTOR RUNS AT LOW LNTIAL PROTEIN CONCENTRATION AVERAGE CELL PRODUCTION (MG/ML) vs DO (% SATURATION). 114 Figure #3.40 FERMENTOR RUNS AT LOW INTIAL PROTEIN CONCENTRATION. MAXIMUM GROWTH RATE (MG/ML x HR) vs DO (% SATURATION). 22 8 CN CD 21 L U CE • J Ocn Ccoo CD~t a" X CC 21 ID ^ a a a <!> 1 1 r 1 1 — 0.0 20.0 4TJ.0 60.0 80.0 100.0 DO % SATURATION be influenced by oxygen. III.6.2.b Protein Consumption Figures 3.41 to 3.96 are plots of protein concentration versus fermentation time. They show that the rate of protein depletion is low at 0% DO, which is expected since no noticable growth was ob-served, but rapid at DO levels above 30% saturation. Mthough Figure 3.34 shows l i t t l e growth when no glucose was present at DO = 80% saturation, Figure 3.43 shows that the most complete removal of protein occured under these conditions. Since no enzyme activity was detected such results are quite surprising. It seems that without glucose in solution, the organism is unable to utilize the proteins, even short chain peptides or amino acid for cell pro-duction, i.e. there is no growth but protein disappears. There appears to be~ no consistent pattern to the protein removal at different DO levels. At DO = 100% saturation and es-pecially at DO = 50% saturation, protein removal is quite complete. However, at the intermediary level of 80% saturation this is not the case. The protein removal is not complete either at DO = 30% saturation. No noticable change in the maximum protein consumptions recorded in Table 3.24.b was observed (Figure 3.47). However, cal-Pm culation of the average protein consumption (-rg^  ^ 9 BSA/ml x hr) with high levels at DO = 50% and DO = 100% saturation, but lower values at DO = 80% and DO = 30% saturation confirm the previous result (Figure 3.48). It was thought that high cell yield would correspond Table 3.24.a Maximum Cell Yields Xm AX ._ . _ APm A Gm D ° Tm AT A P m A G m ATm" &Tm Max Cell Time to Average Cell Max. Growth Prot. Cons. Glucose Cons. Average Average Yield max. Yield rate at Tm at Tm Prot. Cons. Glucose Cons, % Saturation mg/ml hr. mg/ml x hr mg/ml x hr mg BSA/ml mg/ml mg BSA/ml x hr mg/ml x hr 0 0.42 38.0 0.011 0.087 30 3.23 33.0 0.098 0.285 50 2.44 13.0 0.188 0.333 80 . 2.09 50.0 0.042 0.192 100 3.16 24.0 0.132 0.183 80* 0.71 22.25 0.032 0.115 0.980 0.170 0.026 0.004 3.500 - 0.106 3.900 3.870 0.300 0.298 3.440 5.524 0.069 0.110 2.890 2.540 0.120 0.106 3.380 - 0.152 * Without Glucose 'Table 3.24.b Maximum Protein and Glucose Consumption APm APm AGm ATg AGm DO Tp ATp ATg max Prot. Time to Average Max Glucose Time to Average Glucose Consumption max Prot. Cons. Consumption max Consumption % Saturation mg BSA/ml hr mg BSA/ml x hr mg/ml hr mg/ml x hr 0 2.09 61.00 0.034 0.172 14.00 0.012 30 4.18 115.00 0.036 - - -50 4.18 95.00 0.044 4.330 56.00 0.077 80 3.81 93.00 0.041 5.765 81.25 0.071 100 3.31 70.50 0.047 5.380 95.50 0.056 80* 3.75 55.25 0.068 - - -* Without Glucose 118 Figure #3.41 FFIWENTOR RUN #1.1 DISSOLVED OXYGEN FIXED AT DO=100% SATURATION, INITIAL PROTEIN CONCENTRATION CQ=4.14 MG (BSA)/ML TOTAL PROTEIN CONTENT; (MG (BSA)/ML) vs FERMENTATION TIME (HRS.) o to' in 21a & CD CL CO a c o -rn O cn CL o a a A A £ A A A A A A 1 1 1 1 1 1 o n 20 0 40.0 60.0 80.0 100.0 120.0 TIME HRS 119 Figure #3.42 FERMENTOR RUN #1.2 DISSOLVED OXYGEN FIXED AT DO=80% SATURATION INITIAL PROTEIN CONCENTRATION C0=5.31 MG (BSA)/ML TOTAL PROTEIN CONTENT MG (BSA)/ML vs FERMENTATION TIME (HRS.) o to" o in' 21 a CO •. cn A or: °- I A A a h 6 N ' A a o 1 1 1 1 1 I 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 120 Figure #3.43 FERMENTOR RUN #1.3 DISSOLVED OXYGEN FIXED AT DO=80*% SATURATION : (* NO GLUCOSE) INITIAL PROTEIN CONCENTRATION CQ=4.08 MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA)/ML) vs FEPMENTATION TIME (HRS.) i n az r — o A A A A £ A A a 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 121 Figure #3.44 FEFjyENTOR RUN #1.4 DISSOLVED OXYGEN FIXED AT DO=50% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4.88 MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA)/ML vs FERMENTATION TIME (HRS.) o to' o IT)' O CD CC CQ m r — 0_ CC°. fcrrvr a a A . A A ^ A . A A , 1 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 T I M E rIRS 122 Figure #3.45 FERMENTOR RUN #1.5 DISSOLVED OXYGEN FIXED AT DO=30% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.49 MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA)/ML) vs FERMENTATION TIME (HRS.) A A A A A A A A A , — i 1 1 — l 1 0 0 20.0 40.0 60.0 80.0 100.0 120.0 T I M E H R S 123 Figure #3.46 FERMENTOR RUN #1.6 DISSOLVED OXYGEN FIXED AT DO=0% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.74 MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA)/ML) V S FERMENTATION TIME (HRS.) to o in' a CD CE co r — O Ql Q_ CD a a A A A A A A A 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 124 Figure #3.47 FEPMFJNTOR RUNS AT LOW INITIAL PROTEIN CONCENTRATION MAXIMUM PROTEIN CONSUMPTION (MG BSA/ML) vs DO (% SATURATION) o in' co CD O CD i CD 1 CD 21 a turn * CD o X <x 21 S3 a o i 1 1 1 1 r 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION Figure #3.48 FEPJyENTOR RUNS AT LOW INITIAL PROTEIN CONCENTRATION. AVERAGE PROTEIN CONSUMPTION (MG BSA/ML) vs DO (% SATURATION). crft A 4> 1 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION to a faster and more complete removal of proteins. This happens most of the time, and is well illustrated for the runs at DO = 100% and DO = 50% saturation. However, this is not the case for the run made at DO = 30% saturation. There, cell growth is noticed although the protein removal is not complete. However, the cell population starts to decline shortly after the maximum cell production is obtained. This could explain why a complete protein removal is not needed in order to obtain cell growth: the amino acids obtained from a partial protein removal are sufficient to substain some cell growth, but not to keep the population alive later on. This is repeated for the run at DO = 80% saturation which makes any attempt to correlate protein consumption and/or growth to the DO levels above the critical value impossible. For the run made at DO = 0% saturation, the results obtained show clearly that no growth was obtained and no protein was consumed. Because the i n i t i a l protein concentrations were not precisely the same for the different runs, graphs were made to see i f any effect of DO levels could be observed when the protein consumption was con-sidered in terms of % of i n i t i a l protein concentration versus fer-mentation time. This however did not reveal anything different from the information already given by Figures 3.41 to 3.46. III.6.2.c Enzyme Activity Figure 3.49 to 3.53, are plots of enzyme activity as a function of fermentation time. 127 Figure #3.49 FERMENTOR RUN #1.1 DISSOLVED OXYGEN FIXED AT DO=100% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4.14 MG (BSA) / ML ENZYME ACTIVITY (UNIT/10 cc) vs FF^ EMENTATIQN TIME (HRS.) + + + + + + + + + 0.0 ~1— 20.0 -I 1 40.0 60.0 TIME HRS 80.0 _ J 100.0 120.0 128 Figure #3.50 FERMENTOR RUN #1.2 DISSOLVED OXYGEN FIXED AT DO=80% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.31 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) vs FERMENTATION TIME (HRS.) i n CJ O t>i' ' i n CJ CC UJ + in + + a a" 0.0 + + 20.0 4- + + + + 40.0 60.0 80.0 TIME HRS ~1 100.0 -I 120.0 129 Figure #3.51 FERMENTOR RUN #1.4 DISSOLVED OXYGEN FIXED AT DO=50% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4.88 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) vs FERMENTATION TIME (HRS.) + + + + + + + + + + - i I 1 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 130 Figure #3.52 FERMENTOR RUN #1.5 DISSOLVED OXYGEN FIXED AT DO=30% SATURATION INITIAL PROTEIN CONCENTRATION C0=5.49 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) vs FERMENTATION TIME (HRS.) + + + + • + + + + + + + , 1 1 I I I 0 0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 131 Figure #3.53 FFiWLENTOR RUN #1.6 DISSOLVED OXYGEN FIXED AT DO=0% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.74 MG (BSA)/ML ENZYME ACTIVITY (UNIT/lOcc) V S FFJ3MENTATTGN TIME (HRS.) + + + + + + I 1 1 H 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 132 These shew a significant effect of DO level on enzyme activity. Although the data are quite scattered, there does seem to be an overall pattern observable. This indicates that as DO levels in-crease, so does the maximum enzyme activity (Table 3.24.c). It also appears that the activity rises to a peak and then declines. For the run at DO = 80% saturation without glucose, the measurements of tyrosine concentrations did not indicate any noticable enzyme activity. Compared to Wah-On's results, the levels of activity observed are quite low (see Table 1.12 and Appendix II.). Maximum activities were about 2.5 unit/10 cc compared to Wah-On's maximum of 10.52 unit/ 10 cc. This was quite unexpected since larger cell yields are ob-served, with a maximum cell formation of 3.19 mg/ml compared to Wah-On's maximum of 2.05 mg/ml. Wah-On observed a decline of enzyme activity at the end of his run as well. This is believed to be linked to the cell population decline. As activity increases with higher DO levels, this suggests that oxygen is needed to produce enzyme. It does not appear that under insufficient DO conditions, the organism under such stress increases its enzyme activity to improve growth: not only is growth level low, but acitivity is low too. It may be that the proteinaceous material present in the herring solubles is more easily assimilable by the organism than that in the salmon solubles used by Wah-On. Thus the organism has less incentive to produce an extracellular protease. 