Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Protein generation and delignification of alder sawdust by thermophilic microorganisms Wolde-Tsadick, Maheteme Selassie 1978

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

Item Metadata

Download

Media
831-UBC_1978_A6_7 W64.pdf [ 4.64MB ]
Metadata
JSON: 831-1.0094543.json
JSON-LD: 831-1.0094543-ld.json
RDF/XML (Pretty): 831-1.0094543-rdf.xml
RDF/JSON: 831-1.0094543-rdf.json
Turtle: 831-1.0094543-turtle.txt
N-Triples: 831-1.0094543-rdf-ntriples.txt
Original Record: 831-1.0094543-source.json
Full Text
831-1.0094543-fulltext.txt
Citation
831-1.0094543.ris

Full Text

PROTEIN GENERATION AND DELIGNIFICATION OF ALDER SAWDUST BY THERMOPHILIC MICROORGANISMS  by Maheteme Selassie/Wolde-Tsadick M.Sc., Oklahoma StateTTniversity, 1970  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in The Faculty of Graduate Studies (Department of Animal Science)  We accept the thesis as conforming to the required standard  The University of B r i t i s h Columbia May, 1978  ©  Maheteme  SelassieIWolde-Tsadick  In presenting this thesis in p a r t i a l f u l f i l l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the l i b r a r y shall make i t freely available for reference and study.  I  further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of the Department or by his representatives. It  is understood that copying or publication of this thesis for f i n a n c i a l  gain shall not be allowed without my written permission.  Maheteme Selassie Wolde-Tsadick  University of B r i t i s h Columbia Department of Animal Science Vancouver, B.C.  ABSTRACT It  has been indicated through a review of the l i t e r a t u r e that  wood by-products have a potential as a dietary source of energy for the ruminant animal.  However, l i g n i n constitutes a b a r r i e r to the proper u t i l i z -  ation of c e l l u l o s e .  Generally, any treatment to remove or a l t e r  lignin  makes the c e l l u l o s e within 1ignocellulose materials more susceptible to the a c t i v i t y of the c e l l u l o l y t i c enzymes. logical  Therefore, an e f f i c i e n t b i o -  treatment would require a system to s o l u b i l i z e or to remove l i g n i n  from the 1ignin-carbohydrate complex.  Cellulose within ruminant feeds  forms an e f f e c t i v e substrate for eventual conversion to body protein. There are several methods a v a i l a b l e for d e l i g n i f i c a t i o n .  This  study was c a r r i e d out using the thermophilic aerobic oxidation method for which swine manure was used both as the source inocula and i n i t i a l culture media.  In the process of degradation, a part of the energy produced was  u t i l i z e d by the bacteria for c e l l function and m u l t i p l i c a t i o n . mainder of the available energy was released as heat energy.  The r e In  this  method the heat necessary to maintain the temperature in the thermophilic range was derived from both mechanical and from microbial a c t i v i t y . Thermophilic a c t i v i t y i s considered to reduce the time required for organic waste digestion over that experienced by mesophilic digestion.  The rate  of the destruction of pathogenic b a c t e r i a , virus and other organisms is increased as a r e s u l t of the high temperatures fermentation. Batch studies conducted to d e l i g n i f y alder sawdust by the use of the aerobic thermophilic oxidation method demonstrated that the l i g n i n content of sawdust can be reduced by as much as 74%, and crude bacterial protein was generated by approximately 17%.  iii Constant supply of small amounts of swine manure ensures high temperature maintenance.  Periodical addition of 2 kg sawdust within  thermophilic temperature range results in better d e l i g n i f i c a t i o n .  i'Y  TABLE OF CONTENTS Page I. II.  INTRODUCTION  • • •  ix  LITERATURE REVIEW  1  1.  Basic Biological Processes  1  A.  Biochemical Reactions  1  B.  The Energy Relationships in the System  3  C.  Biochemical Transformations  5  2.  a) b) c)  Carbon Nitrogen Nitrification (i) Dissolved oxygen . (ii) Temperature (iii) pH  6 6 .7 8 9 9  d)  Denitrification  10  e)  Phosphorus  10  Thermophilic Microorganisms  10  A.  11  Origin and Distribution a) b) c)  B.  Division of Thermophiles Growth at High Temperature Growth at Low Temperature  11 14 15  . .  Potential of Thermophilic Aerobic Digestion of Organic Solids  16  C.  Nutritional Requirements .  18  D.  The E f f e c t of Temperature on the Nutritional Requirement  21  E.  Energy Metabolism  22  F.  Oxidative Metabolism  23  G.  Transformation of Nitrogenous Compounds  24  H.  Decomposition of Natural High Polymeric Materials  . . . .  25  Page 3.  III.  IV.  Wood and Its  Potential In Ruminant Diet .  26  A.  Lignin Distribution in Wood Tissues  .  B.  Lignin  30  C.  Del i g n i f ication  31  MATERIALS AND METHODS  37  1.  Materials  37  2.  Methods of Culturing the Microorganisms-.  .  . . . .  Stage of Procedure for Substrate Preparation  41  A.  Initial  . .  B.  Culturing Parameters  42  a) b)  Temperature pH  42 43  c)  Dissolved oxygen  43  41  C.  Delignif ication  44  D.  Harvesting  • •  45  E.  Drying  • .  45  F.  Nutritional Value . .  • •  46  G.  Analyses  46  RESULTS AND DISCUSSION  48  1.  Environmental Parameter  48  2.  Effect of Aerobic Thermophilic Bacteria on Lignin Content of Alder  V.  29  55  Relationship Between Lignin Removal and Removal of Cellulose  57  3.  In Vitro D i g e s t i b i l i t y Test  59  4.  Crude Protein  67  SUMMARY AND CONCLUSION BIBLIOGRAPHY VITA  .' . . .  . . . . . .  80 81 93  vi  LIST OF FIGURES Page 1.  Schematic of fermenter  38  2.  Mixing action  39  3.  Mixing action  40  4.  Relevant change in environmental variables during treatment Batch I  49  Relevant change in environmental variables during treatment Batch II  50  Relevant change in environmental variables during treatment Batch III  51  Results of d e l i g n i f i c a t i o n to c e l l u l o s e content and in v i t r o d i g e s t i b i l i t y in terms of treatment duration and amount of sawdust added - Batch I  63  Results of d e l i g n i f i c a t i o n to c e l l u l o s e content and in v i t r o d i g e s t i b i l i t y in terms of treatment duration and amount of sawdust added - Batch II  64  Results of d e l i g n i f i c a t i o n to c e l l u l o s e content and in v i t r o d i g e s t i b i l i t y in terms of treatment duration and amount of sawdust added - Batch III  65  10.  The nitrogen cycle  72  11.  Results of the rate of d e l i g n i f i c a t i o n and environmental variables in terms of the amount of sawdust added and duration of treatment of three batches combined . . . . . . .  78  Results of crude protein value and environmental variables in terms of treatment of the three batches combined .  79  5. 6. 7.  8.  9.  12.  vii  LIST OF TABLES Page 1.  The d i s t r i b u t i o n of l i g n i n in black spruce earlywood  29  2.  Chemical composition and in v i t r o d i g e s t i b i l i t y of supernatant and processed products of alder sawdust - Batch I  60  Chemical composition and in v i t r o d i g e s t i b i l i t y of supernatant and processed products of alder sawdust - Batch II  61  Chemical composition and in v i t r o d i g e s t i b i l i t y of supernatant and processed products of alder sawdust - Batch III  62  Nitrogen composition of the waste and/or wood substrate at i n i t i a l and f i n a l stage of processing  70  3. 4. 5. 6. 7. 8.  Results of crude protein value and environmental variables in terms of sawdust volume and treatment time - Batch I  . . .  75  Results of crude protein value and environmental variables in terms of sawdust volume and treatment time - Batch II  . . .  76  . .  77  Results of crude protein value and environmental variables in terms of sawdust volume and treatment time - Batch III  vi i i  ACKNOWLEDGEMENTS  Dean W. D.  Kitts served as advisor to the author during the entire  period of this study.  His understanding, guidance, and unsurpassing  kindness are deeply appreciated  and w i l l be long remembered.  Special appreciation is also expressed to Dr. D. B. Bragg, Department of Poultry Science for his help in the protein evaluation technique and for the useful suggestions he made in connection with the writing of the manuscript. The author is indebted to the members of my Graduate Studies Committee for t h e i r advice and suggestions in the preparation of the manuscript:  Dr. B. Owen, Department of Animal Science, Dr. P.M. Townsley,  Department of Food Science, and Dr. C R . Animal Science.  Krishnamurti, Department of  Sincere thanks is also expressed to Professor C. L.  Coulthard, Dr. C. Cross, and P. William for t h e i r invaluable counselling in regard of fermentation technique and use of materials and equipment. A note of appreciation is due to the Department of Animal Science for the space and f a c i l i t i e s which have greatly enhanced the nature of the study. The author is very grateful to the governments of Ethiopia and Canada whose collaborated e f f o r t s made t h i s study possible.  ix  I.  INTRODUCTION Lignin present in natural f i b e r s provides both a physical and a  chemical b a r r i e r to enzymes that are able to attack isolated f i b e r s of cellulose.  Physically, penetration by enzyme molecules is  suppressed;  chemically, 1ignin-carbohydrate complexes form metabolic blocks that i n h i b i t polysaccharide hydrolysis  (21).  Therefore, an e f f i c i e n t ' b i o l o g i c a l  treatment may require a system to s o l u b i l i z e or remove l i g n i n thus d i s sociating the 1ignin-carbohydrate complex.  In general, any treatment  to depolymerize and s o l u b i l i z e l i g n i n makes the c e l l u l o s e in  1ignocellulosics  more susceptible to the a c t i v i t y by c e l l u l o l y t i c enzymes. The results presented by Kitts et a]_. (55) and several others have indicated that wood and wood by-products are potential sources of dietary energy for the ruminant animal.  However, change in the chemical compos-  i t i o n of plants owing to maturity and species as well as the presence of natural i n h i b i t o r s to cellulases a preliminary p h y s i c a l , chemical and biological method to a l t e r the structure of plant materials appears to be necessary before c e l l u l o l y t i c enzymes can be e f f e c t i v e . Cellulose is a major constituent of a l l vegetation accounting for from one-fourth to one-half of the plant's dry material.  Cellulose also  constitutes the world's most p l e n t i f u l renewable energy resource.  Use of  c e l l u l o s e as ruminant feed affords an e f f e c t i v e means by which the production of high quality protein can r e s u l t (97).  Incorporation of c e l -  l u l o s i c material as the energy source in ruminant rations w i l l permit a greater use of cereal grains for man and non-ruminant animals. The thermophilic aerobic oxidation is a procedure leading -to the degradation, s t a b i l i z a t i o n , pasteurization and deodorization of waste a r i s i n g from many sources such as animal and plant production.  The  X  bacteria that occur naturally in agricultural wastes (eg. swine manure) under aerobic conditions w i l l oxidize the usable substrate converting i t to carbon dioxide and water.  In order for bacteria to grow and reproduce,  a source of nutrients i s required.  Such nutrients, present in the waste,  include nitrogen, phosphorus, sulfur and minor trace elements a l l of which are essential for bacterial c e l l production.  Bacterial  degradation  of waste releases a part of the energy f o r c e l l function and the remainder of the energy in the form of heat energy, producing an increase in substrate temperature.  This increase in temperature has the e f f e c t of  accelerating the oxidation process by increasing the rate of biochemical reaction (29). Not only does the thermophilic aerobic process greatly reduce the time over that experienced by mesophilic digestion, but i t also leads to the destruction of pathogenic bacteria, viruses, and other organisms as a r e s u l t of the attainment of high temperature (24). The study reported here was designed to investigate possible means of reducing the cost of animal feed by generating the cheapest source of energy and possibly crude protein.  The object of this project was three-  fold: 1.  to examine the apparent capacity of aerobic thermophilic bacteria to d e l i g n i f y alder sawdust (wood).  2.  to subject fermentation samples c o l l e c t e d at d i f f e r e n t periods of fermentation and levels of temperatures to in v i t r o assays designed to evaluate the d i g e s t i b i l i t y of residual  carbohydrates,  and 3.  to study the p o t e n t i a l i t y of this biological method in single c e l l protein production.  II. 1.  Basic Biological  LITERATURE REVIEW  Processes  A microbiological process may be defined as a sequence of biochemical events under the control and direction of microscopically v i s i b l e c e l l s , in p a r t i c u l a r bacteria, fungi, and algae.  living  The important culture  parameters to consider in engineering design in order to maximize the desired biochemical events are oxygen requirements, and whether or not the organism is to be held in suspension within medium or fixed to a surface of supporting structure  (113).  The term aerobic processes refers to a condition in which dissolved oxygen is present within the medium.  The oxidation of organic matter using  molecular oxygen as the ultimate electron acceptor serves as the primary pathway y i e l d i n g useful energy to the microorganisms.  Microbes that use  oxygen as the ultimate electron acceptor are termed "aerobic microorganisms"  (67).  The term suspended growth refers to a culture condition in which the organism is suspended within the medium.  The microorganisms  are  under some culture conditions able to aggregate into masses containing a large number of c e l l s .  Agitation of the l i q u i d medium keeps the microbial  "masses" in suspension thereby maintaining a continuous contact with the substrate and removal of waste products (110 )• A)  Biochemical  Reactions  E f f i c i e n t u t i l i z a t i o n of the substrate for microorganisms  involves  a maximum conversion of energy and carbon into an increase in c e l l Thus the organism i s concerned primarily in c e l l duplication (110).  - 1 -  numbers. The  2. organisms also can use previously accumulated internal or endogenous food supplies for respiration purposes and do so for a limited time in the absence of external or exogenous food sources.  Synthesis and endo-  genous respiration occur, simultaneously in biological "synthesis"  systems with  predominating, when there is an excess of exogenous food and  endogenous respiration dominating when the exogenous food supply is small or nonexistent (67).  The general reactions that occur as i l l u s t r a t e d by  Loehr (67 are as follows: Energy containing metabolizable substrate + microorganisms t end products + additional microorganisms. In explaining the equation, Loehr (67) stated that i t represents reactions in which substrates such as biodegradable wastes are metabolized for energy and for the  synthesis of new c e l l s .  The energy u t i l i z e d in the  equation is obtained during the metabolism of the wastes. growth is affected by the a b i l i t y of the microorganisms and assimilate the food.  