'Table 3.24:d Maximum Enzyme Activity DO Em Te ;-M AG^ &P 7jr~ E E E Max. Enz. Time to Average Glucose used Protein used Activity max Enz. Act. at T„ at T E E % Saturation unit/10 cc hr unit/10 cc x hr mg / ml, mg BSA/ml 0 0.31 43.00 30 0.53, 13.00 50 1.11 23.00 80 0.83 81.25 100 2.47 47.50 0.007 0.160 0.730 0.041 - 2.900 0.048 3.790 3.990 0.010 5.765 3.380 0.052 4.620 3.130 134 III.6.2.d Glucose Consumption Figures 3.54 to 3.57 are plots of glucose concentration versus fermentation time. These shew that glucose consumption is low with 0% DO but is higher at DO levels above 30%. There is no apparent pattern to the results observed at levels of DO above 30%. At these levels, glucose was observed to be rapidly consumed during the i n i t i a l stages of the fermentation. Since there is l i t t l e growth observed in the absence of glucose (see Figure 3.34); i t can be concluded that glucose is a preferred substrate for Sorangium 495 when compared to the protein in herring solubles. However when both are present, both are depleted at fairly rapid rates. Furthermore, in the absence of glucose (see Figure 3.43) the protein content of the medium is again rapidly depleted as this re-presents the only source of nutrient for the organism. It seems that glucose is required i f a high level of bacterial growth is to occur, or i f high levels of enzyme activity are to be found. This creates somewhat of a paradox in as much as one would expect that in the absence of an easily assimilable source of energy such as glucose the organism would produce more enzyme in order to make use of the available protein. But no observable enzyme activity was produced in the fermentation done without glucose although protein consumption was more or less complete (see Section III.6.2.b). The results of the glucose measurements were replotted on the basis of % of i n i t i a l glucose-concentration versus time to incorporate any variations in i n i t i a l glucose levels. However, these provided no. 135 Figure #3.54 FERMENTOR RUN #1.1 DISSOLVED OXYGEN FIXED AT DO=100% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4.14 MG (BSA)/ML GLUCOSE CONCENTRATION (MG/ML) vs FERMENTATION TIME (HRS.) m rn rn 03 to to .10" CD 2Z C J Q C J U J CO O m C J £ ) —I« n " CD to to X X X X X X X o a 0.0 20.0 "I 1 1 40.0 60.0 80.0 TIME HRS n 100.0 120.0 136 Figure #3.55 FERMENTOR RUN #1.2 DISSOLVED OXYGEN FIXED AT DO=80% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.31 MG (BSA)/ML GLUCOSE CONCENTRATION (MG/ML) vs FEPMENTATION TIME (HRS.) o m m en 03 C O co • CD*' CD OLD CJ UJ CO CJfn CD co to a a X X X X X X X X I 1 1 1 1 1 0-0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 137 Figure #3.56 FERMENTOR RUN #1.4 DISSOLVED OXYGEN FIXED AT DO = 50% SATURATION INITIAL PROTEIN CONCENTRATION C =4.88 MG (ESA) / ML GLUCOSE CONCENTRATION(MG/ML)VS FERMENTATION TIME (HRS.) o m 0 3 ' CO to .co"' CD HZ CJ UJ CO Z=>°! CD co co a o X X x x x x x X X X 1 1 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 120.0 TIME HRS 138 Figure #3.57 FERMENTOR RUN #1.5 DISSOLVED OXYGEN FIXED AT DO = 0% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.49 (MG (BSA)/ML) GLUCOSE CONCENTRATION (MG/ML) V S FEPMENTATION TIME (HRS.) o t a -rn 83 onCD r -co ID _ , t o CD 21 CJ cn C J rn CD to to *x xx>< X x x x 0.0 20.0 40.0 60.0 80.0 100.0 120.0 T I M E H R S 139 further information and so were omitted here. III.6.3 Cell Yield and Nutrient Consumption In Table 3.24.a maximum cell yields, time Tm i t took to reach them and maximum growth rates obtained at different DO were reported (see Section III.6.3.a). No definite pattern linking average and maximum cell production to DO levels could be seen. However, the protein consumption was most of the time followed by cell production, which was expected since the organism is known to grow using protein as a substrate (see Section III.6.3.b). Protein consumptions up to Tm were recorded ( Pm mg BSA/ml) as well as glucose consumptions ( Gm mg/ml) and the results show no relationship between nutrient consumption and DO levels. Such a result is consistent with the fact that oxygen did not influence the cell production above the critical level. Maximum protein and glucose consumptions for the complete runs are recorded in Table 3.24.b (Figure 3.47 and Figure 3.58) with the time i t took to reach them. Average values were calculated and plotted for each run (Figure 3.48 and Figure 3.59). The glucose consumption does not follow the same pattern than the protein consumption and is mostly used in a l l cases, confirming the hypothesis that glucose is believed to be a preferred substrate for Sorangium 495 (see Section III.6.d). The organism s t i l l continues to consume protein and glucose well'after the time Tm, although the majority of nutrient is already 140 consumed by then. However, the DO level has no influence on the total nutrient consumption over the whole fermentation process. The naximum rates of protein and glucose consunption per hour of fermentation are shown in Table 3.24.d. They indicate the maximum changes in concentration of protein and glucose. They a l l occur at the beginning of the fermentation process and precede the maximum cell production, which i s expected. The values obtained do not change significantly with the DO levels either. From those results, no relationship between protein and glucose consumption is apparent. Glucose constitutes an easy digestable source of nutrient and was always consumed. At one point, i t appears that protein could not be digested as completely as glucose (run at DO = 80% saturation). However, even in that case, protein was partially consumed. When no glucose was present, protein was removed but no growth was noticed. This leads to the conclusion that, even when short chained amino-acids are available, the organism is unable to utilize them to grow without the presence of glucose. The glucose molecules are broken down into two molecules of lactic acid (Embden-Meyerhof glycolysis). The global reaction: glucose—> 2 lactic acid occurs liberating a great deal of energy: L\G° - -47000 cal/mol (56). It seems that this constitutes an irreplacable source of energy which is necessary in the basic biochemical pathways involved in the transformation of amino acids into cells. Furthermore, the herring solubles used as culture medium is rich in proteins, but. does not con-tain any sugar. Table 3.24.d Maximum Protein and Glucose Consumption Rates DO TP Rp/Tp Rg Tg Rg/Tg Max Protein Time Period Max Rate Cons, rate per hr . % Saturation mg BSA/ml hr Max Glucose Time Period Max Rate Cons, rate per hr mg BSA/ml x hr mg/ml hr mg/ml x hr 0 30 50 80 100 80 1.35 3.62 2.58 2.58 1.47 2.58 3.00 9.25 5.00 5.00 3.00 5.00 0.45 0.39 0.52 0.52 0.49 0.52 0.10 7.66 3.71 8.00 3.00 10.00 5.00 11.25 0.33 0.76 0.74 0.71 142 Figure #3.58 FERMENTOR RUNS AT LOW INITIAL PROTEIN CONCENTRATION. MAXIMUM GLUCOSE CONSUMPTION (MG/ML) vs DO (% SATURATION). o co a in' a to ZD a CO O CD 'CM CE a a i 1 ^ 1 1 1 — r 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION 143 Figure #3.59 FERMENTOR RUNS AT LOW INTIAL PROTEIN CONCENTRATION. AVERAGE GLUCOSE CONSUMPTION (MG/ML) vs DO (% SATURATION) m. 4> , 1 1 1 ~ T 0.0 20.0 40.0 60.0 80.0 100.0 DO X SATURATION 144 Table 3.24.C shows the iteximum enzyme activity obtained for different DO levels. They usually occur before maximum yield is obtained and at this time, a large amount of protein and the majority of glucose available are already consumed. There seems to be a trend to higher activity when DO increases as shown in Figure 3.60, which is somewhat paradoxical since this is not the case neither for cell production nor for protein and glucose con-sumption. The average values of enzyme activity over the complete fermentation process show a similar trend (Figure 3.61). However low values are observed for the run at DO = 80% saturation which is consistent with the cell production, protein and glucose consumptions obtained at this level. Yields for cell production and enzyme activity based on protein and glucose consumption were calculated (Table 3.25) and plotted versus DO levels (Figure 3.62 to 3.65) . No pattern could be observed although maximum values were usually obtained at DO = 100% saturation. This seems to confirm that the efficiency of the organism to digest the available nutrients is not affected by oxygen at concentration above the criti c a l level. Ill.6.4 Conclusion The results obtained for the 7 l i t e r fermentor runs at different DO levels do not indicate any relationship between oxygen levels and cell production. It is confirmed by.the way protein and glucose are consumed, but a trend for higher enzyme activity seems to be observable which is hard to explain. Furthermore i t appears that the glucose and protein consumption are not related to each other. Glucose seems to be 145 23 co o Figure #3.60 FERMENTOR RUNS AT LOW INITIAL PROTEIN CQJCENTRATION. MAXIMUM ENZYME ACTIVITY (UNIT/10 c c ) VS DO (% SATURATION) in CJ CJ o I—to CJ a: | CD UJ UJ CE 5 CP a a ' 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION 146 Figure #3.61 FERffiNTATICJN RUNS AT LOW INITIAL PROTEIN COSICENTRATION. AVERAGE ENZYME ACTIVITY (UNIT/10 cc) vs DO (% SATURATION) X X X X X I I I —1 1 — 0.0 20.0 40.0 60.0 80.0 100.0 DO X SATURATION 'Table 3.25 Yield Coefficients Based on Protein and Glucose Usage DO Y(X/P) Y(X/G) Y(E/P) Y(E/G) Mass Cells Mass Cells Enz. Act. Enz. Act. % Saturation /Protein used /Glucose used /Protein used /Glucose used 0 0.429 2.471 0.425 1.938 30 0.923 - 0.183 -50 0.626 0.630 0.278 0.293 80 0.608 0.378 0.246 0.144 100 1.093 1.244 0.789 0.535 80* 0.210 * Without Glucose 148 4> Figure #3.62 FERMENTOR RUNS AT LOW INITIAL PROTEIN CONCENTRATION. Y(X/P): CELL YIELD BASED ON PROTEIN CONSUMPTION. <!> <!> I 1 1 1 1 °-° 20.0 40.0 60.0 80.0 100.0 DO X SATURATION 149 Figure #3.63 FFJ^ IENTOR RUNS AT LOW INITIAL PROTEIN CONCENTRATION. Y(X/G) : CELL YIELD BASED ON GLUCOSE CONSUMPTION. 23 CQ o 8«. ZDo" UJ CO O CJ _ J C D CO _J _J LLJ CJ22 CO •• C O ° r-a" a ° "i 1 " 1 r 0-0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION 150 Figure #3.64 FERMENTOR RUNS AT LOW INITIAL PROTEIN CONCENTRATION. Y(E/G): ENZYME PRODUCTION BASED ON GLUCOSE CONSUMPTION. I — 1 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION 151 co a' to Oft C C - _ J Figure #3.65 FERMENTOR RUNS AT LOW INITIAL PROTEIN COSICENTRATION. ENZYME PRODUCTION BASED ON PROTEIN CONSUMPTION Y(E/P). C J co . C M UJ X a a ' 0.0 20.0 40.0 60.0 80.0 DO X SATURATION 100.0 a preferred nutrient which is expected since glucose molecules are easily digestable. Glucose appears to be a major energy source for amino acid utilization by the organism. Both nutrients are used right at the beginning of the fermentation process, and at a maximum rate. III.7. Fermentor Runs at High Initial Protein Content 111.7.1 Introduction Although the best condition for producing proteases was de-termined by Wah-On to be using a solution of i n i t i a l protein concentration of around 4 mg BSA/mo, some 7-liter fermentor runs were made at higher i n i t i a l protein contents (from 11 to 16 mg BSA/ml). It was thought that these might provide some more information to clarify the results obtained from the lower protein level 7-liter fermentor runs. The conditions used are specified in Table 3.26. The medium also contained 1% glucose by weight. 111.7.2 Results and Discussion The data are recorded in Appendix III. The growth curves (Figures 3.66 to 3.71) show that growth occurred at these high protein levels. At DO = 0% saturation, some growth was noticed, but i t remained at a lower level than the growth obtained at DO = 20%, 30%, 60% saturation. This confirms the results obtained from the runs at low i n i t i a l protein content. Long chained Table 3.26 Process Conditions of the Experiments DO level Initial Protein Number of Length of Concentration Samples Fermentation % Saturation mg BSA/ml hr 0 13.79 11 26.0 20 15.63 13 27.0 30 11.59 11 28.5 60 15.02 12 33.0 70 11.21 9 11.0 100 15.63 12 29.0 proteins were not broken down at 0% DO (Figure 3.71) which leads to the conclusion that the growth obtained, although quite small, was probably obtained from the use by the organism of the amino acids already existing in this rich medium. Maximum growth was observed at 20% DO. The maximum cell yield was 5.44 mg/ml at this level which was much higher than the level observed at lower protein concentrations. Cell production was not very high at 100% DO and 70% DO (Figure 3.66 and Figure 3.67) although the protein removal was quite efficient at these levels. The maximum cell productions recorded in Table 3.27.a (Figure 3.72) were reached very quickly, compared to the times Tm i t took for the runs at lower protein content. This makes the average cell yield quite high. At those times too, the protein consumptions were not very advanced. For example, at 20% DO only 1.405 mg BSA/ml was consumed at Tm compared to a maximum of 13.450 mg BSA/ml used over the whole fermentation process. At lower i n i t i a l protein content, the majority of the protein was used by the time maximum growth was obtained. However, the amount of protein consumed remain higher than at lower i n i t i a l protein content (Table 3.27.b and Figure 3.73). The maximum protein consumption does not follow maximum growths obtained confirming the importance of i n i t i a l amino acid concentration already present in solution. Although a certain vitality of the culture broth at mid-DO levels was observed, no consistent pattern of growth with DO level could be found. The maximum cell yields noted at 100% 155 Figure #3.66 FFJ<MENTOR RUN #2.1 DISSOLVED OXYGEN FIXED AT DO = 100% SATURATION INITIAL PROTEIN CONCENTRATION C0=15.63 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) V S FERMENTATION TIME (HRS.) CD CD CD CD CD O ^ I 1 1 1 1 u-D B.O 16.0 24.0 32.0 40.0 48.0 TIME HRS 156 Figure #3.67 FERMENTOR RUN #2.2 DISSOLVED OXYGEN FIXED AT DO = 70% SATURATION INITIAL PROTEIN CONCENTRATION C0=11.21 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FERMENTATION TIME (HRS.) o t o ' a in' 2Io CD 21 CO _ ! _ J U J a C J .. m cc a C D a . — i p i ' CD CD CD CD CD CD CD C 7 I I I 1 1 1 0.0 B.D 16.D 24.0 32.0 40.0 48.0 T I M E HRS 157 Figure #3.68 FERMFJSITOR RUN #2.3 DISSOLVED OXYGEN FIXED AT DO = 60% SATURATION INITIAL PROTEIN CONCENTRATION Co=15.02 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FERMENTATION TIME (HRS.) CD CD CD • CD CD £ 3 I I 1 1 "| , °-° B - 0 16.D 24.0 32.0 40.0 4B 0 TIME HRS 158 Figure #3.69 FFJ3MFJSITOR RUN #2.4 DISSOLVED OXYGEN FIXED AT DO = 30% SATURATION INITIAL PROTEIN CONCENTRATION C0=11.39 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) VS FEPMENTATION TIME (HRS.) o o IT) ' 2=o CD 2Z co CC a C_D a. — i r V CD CD CD C§D CD • CD 1 1 : 1 1 1 1 0.D B.O 16.0 24.0 32.D 40.0 4B.D TIME HRS 159 Figure #3.70 FERMENTOR RUN #2.5 DISSOLVED OXYGEN FIXED AT DO = 20% SATURATION INITIAL PROTEIN CONCENTRATION CQ=15.63 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) vs FFJ^ MENTATION TIME (HRS.) o to" a LD ' CD^' 21 CO L J - J C 3 CJ -. m CC a CD°. UJ CD CD • CD CD CD CD CD CD CD CD $ 1 1 1 1 1 1 0 0 B.D 16.0 24.0 32.0 40.0 43.0 TIME HRS 160 Figure #3.71 FERMENTOR RUN #2.6 DISSOLVED OXYGEN FIXED AT DO = 0% SATURATION INITIAL PROTEIN CONCENTRATION CQ=13.79 MG (BSA)/ML WEIGHT DRY CELLS (MG/ML) V S FERMENTATION TIME (HRS.) 1 * Q (T) Q CD CD CD CD CD CD i i i 1 1 1 0.0 a.O 16.0 24.0 32.0 40.0 48.0 TIME HRS Table 3.27.a Maximum Cell Yields DO Max Cell Yield T M Time to Max *M TM Average Yield Cell Protein Cons. At Tm 21P . M Av. Prot. Cons, at Tm. % Saturation mg/ml hr mg/ml x hr mg BSA/ml mg BSA/ml x hr 0 1.92 4.00 0.480 0.000 0.000 20 5.44 8.00 0.680 11.240 1.405 30 5.97 6.67 0.895 3.320 0.498 60 2.72 11.50 0.237 5.720 0.497 70 2.84 6.00 0.473 7.840 1.307 100 2.32 10.00 . 0.232 11.490 1.149 Figure #3.72 FERMENTOR RUNS AT HIGH INITIAL PROTEIN CONTENT. MAXIMUM CELL YIELD (MG/ML) vs DO (% SATURATION) . m cn ^ o o CD 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION Figure #3.73 FFJ^ MENTOR RUNS AT HIGH INITIAL PROTEIN CONCENTRATION. MAXIMUM PROTEIN CONSUMPTION (MG BSA/ML) vs DO (% SATURATION) . <5> I I 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 DO X SATURATION Table 3.27.b Maximum Protein Consumption DO M 4 T P M «P Max Prot. Consumption Time to Max Av. Prot. Cons. Max Prot. Cons. Rate per hr. % Saturation mg BSA/ml H" hr mg BSA/ml mg BSA/ml x hr 0 2.770 14.00 0.198 -0.895 20 13.450 12.00 1.121 -3.080 30 8.760 12.00 0.730 -4.446 60 10.820 33.00 0.328 -2.270 70 9.770 11.00 0.888 -2.400 100 13.390 29.00 0.462 -2.950 165 DO and 70% DO were not much greater than the one observed at 0% DO in contrast with the lower i n i t i a l protein data. These results at high DO levels are similar to those obtained from the flask runs. This is consistent since the flask runs were made under conditions allowing an abundant oxygen supply. This leads to the conclusion that both too much nutrient and oxygen do not favor cell production. The growth pattern was similar to the one observed at lower i n i t i a l protein levels. There was a rapid rise to a maximum cell yield followed by a decline in population. However, the presence of large amount of oxygen did not delay the population decline like i t did at low i n i t i a l protein content. Similarly to the low i n i t i a l protein content runs, the growth rates (see Appendix III.A) were maximum at the beginning of the fermentation, and then reached ne-gative values corresponding to the population decline. The final cell productions were higher at higher i n i t i a l protein level. More protein was used, but the final yields based on protein usage (Table 3.28 and Figure 3.74) were lower. This means that the organism uses more pro-teins when they are available leading to a greater ce l l production, but this is done with a lower efficiency. This is expected i f the organism would tend to use f i r s t the amino acid already in solution. Some enzyme activity was noticed (Figures 3.75 to 3.80). At DO = 0% saturation, enzyme activity was very low, which was expected since at this level, protein was not consumed, and very l i t t l e growth was observed. Enzyme activity was consistently higher at DO = 20% saturation as well as for the runs at 30% and 60% DO. This corresponded to a fast and complete removal of protein at DO = 20%, but that was Table 3.27.C Maximum Enzyme Activity DO i % Saturation EM Max Enz. 'Act. unit/10 cc TE Time to Max hr EM V Average Enz. 