In addition, the  Synthesis or.  to metabolize  presence of toxic materials,  the temperature, the a v a i l a b i l i t y of adequate dissolved oxygen (DO), the pH of the system, and also the presence of adequate accessory nutrients and trace elements a l l determine or l i m i t the extent of microbial a c t i v i t y . Whatever the nature of the substrate, i t must contain a s u f f i c i e n t amount of the major elements such as carbon, nitrogen, phosphorus and minerals to meet the n u t r i t i o n a l requirements of the microorganism. Most of the research investigations  dealing with substrates such as nat-  ural occurring organic wastes indicate that the contained nutrients are available to biodegradation, however, the biological  reactions may be  constrained by environmental factors such as temperature, pH, dissolved oxygen and inhibitory compounds.  3.  In respect to batch systems, Loehr (67) mentioned that in the biological waste treatment system in which the culture medium and the products of microbial metabolism are contained within an enclosed v e s s e l , carbon may become a l i m i t i n g factor as the carbonaceous material  is  metabolized and lost from the systems as carbon dioxide. When growth becomes l i m i t e d , or increases in c e l l mass many of the microorganisms may die and lyse thereby releasing the nutrients of their protoplasm which may be u t i l i z e d by the remaining or scavenging cells. Further in his explanation pertaining to biochemical reactions Loehr (67) stated that in the presence of waste material and congenial  living  conditions, microbial metabolism w i l l occur allowing new c e l l s to be formed; energy and the microbial solids w i l l increase.  In the absence of  food, endogenous respiration w i l l predominate and a reduction of the net microbial solids w i l l r e s u l t .  The depleted c e l l residue w i l l not be  reduced to a zero net weight even with a long endogenous respiration period.  Supporting this statement, Kountz, et_ al_. (61) and Washington,  et al_. ( l i g )  reported that a residue of about 20 to 25% of synthesized  microbial mass w i l l remain. When organic matter is metabolized and resynthesized into microbial c e l l s , the converted waste also being organic matter is p a r t i a l l y ilized.  stab-  As indicated e a r l i e r , the microbial c e l l s are capable of further  degradation.  Only when the biochemical oxygen demand of the waste degrad-  ation has stopped does a s t a b i l i z e d effluent result  B)  The Energy Relationships  (67).  in the System .  A l l c e l l s , whether animal or plant, use similar fundamental mechanisms for t h e i r energy transforming a c t i v i t i e s  (108).  4. Loehr (67) indicated that knowledge of the energy relationships of microbial c e l l s permits an understanding of energy available for synthesis and r e s p i r a t i o n , of production of microbial c e l l s in biological waste treatment units.  This also includes an explanation of the nature  of the expected end products occurring under certain conditions. The energy of the food material u t i l i z e d by the l i v i n g c e l l in part conserved chemically in the compound known as adenosine phosphate (ATP).  is  tri-  ATP is the c a r r i e r of chemical energy from the oxidation  of foods to those processes of the c e l l s which do not occur spontaneously and can proceed only i f chemical energy is supplied.  These processes  are involved in the performance of osmotic, mechanical or chemical work (67).  In the context of this study, the food for the c e l l s could be from  animal wastes (swine) and sawdust (alder). Adenosine triphosphate is formed from adenosine diphosphate during oxidation in the c e l l s .  (ADP)  Adenosine triphosphate is the high energy  form of the energy transporting system and ADP is the lower energy form. A portion of the energy of the oxidation is conserved as the energy of the ATP.  This process operates in a continuous dynamic c y c l e , receiving  energy during the oxidation of foods and releasing energy during the performance of c e l l u l a r work.  A molecule of inorganic phosphate (Pi.)  is  released when ADP is formed and incorporated in ATP when ATP is formed. Although ATP is not the only energy carrying compound in every c e l l u l a r reaction, i t is the common intermediate in the energy transformation in the c e l l s  (67).  The ultimate electron acceptor is oxygen in aerobic organisms. However, electrons are not transferred d i r e c t l y from fuel molecules and t h e i r breakdown product to oxygen.  Instead, these substrates  transfer  electrons to special c a r r i e r s , such as pyridine nucleotides or f l a v i n s . The reduced forms of these c a r r i e r s then transfer their high-potential electrons to oxygen via an electron-transport chain located in the inner membrane of mitochondria.  It was stated e a r l i e r that ATP is formed  from ADP and Pi as a result of this flow of electrons.  This process  called oxidative phosphorylation, is the major source of ATP in aerobic organisms ( 108). In a comparison of aerobic and anaerobic organisms Loehr (67) commented that, for a given organic loading, aerobic conditions w i l l produce a more oxidized end product or effluent than w i l l anaerobic conditions and w i l l permit synthesis of a greater quantity of microbial cells.  These additional c e l l s are an asset because i t i s thus possible  to have a larger amount of active microbial solids to increase the removal of organic-wastes.  Aerobic organisms in a complete oxidation can conserve  f o r themselves a greater portion of the available energy from metabolism of organic matter than can anaerobic organisms.  Thus, anaerobic organisms  must process a greater quantity of food to obtain the same amount of energy.  In further comment, Loehr stated that the energy recovery per  unit of food is small for anaerobic organisms which indicates that the number of microbial c e l l s synthesized per unit of food metabolized w i l l be s i g n i f i c a n t l y less than for aerobic organisms.  C)  Biochemical Transformations  Several studies described in l i t e r a t u r e s (67)  (109) ( i l l )  have ascertained the fact that to achieve satisfactory biological  and (61) break-  down of wastes, the material must contain s u f f i c i e n t carbon, nitrogen, phosphorus and other necessary elements to sustain optimum rates of microb  6.  synthesis.  A problem due to lack of these elements in most biological  wastes has not been indicated, since there usually is more than enough nitrogen, phosphorus, and trace minerals with respect to the carbon used in c e l l  synthesis.  Loehr (67) b r i e f l y described these elements as  fundamental transformations  in a variety of treatment systems.  Loehr also  describes the signficant role that temperature, oxygen and pH play in respect to aerobic biological  a)  systems.  Carbon  The oxidation of organic carbon-containing compounds represents the mechanism by which heterotrophic organisms obtain the energy for synthesis.  In aerobic treatment systems organic carbon is transformed,  via many steps, to synthesized microbial protoplasm, C g H ^ N , and CO,,. Organic Carbon + 0 — • C g H ^ N + C0 2  2  The uptake of oxygen and formation of carbon dioxide represent the effects of r e s p i r a t i o n . b)  Nitrogen  Nitrogen is an important nutrient in biological  systems and is  present at a concentration of about 12% in bacterial protoplasm.  In  waste matter, nitrogen w i l l be present as organic and ammonia nitrogen, the proportion of each depending upon the degradation of organic matter that has occurred.  In biological  systems, organic nitrogen compounds  can be transformed to ammonium nitrogen and oxidized to n i t r i t e and n i t r a t e nitrogen. Organic N  * ammonium N  « nitrite N  * n i t r a t e N.  7. The oxidation of ammonia to n i t r i t e and n i t r a t e is termed n i t r i f i c a t i o n and occurs under aerobic conditions.  A more basic d e f i n i t i o n  of n i t r i f i c a t i o n is the biological conversion of inorganic or organic nitrogen compounds from a reduced to a more oxidized state.  In waste  treatment the term usually is used to refer to the oxidation of ammonia. According to Loehr s statement,, a residual dissolved oxygen concentration 1  of about 2 mg/1 has been found necessary to have optimum n i t r i f i c a t i o n . Autotrophic bacteria, such as Nitrosomonas, which obtain energy from the oxidation of ammonia to n i t r i t e , and Nitrobacter, which obtain energy from the oxidation of n i t r i t e to n i t r a t e , are organisms that in combination can accomplish the complete oxidation of nitrogen.  It has been pointed out  by Loehr that the release of ammonia nitrogen to aerobic treatment units creates an added oxygen demand to these systems.  The oxidation of 1 kg.  of ammonia nitrogen to nitrate nitrogen w i l l require 4.57 kg. of oxygen.  c)  Nitrification  N i t r i f i c a t i o n can be defined b a s i c a l l y as the biological conversion of nitrogen in inorganic or organic compounds from a reduced to a more oxidized state  (67).  Loehr (67) referred to Pasteur's work (1862) which suggested that the oxidation of ammonia was due to microbial a c t i v i t y .  This suggestion  was v e r i f i e d in sewage and s o i l studies which showed that oxygen was essential and that alkaline conditions favored n i t r i f i c a t i o n . Several genera of n i t r i f y i n g organisms have been reported (14). Nitrosomonas, Nitrosospira, Nitrosococcus and Nitrosocystis oxidized ammonia to n i t r i t e .  Nitrosogloea, Nitrobacter and Nitrocystis oxidized  n i t r i t e to n i t r a t e .  Of these genera, only Nitrosomonas and Nitrobacter  8. are generally encountered in  aquatic and s o i l ecosystems and are the  n i t r i f y i n g autotrophs of importance. trophic  Two new genera of obligate auto-  n i t r i t e - o x y d i z i n g bacteria, Nitrospira and Nitrococcus species  have been reported (120).  Factors l i k e dissolved oxygen, temperature  and pH play an important role in the n i t r i f i c a t i o n process. (i)  Dissolved Oxygen:  Since the n i t r i f y i n g organisms  are aerobic, adequate dissolved oxygen (DO) is necessary to support n i t r i f i c a t i o n assuming other environmental conditions are satisfactory (67). Loehr (67) reported that low dissolved oxygen concentration suppressed measurably the growth of Nitrosomonas and Nitrobacter was also affected to an even greater degree.  Further in this comment,  Loehr (67) stated that s e n s i t i v i t y of the Nitrobacter to low dissolved oxygen concentrations is one of the reasons complete n i t r i f i c a t i o n is d i f f i c u l t to accomplish in heavily loaded systems where the oxygen demand is s i g n i f i c a n t and where adequate oxygen does not exist in the microbial f l o e . A c r i t i c a l dissolved oxygen concentration exists below which n i t r i f i c a t i o n does not occur.  The c r i t i c a l dissolved oxygen concentra-  tion has not been precisely determined, but i t appears to be around 0.5 mg/l  (123).  The actual l i m i t is more dependant on the rate of oxygen  d i f f u s i o n to the microorganisms mixed l i q u o r .  than on the oxygen concentration in the  To assure that the dissolved oxygen concentration in a  treatment unit is not  l i m i t i n g n i t r i f i c a t i o n , the dissolved oxygen in  a treatment unit generally is kept above 1.0 mg/l. proceeds at a rate independent  Nitrification  of the dissolved oxygen above the  9.  c r i t i c a l concentration (ii)  (67). Temperature:  temperature of the media.  N i t r i f i c a t i o n is affected by the  Pure culture studies indicated that the  optimum growth of n i t r i f i e r s occurred between 30° and 36°C (1). of the early studies  One  (39) indicated that an exposure for 10 min. at  53°C - 55°C and at 56° - 58°C k i l l e d Nitrosomonas  and Nitrobacter,  respectively. Laboratory activated sludge studies indicated that the rate of n i t r i f i c a t i o n increased throughout the range of 5° - 35°C (i23) Information on n i t r i f i c a t i o n at low temperatures indicates data.  conflicting  Different studies have indicated that n i t r i f i c a t i o n did not  develop below 10°C (96), that i t was possible to maintain n i t r i f i c a t i o n at 8°C (82), that l i t t l e n i t r i f i c a t i o n was achieved at temperatures below 6°C (32) and that n i t r i f i c a t i o n was achieved at temperatures below 1°C (43). (iii)  p_H:  The optimum pH for the growth of the  n i t r i f i e r s is not sharply defined, but in pure cultures i t has been shown to be generally on the a l k a l i n e side (67).  In a detailed study  of n i t r i f i c a t i o n , optimum pH was found to be 8.4  Ninety percent of  the maximum rate occurred in the range of 7.8 - 8.9 and outside the ranges of 7.0 to 9.8 less than 50% of the optimum rate occurred (123). According to Hang's and McCarty's  (43) report n i t r i f y i n g organisms  can adapt to low pH levels and achieve adequate n i t r i f i c a t i o n .  When  the pH was adjusted to a range of 5.5 to 6.0 in a submerged aerobic f i l t e r , the n i t r i f i e r s adapted to the lower pH levels and the rate of ammonia oxidation reached that comparable to what had been achieved at a pH of 7.0.  10.  d)  Denitrification  Microbial d e n i t r i f i c a t i o n takes place under anoxic conditions where n i t r i t e s and nitrates are used as terminal electron acceptors in place of molecular oxygen  (67).  The nitrates and n i t r i t e s are reduced to gaseous nitrogen resulting in a reduced nitrogen content of the treatment unit as the gas escapes from the unit.  The composition of the gas produced is a  function of environment conditions. gaseous end product.  Nitrogen gas (N ) 2  is the primary  Other gases produced include nitrous oxide (N^O),  n i t r i c acid (NO), C 0 and H 2  2  (67).  D e n i t r i f i c a t i o n is brought about by f a c u l t a t i v e heterotrophic bacteria.  Most of the active d e n i t r i f y i n g organisms belong to the genera  of Pseudomonas, Achromobacter, Bacillus and Micrococcus e)  (67).  Phosphorus  Phosphorus is an important nutrient in biological processes. phosphorus content of bacterial c e l l s is about two percent (1).  The  The  sources of phosphorus in waste processes include organic matter, phosphates originating  in cleaning compounds used for cleaning up, and the  urine of animals.  The organic phosphorus is transformed to inorganic  phosphorus during biological  treatment (67).  Aerobic biological  treat-  ment w i l l convert condensed phosphates to orthophosphates.  2.  Thermophilic  Microorganisms  The word thermophilic, derived from Greek, means thermo = heat, phile = loving:  heat loving microorganisms.  In a simple schematic  c l a s s i f i c a t i o n of microorganisms, thermophiles f a l l under bacteria.  11. A)  Origin and  Distribution  The o r i g i n of thermophiles s t i l l remains open for debate among authorities in the f i e l d .  Opinions of various people who conducted a  study at measurable depth are given below b r i e f l y . Imsenecki et al_. (49) speculated that variants of well-known strains of mesophilic bacteria, progressively more completely adapted to higher temperatures  up to the f i n a l obligate stage; whereas MacFadyen  et al_. (71) considered that they had become adapted even more gradually and more reversibly than connoted by the term variant.  