'Activity unit/10 cc 4P Protein Used 'at T E mg BSA/ml 0 0.86 2.0 0.430 0.000 20 1.14 5.0 0.228 6.760 30 1.32 12.0 0.110 8.390 60 1.34 18.0 0.074 8.600 70 0.86 6.0 0.143 7.840 100 1.05 29.0 0.036 13.390 (—1 Ol o^  167 Figure #3.74 FERMENTOR RUNS AT HIGH INITIAL PROTEIN CONCENTRATION. Y(X/P): MASS CELLS/PROTEIN USED. a CM" to U 3 cn CO " v . - . c o -LU C J CO to C O 1 0 , d o ' co cn a 1 —I 1 I I 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION Table 3.28 Yield Coefficients Based On Protein Usage Y(X/P) Y(E/P) Mass Ce l l / Enz. Act./ % Saturation Protein Used Protein Used 0 Y ( X / P ) = V X M F ( T ) M 20 0.484 0.169 Y, ... = EM (E/P) 30 1.798 0.157 60 0.476 0.156 70 0.362 0.110 100 0.202 0.078 P ( T E ) 169 Figure #3.75 FERMENTOR RUN #2.1 DISSOLVED OXYGEN FIXED AT DO = 100% SATURATION INITIAL PROTEIN CCNCENTRATION CQ=15.63 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) VS FERMENTATION TIME (HRS.) 4-4-+ 4- 44-4-. —I 1 1 I i l 3.0 B.D 16.0 24.0 32.0 40.0 48.0 TIME HRS 170 Figure #3.76 FERMENTOR RUN #2.2 DISSOLVED OXYGEN FIXED AT DO = 70% SATURATION INITIAL PROTEIN CONCENTRATION C0=11.21 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) V S FERMENTATION TIME (HRS.) +++ + ++++  a "I I 1 1 1 1 1 0.0 B.O 16.0 24.0 32.0 40.0 4B.0 TIME HRS 171 Figure #3.77 FFJ3MENT0R RUN #2.3 DISSOLVED OXYGEN FIXED AT DO = 60% SATURATION INITIAL PROTEIN CX3NCENTRATION C =15.02 .MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) V S FERMENTATION TIME (HRS.) o + 4 f + + + 4-4-4-+ 4-~ l 1 IB.D 24.0 TIME HRS 0.0 B.O 32.0 40.0 43.0 172 a (V to U3 Figure #3.78 FFJ3MENT0R RUN #2.4 DISSOLVED OXYGEN FIXED AT DO = 30% SATURATION INITIAL PROTEIN CONCENTRATION C =11.39 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) vs FERMENTATION TIME (HRS.) m cn m •a + 4-+ 4- 4- 4-4-+ + + .<£> UJ a a 0.0 8.0 16.0 24.0 T I M E HRS 32.0 40.0 48.0 173 Figure #3.79 FERMENTOR RUN #2.5 DISSOLVED OXYGEN FIXED AT DO = 20% SATURATION INITIAL PROTEIN CONCENTRATION CQ=15.63 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) V S FFJ<MENTATION TIME (HRS.) o i i 1 1 1 1 1 0.0 8.0 16.0 24.0 32.0 40.0 49.0 TIME HRS 174 Figure #3.80 FERMENTOR RUN #2.6 . DISSOLVED OXYGEN FIXED AT DO = 0% SATURATION INITIAL PROTEIN CONCENTRATION CQ=13.79 MG (BSA)/ML ENZYME ACTIVITY (UNIT/10 cc) vs FERMENTATION TIME (HRS.) tn tn CJ O CLr~ .to LU tn tn tn + + + + + + + 0.0 B.O 16.0 2 4.0 TIME HRS 32.0 40.0 4B.0 175 not the case for the runs at 30% and 60% DO. In those runs, protein was removed, but not as fast as at 20% DO. The relatively low values of enzyme activity obtained for DO = 70% are hard to explain, since at that level, protein is rapidly and completely removed. At this level, the growth obtained was not spectacular either. The yield of enzyme based on protein consumption, Y(E/P), was noticed to be decreasing with DO levels (Figure 3.81). Since proteins were broken down for a l l runs (Figures 3.82 to 3.87), this suggests that maybe, less activity is necessary to break the long chained proteins when oxygen level increases. However, this did not create a larger cell production at high DO levels. Compared to the low i n i t i a l protein runs, the enzyme activity was usually but not always higher, but the yields Y(E/P) were much lower. But, then, cell production was higher at high DO levels. For the runs at high i n i t i a l protein content, i t seems that high DO levels did not impair enzyme production and protein breakdown, but somehow those amino acids could not be used by the organism for growth. Ill.7.3 Conclusion The runs at higher i n i t i a l protein concentration show that growth is s t i l l possible under such condition. No obvious relationship between growth and DO levels could be found. However, i t seemed that better growth occurred at lower DO levels in contrast with the low i n i t i a l protein runs. It appeared that the organism could f i r s t use the amino acids available in solution but later on, was not able to Figure #3.81 FERMENTOR RUNS AT HIGH INITIAL PROTEIN CONCENTRATION. Y(E/P) : ENZYME ACTWITY/PROTEIN USED. X X 1 r 1 1 1 0.0 20.0 40.0 60.0 80.0 100.0 DO % SATURATION 177 Figure #3.82 FERMENTOR RUN #2.1 DISSOLVED OXYGEN FIXED AT DO = 100% SATURATION INITIAL PROTEIN CONCENTRATION. CQ=15.63 MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA)/ML) vs FERMENTATION TIME (HRS.) r*—, A A A A A A i r i 1 1 1 0.0 B.O 16.0 24.0 32.0 40.0 4 8 0 TIME HRS 178 Figure #3.83 FERMENTOR RUN #2.2 DISSOLVED OXYGEN FIXED AT DO = 70% SATURATION INITIAL PROTEIN CONCENTRATION C0=11.21 MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA)/ML) vs FERMENTATION TIME (HRS.) a <n <n CD A CO •-I— O CE 0_ r— I L P cr03.. I IT) O A A tn <n oq A A A o.o B.O -| 1 L6.D 24.0 TIME HRS 32.0 ~ ~ 1 — 40.0 4B.0 179 Figure #3.84 FERMENTOR RUN #2.3 DISSOLVED OXYGEN FIXED AT DO = 60% SATURATION INITIAL PROTEIN CONCENTRATION C =15.02'MG (BSA) / ML TOTAL PROTEIN CCMTENT (MG (BSA) / ML) V S FERMENTATION TIME (HRS.) a r-—, £ A A A A A A AAA A A A i i I 1 —I 1 °-0 B.O 16.D 24.0 32.0 40.0 43 0 TIME HRS 180 Figure #3.85 FERMENTOR RUN #2.4 DISSOLVED OXYGEN FIXED AT DO 30% SATURATION INITIAL PROTEIN CONCENTRATION C0=11.39 MG (BSA)/ML TOTAL PROTEIN CONTENT (MG (BSA) / ML) V S FEFMENTATIGN TIME (HRS.) A A !zi A A A A A A A A A A ~i 1 L6.0 24.0 TIME HRS 0.0 a.o 32.0 40.0 48.0 181 Figure #3.86 FERMENTOR RUN #2.5 DISSOLVED OXYGEN FIXED AT DO = 20% SATURATION INITIAL PROTEIN COTCENTRATIGN CQ=15.63 MG (BSA) / ML TOTAL PROTEIN CONCENTRATION (MG (BSA) / ML) V S FERMENTATION TIME (HRS.) r " — i us <n , c n Jen LD CL £ m CD -. CP l— O CC Q_ CL"3. •—m' O cn A A A A A ^ A A A 1 -i 1 i i l 0 0 B.O 15.0 24.0 32.0 40.0 48.0 TIME HRS 182 Figure #3.37 FFJMENTOR RUN #2.6 DISSOLVED OXYGEN FIXED AT DO = 0% SATURATION INITIAL PROTEIN CONC!ENTRATION C0=13.79 MG (BSA) / ML TOTAL PROTEIN CONCENTRATION (MG (BSA) / ML) vs FERMENTATION TIME (HRS.) A A A A A A A A A A A CE cn--0 3 I— o cn Q_ r— _ icp CE 0 3. i—in O m OQ O.D 8.0 -I 1 L6.0 24.0 TIME HRS 32.0 40.0 48.0 utilize the broken down molecules with efficacy. The population decline appeared quite rapidly this time and the presence of large amounts of oxygen did not delay i t . Protein molecules were broken down, and this corresponded to a noticable enzyme production. Enzyme activity was usually higher than for the runs at lower i n i t i a l protein content, but the maximum values obtained remained lower. Sometimes, the scatter of these data did not allow any reliable conclusion. 184 CHAPTER IV  CONCLUSIONS Herring solubles were used for the production of protease using Sorangium 495. Tests done in shake flasks showed that maximum values for enzyme yield were observed at low (ca 4 mg (BSA)/ml) i n i t i a l protein concentrations. This was also observed in the 7-liter fermentor. In the shake flasks, the low protein concentrations gave the higher cell yields whereas in the 7-liter fermentor, cell yields were greater at the higher protein levels (ca 15 mg (BSA) /ml). Fermentation runs were made at different DO levels using herring solubles solution at a protein concentration of about 4 mg (BSA)/ml. It was shown that Sorangium is an aerobic organism since very l i t t l e growth was observed at DO = 0% saturation in contrast with the runs performed at DO levels above 30% saturation. Growth of Sorangium 495 requires glucose since l i t t l e growth was obtained for a run performed at DO = 80% saturation without glucose.. - A c critical DO level between 0% and 30% saturation is suspected. Above DO = 30% saturation, i t appears that the growth of the organism in term of cell mass concentration is independent of DO levels. After reaching a maximum, the cell mass concentration drops off. The rate of decrease of the cell population however is reduced when a high DO level is maintained. Oxygen seems to play an important role in the enzyme formation. As DO levels increase, so does maximum enzyme activity. However, the 185 levels of enzyme activity obtained were considerably lower than Wah-On's (Ca 2.5 unit/10 cc compared to 10.5 unit/10 cc). Glucose seems to be preferred as a substrate when compared to protein or protein like material. It was consumed right away in • -every, run performed. The majority of enzyme activity appears right after this stage, once the glucose is consumed a lower rate of cell production can be observed. At low DO levels, the cell population will subsequently decline. When the oxygen concentration is higher, this decline is delayed .suggesting that the enzyme utilizes the available nutrients better in a medium rich in oxygen. However, no consistent pattern to the level of cell mass concentration as. a function of DO level could be found. Protein was consumed most of the time, but at a lower rate than glucose. For each run, both nutrients were consumed at their highest rate at the beginning of the fermentation process. However, a l l attempts to correlate enzyme and cell production to protein and glucose consumption failed. It appears that growth and protease synthesis are independent. Furthermore, no relationship between glucose and protein usage could be determined. Above the critical level, oxygen concentration did not influence the nutrient consumption pattern, and seemed to have a greater effect on protease formation than on cell production. Fermentor runs were made at different DO levels using herring solubles solution at higher i n i t i a l protein concentration (11 to 16 mg (BSA)/ml). Here too, growth was low at DO = 0% saturation compared to the cell .production at higher DO levels. However, at DO levels above 186 60% saturation, cell production was not as great as was obtained in the 30-60% range. This contrasts with the low i n i t i a l protein runs where cell production did not seem to vary significantly with LX) above 30% saturation. Protein was consumed, and a noticable degree of enzyme production was observed. Although the enzyme activities found were sometimes higher than those noticed at the lower i n i t i a l protein content runs for similar conditions, the maximum values obtained were lower. This leads to, the conclusion that a herring solubles solution at i n i t i a l protein concentration of 4 mg (BSA)/ml and kept at a high DO level constitutes the most favorable culture medium for protease formation. At higher i n i t i a l protein concentra-tions, i t appeared that the organism could f i r s t use the amino acids available in solution, but later on was not able to utilize them effectively. In these studies too, any attempt to correlate enzyme and cell production to DO levels failed. Usually, the data obtained for these runs were more scattered, and although i t confirmed some results from the lower level protein experiments, they did not clarify very much the obscure points. From the results obtained, the major steps of the fermentation process can be described as- follows. A culture medium containing glucose and proteinaceous matter is innoculated with Sorangium 495. The organism will consume the glucose preferably, and at this stage when enough oxygen is available, yields of cells, maximum level of growth and protease formation are determined. Then, when the majority of the glucose present in solution is consumed, i t seemed that growth started to decline as the protein source was simultaneously consumed. 