Gaughran (38) -  in his thermophilic microorganisms review indicated several p o s s i b i l i t i e s  -  that spontaneous adaptation or mutation, tropics as the central part of the evolution, propelled by the radiation pressures of the sun from Venus to Earth.  Nevertheless, most of the recent authorities in the microbio-  l o g i c a l area seem to accept Imsenecki's ejt al_. (49) assumption.  Along  the same l i n e Loginova and Tsaplina (70) assumed that most of the thermop h i l i c organisms developed from mesophilic species close to them in such a way that the enzymes and other c e l l components of the thermophiles acquired heat resistance. a)  Division of thermophiles  According to Smith et al_. (104), thermophilic bacteria are c l a s s i f i e d into two well defined species: 1.  Bacillus coagulans - the majority of these strains grow between 33° and 60°C, none above 60°C, and  2.  Bacillus stearothermophilus - growing between 37° and 65°C, with many growing above 70°C.  Definite nomenclature is not yet given to thermophiles with s u f f i c i e n t data to make possible a comparison among organisms. recent designations  reviewing the early descriptive terms of  The most  12.  Fischer (36) and the animal physiologists,  are those of Imsenecki and  Solnzeva (49). True thermophilic bacteria are those species whose optimal range of growth l i e s between 55°C and 60°C. a)  They may be divided into two groups:  Stenothermal thermophiles - develop at 60°C but show no growth after many days at 28-30°C.  b)  Eurithermal thermophiles - develop at 60°C, and show s l i g h t to abundant growth at 28-30°C.  Thermophiles constitute a very heterogenous group, i f a l l organisms are included with an optimum temperature for growth above 50°C. Their morphology and staining reactions are varied.  Fundamental d i f f e r -  ences appear in t h e i r nutritional requirments and metabolic a c t i v i t i e s  (38).  Egorova (35) found that the growth of the thermophilic bacteria was considerably poorer when the medium was based on tap water rather than some s o l i d material which in his case was 20% potato broth. The optimum growth temperature for thermophilic bacteria is usually somewhat lower in l i q u i d medium than on an agarized medium. . This has been confirmed by Loginova (68).  Based on this f a c t , Egorova (35) assumed that  thermophilic bacteria are no exception in t h i s respect. According to Egorova's studies, no differences in c e l l were observed during the  morphology  period of active growth (the f i r s t 2-5 h), when  the cultures were incubated in the shaker and in the freezer apparatus. However, the same author stated that the extremely thermophilic bacterium, Thermus flavus, exhibited marked morphological differences during d i f f e r e n t stages of i t s growth.  This microorganism is distinguished from obligative  thermophilic sporogenous'bacteria by a lower growth rate.  13. Thermophilic bacteria growing in an aerobic environment, reproduce at a maximum rate (in the exponential phase of growth) for a period of 2-3 hours.  In the l i n e a r phase of growth, thermophiles metabolize the  substrate (glucose) by using both aerobic and anaerobic pathways, which is evident by the increasing values of the respiratory quotient and the v o l a t i l e acids liberated into the external medium (91).  Pozmogova  (93),  in his e a r l i e r work has also indicated that an organism may experience a limited oxygen supply during the stage of rapid growth.  As a result  of this deprivation, there is a sharp drop in the maximum rate of growth; with a result that there is a changeover to anaerobic metabolism.  This  observation has been supported by M i l l e r et al_. (79). The a b i l i t y of thermophiles to survive at higher temperatures still  remains unanswered.  Among p o s s i b i l i t i e s that are being envisaged  by various investigators . 1.  Suitable media may increase heat resistance by providing  favorable conditions for active metabolism sp that the organism is better able to repair damage done by heat or they may aid growth at high temperatures because they contain substances which protect proteins from thermal denaturation 2.  (2).  The effect of divalent ions i . e . Ca or Mg ions is believed to  play an important role on thermal resistance aspect, p a r t i c u l a r l y when considered together with the finding that higher calcium content is one of the few chemical differences which have been found between bacterial spores, which are the most thermoresistant l i v i n g objects known, and vegetable c e l l s Regarding  (27). the growth l i m i t a t i o n of thermophilic bacteria Allen  (3)  14.  reported that i t is due to exhaustion of the dissolved oxygen a v a i l a b i l i t y at elevated temperatures.  This statement has been backed by results of  Tanner et al_. ( I l l ) , but neither has mentioned nutrient exhaustion e f f e c t . b)  Growth at high temperature  Tanner and Wallace (111) were the f i r s t to apply the quantitative growth-curve method to the thermophilic bacteria; this is according to Gaughran's (38) report. increase in c e l l  At 55°C, Tanner and Wallace observed most rapid  numbers and, a f t e r the period of active growth, a rapid  death; and since cultures often became s t e r i l e , i t may be inferred that these thermophiles did not sporulate at this temperature (38).  Casman  and Rettger (20) assume that the absence of spores may have been the result of low oxygen tension in a l i q u i d medium at an elevated temperature, in accord with the observed relationship of oxygen tension and the capacity to sporulate among the aerobes, as well as f a c u l t a t i v e and s t r i c t anaerobes. In Gaughran's (38) report,  i t was stated that the generation time  of thermophiles isolated at 55°C was 16 minutes.  In the same i n v e s t i -  gation, i t was mentioned that calcium carbonate was added in order to 1  neutralize acid production.  8 The maximum viable c e l l y i e l d (6 x 10 per  ml) was obtained at 42°C, rather than at 55°C; with a second optimum c e l l y i e l d at 20°C. A study of the rate of biochemical a c t i v i t y of a number of thermophiles indicated that the high reproductive rate is inadequate in explaining the intense biochemical a c t i v i t i e s of the thermophilic bacteria. In some thermophilic cultures the progress of proteolysis may, of course, be related to reproduction r a t e , death and a u t o l y s i s , and a resultant i l  '  15.  accumulation of proteolytic enzymes in the medium.  Thus Imsenecki  was led to the conclusion that a possible explanation of the high b i o chemical a c t i v i t y of thermophilic bacteria may be found in the very intense metabolic a c t i v i t y of these organisms, and not merely in t h e i r rapid p r o l i f e r a t i o n It  (38).  has been suggested that rapidly metabolizing bacterial  vegetatiye c e l l s growing in a depleted medium with an accumulation of waste products of metabolism would l i m i t bacterial survival were capable of existing into a dormant or resistant stage.  unless they The a b i l i t y  to form spores has been suggested as a means by which the organism can overcome unfavorable culture conditions.  Thermophiles with a narrow  temperature range, however, do produce spores in t h e i r optimum growth range, but experience a depression in this a c t i v i t y as the maximum growth temperature l i m i t is reached (38). c,)  Growth at low temperature  Thermophilic microorganisms,  a r b i t r a r i l y characterized by an  optimum temperature above 45 - 50°C exhibit considerable latitude in their overall temperature range for growth.  A large number of thermo-  p h i l i c bacteria have been found to grow at 37°C, and even at 20°C (38). Evidence has been presented (81) to show that the environment (culture medium) exerts an important influence on the temperature l i m i t s fj.or growth.  Morrison and Tanner (81) have suggested that during  the growth of thermophilic bacteria at lower temperatures, the time of observation i s of greatest  significance.  Foter and Rahn (37) in t h e i r analysis of the l i m i t i n g minimum temperature of thermophilic growth, stated that the most common explanation  16. of cessation of growth at low temperatures is the assumption that the numberous interlinked reactions of the c e l l are influenced d i f f e r e n t l y by a change in temperature, with the result that the growth mechanism is upset.  In t h e i r further comment, they mentioned that the accumulation  of toxic metabolites within the c e l l  is not considered as a possible  cause f o r a disturbance of the growth mechanism in the case of bacteria or other c e l l s with large surface area.  Contrary to t h e i r comment,  Surucu (110) reported that the growth of microbial populations is normally limited either by the exhaustion of available nutrients or by the accumulation of toxic metabolic products.  It should be noted that other  environmental changes such as dissolved oxygen l e v e l , temperature and pH  are also important, since these changes in the environment may be  produced by the microorganisms themselves, thus l i m i t i n g the development of the microbial population. Brock and Darland (15), in t h e i r study pertaining to e f f e c t of temperature and pH on microbial existence, concluded that bacteria have the a b i l i t y to grow at either high temperature or high a c i d i t y , but not at both high temperature and high a c i d i t y .  B)  Potential of Thermophilic Aerobic Digestion of Organic Solids  Thermophilic aerobic digestion is a process which appears to have, a great potential for the degradation of organic solids  (51).  One of the  major factors to be considered in the use of thermophilic process  is  the heat energy  However,  required to maintain thermophilic temperatures.  heat can be produced by microbial thermogenesis as evidenced by the autothermal nature of composting thermophile range (51).  processes which usually operate in the  17.  Simulation studies have shown that s u f f i c i e n t heat can be generated in the aerobic digestion of organic solids to make the process self-sustaining  in the thermophilic range (45 - 65°C).  Kambhu and Andrews (51) l i s t e d the possible advantages of thermophilic aerobic digestion over the corresponding mesophilic process: a)  Increased reaction rate.  According to the c l a s s i c  thermodynamic rule of Van't Hoff-Arrhenius, the biochemical reaction rates w i l l double with each 10°C temperature increase (109). b)  Increased e f f i c i e n c y .  c)  Improved solids - l i q u i d separation.  d)  Increased destruction of pathogenic  e)  Biomass harvested from thermophilic aerobic digestion  organisms.  process may have r e l a t i v e l y high protein and vitamin content ( 109) • f)  The single c e l l protein produced by the thermophilic aerobic digestion process in some cases has been found to be composed of a better balanced amino acids composi t i o n for n u t r i t i o n a l purposes  Although many thermophiles had  (73).  been isolated from canned food  early in the history of the canning industry, the canned food spoilage e f f e c t did not receive s u f f i c i e n t attention until Barlow's published.  (7) work was  The significance of these bacteria l i e s in t h e i r a b i l i t y to  ferment lactose, or less commonly decompose proteins, and cause undesirable flavours or odours  (38).  Apart from t h e i r undesirable activities,thermophiles do have some  18. beneficial aspects as w e l l . a)  They are considered as potential agents:  in controlled fermentation of c e l l u l o s e to useful products  (38);  b)  in the recovery of vegetable o i l s and fats  (8);  c)  in the degumming of s i l k  d)  in enzyme a p p l i c a t i o n , i . e . amylase (38) and degummase  (52);  (48) as well as in pharmaceutical products.  Certain  enzymes of thermophilic microorganism are being tested as drug preparation. A determination of the proteolytic a c t i v i t y in d i f f e r e n t species of the isolated spore-forming bacteria showed that the largest zones of casein hydrolysis are c h a r a c t e r i s t i c of B. Stearothermophilus, B. B. brevis and B. megaterium (69).  substitis  The authors (Loginova, et a l . )  also refer to the role not only of the above l i s t e d organisms but also to B. cereus in the decomposition of organic  C)  Nutritional  substance.  Requirements  Early workers indicated that thermophilic bacteria required organic nitrogen (3).  Such statements have arisen from the fact that from the  limited studies to date, very few thermophilic sporeformers can develop in a simple medium with ammonia nitrogen and a single carbon source  (3).  Cleverdon et al_. (22) studied the minimal n u t r i t i o n a l requirements of 12 strains of B. stearothermophilus and they responded wel 1 with the addition of thiamine, n i c o t i n i c acid and biotin to the growth medium. However, they did not determine whether these vitamins were absolute requirements for a l l strains used in t h e i r study. have been carried to a greater depth by  Similar experiments  Cambell and Williams  (18) which  19.  were based on essential ature (55°C).  amino acids and vitamins as a function of temper-  In general, their findings  indicate that the complexity  of nutritional requirements increase with temperature, even though this does not always hold true.  Baker et a]_. (5) stated that the increased  complexity of the nutritional requirements of the thermophilic bacteria may r e f l e c t the i n a b i l i t y of c e l l ahead of t h e i r destruction.  to synthesize these v i t a l  components  Furthermore, Baker ejt al_, (6), a f t e r  studying  strains of B. coagulans and 68 strains of B. stearothermophilus, reported that these organisms f e l l  into four main n u t r i t i o n a l patterns,  i.e.:  a)  methionine;  b)  methionine plus other metabolites;  c)  methionine but c l e a r l y stimulated by other factors and,  d)  no methionine.  These results are also in agreement with (18), and Bhat and B i l l i m o r i a Several investigators  (11). have indicated that the three vitamins:  b i o t i n , niacin and thiamin are essential S i m i l a r l y , the same authors  Cambell and Williams  for thermophiles growth  (3).  (3) reported that glucose, lactate and malate  were the best carbon sources f o r growth at temperatures of 20 - 37°C. However, addition of 0.3% monosodium glutamate to the simple medium permitted growth at 55°C for some thermophiles.  glucose  The addition  of 0.1% casein acid or enzyme hydro!ysat.eto the simple medium described above stimulated growth. As Baker (5) indicated, thermophiles would appear to require high concentrations of nutrients for optimal growth.  Such culture media  exerts a rather high osmotic pressure on the c e l l s , which in turn suggests that thermophiles may be halophilic ( s a l t loving).  Contrary to  20.  this conclusion, Allen (3) in his review stated that recent data would indicate that the growth factor requirements of the thermophilic b a c i l l i are in r e a l i t y quite simple and that they do not need extensive vitamin and amino acid supplement - and that past culture d i f f i c u l t y may be traced to the use of an inadequate mineral base and, possibly, to the use of unsuitable carbon source or substrate concentration. Thermophily was considered to depend not only on the presence of unusually thermostable enzymes but also on the functioning of greatly expanded biosynthetic systems.  The r e l a t i v e l y high permeability and high  osmotic tolerances of thermophiles permit the e f f i c i e n t provision of c a t a l y s t s , fuel and building blocks to these systems  (5).  Based on t h e i r findings, Baker ejt aj_. (5) concluded that "high temperature" strains u t i l i z e a wider range of substrates, have a higher osmotic tolerance and also a heightened permeability than do "low temperature" s t r a i n s . Various thermophilic organisms are unable to u t i l i z e the simple mono and disaccharides (121).  Prickett (94) indicated that some of the  thermophiles are also unable to u t i l i z e starch and c e l l u l o s e . It has been pointed out that, although some thermophiles are proteolytic (8) and several are n o n - c e l l u l o l y t i c , the a c t i v i t y of these organisms in symbiotic relationships in nature would suggest that they are, at most, feebly capable of attacking native proteins (38). has been found to be true with pure cultures of food spoilage  This  organisms,  such as the hydrogen s u l f i d e or sulfur producing stinker organisms in canned corn (122) and the thermophiles found in milk (34). s u l f i d e and  Hydrogen  indole production were the only end products of organic  nitrogen metabolism studied (38).  It was also pointed out by the same  21.  author that both indole and hydrogen s u l f i d e were produced only by a limited number of organisms.  Gaughran (38) in his review, c i t e d types  of thermophilic microorganisms  according to their s p e c i f i c a c t i v i t i e s ,  - i . e . nitrogen f i x i n g , n i t r o s i f y i n g , d e n i t r i f y i n g , sulfate reducing, etc. Suitable growth of a number of thermophiles has been achieved by using basal medium which contains several carbon sources, a richer complement of mineral nutrients, biotin and r i b o f l a v i n . growth also resulted, s u r p r i s i n g l y ,  Excellent  i f instead of the vitamins a large  quantity of calcium ions (12 mg%) was added to the culture solution D)  The E f f e c t of Temperature on the Nutritional  Requirement  Cleverdon et a_l_. (22) studied the vitamin requirements of coagulans at 37°C and 55°C.  (3).  Bacillus  Employing a vitamin-free casein hydrolyzate  medium they found that n i a c i n , thiamin and b i o t i n were required for growth at both temperatures, growth and sporulation being most abundant at lower temperature of incubation.  In a study of the vitamin requirements of  twelve obligate thermophilic strains of Bacillus  stearothermophilus,  these same w o r k e r s ( 2 2 ) noted that b i o t i n , niacin and thiamin were the only vitamins found to be essential  for continued growth of the cultures  at 55°C and 65°C in the casein digest medium employed. These workers(22)  followed the c l a s s i f i c a t i o n proposed by Cameron  and Esty (19) in order to avoid confusion.  Their c l a s s i f i c a t i o n  is:  Facultative thermophiles - growth at 37° and 55°C Obligate thermophiles - growth at 55° but not at 37°C. Campbell et al_. (18) in their study dealing with the e f f e c t of temperature on n u t r i t i o n a l requirements commented, in agreement with the  22. majority bf the investigators,  that at higher temperature the enzymes  responsible for the synthesis of a p a r t i c u l a r metabolite required, for example, compound X.  This compound may undergo thermal  inactivation  and thus the organism would require an exogenous source before growth can take place. In an attempt to explain his data, Cambell ert aj_. (18) considered several p o s s i b i l i t i e s and concluded that the genetic material was responsible for the potential capacity of an organism to produce enzymes, which, in turn, synthesizes the metabolites necessary for growth.  Thus,  the complete inactivation of a gene responsible for the synthesis of an essential metabolite imposes upon the organism a requirement of an external source of the substance which i t can no longer produce.  One of the  bacterial strains Cambell (18) used in his studies f a i l e d to synthesize h i s t i d i n e at culture incubation temperatures of 20 - 37 - and 55°C. Histidine therefore was required for growth.  In interpreting this  p a r t i c u l a r f i n d i n g , Cambell (18) assumed that the gene necessary for the production of the enzyme responsible for the synthesis of h i s t i d i n e , i f present, has been inactivated; thus this strain can no longer make h i s t i d i n e and now requires an external source of this metabolite before growth can occur. E)  Energy Metabolism  During growth, thermophilic organisms lose a large quantity of energy in the form of heat (70).  The maximum weight of the c e l l biomass  at f i r s t increases with increasing concentration of the carbon source; however, a f t e r a maximum is reached, the weight of the biomass remains constant, limited not on the increase in the concentration of the carbon  23. source, but only on the concentration of oxygen dissolved in the medium [', Regarding the e f f e c t of dissolved oxygen in this respect, Loginova (70).stated that the concentration of dissolved oxygen is lower at higher temperatures even though the oxygen demand by the organism to meet the metabolism is increased.  A portion of the energy requirement of certain  thermophilic organisms for the production of supplementary energy of dissimilation  (catabolism) may be n i t r a t e reduction.  In a comparison of the amino acid composition of some enzymes, i t was shown by O'Brian et a]_. (70) that a correlation is observed between the thermal s t a b i l i t y and the increase in the content of isoleucine, arginine and tryptophan, and a decrease in the content of phenylalanine and aspartic a c i d .  The authors suggest that there are stronger hydrophob  bonds between the subunits in the enzyme molecules of the thermophiles, which is responsible for t h e i r thermal s t a b i l i t y .  It has been reported  by Heinen (70) that the enzyme amylase a c t i v i t y of the extreme thermop h i l i c bacterium (70°C optimum for growth), plays an important role in thermal s t a b i l i t y whereas Ca and Mg ions are essential for ensuring the thermal s t a b i l i t y of amylase and i t s high a c t i v i t y . F) It  Oxidative Metabolism has been reported by Allen (3) that the d i f f i c u l t y of growing  thermophilic organisms in chemically defined media with a single carbon source has inhibited research progress.  Knowledge of the types of com-  pounds which can be oxidized by these sporeformers and the pathways of oxidation of those compounds to be u t i l i z e d remain completely unknown. However, Allen (3) managed to extract some general conclusions on the oxidative a b i l i t i e s of the thermophiles from the l i t e r a t u r e and  24. from his personal observations. In his general conclusion he stated that: a) Sugars and polyhydric alcohols are usually readily oxidized, as are salts of lactic acid and the common  dicarboxylic  acids; b) Some thermophiles do utilize citrate; c) Utilization of fatty acid salts in pure cultures has been noted,  although available information is limited;  d) Amino acids are excellent substrates for oxidation. Evidence has been reported by Pozmogova and his associates (93) that thermophilic sporeforming bacteria possess both aerobic and anaerobic metabolic pathways. During the growth of the bacilli in a well aerated media the organisms displayed a predominantly aerobic type of metabolism during the first 1 - 3 hours, after which followed an increasingly active anaerobic pathway. Further in their report, they mentioned that the reason for such a change in substrate metabolism was due to the presence in the thermophiles of more active anaerobic dehydrogenases. The data of Pozmogova et al_. (93) showed that development at submaximal temperature leads to the activation of enzymes in pathways for the cleavage of glucose (glucose 6-phosphate dehydrogenase),for the anaerobic transformation (alcohol dehydrogenase), and for the reduction in the rate of amination of a - ketoglutarate (a fall in activity of glutamate dehydrogenase). G) Transformation of Nitrogenous Compounds The most conspicuous conversions of nitrogenous compounds effected by thermophilic aerobic sporeforming bacteria are proteolysis, ammonifica-  25. tion and d e n i t r i f i c a t i o n (3). Oxidation of ammonia to n i t r i t e by cultures of thermophilic sporeformers has been reported by  Cambell  (17).  Allen in his review (3), stated that the nitrogen f i x a t i o n by thermophilic bacteria in crude culture may proceed at a rate of three to six mg nitrogen per gram of sugar u t i l i z e d ; however, the mechanism is not well understood. " N i t r i f i c a t i o n " and  "nitrogen f i x a t i o n " by cultures of thermo-  p h i l i c organisms, although reported by few investigators  (78, 72), have  not been extensively studied. H)  Decomposition of Natural High Polymeric Materials  It aerobic  has been noted as early as 1894 (71) that the thermophilic sporeforming bacteria have the a b i l i t y to decompose natural high  polymeric substances found in plants.  The production of important enzymes  such as amylase, proteinases, and lipase as by-products was recorded by many early researchers.  Several investigators  have obtained thermostable  hydrolytic enzyme preparations from the culture f i l t r a t e s of thermophilic sporeformers; however, the properties of these enzymes have not been studies in d e t a i l . Viljoen et a l . (117) have isolated the organisms responsible for the primary attack on c e l l u l o s e and have reached the conclusion that the thermophilic bacteria which actually decompose c e l l u l o s e are obligate anaerobes.  Tetrault (112) has reported that the aerobic and f a c u l t a t i v e l y  anaerobic sporeformers associated with the anaerobes play a secondary r o l e . McBee (75) in his study using pure cultures also obtained a similar r e s u l t . Claims have also been made, however, for c e l l u l o s e decomposition by  26. aerobic thermophiles. and McBeth (53).  The f i r s t of these claims came from Kellerman  Allen (3) in his review reported that results of  several studies have evidence for aerobic thermophilic bacteria's  ability  to decompose c e l l u l o s e . Linder natural conditions, anaerobic c e l l u l o s e breakdown may be considered to be more prevalent (107)  (3).  Hydrogen, carbon dioxide, ethyl  a l c o h o l , acetic acid and occasionally butyric acid have been found as products of high temperature c e l l u l o s e breakdown (100). Murray (85) reported on the aerobic growth of thermophilic c e l l u l o s e bacteria in atmospheres where the humidity was maintained at 98 to 100 percent.  He obtained l i t t l e or no growth when the humidity f e l l  percent or less.  to 90  He concluded that the many f a i l u r e s to obtain pure  cultures of thermophilic aerobic c e l l u l o s e decomposers may not be due to the anaerobic nature of the c e l l u l o l y t i c organisms, but due to insuff i c i e n t water vapor in the a i r .  Still  less is known regarding the  decomposition of other constituents of plant material by thermophilic bacteria.  There is almost no l i t e r a t u r e on the breakdown by thermophiles  of such materials as hemicellulose, pectin, l i g n i n and c h i t i n . 3.  Wood and Its  Potential in Ruminant Diet  Wood residues, of  70 to 80% carbohydrate, are a potential source  of dietary energy for ruminants.  However, only a minor percentage of this  carbohydrate can be u t i l i z e d by rumen microflora without some form of pretreatment (99).  The high l i g n i n and .the low c e l l contents are believed  to be the c o n t r o l l i n g factors (114, 115). The purpose of preliminary physical and/or chemical treatment of wood and wood by-products as explained by Kitts et  (55), is to  •  27. remove factors which contribute a negative impact toward n u t r i t i v e value. These are due to changes in the chemical composition of the plants owing to age and species and  the presence of natural i n h i b i t o r s of c e l l u l o s e s .  It was suggested by Kitts et al_. (55) that before a decision is made to use wood in practical feeding to ruminants, the following factors must be closely observed: a)  Economic consideration;  b)  Nature of treatment applied should allow more nutrients to become available to the rumen microorganisms  without  causing excessive depolymerization of polysaccharides or loss of micronutrients, and c)  Effect of treatment should not  leave any residue which  w i l l be toxic to the microorganisms or the host while removing the natural inhibitors of polysaccharides, i f any The ruminant is adapted physiologically and anatomically to convert fibrous material containing large amounts of c e l l u l o s e into food that is acceptable to man.  Since wood and wood by-products contain mainl  free c e l l u l o s e , c e l l u l o s e chemically associated with l i g n i n and nitrogen, and pentosans often referred to as "hemicelluloses," i t is reasonable to speculate that the rumen microflora and fauna could u t i l i z e these substances and in turn have the end-products of this fermentation transformed into human food (55). The value of forages for meeting the energy needs of ruminant animals is mainly a function of the amount of feed consumed and the d i g e s t i b i l i t y of the feed's energy.  These factors are i n t e r - r e l a t e d  and depend to a large extent on the chemical composition of the herbage. The r e l a t i v e amounts of solubles, c e l l u l o s e , hemicellulose and l i g n i n  28.  are a l l aspects of composition that influence d i g e s t i b i l i t y .  Intake is  related to these composition factors as well as to the rate of digestion and rate of passage of the f i b e r mass (118). Smith et a K  (105) reported that there is a s i g n i f i c a n t correlation  between the rate constant and percent l i g n i n in the plant tissue of several grass and legume forages.  Lignin c e l l u l o s e r a t i o and soluble  dry matter were also correlated with the rate constant. It  has been pointed out by Lechtenberg (64) and his  associates  that i t is d i f f i c u l t to study the e f f e c t of plant composition parameters such as intake, d i g e s t i b i l i t y , or digestion rate. Because of the fact that plant materials that d i f f e r only in the component of i n t e r e s t , i . e . differences in l i g n i n percentages are generally confounded with maturity, or differences in the content of other fibrous components.  They (64) also indicated that occasionally genetic mutants  o f f e r an opportunity to obtain samples of plant tissue which d i f f e r appreciably in only one chemical component.  In order to substantiate  t h e i r statement, they referred to Muller's et al_. (83,84) work which v e r i f i e d the fact that plant material which has less l i g n i n content has greater c e l l wall d i g e s t i b i l i t y and intake. The results of Riquelme et al_. in lamb fattening rations  (97)  indicated that the c e l l u l o s e f i b e r s (bleached hardwood kraft) supported animal weight gain e s s e n t i a l l y equal to the control ration (70% wheat + 24% a l f a l f a hay + 4% molasses + 2% premix) and did so e f f i c i e n t l y .  