187 Oxygen was s t i l l needed at this time, because a large supply of oxygen seems to delay the population decline at low i n i t i a l protein content. Eventually, cell population declined which was characterized by a change of color of the culture medium from light brown to dark brown. The whole process is a complex result of i n i t i a l amino acid utilization, enzymatic digestion of larger polypeptide units plus enzymatic secretion, and oxygen limitation. Oxygen obviously plays an important role, but the kinetic patterns are quite complex, and no simple correlation between the different parameters involved could be accurately determined. 188 BIBLIOGRAPHY 1. Claggett F.G. 1972 "Clarification of Fish Processing Plants by Chemical Treatment and Air Flotation." Fisheries Research Board of Canada: Technical Report No. 343. 2. Claggett F.G'J 1972 "Cemonstration Wastewater Unit. Interim Report 1971 Salmon Season" Fisheries Research Board of Canada, Vancouver Laboratory Technical Report No. 286. 3. Claggett F.G. 1970 "A Proposed Demonstration Plant for Treating Fish Processing Plant Wastewater." Fisheries Research Board of Canada, Vancouver Laboratory Technical Report No. 197. 4. Claggett F.G. and J. Wong, 1969 "Salmon Canning Wastewater Clarification. Part II. A Comparison of Various Arrangements for Flotation and Some Observations Concerning Sedimentation and Herring Pump Water Clarification." Fisheries Research Board of Canada, Vancouver Laboratory, Circular No. 42. 5. Claggett F.G. and J. Wong, 1968 "Salmon Canning Wastewater Clarification. Part I: Flotation by Total Flow Pressuration." Fisheries Research Board of Canada, Vancouver Laboratory Circular No. 38. 6. Burgess G.H.O., Cutting C.L., Lovern J.A., Watterman J.J., 1965, "Fish Handling and Processing." Her Majesty's Stationary Office, Edinburgh U.K. 7. Larsen S., 1965 "Fish Solubles" in Fish as Food Vol. I l l G. Borgstrom (Ed.) Academic Press N.Y. pp. 281-299. 8. Soderquist, M.R., 1970 "Current Practice in Seafood Processing Waste Treatment," Department of Food Science, Oregon State University, Corvallis, Oregon. Report prepared for EPA Water Quality Office, Washington, D.C. 9. Lehminger A.L., 1975, Biochemistry 2nd Edition, Worth Publishers, N.Y. 10. Neurah M., 1964, "Protein-digesting Enzymes" Scientific American Vol. 211.6 pp. 68-79. 11. Stephenson M., 1939 Bacterial Metabolism. 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Technol. Today, 1972 pp 289-298. 20. Kline L., McDonnell L.R., Linewater H., "Bacterial Proteinase from Waste Asparagus Butts." Ind. and Eng. Chemistry, 1944, Vol. 30, pp 1152. 21. Krishnaswamy M.A. and Lahiry N.L., 1963, "Fish Hydrolystates: Microbial Evaluation." J. Food Sciences., Vol. 28, pp 358-364. 22. Snieszko, S.F., Griffin P.J., and Friddle S.B., 1950. "A New Bacterium (Hemophilius Piscium Nsp) from ulcer disease of trout." J. Bacterid., Vol. 59., pp 599-710. 23. Green J.H., et.al. "Exploration of Experimentally Produced Fish Peptone for Growth of Microorganisms." Development in Industrial Microbiolgoy, Vol. 14, Proc. 29th Meeting Society of Indus. Microbiolgoy. 24. Burkholder L., Burkholder, P.R., Chu A., Kostyk N., and Reels O.A., "Fish Fermentation". Marine Biology Division, Lamont Geological Observatory of Columbia University Palissades, N.Y. 25. Saisithi P., Kasemsarn B.O., Liston J. and Dollar A.M., 1966 "Microbiology and (^emistry Fermented Fish". J. Fond. Sc., Vol. 31., pp 105 190 26. Strasdine G.A. and Melville J.M. 1972 "Salmon Canning Waste-water as a Microbial Growth Medium". J. Fish. Res. Bd. , Canada 29. No. 12. 27. Wah-On H.C., 1974, "Proteolytic Enzymes from Fermentation of Fish Plant Wastes". M.A. Sc. Thesis. University of British Columbia Vancouver, B.C. 28. Gillespie, D.C. and Cook F.D., 1965 "Extracellular Enzymes from Strains of Sorangium", Can. J. Microbiol., Vol. II, pp 109-118. 29. Whitaker, D.R., Cook F.D., and Gillespie D.C., 1965. "Lytic Enzymes of Sorangium sp. Some Aspects of Enzyme Production in Submerged Culture," Can. J. Biochem., Vol. 43, pp 1927-1933. 30. Blakebrough N. Biochemical and Biological Engineering Science 1967, Vol. 1, pp 69-99 Academic Press N.Y. 31. Bailey J.E., Ollis D.F. Biochemical Engineering Fundamentals. 1977. McGraw H i l l N.Y. Capt. 8. 32. Calderbank P.H. "Mass Transfer in Fermentation Equipment," p. 102 in Blakebrough N. (Ed.) Biochemical and Biological  Engineering Sciences, 1967, Vol. 1 Academic Press N.Y. 33. Calderbank P.H., 1958, "Physical Rate Processes in Industrial Fermentation. Part I. The Interfacial Area in Gas. -Liquid Contacting with Mechanical Agitation." Trans. Instn. Chem. Engrs. Vol. 36, pp. 443. 34. Aiba S., Humphrey A.E., Millis N.F., 1973, Biochemical Engineering (2nd Ed.), Academic Press N.Y. 35. Finn R.K. "Agitation and Aeration" in Blakebrough N. (Ed.) Biochemical and Biological Engineering Sciences, 1967, Vol. 1, Academic Press, N.Y. 36. Bird R.B., Stewart W.B. and Lightfoot E.N., 1960, Transport Phenomena John Wiley & Son Inc. N.Y. 37. Carpenter, P.L., 1972. Microbiology, 3rd Ed., W.B. Saunders Co., Toronto. 38. Eworkin, M., 1966. "Biology of the Myxobacteria", Ann. Rev. Microbiol., Vol. 20, pp 75-106. 39. Henrici, A.T. and Ordal E.J., 1948 The Biology of Bacteria, 3rd Ed., D.C. Heath & Co., Boston Mass. 151 40. Johnson, M.J., Borkowski, J. and Engblom, C., 1964, Biotech. Bioeng. Vol. 6, pp. 457-68. 41. Fergusson, D.K., 1972, "Manufacture of Vitamin B.12 from Sulfite Spent Liquor," M.A. Sc. Thesis, University of British Columbia, Vancouver, B.C. 42. Gavin, J.J., 1957, "Microbiological Process Report. Analytical Microbiology III Turbidimetric Methods." Appl. Microbiol., Vol. 5., pp. 235-242. 43. Monod, J., 1949, "The Growth of Bacterial Cultures", Ann. Rev. of Microbiology, Vol. 3, pp. 371. 44. Cavin, J.J., 1957, "Microbiological Process Report" Applied Microbiology, Vol. 5. 45. Report of The Commission on Enzymes of the UIB, 1961, Oxford Pergamon Press. 46. Anson, M.L., 1938, "The Estimation of Pepsin, Trypsin, Papain, and Cathespin with Hemoglobin" J. Gen. Physiol. Vol. 22., pp. 79. 47. Wirnt, R., 1974, Method of Enzymatic Analysis, "Trypsin", Bergmeyer, H.U. (Ed.), Vol. 2, pp. 1013-1022. 48. Wirnt, R., 1974, Method of Enzymatic Analysis, "Chymotrypsin" Bergmeyer, H.U. (Ed.), Vol. 2, pp. 1006-1012. 49. Wirnt, R. and Fritsh, W.P., 1974, Method of Enzymatic Analysis, "Pepsin", Bergmeyer, H.U. (Ed.), Vol. 2, pp. 1046. 50. Hazen, G.G., 1974, Method of Enzymatic Analysis, "Proteases", Bergmeyer, H.U. (Ed.), Vol. 2, pp. 1000-1005. 51. Petrova, L.S., and Vintsyuanaite M.M., 1966, "Determination of Proteolytic Activity of Enzyme Preparation of Microbial Origin" Prikl. Biokhim. I. Mikrobiol. (Applied Biochem. and Microbiol.) Vol. 2, pp. 322-327. 52. Mahler, H.R., and Cordes E.H., 1971, Biological Chemistry, 2nd Ed., N.Y. Harper & Row. 53. Folin, O./ and Ciocalteau V., 1927, "On Tyrosine and Tryptophan Determination in Proteins." J. Biol. Chem., Vol. 73, pp. 627. 54. Summer, J.B., "Determination of Glucose by DNS Method". J. Biol. Chem. Vol. 65, pp. 393 (1925). 192 55. Snell, F.D., & Snell C.T., Colorimetric Methods of Analysis Vol. 3.a. pp. 202. 56. Skerman, V.B.D. and McCrae, I.C., (1957), Can. J. Microbiol. Vol. 3 pp. 215-30, and pp. 505-30. APPENDIX I. FLASKS RUNS: EXPERIMENTAL DATA 194 APPENDIX 1.1 FLASKS RUNS: SALMON SOLUBLES AT HIGH INITIAL PROTEIN CONCENTRATION FLASKS RUN: SALMON SOLUBLES AT HI'GN INITIAL PROTEIN CONCENTRATION (1) Time Hrs. W.eight dry mg/ml cells ,absorbance 540 at total proteins mg BSA/ml absorbance 660 iy at tyrosine pg/ml cone. .direct iturbidity Mean Mean Mean Mean 5 .0 - 1.70 1.70 31.30 31.30 31.30 1.20 1.20 56.40 56.40 56.40 -15 .0 2.1900 2.0500 2.1950 3.4700 . 2.48 1.70 1.70 1.70 31.30 31.30 31.30 31.30 1.68 1.66 1.66 77.56 77.56 77.56 77.56 1.60 ' 1.60 ! 1.60 i 1.60 20 .0 3.9450 2.5000 2.2650 1.9750 2.67 1.50 1.50 1.50 27.62 27.62 27.62 27.62 1.86 1.86 85.49 85.49 85.49 ' 2.10 . 2.10 2.10 ! 2.10 25 .0 1.7500 1.7300 1.7350 1.6550 1.72 1.70 1.70 1.70 31.30 31.30 31.30 31.30 1.10 1.08 51.99 51.11 51.55 3.00 3.00 3.00 29 .0 1.6000 1.7450 1.6700 1.5650 1.65 1.70 1.70 1.70 31.30 31.30 31.30 31.30 1.20 1.20 1.20 56.40 56.40 56.40 56.40 3.60 3.60 3.60 35 .0 2.8700 3.1750 2.6550 2.90 1.90 1.90 34.99 34.99 34.99 2.25 2.25 102.68 102.68" 102.68 2.55 2.55 2.55 . 2.2050 FLASKS RUN: SALMON SOLUBLES AT HIGH INITIAL PROTEIN CONCENTRATION (2) Time Hrs. Weight dry cells absorbance at vtotal proteins absorbance at tyrosine cone mg/ml 540 mu mg BSA/ml 660 mp Vg/ml Mean Mean Mean direct turbidity Mean 40.0 50.0 5.5400 4.1100 3.6450 3.3150 2.0300 2.0500 2.0750 2.0550 4.15 2.05 86 86 2.56 2.56 2.40 34.25 34.25 47.16 47.16 44.21 34.25 43.20 .92 .00 1.28 2.48 2.48-88.13 91.66 59.93 112.81 112.81 89.90 95.18 2.55 2.55 2,55 3.40 3.27 3.40 3.00 60.0 85.0 2.1400 2.6350 2.2500 2.0550 2.0850 2.27 0400 0550 1050 2.17 2.55 2.50 2.10 2.10 46.97 46.05 38.68 38.68 46.51 38.68 20 20 60 60 70 56.40 56.40 74.03 74.03 74.03 56.40 74.03 3.80 3 ,,60 3.80 3.73 3.40 3. 3.40 40 90.0 2350 2800 3200 3600 2.30 2.40 2.40 2.40 44.21 44.21 44.21 44.21 ,60 ,60 ,40 74.03 74.03 65.27 71.09 3.40 3.40 3.40 3.40 h-1 VD ON ' APPENDIX 1.2 FLASKS RUNS: HERRING SOLUBLES FLASKS RUN: HERRING SOLUBLES Co=3.65 mg BSA/ml. 1% GLUCOSE ADDED. MEAN VALUES (1) Time T A T weight dry cells A X Growth rate — "Total proteins^P --AP E^nzyme activity X hrs. hrs mg/ml mg/ml mg/ml hr .-mg BSA/ml mg BSA/ml unit/10 cc 0.00 6.75 1.93 0.29 3.65 +3.20 — 6 .75 3.75 1.93 0.46 0.12 0.45 -3.69 0.24 10.50 12.75 2.39 1.85 0.15 4.14 -0.38 0.26 23.00 7.00 4.24 0.38 0.05 4.82 +1.78 0.34 30.00 6.00 4.62 0.25 0.04 3.04 ' 1.36 0.44 36.00 12.00 4.87 -0.10 -0.01 1.68 0.24 0.37 48.00 12.00 4.77 -1.05 -0.09 1.44' . 0.49 0.32 60.00 17.00 3.72 -0.85 -0.05 0.94 0.43 0.30 77.00 18.00 2.87 -2.32 -0.05 1.38 0.99 0.28 125.00 0.55 0.39 0.38 FLASKS RUN: HERRING SOLUBLES: Co=3.65 mg BSA/ml. 1% GLUCOSE ADDED (2) Time T weight dry cells absorbance at total proteins absorbance at tyrosine cone. enzyme activity hrs mg/ml 540 mp mg BSA/ml 660 mp Ug/ml units/10 cc 6.75 1.9000 0.04 0.70 0.04 5.28 0.23 1.9050 0.02 0.33 0.04 5.28 0.23 1.9200 0.02 0.33 0.05 5.72 0.25 1.9200 10.50 2.3650 0.23 4.20 0.06 6.16 0.27 2.3950 0.22 4.02 0.06 6.16 0.27 2.3800 0.23 4.20 0.05 5.72 0.25 2.4050 23.00 4.2300 0.25 4.57 0.08 7.04 0.31 4.2350 0.27 4.94 0.10 7.92 0.35 4.2650 0.27 4.94 0.10 7.92 0.35 4.2350 30.00 4.6100 0.16 2.91 0.14 9.69 0.43 4.6050 0.17 3.10 0.15 10.13 ' 0.45 4.6200 0.17 3.10 0.14 9.69 0.43 4.6350 36.00 4.8900 0.09 1.62 0.11 8.36 0.37 4.8350 0.09 ,1.62 0.15 10.13 . 0.45 4.8950 0.10 1.81 0.07 6.60 0.29 4.8700 00.00 _ 0.20 3.65 — — -0.20 3.65 0.20 3.65 •FLASKS RUN: HERRING SOLUBLES: Co=3.65 mg BSA/ml. 1% GLUCOSE ADDED (3) Time T vweight dry cells -absorbance at 'Total proteins absorbance at 'Tyrosine cone. Enzyme activity hrs mg/ml 540 mjj mg BSA/ml 660 mp p g/ml V"' ; _ / > 48.00 4.7400 0.07 1.25 0.07 6.60 0.29 4.7800 0.08 1.44 0.09 7.48 0.33 4.7600 0.09 1.62 0.09 7.48 0.33 4.7950 60.00 3.7200 0.05 0.89 0.05 5.72 0.25 3.7350 0.06 1.07 0.08 7.04 0.31 3.7300 0 .05 0.89 0.09 7.48 0.33 3.6750 77.00 2.6850 0.08 1.44 0.08 7.04 0.31 2.9450 0.08 1.44 0.06 6.16 0.27 2.9700 0.07 1.25 0.05 5.72 0.25 125.00 0.5550 0.02 0.33 0.14 9.69 0.43 0.5850 0.03 0.52 0.10 7.92 0.35 0.5300 0.02 0.33 0.10 7.92 0.35 0.5200 FLASKS RUN: HERRING SOLUBLES. Co=4.45 mg BSA/ml. 1% GLUCOSE ADDED MEAN VALUES (1) lime T hrs. AT hrs weight dry cells mg/ml X AX mg/ml Growth rate mg/ml hr AX AT Total Protein P mg BSA/ml -AP mg BSA/ml • Enzyme activity unit/10 cc 0.0 4.45 0.00 7.0 0.51 0.07 2.52 7.0 0.51 1.93 0.24 3.0 0.41 0.14 0.98 10.0 0.92 0.95 0.26 5.0 1.51 0.30 -0.30 15.0 2.43 1.25 0.20 6.0 -0.18 -0.03 0.18 21.0 2.25 1.07 0.32 4.5 0.51 0.11 -0.86 25.5 2.82 1.93 0.22 4.5 -0.8 -0.18 -0.31 30.0 2.02 2.24 0.77 18.5 -0.05 -0.002 -0.49 48.5 1.97 2.73 0.63 9.5 -0.23 -0.02 0.25 58.0 1.74 2.48 0.64 23.0 -0.88 -0.04 -0.25 81.0 0.86 2.73 0.76 FLASKS RUN: 1% GLUCOSE, INITIAL CONCENTRATION Co = 4.45 mg BSA/ml (2) Time T .weight dry cells ^absorbance at Total proteins absorbance at Tyrosine cone. E^nzyme activity hrs mg/ml 540 mp mg BSA/ml 660 mp ug/ml units/10 cc 0.0 0.25 4.57 0.00 0.00 0.00 0.23 4.20 0.00 0.00 0.00 0.25 4.57 0.00 0.00 0.00 7.0 0.60 0.13 2.36 0.04 5.28 0.23 0.57 0.10 1.81 0.05 5.72 0.25 0.32 0.09 1.62 0.05 5.72 0.25 0.54 10.0 0.87 0.04 0.70 0.05 5.72 0.25 0.88 0.05 0.89 0.06 6.16 0.27 0.91 0.07 1.25 0.05 5.72 0.25 1.01 15.0 2.40 0.07 1.25 0.03 4.84 0.21 2.45 0.06 1.07 0.02 4.40 0.19 2.46 0.08 1.44 0.03 4.84 0.21 2.42 21.0 2.29 0.04 0.70 0.09 7.48 0.33 2.21 0.06 1.07 0.08 7.04 0.31 2.24 0.08 1.44 0.09 7.48 0.33 2.25 #1 FLASKS RUN: 1% GLUCOSE, INITIAL CONCENTRATION Co =4.45 mg BSA/ml (3) Time T weight dry cells absorbance at Total proteins absorbance at hrs mg/ml mean 540 mp mg BSA/ml mean 660 ml 'Tyrosine cone, pg/ml Enzyme activity units/10 cc mean 25.5 2.81 2.83 2.83 2.79 0.10 0.12 0.10 1.81 2.18 1.81 1.93 0.03 0.04 0.03 4.84 5.28 4.84 0.21 0.23 0.21 0.22 30.0 2.03 1.95 2.04 2.07 2.02 0.12 0.13 0.12 2.18 2.36 2.18 2.24 0.32 0.35 0.28 17.62 18.94 15.86 0.78 0.84 0.70 0.77 48.5 1.97 1.97 1.99 1.97 1.975 0.15 0.15 0.15 2.73 2.73 2.73 2.73 0.47 0.16 0.10 24.23 10.57 7.92 1.07 0.47 0.35 0.63 58.0 1.51 1.52 1.56 1.46 1.74 0.15 0.13 0.13 2.73 2.36 2.36 2.48 0.35 0.20 0.20 18.94 12.33 12.33 0.84 0.54 0.54 0.64 81.0 0.86 0.89 0.85 0.82 0.86 0.15 0.15 0.15 2.73 2.73 2.73 2.73 0.31 0.32 0.30 17.18 17.62 16.74 0.76 0.78 0.74 0.76 O O J 'FLASKS RUN: HERRING SOLUBLES Co=5.59 mg BSA/ml. 1% GLUCOSE ADDED. MEAN VALUES (1) Time T AT .weight dry cells AX hrs hrs mg/ml mg/ml mg/ml hr A X Growth rate .Total Protein P -AP ^Tyrosine Cone. mg BSA/ml mg BSA/ml g/ml 0.0 4.0 12.0 24.0 29.0 36.0 53.0 60.0 89.5 97.0 109.0 4.0 8.0 12.0 5.0 7.0 17.0 7.0 29.5 7.5 12.0 0.74 2.61 3.70 4.04 4.17 4.21 3.89 2.86 2.39 2.17 0.74 1.87 1.09 0.34 0.13 0.04 -0.32 -1.03 -0.47 -0.22 0.19 0.23 0.09 0.07 0.02 0.002 -0.05 -0.03 -0.06 -0.02 5.59 4.66 4.51 4.57 3.65 1.99 0.83 1.01 0.89 0.64 1.56 0.93 0.15 -1.06 0.92 1.66 1.16 -0.18 0.12 0.25 -0.92 84.18 77.41 77.86 78.60 77.42 77.87 79.18 80.51 77.71 79.44 76.83 O FLASKS RUN HERRING SOLUBLES. 1% GLUCOSE ADDED: INITIAL CONCENTRATION Co=5.59 mg BSA/ml (2) Time weight dry cells absorbance at total proteins absorbance at Tyrosine cone. hrs mg/ml 540 mp mg BSA/ml 660 m|J /Jg/ml 0.0 0.30 5.49 0.28 84.18 0.31 5.68 0.28 84.18 4.0 0.7750 0.25 4.57 0.13 77.57 0.7300 0.26 4.75 0.15 78.45 0.7600 - - 0.10 76.21 0.6950 12.0 2.3750 0.26 4.75 0.17 79.33 2.3200 0.24 4.39 0.15 78.45 3.4200 0.24 4.39 0.09 75.80 2.3100 24 3.6950 0.25 4.57 0.12 77.13 3.7300 0.25 4.57 0.16 78.89 3.7300 0.25 4.57 0.18 79.77 3.6300 29 4.0100 0.20 3.65 0.10 76.24 4.0450 0.21 3.65 0.10 76.24-3.9950 0.20 3.65 0.18 79.77 4.1050 36 4.1250 0.10 1.81 0.14 78.04 4.1800 0.12 2.18 0.15 78.45 4.2500 0.11 1.99 0.12 77.13 4.1350 FLASKS RUN HERRING SOLUBLES. 1% GLUCOSE ADDED: INITIAL CONCENTRATION Co=5.59 mg BSA/ml (3) Time .weight dry cells absorbance at Total proteins absorbance at Tyrosince cone. hrs mg/ml 540 mp mg BSA/ml 660 mjJ jj g/ml 53 4.30 0.05 0.89 0.10 76.24 4.320 0.04 0.70 0.20 80.65 3.835 0.05 . 0.89 0.20 80.65 4.370 60 3.845 0.05 0.89 0.17 79.33 3.890 0.06 1.07 0.25 82.86 3.925 0.06 1.07 0.17 79.33 3.880 89.5 2.905 0.06 1.07 0.18 79.77 2.875 0.06 1.07 0.10 76.24 2.800 0.03 0.52 0.12 77.13 2.845 97 2.340 0.04 0.70 0.23 81.87 2.445 0.03 0.52 0.17 79.33 2.435 0.04 0.70 0.12 77.13 2.320 " L09 2.155 0.09 1.62 0.16 78.89 2.195 0.09 1.62 0.10 . 76.24 2.150 0.08 1.44 0.08 75.36 2.160 'FLASKS RUN: HERRING SOLUBLES Co=17.48 mg BSA/ml 1% GLUCOSE ADDED (I) AX Time T AT weight dry c e l l s AX Growth rate — v \ \ AT hrs 0.0 4.0 7.0 8.0 10.0 11.0 12.0 24.0 25.0 27.0 28.0 hrs 4 3 1 2 1 1 12 1 2 1 mg/ml 1.34 1.96 1.96 2.39 2.46 2.63 3.42 3.47 3.37 3.66 mg/ml mg/ml hr Total Proteins P -&P Tyrosince Cone, mg BSA/ml mg BSA/ml j/g/ml 1.34 0.62 0.00 0.43 0.07 0.17 0.79 0.05 0.29 0.34 0.21 0.00 0.22 0.07 0.17 0.07 0.05 0.29 17.48 15.63 15.63 15.63 15.63 15.63 15.27 13.24 13.79 13.36 12.87 1.85 0.00 0.00 0.00 0.00 0.36 2.03 0.43 0.49 17.48 19.67 17.33 17.33 20.55 21.91 21.73 19.09 22.32 21.88 23.06 O -J FLASKS RUN: HERRING SOLUBLES: Co=17.48 mg BSA/ml 1% GLUCOSE ADDED (2) Time weight dry cells '(Protein) abs. Total proteins absorbance at Tyrosine cone. Enzyme activity hrs mg/ml 540 mp mg BSA/ml 660 mp pg/ml units/10 cc 0.0 .95 17.48 0.32 17.62 0.78 .95 17.48 0.33 18.06 0.80 .95 17.48 0.36 19.38 0.86 4 1.31 0.85 15.63 0.38 20.26 0.90 1.31 0.85 15.63 0.34 18.50 0.82 1.35 0.85 15.63 0.38 20.26 0.90 1.37 7 1.95 0.85 15.63 0.30 16.74 0.74 1.98 0.85 15.63 0.32 17.62 0.78 1.97 0.85 15.63 0.32 17.62 0.78 1.94 8 1.93- 0.85 15.63 0.30 16.74 0.74 1.93 0.85 15.63 0.32 . 17.62 0.78 2.01 0.85 15.63 0.32 17.62 0.78 1.95 10 2.38 0.85 15.63 0.38 20.26 0.90 2.42 0.85 15.63 0.38 20.26 0.90 2.35 0.85 15.63 ' 0.40 21.14 0.93 2.39 to o 00 FLASKS RUN: HERRING SOLUBLES: Co=17.48 mg BSA/ml 1% GLUCOSE ADDED (3) Time weight dry cells • (Protein) abs. * Total Proteins 'absorbance at •Tyrosine cone. 'Enzyme activity hrs mg/ml 540 mp mg BSA/ml 660 mjJ pg/ml units/10 cc 11 2.34 0.85 15.63 0.42 22.03 0.24 2.54 0.85 15.63 0.45 23.35 2.52 0.85 15.63 0.45 23.35 2.42 12 2.47 0.83 15.27 0.40 21.14 0.19 2.68 0.83 15.27 0.42 22.03 2.68 0.83 15.27 0.42 22.03 2.69 24 3.40 0.70 12.87 0.34 18.50 0.07 3.43 0.73 13.42 0.34 18.50 3.38 0.73 13.42 0.38 •20.26. 3.45 25 3.54 0.75 13.79 0.40 21.14 0.21 3.51 . 