Further-  more, the carcasses of the f i b e r fed lambs exhibited a conformation and quality comparable to the control group, and because of the reduction in backfat thickness, kidney and pelvic f a t , the calculated y i e l d grade was improved, indicating more salable meat cuts per carcass.  29. A.  Lignin Distribution in Wood Tissues  U l t r a v i o l e t microscopy has been used by Goring et_ al_. (40) to measure the d i s t r i b u t i o n of l i g n i n in spruce and birch wood.  The spruce  xylem was considered to consist predominantly of three d i s t i n c t morphological  regions: 1.  Secondary wall  (S) of the f i b e r (tracheid);  2.  The compound middle lamella (ML) between f i b e r s  3.  Middle lamella (ML  ) at the c e l l corners. cc  Table 1.  The d i s t r i b u t i o n of l i g n i n in black spruce earlywood  Tissue type S  Tissue volume {%)  Lignin % total  Lignin concn. ,g/g  87  72  0.22  ML  9  16  0.50  ML_  4  12  0.85  As shown in Table 1 most of the l i g n i n in spruce earlywood exists in the secondary wall of the f i b e r even though the concentration of in the middle lamella is high.  lignin  It was also mentioned in t h e i r report  that similar results have been obtained in latewood studies.  The d i s t r i -  bution of l i g n i n found in these three areas supports the prediction made by Berlyn and Mark (10). Goring et a l . (40) concern in this p a r t i c u l a r study was to remove l i g n i n and record the rate of removal from various regions in the wood tissue during chemical pulping (Kraft and s u l f i t e pulping). to t h e i r  According  results the secondary wall loses l i g n i n f a i r l y rapidly in the  30. early stages of the work while the middle lamella shows strong u l t r a v i o l e t (UV) absorption u n t i l l a t e in the cook.  They also reported that  similar trends were found in acid s u l f i t e pulping. Both in kraft and s u l f i t e pulping, i t has been noted (40) that low molecular weight lignins are f i r s t extracted with higher molecular weight material being made soluble l a t e r in the cook.  This extraction sequence  of spruce lignins and the microscopic pattern of l i g n i n removal have been interpreted in terms of a theory of d e l i g n i f i c a t i o n based on the degradation of a polymer g e l . B)  Lignin  Lignin is located in the woody parts of plants, such as bobs, and the fibrous portions of roots, stems and leaves. structure remains uncertain.  hulls  Its chemical  Due to this uncertainty, i t has been  suggested (74) that the term must be considered to designate a group of substances having a common basic structure but d i f f e r i n g as attached units.  regards  The substances contain carbon, hydrogen, and oxygen,  but the proportion of carbon is much higher than in carbohydrates. Nitrogen is also present, ranging from 1 to 5 percent in d i f f e r e n t products isolated.  Methoxy groups have been reported to occur in percentages  ranging from 5 to 15 or more. matures.  The percentage increases as the plant  The nucleus is a polyhydrate aromatic compound.  Thus, l i g n i n  cannot be classed as a carbohydrate, but i t is discussed along with this group of compounds because i t occurs in intimate association with c e l l u l o s e and is included with the carbohydrates in the conventional methods of feed analysis.  Its  recognition as a separate entity is important because  of i t s dominant influence on the degree of d i g e s t i b i l i t y of many feeds  (74).  31. Lignin occurs in plants c h i e f l y as 1 i g n o c e l l u l o s e .  There i s sup-  port to the b e l i e f that substances of the glucosanxylan series are the forerunners of l i g n i n , but neither i t s exact chemical structure nor the manner in which i t i s combined with c e l l u l o s e are f u l l y understood.  Its  behavior in n u t r i t i o n i s l i k e w i s e u n s e t t l e d , d i f f e r e n t feeding tests yielding conflicting results.  Proof f o r or against i t s u t i l i z a t i o n by  the animal i s d i f f i c u l t to e s t a b l i s h , f o r u n t i l i t s molecular s t r u c t u r e i s known, no c r i t e r i o n of the accuracy of a q u a n t i t a t i v e t e s t f o r l i g n i n i s possible (26).  Crampton and Maynard (26) have shown in t h e i r  studies that l i g n i n i s not appreciably metabolized by animals. The work of Woodman ejt al_. (125) has shown that l i g n i f i e d plant t i s s u e s apparently are not attacked by alimentary b a c t e r i a .  According  to t h e i r assumption, t h i s might be due to a c e r t a i n degree of a n t i s e p t i c action of the C)  l i g n i n r e s u l t i n g from i t s phenolic nucleus.  Delignification  In one of t h e i r studies, K i t t s e_t al_. (58) reported that unprocessed sawdust in ruminant d i e t has been able to provide energy as high as 244 kcal/kg d i g e s t i b l e energy, and 22.7% of d i g e s t i b l e p r o t e i n .  In a  f u r t h e r statement, i t was mentioned that because the actual u t i l i z a t i o n of the untreated sawdust by rumen microorganisms per se i s poor, i t has not been p a r t i c u l a r l y b e n e f i c i a l to u t i l i z e wood wastes without f u r t h e r processing to remove or lessen the l i g n i n content. Untreated sawdust  has  been included in production rations at low  l e v e l , and i t would appear that i t serves mainly as n o n - n u t r i t i v e bulk in r a t i o n s containing otherwise high l e v e l s of energy feeds (4,55). l i q u i d by-product,, a hemicellulose e x t r a c t prepared from hardboard  One  32. manufacture, has proved commercially successful as a feedstuff of high n u t r i t i v e value  (89).  Sodium hydroxide has been one of the more popular agents for delignification  and has been applied to a variety of materials to  improve t h e i r d i g e s t i b i l i t y for use as a ruminant feed. Sodium hydroxide would appear to be the most e f f e c t i v e and economical, p a r t i c u l a r l y under mild conditions of atmospheric temperature and pressure.  Optimum treatment levels of sodium hydroxide would appear  to be less than 8 g of  chemical/100 g of substance, an indication that  mild heat can be used to reduce chemical levels and thus minimize physiol o g i c a l stress on animals consuming large quantities of treated feedstuffs  (31). Smith et  aJL (103) compared a variety of agents for improving  d i g e s t i b i l i t y of ruminant fecal material. peroxide substantially the material.  Sodium hydroxide and sodium  improved the a v a i l a b i l i t y of carbohydrate in  The increase resulted from the improvement in the a v a i l -  a b i l i t y of insoluble residual c e l l wall as well as the formation of s o l uble matter. Brownell  (16) found that when 20 mesh wood was subjected to  various chemical pretreatments, the amount of l i g n i n obtainable a f t e r subsequent ball m i l l i n g was greatly increased above that obtained without milling.  The  most s t r i k i n g increase occurred a f t e r treatments with  sodium hydroxide or ammonium hydroxide. Pew and Weyna (88) in t h e i r study of enzymatic l i b e r a t i o n of l i g n i n , observed a large increase in the extent of enzymatic digestion following mild treatment witha base. Similar to Brownell (16), Bland  33.  and Menshum (12) reported that the y i e l d of milled wood l i g n i n was increased by a mild a l k a l i treatment before m i l l i n g .  This suggested  that l i g n i n was attached to the carbohydrate by a l k a l i sensitive bonds. Further in t h e i r report (12) they mentioned that prolonged standing of milled wood solutions of sodium or ammonium hydroxide at poor temperature f a i l e d to increase the y i e l d of l i g n i n .  The y i e l d , however, did i n -  crease with increasing temperature of a l k a l i treatments between 48° and 86°C.  F i n a l l y , they (12) concluded that the l i g n i n carbohydrate bond, i f  such bond does e x i s t , is stable to a l k a l i treatment below approximately 100°C and that the breakup of the complex is due to a l k a l i peeling of the hemicellulose.  Above 100°C hydrolysis of l i g n i n begins and compli-  cates further measurements of l i g n i n recovery.  It would appear,however,  that the 1ignin-carbohydrate bond cannot be more a l k a l i - l a b i l e than bonds within the l i g n i n . The objective of treating 1ignocellulosic materials is to remove or dis^ rupt l i g n i n s o that  t h e i r energy potential as a ruminant feedstuff  is  enhanced severalfold compared with t h e i r untreated state. As i t is mentioned in several references, c e l l u l o s i c materials contain a vast store of energy.  Residues from the wood industry,  animal wastes, municipal organic wastes, etc. are being investigated for cheap energy sources for livestock feed. The energy contained in c e l l u l o s i c waste can be converted to food use by means of three general systems  (116);  1.  Chemical or enzymatic hydrolysis to produce sugar;  2.  Microbial fermentation with added nitrogen source to produce s i n g l e - c e l l protein;  3.  Feeding to ruminants to produce meat and milk.  34. E a r l i e r , i t was mentioned that ruminants have the a b i l i t y to u t i l i z e c e l l u l o s i c materials through the rumen fermentation. The nature of fermentation is anaerobic.  The anaerobic microorganisms  convert  carbohydrate to microbial proteins, various v o l a t i l e fatty acids, carbon dioxide and methane (46). Microbial proteins, microbial soluble matter and v o l a t i l e fatty acids are u t i l i z e d by the ruminant, while methane is a c a l o r i c l o s s . For adequate fermentation the nitrogen requirement of the microbes must be met.  Since c e l l u l o s i c wastes are usually very low in nitrogen, these  feedstuffs must be supplemented with nitrogen.  Digestion of c e l l u l o s e  is a function of the time the material resides in the rumen.  Thus, feeding  c e l l u l o s i c materials in rations containing high levels of concentrates may decrease c e l l u l o s e u t i l i z a t i o n because of the faster rates of passage associated with high concentrate rations. material might obtain a similar r e s u l t .  Fine grinding of the c e l l u l o s i c It  has also been postulated that  high levels of soluble carbohydrate might reduce the digestion of c e l l u l o s i c materials  (46).  Kitts and Underkrofler (59) investigated the mechanism of breakdown of c e l l u l o s e to develop methods that would increase the a v a i l a b i l i t y of c e l l u l o s e in low quality roughages dealing with c e l l u l o l y t i c enzymes from mixed rumen microorganisms by grinding the bacterial c e l l s from strained rumen f l u i d with alumina.  They could not detect c e l l u l o l y t i c  a c t i v i t y in the centrifuged and f i l t e r e d rumen f l u i d , suggesting that the c e l l u l o l y t i c enzymes were not present as in the rumen f l u i d but were associated with the bacterial c e l l s .  Being aware of the fact that glucose  was the only major product of hydrolysis of carboxymethyl c e l l u l o s e  (CMC)  35. they hypothesized that the c e l l u l o s e degrading enzyme of the rumen microorganisms was a "celloglucosidase." The rumen microorganisms are limited by the composition of the c e l l u l o s i c matter.  They are unable to degrade l i g n i n , which exerts a  protective action on the c e l l u l o s e with which i t is combined.  This  imposes a l i m i t of d i g e s t i b i l i t y on the structural matter of a l l  plant  materials fed to ruminants, d i g e s t i b i l i t y being negatively related to l i g n i n content (117).  Therefore, removing l i g n i n by the cheapest means  possible is a v i t a l concern for ruminant n u t r i t i o n i s t s .  Various methods  for d e l i g n i f i c a t i o n are being used by concerned investigators.  Some  are 1isted below: a)  Sodium hydroxide (30, 60, 124).  b)  Methods similar to those used in the pulping of wood for paper (88).  c)  The use of white rot fungi that can aerobically ferment lignin  (54) and c e l l u l o s e .  d)  Chlorite treatment (98).  e)  Sodium peroxide treatment  f)  Fermentation (54).  g)  Steaming under d i f f e r e n t combinations of time and pres-  (103).  sure [temperature] (9). h)  Irradiation  treatment (55).  i)  Ball m i l l i n g  j)  Acid hydrolysis  (28). (45), etc.  Kirk and Moore (54) have examined nine white-rot fungi for t h e i r a b i l i t y to remove l i g n i n from bigtooth aspen (Populus grandidentata Michx)  and yellow birch (Betula a l l g h a n i e n s i s ) .  During decay most of the fungi  removed a larger percentage of the l i g n i n than the polysaccharides. Lignin removal was always accompanied by removal of polysaccharides, but l i g n i n removal did not corrolate with removal of any p a r t i c u l a r component of the polysaccharides.  During decay l i g n i n was usually more  s e l e c t i v e l y removed in the f i r s t few percentages of weight loss than were the polysaccharides.  The decayed wood with less l i g n i n were more  digestible by a mixture of polysaccharidases and by rumen f l u i d than were the control samples.  It was known from this type of study that  in general the more l i g n i n removed from wood, the more digestible  is  the residual material. Although some white-rot fungi are e f f e c t i v e in removing l i g n i n faster than polysaccharides from wood, i t was mentioned by the authors that the process was r e l a t i v e l y slow. fungi  For practical use of white-rot  i t would be desirable to speed up the process.  Therefore, they  suggested meeting the following c r i t e r i a which are believed to be optimizing f a c t o r s : 1.  Aeration, moisture, temperature and source and amount of nutrient nitrogen should  2.  enhance the rate of decay.  To f i n d conditions that would improve the s e l e c t i v i t y of l i g n i n removal.  3.  To f i n d better fungus species or obtain desirable mutants, as is common in industrial  microbiology.  37.  III.  1.  MATERIALS AND METHODS  Materials Treatment tank:  A f i b e r glass fermentation tank (Figure 1) with  a total capacity of 1690 1 itres(operating volume of 1362 l i t r e s ) one inch thick styrofcam blanket bonded to the side for insulation purposes and the entire outer surface covered with a protective coat of canvas and epoxy resin (25) was u t i l i z e d in the study. Aerator:  Aeration was accomplished with a commercial compressor.  A four inch diameter rotor driven by a 1/2 H.P. e l e c t r i c motor at 1725 RPM delivered approximately 1400 gms of oxygen per day.  Aeration and  subsequent dissolved oxygen in the substrate was a c r i t i c a l thermophilic waste treatment processes Mixer:  Active  factor in  (25).  agitation of the substrate was considered to be  essential and was achieved by a 1 HP 1725 RPM motor d i r e c t l y connected to 7.62 cm - 3 blade propellor mounted on a 20.32 cm. diameter steel  draft tube (Figure 1).  stainless  The mixer provided a small amount of  aeration to the substrate (supplying approximately 0.54 kg. of oxygen per day).  In combination with the aerator, a previous t r i a l  the two units supplied a total of over 1.81 substrate  showed that  kg per day of oxygen to the  (25).  