0.75 13.79 0.42 22.03 3.29 0.75 13.79 0.46 23.79 3.53 27 3.41 0.70 12.87 0.45 23.35 0.19 3.40 0.75 13.79 0.40 21.14 3.35 0.73 13.42 0.40 21.14 3.31 28 3.68 0.70 12.87 0.43 22.47 0.25 3.63 0.70 12.87 0.45 23.35 3.61 0.70 12.87 0.45 23.35 'FLASKS RUN: HERRING WASTE AT INITIAL CONCENTRATION Co ABOUT 20 MG (BSA)/ML (1) -Time T AT iweight dry cells A X , : Growth rate AX ,Total Proteins ( -&P v Tyrosince cone, direct tu: AT hrs hrs mg/ml mg/ml mg/ml hr mg BSA/ml mg BSA/ml Ug/ml 0.0 - 1.44 0.29 - - -5.0 1.44 18.65 40.53 0.33 5 -0.62 -0.12 -0.04 10.0 0.82 18.89 42.89 1.27 5 0.41 0.08 0.00 15.0 1.24 18.89 47.59 1.73 5 -0.01 -0.002 0.98 20.0 1.25 19.87 44.94 1.80 4.5 0.37 0.08 0.73 24.5 1.62 19.14 43.18 2.64 5.5 0.09 0.02 -0.60 30.0 1.71 19.74 59.33 _ 6.0 -0.11 -0.02 -0.13 36.0 1.60 19.87 44.94 _ 4.0 0.26 0.07 1.84 40.0 1.86 18.03 43.62 3.20 20.0 0.41 0.02 2.40 60.0 2.27 15.63 45.51 4.00 25.5 0.39 0.02 1.84 85.5 2.66 13.79 52.88 4.00 58.5 -2.18 -0.04 11.92 144.0 0.48 1.87 82.85 1.12 6.0 2.68 0.45 -7.31 150.0 3.16 9.18 - 3.44 1 FLASKS RUN: HERRING WASTE HIGH INITIAL PROTEIN CONCENTRATION (2) Time hrs weight'dry cells absorbance at Total proteins absorbance at Tyrosine cone. Enzyme direct activity mg/ml 540 mp mg BSA/ml 660 mp pg/ml units/lOcc turbidity 5.0 1.2350 1.2900 1.4800 1.7500 04 00 00 19.14 18.40 18.40 .80 .84 .88 38.77 40.51 42.30 1. 1. 1. 628 701 777 0.34 0.34 0.32 10.0 0.7800 0.8000 0.8300 0.8700 .04 ,04 ,00 19.14 19.14 18.40 0.90 0.88 0.90 43.18 42.30 43 .18 ,814 ,777 ,814 1.24 1.28 1.28 15.0 1.3050 1.3100 1.1300 1.2200 1, 1. 1. 08 00 00 19.87 18.40 18.40 1.00 1.00 47.59 47.59 999 999 1.70 1.80 1.70 20.0 1.3050 1.2550 1.2350 1.2150 08 08 19.87 19.87 0.96 0.92 45.82 44.06 1.924 1.851 1.80 1.80 24.5 1.4550 1.8050 1.7100 1.5200 ,04 ,04 19.14 19.14 0.90 0.90 43.18 43.18 1.814 1.814 2.64 2.64 30.0 1.7650 1.7100 1.6400 1. 1. 12 08 20.61 19.87 1.00 1.04 1.08 47.59 49.35 51.11 1.999 2.073 to FLASKS RUN: HERRING WASTE HIGH INITIAL PROTEIN'" CONCENTRATION (3) Time hrs. weight dry cells •absorbance at 'Total proteins 'absorbance at 'Tyrosine cone. 'enzyme direct activity mg/ml 540 mp mg BSA/ml 660 mp pg/ml units/lOcc turbidity 36.0 1.5100 1.08 19.87 0.96 45.82 2.147 1.6600 1.08 19.87 0.92 44.06 1.851 1.6300 - - - -1.5950 40.0 1.7550 0.96 17.66 0.90 43.18 1.814 3.20 2.0950 1.00 18.40 0.92 44.06 1.851 3.20 1.9850 - - - - 3.20 1.6150 60.0 2.2050 0.85 15.63 1.00 47.59 1.999 4.00 2.1750 0.85 15.63 0.96 45.82 2.147 4.00 2.1050 - - 0.90 43.18 1.814 -2.6100 85.5 2.6350 0.75 13.79 1.12 52.88 2.221 4.00 2.6400 0.75 13.79 1.12 52.88 2.221 4.00 2.7450 - - - - - -2.6150 144.0 0.4800 0.11 1.99 1.80 82.85 3.480 1.12 0.4750 0.10 1.81 1.80 82.85 3.480 1.12 0.5000 0.10 1.81 - - - -- 0.4650 150.0 3.1150 0.50 9.18 _ _ 3.44 3.2250 0.50 9.18 3.44 3.1500 0.50 9.18 3.44 3.1400 213 APPENDIX II 7-I FERMENTOR RUNS INITIAL PROTEIN CONCENTRATION AT ABOUT 4 mg (BSA) /ml EXPERIMENTAL DATA RUN FERMENTOR // 1.1 DISSOLVED OXYGEN FIXED AT DO=100% SATURATION INITIAL PROTEIN CONCENTRATION Co=4.14 mg(BSA)/ml T AT X AX AX/AT P AP G AG E AE PH Fermentation Change -in Weight Change in Growth Total Change in Glucose Change in Enzyme Change in Time Time dry weight rate Protein Protein Content Glucose Activity Enz. Act. cells .  hrs hrs mg/ml mg/ml mg(BSA) mg(BSA)/ mg/ml mg/ml unit/ unit/ /ml ml 10 cc 10 cc  0.00 0.00 4.14 6.28 0.00 7.8 3.00 +0.41 +0.137 -1.47 +0.56 +0.19 3.00 0.41 2.67 6.84 0.19 7.7 2.00 +1.01 +0.505 -1.05 -0.88 +0.07 5.00 1.42 1.62 5.96 0.26 7.4 7.75 +1.42 +0.183 -0.06 +5.88 +0.73 12.75 2.84 1.56 11.74 0.99 6.7 11.25 +0.32 +0.028 -0.31 (10.0) -8.00 +0.06 24.00 3.16 1.25 3.74 1.05 6.3 5.00 -0.24 -0.048 +0.00 -0.98 +0.10 29.00 2.92 1.25 2.76 1.15 6.8 5.00 +0.00 +0.000 -0.36 -0.41 +0.14 34.00 2.92 0.89 2.35 1.29 7.0 13.50 -0.08 -0.006 +0.12 -0.69 +1.18 47.50 2.84 1.01 1.66 2.47 7.1 8.50 -0.24 -0.028 +0.24 -0.27 -0.58 56.00 2.68 1.25 1.39 1.89 7.1 14.50 +0.05 +0.003 -0.42 -0.29 -0.04 70.50 2.73 0.83 1.10 1.85 7.5 9.00 -0.61 -0.068 -0.00 -0.14 -1.01 79.50 2.12 0.83 0.96 0.84 4.0 16.00 -0.52 -0.033 +0.85 -0.06 +0.15 95.5 1.60 1.68 0.90 0.99 8.0 RUN FERMENTOR #1.2 DISSOLVED OXYGEN FIXED AT DO=80% SATURATION INITIAL PROTEIN CONCENTRATION Co=5.31 mg BSA/ml , ; r T AT -X AX AX/AT ,P AP G VAG ,E VAE PH Fermentation Change in Weight Change in Growth Total Change in Glucose Change in Enzyme Change in Time Time dry weight rate Protein Protein Content Glucose Activity Enz. Act. cells hrs hrs mg/ml mg/ml mg(BSA) mg(BSA)/ mg/ml mg/ml unit/ unit /ml ml 10 cc 10 cc . 0.00 0.00 5.31 6.325 0.00 7.0 5.00 +0.96 +0.192 -2.58 -0.235 +0.18 5.00 0.96 2.73 6.015 0.18 7.7 5.00 +0.71 +0.142 +0.06 -3.710 +0.12 10.00 . 1.67 2.79 2.305 0.30 5.0 11.50 +0.33 +0.029 -0.37 +0.225 +0.04 21.50 2.00 2.42 2.530 0.34 7.8 6.50 +0.07 +0.011 +0.00 -0.730 -0.08 28.00 2.07 2.42 1.800 0.26 7.9 12.00 -0.04 -0.003 -0.18 -0.740 +0.02 40.00 2.03 .2.24 1.060 0.28 "" 8.1 10.00 +0.06 +0.006 -0.37 -0.259 -0.01 50.00 2.09 1.87 0.801 0.27 9.00 -0.30 -0.033 +0.49 -0.111 +0.08 59.00 1.79 2.36 0.690 0.35 8.15 11.00 -0.21 -0.019 -0.37 +0.070 +0.24 70.00 1.58 1.99 0.760 0.59 8.25 11.25 -0.17 -0.015 -0.06 -0.200 +0.24 81.25 1.41 1.93 0.560 0.83 11.50 -0.09 -0.008 -0.43 +0.010 0.51 93.00 1.32 1.50 0.570 "' 0.32 7.0 r - 1 RUN FERMENTOR #1.3 NO GLUCOSE ADDED DISSOLVED OXYGEN FIXED AT D0=80% SATURATION INITIAL PROTEIN CONCENTRATION Co=4.08 mg BSA/ml T AT X AX AX/AT ,P AP PH Fermentation Change in Weight Change in Growth Total Change in Time Time dry- weight rate Protein Protein cells hrs hrs mg/ml mg/ml mg(BSA) mg (BSA)/ /ml ml 0.00 0.00 4.08 7.0 5.00 +0.58 0.115 -2.58 5.00 0.58 1.50 7.8 4.25 -0.01 -0.002 -0.67 1 i 9.25 0.57 0.83 i 8.2 13.00 +0.14 +0.011 -0.13 22.25 0.71 0.70 8.6 5.00 -0.07 -0.014 +0.00 27.25 0.64 0.70 8.5 4.75 -0.11 -0.023 -0.06 32.00 0.53 0.64 8.3 4.00 -0.10 -0.025 -0.12 36.00 0.43 0.52 8.5 10.00 -0.03 -0.003 +0.18 46.00 0.40 0.70 8.4 4.00 -0.05 -0.013 -0.31 50.00 0.35 0.39 7.8 5.25 -0.01 -0.002 -0.06 55.25 0.34 0.33 7.8 15.00 +0.09 -0.006 -0.06 70.25 0.43 0.39 8.2 5.00 -0.16 -0.032 +0.31 75.25 0.27 0.70 6.2 j 19.75 +0.00 +0.000 -0.13 | j 95.00 0.27 0.57 7.3 ' 7.25 +0.15 +0.021 -0.18 ; 102.25 0.42 0.39 i 7.7 ! RUN FERMENTOR #1.4 DISSOLVED OXYGEN FIXED AT DO=50% SATURATION INITIAL PROTEIN CONCENTRATION Co=4.88 mg BSA/ml •T A T X A X A X / A T P A P G A G E A E PH .Fermentation Change in Weight Change in Growth Total Change in 'Glucose (Change in xEnzyme 'Change in Time Time dry weight rate Protein Protein Content Glucose Activity Enz. Act. cells hrs hrs mg/ml mg/ml mg(BSA) mg(BSA)/ mg/ml mg/ml unit/ unit/ /ml ml 10 cc 10 cc 0.00 0.00 4.88 5.55 0.00 6.2 5.00 0.67 0.134 -2.58 -1.08 0.00 5.00 0.67 2.30 4.47 0.00 6.85 3.00 +0.99 0.333 -1.17 +0.62 +0.22 8.00 1.66 1.13 5.09 0.22 7.3 5.00 +0.78 0.156 -0.15 +4.33 +0.07 13.00 2.44 0.98 9.42 0.29 6.75 10.00 +0.00 0.000 -0.09 -7.66 +0.82 23.00 2.44 0.89 1.76 1.11 6.0 5.00 -0.04 -0.008 -0.06 -0.21 -0.28 28.00 2.40 0.83 1.55 0.83 6.3 7.00 -0.27 -0.039 -0.25 -0.22 -0.18 35.00 2.13 0.58 1.35 1.01 6.6 11.00 +0.000 +0.000 +0.31 -0.11 -0.26 47.00 2.13 0.89 1.24 0.75 7.0 9.00 +0.14 +0.016 +0.24 -0.02 -0.31 56.00 2.27 1.13 1.22 0.44 7.1 15.00 +0.00 +0.000 -0.55 +0.09 +0.04 71.00 2.27 0.58 1.31 0.48 7.4 12.00 -0.14 -0.012 +0.18 -0.01 -0.14 83.00 2.13 0.76 1.30 0.34 7.45 12.00 +0.06 +0.005 -0.06 +0.00 +0.00 95.00 2.19 0.70 1.30 0.34 7.60 HUN FERMENTOR #1.5 DISSOLVED OXYGEN FIXED AT DO=30% SATURATION INITIAL PROTEIN CONCENTRATION Co=5.49 mg BSA/ml T AT X AX AX /AT P AP E PH Fermentation Change i n Weight Change in Growth Total Change in Enzyme Change in Time Time dry- Cells rate Protein Protein Activity Enz. Act. cells hrs hrs mg/ml mg/ml mg(BSA) mg(BSA)/ unit/ /ml ml 10 cc 0.00 0.00 5.49 0.00 7.0 9.25 2.64 0.285 . -3.62 +0.26 9.25 2.64 1.87 . 0.26 5.3 3.75 -0.42 -0.112 +0.72 +0.27 13.00 2.22 2.59 0.53 4.66 4.50 +0.29 0.064 ; -0.32 -0.06 17.50 2.51 2.27 0.47 6.47 15.50 +0.72 0.046 1 -0.28 +0.04 33.00 3.23 1.99 0.51 7.77 6.00 -0.15 -0.025 -0.09 -0.23 39.00 3.08 1.90 0.28 7.8 7.50 -0.15 -0.020 +0.19 +0.00 46.50 2.93 2.09 0.28 7.8 12.50 -0.16 -0.013 -0.71 -0.02 59.00 2.77 1.38 0.26 7.10 5.00 -0.09 -0.018 +0.006 +0.15 64.00 2.68 1.44 * 0.41 7.3 4.25 +0.06 +0.014 -0.19 +0.04 68.25 2.74 i.25 : 0.45 7.5 14.25 -0.73 -0.053 i +0.19 -0.21 82.50 2.01 1.44 0.24 7.5 7.50 +0.44 -0.059 +0.18 +0.08 90.00 2.45 1.62 0.32 7.6 15.00 0.00 0.00 i -0.13 .-0.02 ,RUN FERMENTOR #1.6 (DISSOLVED OXYGEN FIXED AT D0=30% SATURATION INITIAL PROTEIN CONCENTRATION Co=5.49 mg BSA/ml T AT X AX 4X/AT P AP G AG E 4E PH Fermentation Change in Weight Change in Growth Total Change in Glucose Change in Enzyme Change Time Time dry cells Cells rate Protein Protein content Glucose Acti-vity in enz. act. hrs. hrs mg/ml mg/ml X/ T mg(BSA) /ml mg(BSA) 7ml mg/ml mg/ml unit/ 10 cc unit/ 10 cc 105.00 10.00 2.45 -0.39 -0.039 1.49 -0.18 0.30 +0.04 7.8 115.00 19.00 2.06 -0.32 -0.017 1.31 +0.00 0.34 -0.06 7.15 134.00 1.74 1.31 0.28 — RUN FERMENTOR #1.7 DISSOLVED OXYGEN FIXED. AT D0=0% SATURATION INITIAL PROTEIN CONCENTRATION Co=5.49 mg BSA/ml T AT X AX AX/AT P AP G AG E &E PH Fermentation Change in 'Weight Change in Growth Total Change in Glucose Change in Enzyme Change ' v Time Time dry Cells rate Protein Protein content Glucose Acti- in Enz. cells vity act. hrs. hrs mg/ml mg/ml X/ T mg(BSA) mg(BSA) mg/ml mg/ml unit unit : /ml /ml 10 cc 10 cc  0.00 0.00 5.49 0.942 -0.10 0.00 6.8 3 +0.26 0.087 -1.35 0.00 3 0.26 4.14 0.84 0.00 11 +0.10 0.009 +1.60 -0.07 0.23 14 0.36 5.74 0.77 0.23 4.5 6 -0.05 -0.008 -0.37 +0.05 -0.01 20 0.31 5.37 0.82 0.22 . 