Pumps:  a)  Charge Pump - A "Moyno" charge pump with a 10.16 cm.  diameter intake and 3.81 cm. discharge was employed.  The pump was capable  of delivering 68.10 l i t r e s per minute. b)  Transfer Pump - The transfer pump is a smaller rever-  s i b l e action "Myno" screw pump.  This unit allowed transfer of the sludge  from the c l a r i f i e r unit to the digester or from the digester to the screening unit.  38.  00  39  CT) CO  FIGURE:'^  MIXING  ACTION  S H E P H E R D , 1977.  40,  41.  Transfer l i n e s :  A l l transfer lines were of 5.08 cm. diameter  (minimum) rubber tubing, equipped with quick coupling Screening:  fittings.  Processed mixed liquor and suspended solids  (MLSS)  were screened into three major fractions by a "Sweco" vibrating screen which was driven a)  by a 1/4 HP e l e c t r i c motor.  The coarse sized material was retained on a 30-mesh sieve (30 holes/2.54 sq. cm.).  b)  The f i n e r sized material was collected on a 150-mesh sieve (150 holes/2.54 sq. cm.).  c)  The l i q u i d f i l t r a t e was collected and subjected to a "baskettype" centrifugation for further solids  Drying:  recovery(56).  Process MLSS was subjected to drying using two d i f f e r e n t  sources of energy depending on the weather condition. a)  Solar energy was u t i l i z e d when weather permitted.  A thin  p l a s t i c sheet was spread over a dry ground surface on which the processed sawdust was evenly spread for drying. b)  An e l e c t r i c heater blanket covering a surface area of 5.95 square meters was used as the heating surface to supply drying conditions when the weather did not permit sun drying.  2.  Methods of Culturing the A)  Initial a)  Microorganisms  Stage Procedure for Substrate Preparation  The digester was washed, cleaned, and f i l l e d with tap-  water to a height at which the mixer propeller was just submerged. b)  Mixer and aerator were turned on.  Assuming a regulator  flow of a i r is of v i t a l importance for a successful aerobic.fermentation, a constant flow of fine a i r bubbles at the overhead of the sparger (Figure 1)  42.  was maintained. c)  One hundred and t h i r t y to one hundred and forty  liters  of fresh and p a r t i a l l y decomposed (from swine waste sump) swine manure (50:50 v/v) was added to the digester. d)  Additional water was added in order to adjust the working  volume to three-fourths of the digester capacity.  The remaining one-  fourth of the fermentation volumes provided space for foam insulation and for sawdust addition. , e)  Two to four kilograms of sawdust were added to the digester  in amounts a f t e r the temperature of the substrate within the digester reached 55°C. f)  Imhoff cone (volume of settleable solids) and i n i t i a l  temperature ( °C) measurements were taken immediately a f t e r completion of the f i l l i n g of the digester with substrate.  Coulthard (25)  stated  that the desirable quantity in the slump for thermophilic treatment process is when s e t t l e a b l e s o l i d s or p a r t i c l e s of the substrate are between 50 and 75%. B)  Culturing a)  Parameters  Temperature - temperature is one of the most important  environmental parameters affecting the growth, a c t i v i t y and evolution of organisms (3).  Surucu (110) stated that growth rate of  microorganisms  increases with temperature up to certain points and a f t e r that point, begins to decrease.  it  This would indicate that temperature can be  expressed as the resultant of two opposing a c t i v i t i e s on microorganisms which are: i)  rate of enzymatic a c t i v i t y is low at low temperatures and accelerates as the temperatures r i s e , and  43.  ii)  denaturation of c e l l  proteins (or enzymes) is not  s i g n i f i c a n t until moderate temperatures are attained. Protein inactivation increases rapidly with further increase in temperature. b)  pH - the [H] ion concentration e f f e c t is one of the factors  which has a very pronounced e f f e c t on enzyme reactions (77).  A change  in pH of the substrate or medium frequently occurs as the r e s u l t of microbial growth  (66).  One of the conditions which l i m i t s microbial growth is the hydrogen ion concentration of the medium. enzyme proteins w i l l occur.  At low pH denaturation of the key  With the exception of a few bacterial species,  such as the sulfate oxydizers, most bacteria are not active below pH 4.0.  The same is true at pH values greater than n e u t r a l i t y .  As pH  rises over 9.5, the hydroxyl-ipn begins to exert a toxic e f f e c t . i f any, microorganisms can survive above pH H.O.  Few,  Control of pH at  either a high or low range can be used to prevent or retard bacterial decomposition of p o t e n t i a l l y degradable materials c)  (77).  Dissolved oxygen - aeration in biological  processes  serves two important functions: i)  provides adequate oxygen to meet the demand of the microorganisms and  ii)  provides adequate means to ensure mixing in order to prevent s e t t l i n g of the solids  (23).  Aeration and subsequent dissolved oxygen in the substrate is a critical  factor in the thermophilic type of waste digestion  (25).  A  low level of dissolved oxygen in the medium tends to favour low thermal energy release, a low operating temperature and the development of  44.  undesirable microflora (24).  The aerobic thermophilic process demands  a high oxygen infusion due to the high rate of biological activity  oxidative  (24). d)  Imhoff cone measurement (Amount of Settleable  Solids)-  the settleable solids most frequently encountered in waste treatment practice are of such p a r t i c l e sizes that they tend to s e t t l e through water at constant rates depending on the e f f e c t i v e sizes and r e l a t i v e densities of the individual p a r t i c l e s  (47).  While a certain r a t i o of degradable organic wastes to water are necessary for the normal growth of microorganisms, an excessively high organic matter to water content of the medium can result in a depressed fermentation (77). settleable solids  Coulthard (25) indicated that 50 to 75%  is a desirable quantity in the slurry for the thermo-  p h i l i c treatment process.  Large volumes of water to solids r a t i o reduces  the level of heat energy derived from oxidation while an excessively high s o l i d content reduces the e f f i c i e n c y of mixing and aeration. C)  Del i g n i f i c a t i o n  As stated above, the digestor was f i l l e d to three-fourths of capacity.  its  Aeration and mixing (25) continuously creates a foam blanket  on the surface; thus, insulating the medium and preventing the heat being produced by the reaction from dissipating to the atmosphere. With a suitable physical and chemical environment the thermophilic bacteria multiply and a portion of the energy in the carbonaceous material is released to heat the medium to temperatures of 55° to 65° (25). A portion of 2-4 kg. of alder (Alnus rubra) was added to the digestor a f t e r the temperature of the substrate had reached 55°C.  The  45.  Imhoff cone percentage was used as a guide in order to decide when to add the sawdust, i . e . the lower the percent, the greater the amount of material that has been degraded and vice versa. For several days the temperature remained at the optimum range, resulting in a rapid d e l i g n i f i c a t i o n of the sawdust.  The declining  phase of the microbial c e l l growth was accompanied by a gradual decline in temperature of the mixed liquor and suspended sludge (M.L.S.S.), a Tow biological oxygen demand value (B.O.D.) and a s t a b i l i z e d sludge. A sample was collected from the vat before adding new unprocessed sawdust.  Excessive foam formation and low Imhoff cone percentage  were the basic guides for sample c o l l e c t i o n . stored in a deep-freezer until analyzed. used as a control for  Collected samples were  Unprocessed raw sawdust was  analysis.  The collected samples and unprocessed (control)  sawdust were  analyzed or tested for: a)  acid detergent f i b e r  b)  cellulose  c)  lignin  d)  crude protein and  e)  digestibility  D)  Harvesting  (in v i t r o  digestion)  Once the microbial growth ceased, the M.L.S.S. was pumped by a transfer pump to a screening unit.  The harvested products were collected  in three major fractions as described above. E)  Drying  The objectives of drying are to remove moisture, eliminate f l i e s  46.  and odours, and to f a c i l i t a t e the storage and handling of the processed product.  Regarding the odour and f l y problem, the processed product  had a negligible e f f e c t once i t has reached this dry stage. A moisture content in the 10-15% range w i l l  i n h i b i t mold and  microbial growth and minimize the dust problem (106). Solar energy (with one exception) was the only type of energy used for drying purposes throughout the experimental period. F)  Nutritional Value  The processed sawdust as well as unprocessed sawdust (control) was subjected to the following analysis  in order to determine the  n u t r i t i v e value of the product.  G)  1.  Dry matter  2.  Crude protein  3.  Bomb calorimetry  4.  Ash  gross energy  Analyses  Frozen processed sawdust (supernatant) dried in an oven at 65°C.  samples were thawed and  Similar drying treatment was carried out on  unprocessed sawdust (control).  Dried samples were ground to pass a  40-mesh screen and analyzed f o r : a) b) c) d) e) f) g)  dry matter acid detergent f i b e r cellulose lignin crude protein gross energy and ash  In v i t r o digestion was also conducted to determine the.effect of such microbial treatment towards dry matter d i g e s t i b i l i t y .  47. Procedures as described in the Association O f f i c i a l s of A g r i c u l tural Chemists (A.O.A.C.),  1960, 9th edition were used for a l l the  analyses with the exception of crude protein and gross energy.  Crude  protein was determined according to the Triebold Modification of A.O. A.C.  Gross energy determinations were made by the use of the Parr Bomb. The procedure for vn v i t r o digestion was a development of the  rumen liquor and pepsin-acid procedures from Larsen and Jones (63) with s l i g h t modification.  The source of inoculum was from a rumen of f i s t u l a t e d  steer which was fed a diet of a l f a l f a hay with premix mineral choice, and water available at a l l  times.  salt-free  48.  IV. 1.  Environmental  RESULTS AND DISCUSSION  Parameter  Studies were conducted in order to examine the p o s s i b i l i t y alder sawdust d e l i g n i f i c a t i o n  of  by aerobic thermophilic fermentation.  The purpose of the studies using the batch technique was to obtain i n formation r e l a t i n g to the environmental variables thought necessary to ensure the growth of microorganisms delignificantion.  and to t h e i r effectiveness in  The variables examined in the environmental  parameter  study included temperature, dissolved oxygen and pH. The relevant changes in temperature, pH and dissolved oxygen including ambient temperature of Batch I, 4, 5, and 6 respectively.  II  and III  are shown in Figures  In a l l three batches the pH increased as the  fermentation period progressed.  In most cases, in 7 to 10 days of proces-  sing the pH range was between 7.5 and 8.3.  McKinney (77) assumed that  the increase of pH in organic fermentation may be due to some ammonia production and also possibly some increase in amino acid content although analyses to support this assumption have not yet been reported.  The  l i t e r a t u r e on the growth of thermophilic organisms in pure cultures contains s u f f i c i e n t information to indicate that many thermophilic organisms grow best when the pH of the medium i s between 6.5 to 7.5  (67).  Regarding mixed liquor and suspended s o l i d s , Bragg, et al_. (13) reported that for the best result the aerobic thermophilic processing should be in the range of 7.5 to 8.5 pH value.  alkalinity  Brock and Darland  (15)  have shown in their work that the bacteria have the a b i l i t y to grow at either temperature or high pH, but not at both high temperature and high pH.  These workers were not s p e c i f i c as to the genus or species of  FIG. 4. RELEVANT-CHANGE  JN ENVIRONMENTAL VARIABLES BATCH X .  DURING  TREATMENT. I  45  70  56  o o UJ  42  rr ZD h<  or LL!  0_  28  Iii I-  14  5 °  ambient  temp.  Oj 5  PERIOD  10  15  OF T R E A T M E N T  20 (day).  25  0 35  X a.  FIG. 5. R t L E V A N T ' CHANGE  5 0  IN  ENVIRONMENTAL BATCH  VARIABLES  DURING  TREATMENT  TX  H  7.5  15 PERIOD  22.5 OF  30 TREATMENT  37.5 (day).  4  5  5 2  .5  60)  -  51  52. bacteria studied.  Nevertheless, these results suggest that there are  physico-chemical limitations of the environment beyond which l i f e impossible.  is  However, Surucu (110) indicated that there is l i t t l e d i f -  ference in the  pH requirements between thermophilic groups.  In case  the pH becomes a problem, he suggested the use of a phosphate buffer to maintain the pH of the medium at approximately n e u t r a l i t y . Temperature changes in Batch I, 4, 5, and 6 respectively.  II,  and III  are shown in Figures  These results show a relationship between high  rate of d e l i g n i f i c a t i o n and high temperature (Tables 2, 3, and 4). Surucu (110) reported that 55°C is the temperature which the highest observed net c e l l mass production occurred while the maximum growth and substrate u t i l i z a t i o n rates were noted at 58°C. did not d i f f e r s i g n i f i c a n t l y  However, these results  between 55 to 58°C (110).  Samples collected  within this range of temperature are low in l i g n i n and acid detergent f i b e r (Tables 2, 3, and 4).  Once the vat temperature reached the optimum  range (55° - 60°) i t remained isothermal for 4 to 8 days resulting in high d e l i g n i f i c a t i o n .  The foam produced as a result of the biochemical  a c t i v i t y of the vat, aids to conserve the released heat.  The temperature  generated in a l l the "batch" studies was followed by a decrease in temperature.  The addition of about 14 to 23 l i t e r s of fresh swine manure  caused the temperature to return to the optimum range within 24 to 48 hours' time.  These results indicate that swine manure in small quantities provides  adequate nutrient supply to provide optimum bacterial a c t i v i t y .  Regarding  the e f f e c t of temperature towards the growth of microorganisms, Cambell and Williams  (18) commented that in case the incubation temperature is  increased there is an increase in the growth requirements of the p a r t i c u l a r  53.  organism.  Further in their comment, they stated that at a higher  temperature the enzyme(s) responsible for the synthesis of a p a r t i c u l a r metabolite(s)  required, undergoes thermal inactivation and thus the  organism requires an exogenous source of compound before growth can be restored. In aerobic thermophilic type of processing, high temperature is achieved as a result of oxidizing the organic portion of the substrate by microorganisms.  A portion of the energy of catabolism is u t i l i z e d  by microorganisms and the surplus energy is released to the substrate in the form of heat and consequently an elevated temperature.  The  increase of temperature accelerates the biochemical reaction proceeding to a biological s t a b i l i z a t i o n of the waste material (24). of Popel's  The result  (90) work has indicated that 3.55 kcal of heat is produced  per gram of organic s o l i d s .  