2.7 7 -0.03 -0.004 -0.18 +0.13 27 0.29 5.19 0.95 0.18 2.7 11 +0.13 +0.012 -0.68 +0.16 +0.05 38 0.42 4.51 1.11 0.23 3.8 5 -0.14 -0.028 +0.25 -0.16 +0.08 43 0.28 4.76 0.95 0.31 4.4 8 +0.04 +0.005 -0.50 ^0.00 -0.12 51 0.32 4.26 0.95 0.19 4.5 10 +0.02 +0.002 -0.86 -0.09 -0.19 61 0.34 3.40 0.86 0.00 4.5 APPENDIX III 7-liter FERMENTOR RUNS HIGH INITIAL PROTEIN CONCENTRATION EXPERIMENTAL DATA 'RUN FERMENTOR #2.1 DISSOLVED OXYGEN FIXED AT DO=100% SATURATION INITIAL PROTEIN CONCENTRATION Co=15.63 mg BSA/ml : : LA T AT X AX AX/AT P AP AP/AT E AE AE/AT Tb 1 Fermen- Change Weight: Change Growth1 Total 'Change i n 1 Change in'Prot. Enzyme 1 Change iii Change iii Direct tation in dry in rate Proteins Protein per hour Activity Enz. Act. Enz. Turbidity Time Time Cells Weight per hr hrs hrs mg/ml mg/ml mg/ml mg BSA/ml mg BSA/ml mg BSA/ml x hr unit/lOcc unit/lOcc unit/lOcc x hr x hr 0 15.63 0.73 0.54 4 - - -0.920 -0.230 0.040 0.010 4 - 14.71 0.77 1.50 2 1.14 0.570 -2.210 -1.105 0.000 0.000 6 1.14 12.50 0.77 1.86 1 0.58 0.580 -2.950 -2.950 -0.040 -0.040 7 1.72 9.55 0.73 2.40 1 0.35 0.350 -1.960 -1.960 0.010 0.010 8 2.07 7.59 0.74 2.40 2 0.25 0.125 -3.450 -1.467 -0.200 -0.100 10 2.32 4.14 0.54 2.40 2 -0.36 -0.180 0.000 0.000 0.040 0.020 12 1.96 4.14 0.58 2.55 2 -0.15 -0.075 -1.110 -0.555 0.000 0.000 14 1.81 3.03 0.58 1.80 1 -0.64 -0.64 0.130 0.130 0.000 0.000 15 1.17 3.16 0.58 1.80 1 0.56 0.560 -0.060 -0.060 0.000 0.000 16 1.73 3.10 0.58 1.80 11 -0.33 -0.033 -0.560 -0.051 0.300 0.027 27 1.40 2.54 0.88 1.80 2 -0.39 -0.195 -0.340 -0.170 0.170 0.085 29 1.01 2.24 1.05 2.10 RUN FERMENTOR #2.2 DISSOLVED OXYGEN FIXED AT DO=70% SATURATION INITIAL PROTEIN CONCENTRATION Co=11.21 mg BSA/ml T AT. X AX AX/AT P AP AP/flT E AE AE/AT T b Fermen- Change Weight Change Growth Total ,Change in Change in Prot. Enzyme Change in Change in Direct tation in dry in rate Proteins Protein per hour Activity Enz. Act. Enz. Turbidity Time Time Cells Weight per hr hrs hrs mg/ml mg/ml mg/ml mg BSA/ml mg BSA/ml mg BSA/ml x hr unit/lOcc unit/lOcc unit/lOcc x hr x hr 0 _ 11.21 0.05 2 0.930 0.465 -1.380 -0.690 0.000 0.000 2 0.93 9.83 0.05 1.05 1 0.710 0.710 -2.120 -2.120 0.010 0.010 3 1.64 7.71 0.06 1.80 1 0.670 0.670 -2.400 -2.400 -0.020 -0.020 4 2.31 5.31 0.04 2.25 2 0.530 0.265 -1.940 -0.970 0.020 0.010 6 2.84 3.37 0.06 2.65 2 -0.350 -0.175 -0.640 -0.320 0.050 0.025 8 2.49 2.73 0.11 2.50 1 -0.140 -0.140 -0.550 -0.550 0.700 0.700 9 2.35 2.18 0.81 2.40 1 -0.170 -0.170 -0.280 -0.280 0.050 0.050 10 2.18 1.90 0.86 2.25 1 -0.330 -0.330 -0.460 -0.460 0.020 0.020 11 1.85 1.44 0.84 1.82 RUN FERMENTOR #2.3 DISSOLVED OXYGEN FIXED AT DO=60% SATURATION INITIAL PROTEIN CONCENTRATION Co=15.02 mg BSA/ml T AT X AVX AX/AT (P AP AP/AT E AE AE/AT Tb Fermen- Change Weight -Change Growth (Total Change in phange in Prot. Enzyme Change in Change in Direct tation- in dry in Rate Proteins Protein per hour Activity Enz. Act. Enz. Turbid: Time Time Cells Weight per hr hrs hrs mg/ml mg/ml mg/ml mg BSA/ml mg BSA/ml mg BSA/ml x hr unit/lOcc unit/lOcc unit/lOcc x hr x hr 0.0 15.02 0.87 0.09 8.5 1.35 0.159 -0.800 -0.094 0.120 0.014 8.5 1.35 14.22 0.99 0.96 0.5 0.350 0.700 -0.430 -0.860 0.020 0.040 9.0 1.70 13.79 1.01 1.47 1.0 0.740 0.740 -2.270 -2.270 -0.060 -0.060 10.0 2.44 11.52 0.95 2.25 1.5 0.280 0.187 -2.220 -1.480 -0.040 -0.027 11.5 2.72 9.30 0.91 2.85 1.5. -0.250 -0.167 -1.720 -1.147 0.040 0.027 13.0 2.47 7.58 0.95 2.85 1.0 -0.300 -0.300 -0.490 -0.490 -0.170 -0.170 14.0 2.17 7.09 0.78 2.55 1.0 -0.470 -0.470 -0.120 -0.120 0.020 0.020 15.0 •1.70 6.97 0.80 0.60 1.0 -0.070 • -0.070 0.000 0.000 0.120 0.120 16.0 1.63 6.97 0.92 1.05 2.0 -0.130 • -0.065 -0.550 -0.275 0.420 0.210 18.0 1.50 6.42 1.34 1.05 2.0 -0.300 • -0.150 0.000 0.000 -0.470 -0.235 20.0 1.20 6.42 0.87 0.90 13.0 0.090 0.007 -2.220 -0.171 -0.040 -0.003 33.0 1.29 4.20 0.83 0.90 rRUN FERMENTOR #2.4 'DISSOLVED OXYGEN FIXED AT DO=30% SATURATION INITIAL PROTEIN CONCENTRATION Co=11.39 mg BSA/ml t AT X- AX/AT P AP AP/AT E AE AE/AT Tb Fermen- Change Weight Growth Total Change in Change in Prot. Enzyme Change in Change in Direct tation in Dry Rate Proteins Protein per hour Activity Enz. Act. Enz. Turbid: Time Time Cells per hr. hrs hrs mg/ml • mg/ml mg BSA/ml mg BSA/ml mg BSA/ml x hr unit/lOcc unit/lOcc unit/lOcc x hr x hr 0.00 11.39 0.93 0.12 3.00 0.120 0.000 0.000 0.200 0.067 3.00 0.36 11.39 1.13 0.10 3.00 0.873 2.400 0.800 -0.200 -0.067 6.00 2.98 13.79 0.93 0.70 0.67 4.463 0.920 1.373 0.200 0.299 6.67 5.97 14.71 1.13 0.95 0.830 -4.482 -3.690 -4.446 -0.100 -0.120 7.50 2.25 11.02 1.03 1.11 1.500 0.840 -2.950 -1.967 -0.040 -0.027 9.00 3.51 8.07 0.99 2.70 0.75 0.040 -0.830 -1.107 0.130 0.173 9.75 3.54 7.24 1.13 2.85 1.25 0.696 -0.890 -0.712 0.050 0.040 11.00 4.41 6.35 1.18 3.00 1.00 -0.160 -0.030 -0.030 0.140 0.140 12.00 4.25 6.32 1.32 1.32 1.50 0.440 -0.370 -0.247 -0.190 -0.127 13.50 4.91 •5.95 1.13 3.30 15.00 -0.050 -1.560 -0.104 0.100 0.007 28.50 4.16 4.39 1.23 2.85 N) ' RUN FERMENTOR #2.5 DISSOLVED OXYGEN FIXED AT DO=20% SATURATION INITIAL PROTEIN CONCENTRATION Co=15.63 mg BSA/ml T AT X AX/AT P AP AP/AT ;E AE AE/AT Tb Fermen- Change Weight 'Growth Total Change in Change in Prot. Enzyme 'Change in Change in Direct tation in Dry- Rate Proteins Protein per hour Activity Enz. Act. Enz. Turbid: Time Time Cells per hr. hr-s hrs mg/ml mg/ml mg BSA/ml mg BSA/ml mg BSA/ml x hr unit/lOcc unit/lOcc unit/lOcc x hr x hr 0.0 15.63 0.97 2.0 0.725 -0.920 -0.460 -0.090 -0.045 2.0 1.45 14.71 0.88 0.94 1.0 0.580 -1.470 -1.470 0.010 0.010 3 2.03 13.24 0.89 0.78 1.0 0.900 -3.080 -3.080 0.000 0.000 4 2.93 10.16 0.89 1.56 1.0 0.800 -1.290 -1.290 0.250 0.250 5 3.73 8.87 1.14 2.55 1.0 0.660 -2.040 -2.040 -0.140 -0.140 6 4.39 6.83 1.00 3.00 2.0 0.525 -2.440 -1.220 -0.140 -0.070 8 5.44- 4.39 0.86 6.12 1.0 -0.350 -1.050 -1.050 -0.030 -0.030 9 5.09 3.34 0.83 6.12 2.0 -0.070 -0.610 -0.305 -0.140 -0.070 11 4.95 2.73 0.69 6.12 1.0 -0.310 -0.550 -0.550 -0.020 -0.020 12 4.64 2.18 0.67 5.22 12.0 -0.094 0.180 0.015 -0.020 -0.002 24 3.51 2.36 0.65 4.86 1.0 -0.490 0.000 0.000 0.050 0.050 25 3.02 2.36 0.70 4.86 2.0 0.010 0.000 0.000 -0.030 -0.015 27 3.04 2.36 0.67 4.86 RUN FERMENTOR #2.6 DISSOLVED OXYGEN FIXED AT DO=0% SATURATION INITIAL PROTEIN CONCENTRATION Co=13.79 mg BSA/ml >T AT X AX/AT P ' AP AP/AT E AE AE/AT T, D Fermen- Change Weight Growth Total Change in ,Change in Prot. Enzyme Change in Change in Direct t a t i o i in Dry Rate Proteins Protein per hour Activity Enz. Act. Enz. Turbidity Time Time Cells per hr hrs hrs mg/ml mg/ml mg BSA/ml mg BSA/ml mg BSA/ml x hr unit/lOcc unit/lOcc unit/lOcc x hr x hr 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 15.0 25.0 26.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 1.0 10.0 1.0 0.83 1.92 1.64 1.83 1.41 0.45 0.43 0.43 0.45 1.42 0.415 0.545 -0.140 0.095 -0.210 -0.480 -0.010 0.000 0.010 0.970 13.79 13.79 13.79 12.87 13.42 12.87 12.81 11.02 11.02 11.02 11.02 0.000 0.000 -0.920 0.550 -0.550 -0.060 -1.790 0.000 0.000 0.000 0.000 0.000 -0.460 0.275 -0.275 -0.030 -0.895 0.000 0.000 0.000 0.81 0.86 0.83 0.81 0175 0.79 0.70 0.74 0.70 0.70 0.70 0.050 -0.030 -0.020 -0.060 0.040 -0.090 0.040 -0.040 0.000 0.000 0.025 -0.015 -0.010 -0.030 0.020 -0.045 0.020 -0.040 0.000 0.000 0.58 0.54 0.48 0.54 0.50 0.56 0.54 0.53 0.54 0.56 APPENDIX IV 7-liter Fermentor Runs High Initial Protein Concentration TURBIDITY MEASUREiyLFJSITS 229 FERMENTOR RUN #2.1 DISSOLVED OXYGEN FIXED AT DO = 100% SATURATION INITIAL PROTEIN CONCENTRATION C Q=15.63 MG (BSA)/ML DIRECT TURBIDITY vs FFINMENTATION TIME (HRS.) a cn cn co in' r -tp to m tn " a ^ t n CQcn O - t n cp X X X X X X X X X X X X a 0.0 B.O 16.0 24.0 TIME HRS 32.0 40.0 4B.0 230 FERMENTOR RUN #2.2 DISSOLVED OXYGEN FIXED AT DO = 70% SATURATION INITIAL PROTEIN CONCENTRATION C =11.21 MG (BSA) /ML DIRECT TURBIDITY vs FERMENTATION TIME (HRS.) X X X 0.0 B.D 16.D 24.0 TIME HRS 32.D 40.0 48.D 231 FERMENTOR RUN #2.3 , DISSOLVED OXYGEN FIXED AT DO = 60% SATURATION INITIAL PROTEIN CONCENTRATION CQ=15.02 MG (BSA)/ML DIRECT TURBIDITY vs FERMENTATION TIME (HRS.) o X X X X X X X X X X X 0.0 a.o ~i 1 16.0 24.0 TIME HRS 32.0 40.0 48. FERMENTOR RUN #2.4 DISSOLVED OXYGEN FIXED AT DO = 30% SATURATION INITIAL PROTEIN CONCENTRATION C0=11.39 MG (BSA)/ML DIRECT TURBIDITY V S FERMENTATION TIME (HRS.) X X B.O 16.0 24.0 TIME HRS 32.0 40 233 o «n LT) ' to U 3 m tn' a — ' m ca<n to FERMENTOR RUN #2.5 DISSOLVED OXYGEN FIXED AT DO = 20% SATURATION INITIAL PROTEIN CONCENTRATION CQ=15.63 MG (BSA)/ML DIRECT TURBIDITY V S FERMENTATION TIME (HRS.) X X X X X X X X X a -I- 1 1 1 I I I 0 0 B.D 16.0 24.0 32.0 40.0 48.0 TIME HRS 234 o FFJ3MENT0R RUN #2.6 DISSOLVED OXYGEN FIXED AT DO=0% SATURATION INITIAL PROTEIN CONC0SITRATION C0=13.79 MG (BSA)/ML DIRECT TURBIDITY vs FERMENTATTON TIME (HRS.) x x x x x x x x X X 1 1 1 r i i 0 0 B.O 16.0 24.0 32.0 40.0 48.0 TIME HRS APPENDIX V PH CONTROL 7-liter FERMENTOR RUNS RUNS AT LOW INITIAL PROTEIN CONTENT (around 4 MG (BSA)/ML) FERMENTOR RUN #1.1 DISSOLVED OXYGEN FIXED AT DO = 100% SATURATION INITIAL PROTEIN CONCENTRATION C 0=4.14 MG (BSA)/ML PH versus FFJNMENTATICN TIME (HOURS) o.o 40.0 — ! 80.0 TIME HRS i 120.0 160.0 237 FERMENTOR RUN #1.2 DISSOLVED OXYGEN FIXED AT DO = 80% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.31 MG (BSA)/ML PH versus FERMENTATION TIME (HOURS) a a-, . <!> O O <J> <!><!> <!> a to " a a CM ' 1 1 1 1 0.0 40.0 80.0 120.0 160.0 TIME HRS FEPJyENTOR RUN #1.3 DISSOLVED OXYGEN FIXED AT DO = 80% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4.08 MG (BSA)/ML PH versus FERMENTATION TIME (HOURS) •RUTS' WITHOUT GLUCOSE o o v o o oo o o o - I I I 1 0.0 40.0 80.0 1 20.0 160 0 TIME HRS FERMENTOR RUN #1.4 DISSOLVED OXYGEN FIXED AT DO = 50% SATURATION INITIAL PROTEIN CONCENTRATION CQ=4..88 MG (BSA)/ML PH versus FERMENTATION TIME (HOURS) 0 <J> <!> <t> <j> <$> o.o 40.0 "I 80.0 TIME HRS 120.0 150.0 240 CD a a*-I ZTL a_ a cn"' a co" 0 FERMENTOR RUN #1.5 DISSOLVED OXYGEN FIXED AT DO = 30% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.49 MG (BSA)/ML PH versus FERMENTATION (HOURS) <!> <!> <!> <!> <!> 1 1 1 1 0.0 40.0 80.0 120.0 160.0 TIME HRS FERMENTOR RUN #1.6 DISSOLVED OXYGEN FIXED AT DO .= 0% SATURATION INITIAL PROTEIN CONCENTRATION CQ=5.74 MG (BSA)/ML PH versus FERMENTATION TIME (HOURS) "1— 40.0 1 160.0 

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