Observations of these studies and several  other "batch" process t r i a l s showed that the more heat produced, the greater the degradation of organic solids by the a e r o b i c bacteria. Therefore, the maintenance of high temperature during processing is of vital  importance for increasing the rate of biodegradation.  To achieve  t h i s , the following factors must be c l o s e l y observed: a)  Increasing oxygen transfer e f f i c i e n c y (51),  b)  Supplying high nutrient content of organic matter, and  c)  Insulation of  the reactor.  Aeration of a biological system serves two separate physical functions; oxygen transfer and mixing.  These two functions are of  primary importance in waste treatment systems.  Oxygen is essential  for  proper functioning of the aerobic organisms and mixing is required for transfer of oxygen into the l i q u i d phase (111), for uniform contact of  54.  microorganisms with waste material and also for preventing settlementation of sludge in the aeration vat. In these studies, dissolved oxygen was monitored by using a YSI Model-dissolved oxygen meter.  The results of a l l three batches showed  the decrease in dissolved oxygen (Tables 6, 7, and 8 ) ; i . e . i t was inversely related to temperature. Harrison (42) showed in his experiment that maximum growth can be obtained with an oxygen concentration of 1.4 to 4.6 mg/liter. Harrison claimed that an increase in oxygen concentration beyond t h i s range retarded the growth of the thermophiles.  In a "batch" experiment  performed in this study, the dissolved oxygen concentration of the medium was carried out between 2 and 5 mg/liter in order to provide a condition in which oxygen was not l i m i t i n g . It  has been pointed out by Coulthard (24) that the oxygen transfer  c o e f f i c i e n t is affected by the physical and chemical c h a r a c t e r i s t i c s of the system under aeration.  Along the same point Eckenfelder (7)  lists  the variable c h a r a c t e r i s t i c s as temperature, turbulent mixing, l i q u i d depth and waste composition.  This observation can be explained by the  fact that the concentration and the biological a c t i v i t y of mixed liquor suspended solids decreased with increasing treatment period.  This has  been demonstrated also by Surucu (110) that at higher s o l i d retention time values, autoxidation of biological solids becomes very important and increases the apparent amount of oxygen u t i l i z e d per quantity of organic matter removed.  As another p o s s i b i l i t y , the increase in oxygen  u t i l i z a t i o n may be the result of an increase in rate of endogenous respiration.  An increase in a i r requirement per unit organic-matter  55. removed with longer aeration periods is well documented in the l i t e r a t u r e . For example, a i r requirements for a conventional biological  process are  about 14 to 26 cu. meters/kg. of B.O.D. removed and i t increases to 34 to 51 cu.m./kg. of B.O.D. removed for the extended aeration process, where s o l i d retention time is longer  (78).  The oxygen input and output were expressed as functions of the a i r flow rate and oxygen transfer e f f i c i e n c y since these are of importance in the heat balance.  A study was conducted by Kambhu, et al_. (51) to  determine the quantity of oxygen leaving the reactor as dissolved oxygen in the medium; the r e s u l t they found was n e g l i g i b l e .  Therefore,  according to these studies reported in this t h e s i s , the results  (Tables  6, 7, and 8) showing the decrease of dissolved oxygen as the temperature rises should not be concluded as an increase of oxygen dissipation to the open atmosphere.  2.  Effect of Aerobic Thermophilic Bacteria on Lignin Content of Alder Alder (Alnus rubra) sawdust  1  was obtained from the lower Fraser  Valley, B r i t i s h Columbia and was c l a s s i f i e d as matured wood.  The size  of the sawdust p a r t i c l e was approximately 2.5 x 3 x 10.15 millimeters. The chemical composition and d i g e s t i b i l i t y of the untreated alder sawdust (control) the fresh swine manure and the processed samples of all  batches are given in Tables 2, 3, and 4. In the case of "batch" I and II,  sawdust was added each time the  percent of Imhoff read low which was the indication of a high percentage degradation or digestion, and when the vat temperature was within the optimum growth range (55° - 60°C) of aerobic thermophilic bacteria.  Purchased from Spruce Specialties Ltd.  Batch  III  56.  was designed to investigate: a)  Whether d e l i g n i f i c a t i o n was taking place before vat temperature reaches 55°C or not;  b)  Whether the stage of processing would y i e l d maximum d e l i g n i f i c a t i o n .  However, the major system of processing was not d i f f e r e n t from Batch I and II  except that 22 kg. of sawdust was added along with fresh swine  manure at the i n i t i a l stage of processing. Batch I has shown about 41% of d e l i g n i f i c a t i o n with a 24 hour period (Table 2).  This was with a limited sawdust load of 22 kg.  On  the other hand, 59% of d e l i g n i f i c a t i o n was achieved within 22 days of processing having a total load of 99 kg. of sawdust (Table 2). The result of Batch II within 13-17 days of  showed s l i g h t l y over 56% of d e l i g n i f i c a t i o n  processing with 44 kg of sawdust.  There was an  indication of poor d e l i g n i f i c a t i o n as the vat temperature drops (Table 3). This can be explained by the p o s s i b i l i t y that aerobic thermophilic bacteria and temperature play an important role in d e l i g n i f i c a t i o n . With Batch III,  the highest d e l i g n i f i c a t i o n rate (74%) was achieved  within 15 days of processing (Table 4).  As i t was mentioned e a r l i e r ,  this batch was treated only with 22 kg of sawdust throughout the processing period.  Again, as the vat temperature dropped there was a  lower d e l i g n i f i c a t i o n rate (Table 4). In most cases the extended rate of d e l i g n i f i c a t i o n was not as e f f i c i e n t as i t was during the f i r s t 24 hours (Batches I and II).  Pre-  sumably, this was mainly because of low load at the beginning and at this p a r t i c u l a r stage, the microorganisms are within t h e i r optimum growth range, accounting for the high chemical a c t i v i t y and measurable degree of degradation.  As the period of processing progressed, there was an  57. increase of sawdust load p e r i o d i c a l l y and exhaustion of the nutrients from the medium resulting in poorer d e l i g n i f i c a t i o n .  Under conditions  described in these studies 10 to 15 days of processing appears to be s u f f i c i e n t time to d e l i g n i f y 20 to 25 kg of alder sawdust. Relationship between l i g n i n removal and removal of c e l l u l o s e Due to the p a r t i a l u t i l i z a t i o n of c e l l u l o s e by microorganisms, i t was d i f f i c u l t to correlate the rate of d e l i g n i f i c a t i o n with the amount of c e l l u l o s e release.  As the amount of raw sawdust increased within  the fermenter, the amount of free c e l l u l o s e in the medium and rate of d e l i g n i f i c a t i o n decreased (Figures 7 and 8).  Low level of total  corresponded with high vat temperature (Table 2).  cellulose  Since the higher vat  temperature promoted a general increase in chemical a c t i v i t y and growth, these data might indicate that the released c e l l u l o s e was being u t i l i z e d by microorganisms  as a nutrient source.  In most experiments, low  analytical values for c e l l u l o s e was related to high vat temperature. However, this low cellulose-high processing temperature relationship was connected to the fermentation period of 3 to 7 days from the start of processing.  During the f i r s t few days a f t e r sawdust addition, percent  of c e l l u l o s e remained r e l a t i v e l y high (Table 2).  The high content of  c e l l u l o s e at an early stage of processing may be due to readily available nutrients from swine manure which is preferred to the c e l l u l o s e by microorganisms.  However, as the period of processing advanced, this  would be followed by exhaustion of preferred nutrients, and thus c e l l u l o s e would become the predominate medium.  Consequently, more c e l l u l o s e  u t i l i z a t i o n took place resulting in reduced c e l l u l o s e content. Most workers who attempted to isolate the organisms responsible  58.  for the primary attack on c e l l u l o s e have reached the conclusion that the thermophilic bacteria which actually decompose c e l l u l o s e to carbon dioxide and short chain organic compounds are obligate anaerobes  (38).  Claims have also been made, however, for c e l l u l o s e decomposition by aerobic thermophilic bacteria. Kellerman and McBeth  The f i r s t of these claims came from  (53).  Cellulose content decreased as processing time increased.  This  decrease was accompanied by .an increase in l i g n i n especially when the vat temperature was high (approximately 66°C) as shown in Figure 2. A r e l a t i v e l y high percentage of ADF depletion had also been exhibited at this temperature (Table 2). Under conditions described in these studies 10 to 15 days of processing seems to be a reasonable period of time to remove 5 0 - 7 $ of l i g n i n from a 20 to 25 kg load of alder sawdust. Percent of ash was taken to represent the inorganic constituents of the samples.  It  (mineral)  has been indicated by Macdonald, et a l .  (76) that the ash may, however, contain material of organic o r i g i n such as sulfur and phosphorus from proteins and some loss of v o l a t i l e material in the form of sodium, c h l o r i d e , potassium, phosphorus, and sulfur w i l l take place during i g n i t i o n .  The ash content is thus not  t r u l y representative of the inorganic material in a given sample quant i t a t i v e l y or q u a l i t a t i v e l y . Results of percent of ash of Batches I, Tables 2, 3, and 4 respectively.  II,  and III  In most cases the results  are given in indicate  that as the period of treatment progressed the percent of ash increased. Processed products have shown higher mineral content than supernatant samples.  Negative relationship between mineral concentration and gross  59. energy was observed (Tables 2, 3, and 4); i . e . the higher the mineral content the lower the gross energy and vice versa. Since the major constituent of the substrate  (swine manure and  alder sawdust) was predominantly carbohydrate in nature, results of supernatant as well as processed products showed l i t t l e variation in gross energy content (Tables 2, 3, and 4). The value of gross energy is not consistently a good indicator of the quality of the feed product in this regard.  A substantial  quantity  of heat can be contributed from l i g n i n by complete oxidation of unit weight of a sample and does not occur (complete oxidation of l i g n i n ) in. the animal's  body.  The y i e l d of processed product obviously depends on the volume of sawdust added and to a c e r t a i n extent to the amount of swine waste used in the process.  3.  In Vitro D i g e s t i b i l i t y Test It has been documented in a number of reports that wood residues  of 70 to 80% carbohydrate, are potential sources of dietary energy for ruminants.  However, the intimate association of carbohydrates with the  l i g n i n renders most wood p r a c t i c a l l y i n d i g e s t i b l e .  It  is known from this  and other types of study that in general the more l i g n i n removed from wood, the more digestible is the residual material by domestic animals. The chemical analyses and in v i t r o dry matter d i g e s t i b i l i t y for alder sawdust are given in Tables 2, 3, and 4 for Batch I, III,  respectively.  II,  results and  Values for d i g e s t i b i l i t y are averages of duplicates.  As the volume of sawdust added to the treatment vat increased, the percent of d i g e s t i b i l i t y  increased to a certain point and declined  o  60. Table 2.- Chemical composition and i n v i t r o d i g e s t i b i l i t y o f supernatant and processed product of alder s a w d u s t M - - Batch j " p  sawdust added(b) (kg.) Raw sawdust (control)  -  (%)  1  66 52 49  4 44 (66) 9 n 22 (88) 14 11 (99) 22  46 45 49 49 44  Fresh swine manure Supernatant:  (day)  22  {%)  (%)  34 52 19 (44) 44 20 (41) 43 20 (41) 26 19 (44) 14 . 17 (50) 14 16 (53) 22 14 (59) 16  (cal/q)  (%) \ JO j  6298 5989 5970 6161 6309 6044 6096 4338  1 12 9 10 8 10 10 10  6214 5584 5538  6 13 18  :  Processed products: 30 mesh 150 mesh 150 mesh f i l t r a t e  22 22 22  48 • 20 (41) 13 42 33 (.3) 27 39 • 28 (18) 20  e  r  n  a  t  a  n  t  In vitro dry matter digestibility \ Io  1  19 41 43 - 45 47 53 50 55 35 29 24  Vat temperature ( C)  Yield of tt) processed product (kg)  59 49 60 66 67 41 144.0 17.0 2.0  Table  C h e m i c a l c o m p o s i t i o n and i n v i t r o d i g e s t i b i l i t y o f and p r o c e s s e d p r o d u c t o f a l d e r s a w d u s t ( ) — B a t c h  3.  a  Amount o f T r e a t m e n t A c i d d e t . sawdust time fiber Added ( )  Lignin^ content  Cellulose content  supernatant II.  Ash  (cal/g)  {%)  (%)  6129 6285 5872 5790 5852 5919  1 8 12 12 n 13  19 48 61 63 60 58  5883 5531 5505  8 17 23  37 28 24 71  b  (kg.) Raw s a w d u s t Fresh  swine  Processed 30  (%)  -  13 17 11 (55) 26 .11 (55) 37  71 60 49 53 55 55  32 22 (31) 14 (56) 14 (56) 17 (47) 19 (41)  37 37 37  60 52 53  17 (47) 38 26 (19) 30 30 (6) 25  (control)  -  manure  Supernatant:  44  mesh  150  mesh  Solka  (%)  Vat temperature (°C)  Yield ofidl processed product (kg.)  -  55 56 43 25  products:  mesh  150  (%) 48 40 31 34 35 32  (day)  In v i t r o dry matter digestibility  Gross energy  filtrate  (purified  cellulose)  (a) (b)  Dry b a s i s Accumulated  (c)  Figures  in parentheses  (d)  Air  basis  dry  amount o f  sawdust  (kg.)  represent  percent of  delignification  49.0 11.0 2.0 -  CM  62. Table 4.  Chemical composition and in vitro digestibility of supernatant and processed product of alder sawdust( )- - Batch III. a  Amoung of Treatment Acid det. L i g n i n ^ Cellulose sawdust. . time • fiber content content Added ( ) (day) (kg.) (%) (%) (*)  Gross energy  Ash  (cal/g)  (%)  (*)  b  Raw sawdust (control)  -  -  73  35  Fresh swine manure  -  -  52  23  Supernatant:  20  3 '  -  In vitro Vat dry matter temperd i g e s t i b i l i t y ature  rc)  Yield of (d) processed product (kg.)  53  6278  3  16  .  43  6023  8  50  51  21 (40)  30  5617  17  41  46  9  47  17 (51)  33  5798  16  48  44  12  50  11 (69)  34  5497  15  . 50  43  15  45  9 (74)  35  5639  17  55  57  21  43  10 (71)  24  5292  19  54  47  30 mesh  21  -  -  -  -  150 mesh  21  45  25 (29)  28  5235  17  30.  43.0  150 mesh f i l t r a t e  21  37  28 (20)  23  4767  30  25  5.0  Processed products:  -  Solka (purified cellulose)  (a) (b) .(c) (d)  Dry basis Accumulated amount of sawdust (kg.) Figures in parentheses represent percent of delignification Air dry basis  -  -  73  63.  FIG. 7  to  R E S U L T S OF DELIGNIFICATION T O C E L L U L O S E CONTENT D I G E S T I B I L I T Y IN T E R M S OF T R E A T M E N T DURATION. B AT C H  AND  in vitro  I .  55 control  \v  in vitro d.m. d i g e s t i b i l i t y  cellulose' content  y-'~>-,~l* lignin content  Ol 1 0  T R E A T M E N T  15 • PERIOD  20" (doy).  22  FIG. 8 R E S U L - T S OF DELIGNIFICATION T O C E L L U L O S E D I G E S T I B I L I T Y IN T E R M S OF TREATMENT* BATCH  64. C O N T E N T in vitro  H .  55 c ontrol  in vitro d.m. digestibility  44  33  control cellulose  content  2 2 control lignin  II  0  8  16 T R E A T M E N T  24 (day)  content  32  ' 40  0  R E S U L T S  OF  DELIGNIFICATION  DIGESTIBILITY  IN  TERMS  5  OF  TO C E L L U L O S E  TREATMENT  10 TREATMENT  DURATION^.  15 PERIOD  CONTENT  BATCH  2 0 (day).  in vitro HL•  25  as tn  66. as the load of sawdust further  increased (Figures 7 and 8).  A parallel  relationship was detected between high d e l i g n i f i c a t i o n and high d i g e s t i bility.  However, low rate of d e l i g n i f i c a t i o n and in v i t r o  digestibility  was experienced along with low vat temperature a f t e r the sawdust volume reached 88 kg within two weeks of treatment. (Figure 7). In Batch II,  the periodical addition of small amounts of sawdust  accounted for an improvement in d i g e s t i b i l i t y as well as d e l i g n i f i c a t i o n (Figure 8). The result obtained from Batch III  of which only 22 kg of sawdust  was added at the i n i t i a l stage of treatment, c l e a r l y indicate the effectiveness of microorganisms  in d e l i g n i f i c a t i o n .  Progressive in v i t r o  digestion corresponding with the improvement of d e l i g n i f i c a t i o n was observed (Figure 9). The f i r s t 10 to 15 days of processing appears to have the major influence on d i g e s t i b i l i t y .  High d i g e s t i b i l i t y  (%) was attained beyond  which additional processing is of no value mostly in terms of d e l i g n i f i c a t i o n In v i t r o d i g e s t i b i l i t y test showed that processed sawdust is 55% digesti b l e which is equivalent to high quality a l f a l f a hay in terms of animal feeding value.  As a result of aerobic thermophilic bacteria treatment  almost threefold increase of alder sawdust dry matter d i g e s t i b i l i t y has been achieved compared to the control (19%).  Some of the samples which  showed low l i g n i n content f a i l e d to show high d i g e s t i b i l i t y and 4).  (Tabes 2, 3,  It seems reasonable to expect that the rate constant for digestion  of a forage is related to the structural composition of the m a t e r i a l ; however, factors other than l i g n i n content were involved to lower the d i g e s t i b i l i t y value.  67.  Although the experimental design did not allow the interaction between samples and other environmental variables  (in addition to swine  manure dosage and volume of sawdust added] to be s t a t i s t i c a l l y analyzed, there was an impressive trend which indicates that aerobic thermophilic bacteria treatment was more e f f e c t i v e in increasing the in v i t r o dry matter d i g e s t i b i l i t y of alder sawdust. P a r t i c l e size in a l l batch studies reduced [150 mesh, 150 mesh f i l t r a t e (centrifuged product)] the in v i t r o d i g e s t i b i l i t y value as well as the gross energy value, but the ash value increased.  These findings  indicate high mineral and less organic matter concentration in these p a r t i c u l a r samples. Based on these in v i t r o d i g e s t i b i l i t y observations, i t can be concluded that aerobic thermophilic treatment appears to be very e f f e c t i v e in making the nutrients in alder sawdust available to rumen microorganisms. 4.  Crude Protein Results of crude protein value of Batch I,.  Tables 6, 7, and 8, respectively.  II  and III  are shown .in  The crude protein value of the pro-  cessed sawdust was improved from 0% (control) to 17% (Table 8). The protein values for Batch I was low.(Table 6) compared to Batch II Before Batch II  and  III.  was conducted, i t was assumed that some loss of nitrogen  might have taken place during the drying process.  To confirm t h i s ,  samples were freeze dried and analyzed for crude protein value. no difference was observed.  Therefore, Batch II  However,  was designed to determine  whether long term (37 days) was a major contributing factor for the improvement of the crude protein percent or not. processing  (Table 7) has shown a substantial  The result of long  increase in the crude protein  68.  value compared to Batch I;  thus length of processing has an important  role to play in regards to nitrogen value. . Figure 12 was plotted to show the level of crude protein as may be affected by environmental variables.  it  In most cases, high crude  protein value p a r a l l e l s the sharp temperature r i s e and the high bacterial a c t i v i t y at the same time. Comparatively, 150 mesh s o l i d material and 150 mesh-filtrate have shown high crude protein value from supernatant samples. value of Batch III  The crude prote  was r e l a t i v e l y high (Table 8) compared to other  batches; this was mainly due to the addition of about 160 l i t e r s of p a r t i a l l y pre-processed substrate (after the e f f e c t of 22 kg of raw sawdust towards d e l i g n i f i c a t i o n study was completed). The percent of crude protein content (see Tables 6, 7, and 8) of the 150 mesh f i l t r a t e is higher than that of the 30 mesh and 150 mesh solids.  This indicates that most of the crude protein is in the fines  not captured by the 30 and 150 mesh screens.  Thus, i t is obvious that  to recover more protein the 150 mesh f i l t r a t e is the f r a c t i o n requiring further attention, since i t contains the highest percentage of crude protein.  In most cases, supernatant samples had higher crude protein  value than 30 and 150 meshes. An additional study was also carried out to determine the amount of s o l i d material that remains in the supernatant a f t e r centrifugation. This was found to be only 0.7%. could become a substantial  However, on a large scale basis this  loss in crude protein.  The data of Batch I shows the crude protein value of the sludge reached a maximum of 5% during the 22-day period with 99 kg sawdust load (Table 6).  In the case of Batch III  a maximum of 17% crude protein  69.  was achieved during the 21-day period with only 20 kg sawdust load which was added at the i n i t i a l stage of processing  (Table 8).  As i t was  mentioned e a r l i e r , the main contributing factor for high crude protein value in this p a r t i c u l a r batch was the addition of pre-processed.sludge. Batch II  required a 37 day period to reach the highest crude protein  value which was 12% with 66 kg sawdust load (Table 7).  Each time a given  amount of sawdust was added there was a drop of 5-10°C in vat temperature; and in some cases a total loss of foam which results in a 15-20°C drop in temperature. Similar batch treatment studies were conducted by Coulthard and Townsley (24) using i d e n t i c a l materials and culturing media (swine manure). They reported on 8% crude protein as a maximum value after a 7 to 9 day period.  The data of these studies indicated that the addition of sawdust  in a larger volume w i l l increase d i l u t i o n rates apart from creating a disfavourable environment to the microorganisms.  Thus, less c e l l y i e l d  and consequently longer periods of time for a given percent of crude protein (102); i . e . the percent protein of the biomass decrease within increasing d i l u t i o n rate. Regarding the quality of the single c e l l et alk  (S.C.) protein, Mateles,  (73) reported that the amino acid composition of thermophilic  organisms protein is of r e l a t i v e l y high q u a l i t y .  Surucu (110) commented  that the n u t r i t i o n a l value of S.C. protein is d i r e c t l y proportional to the protein content and inversely proportional to the nucleic acids content of the biomass.  Since the percentage of protein in the biomass  was higher and the nucleic acids were lower at the lower d i l u t i o n rate (44), i t would seem desirable to operate the system accordingly.  There-  f o r e , operation of the system at a lower d i l u t i o n rate is preferable from  70. the standpoint of the protein and nucleic acid content in the biomass, i . e . tradeoffs of d i l u t i o n rates against protein should be made i f maximum protein production is to be achieved. The most common sources of nitrogen for microorganisms are organic nitrogen, n i t r a t e , n i t r i t e , and ammonium compounds.  There are, however,  some species which can u t i l i z e atmospheric nitrogen; v i z . some bacteria and blue-green algae.  Table 5.  Nitrogen composition of the waste and/or wood substrate at i n i t i a l and f i n a l stage of processing  Total Nitrogen Composition g  Substrate at i n i t i a l stage  a t c h  (%)  Processed product H L  I  1.28  1.12(47)  0.16(11)  II  1.12  2.08(65)  1.44(56)  III  1.28  3.36(72)  2.08(62)  * Dry matter basis  . H = Highest L = Lowest  Figures in parentheses implies total nitrogen in percent  Results of Batches II  and III  indicated an increase of 65% to  72% (Table 5) respectively of total nitrogen in the f i n a l processed product.  The organic nitrogen contribution from the swine waste was  only 35% for Batch II of Batch I,  and 28% f o r Batch III  (Table 5).  But in the case  the results showed a 6% reduction in total nitrogen content  (Table 5) from the i n i t i a l nitrogen content of the substrate.  Thus,  71. d e n i t r i f y i n g organisms might have been involved in the fermentation process compared to n i t r i f y i n g organisms.  Since the crude protein per-  cent value of the sawdust was n i l (Tables 6, 7, and 8) the supply of organic nitrogen from this source was n e g l i g i b l e . The data of this study (Batch II  and III)  indicate that micro-  organisms p a r t i c u l a r l y those capable of f i x i n g atmospheric nitrogen have contributed a substantial amount of nitrogen to the total nitrogen content of the processed product.  Linday (66) stated that atmospheric  nitrogen f i x a t i o n by microorganisms together with e l e c t r i c a l  discharge  in the atmosphere, by which molecular nitrogen can under normal circumstances, be made available f o r the majority of l i v i n g  things.  The general mechanism of the nitrogen cycle is shown in Figure 10. Nitrate is the most common inorganic source of nitrogen in the aerobic environment.  This is due to the rapid oxidation of ammonia to n i t r a t e  by bacteria (lithotrophic) Levy, et al_. (65). accomplished both by aerobes and anaerobes.  Nitrogen f i x a t i o n is Levy et al_. (65) stated  that a key nitrogenous compound in microbial n u t r i t i o n is  inorganic  ammonium, for i t combines with organic acids (d-keto acids) to form amino acids.  The p r i n c i p l e product of this event is glutamic a c i d .  Further  in t h e i r statement regarding the mechanism of n i t r i f i c a t i o n , they also mentioned that many bacteria possess this a b i l i t y and have the additional enzymes to form a l l of t h e i r nitrogen-containing c e l l u l a r constituents from glutamic a c i d .  Microorganisms, therefore, can use inorganic  ammonium salts as t h e i r sole source of nitrogen.  The u t i l i z a t i o n of  n i t r a t e by microorganisms as a source of nitrogen f o r c e l l u l a r synthesis e n t a i l s the reduction of n i t r a t e to the oxidation level of ammonia; at which i t can be incorporated into amino acids and other nitrogen-  72.  F i g . 10.  THE  NITROGEN  CYCLE  _ LEVY  * i al. I  9  7  CN  Dissimilatory reduction I De ni t ri fication )  N fixation ( Nitrification )  Nitrosomonas  NH.  \ \  \  , Microbial degradation  v  - > N 0  \  Immobilization  \  ...  \  Nitrobacter  2  —  V \  2^.  Assimilatory 0  r g a  n  i c  reduction  ( 1 m m o bi Ii z a ti on )  _ Reduction Oxidation No valency' change  3  73. containing c e l l  constituents.  As shown in the nitrogen cycle (Figure 10),nitrogen f i x a t i o n a reduction process required.  is  and a supply of potential reducing agents is  Levy (65) stated that the nitrogen-fixing aerobes (the  hetertrophic Azotobacter species, the symbiotic Rhizobia, and the photosynthetic blue-green algae) carry out the reaction in an e s s e n t i a l l y anaerobic microenvironment.  The nitrogenase enzyme system that performs  the reduction is in a l l cases oxygen sensitive.  It seems l i k e l y that  the nitrogenase i s protected from oxygen by a very high rate of r e s p i r ation in Azotobacter, which l i m i t s the access of oxygen to the s i t e of nitrogen f i x a t i o n . As i t was mentioned e a r l i e r , the fixed nitrogen f i n a l l y becomes ammonia, and is then combined to form amino acids, and thence to protein. Degradation of nitrogenous material, for example, dead c e l l s , follows the reverse pathway down to ammonia; in which form i t may be released or rebound into protein again. when they decompose. no valency  Organic nitrogen compounds y i e l d ammonia  T h i s . i s primarily a hydrolytic reaction involving  (65).  Mischustin and Shilnicova (80) have reported that the nitrogen f i x i n g bacteria (Azotobacter chrooccum) is e f f e c t i v e within pH range (6.2 to 8.8) which in the case of a l l batches of this study the pH was within the reported range. There are also present d e n i t r i f y i n g organisms which reverse this process, releasing atmospheric nitrogen.  Thus some organisms f i x nitrogen  for themselves, and this becomes available for others a f t e r c e l l and breakdown.  death  Whilst others d i r e c t l y convert i t to a form more available  for other organisms (66).  Regarding d e n i t r i f i c a t i o n , Levy (65)  stated  74. that d e n i t r i f i c a t i o n reaction c h a r a c t e r i s t i c a l l y carried out by aerobes that find themselves in an anaerobic environment.  If  there are oxidizable  carbon compounds present these w i l l be oxidized and n i t r a t e is u t i l i z e d as the terminal electron acceptor instead of oxygen.  There is a pos-  s i b i l i t y that such a reaction took place in Batch I;  because the  final  processed product has shown a decrease of 13% to 88% in total  nitrogen content from the i n i t i a l  stage (Table 5).  75. LO  Table 6.  Results of crude p r o t e i n value and environmental v a r i a b l e s i n terms o f sawdust volume and treatment time - - Batch I Amount o f sav/dust added (kq.)  Treatment time (day)  Raw sawdust ( c o n t r o l )  Crude (GT Vat temperature Dry m a t t e r ( ) p r o t e i n b  (%)  (X)  pH  Dissolved oxygen  (mq/1)  (°C)  86  0  -  -  -  Fresh swine manure  -  -  97  8  -  -  -  Supernatant:  22  1  97  3  59  • 8.2  2.4  _  4  . 96  4  49  8.4  3.0  9  97  • 3  60  6.8  0.4  .11  96  4  66  7.2  0.6  22 (88)  14  • 96  4.  67  7.7  0.8  11 (99)  22  94  5  41  7.8  .2.0-  22  93  1  4A (66)  -  c  Processed products: 30 mesh 150 mesh  -  22  89  2  150 mesh f i l t r a t e  -  22  93  7  fa] (b) (c)  Accumulated amount o f sawdust (kg.) Average o f t r i p l i c a t e sample. . Dry matter basis and average of t r i p l i c a t e sample  76.  e 7.  R e s u l t s o f crude p r o t e i n v a l u e and e n v i r o n m e n t a l v a r i a b l e s terms o f sawdust volume and t r e a t m e n t t i m e - - B a t c h II  Amount  of  sawdust Treatment a d d e d (a) time (kg.) Raw  sawdust  Fresh  swine  (dav)  (b) C r u d e ^ protein Dry  matter  '  (%)  (%)  PH  (°0  Dissolved oxygen (ma/1)  -  -  86  0  manure  -  -  95  7  44  13  93  9  55  8.34  0.7  -  17  93  9  56  8.47  3.9  26  92  11  43  8.29  5.5  .37  93  12  25  7.36  5.0  C55)  1 1  " (66)  '  products:  30  mesh  -  37  93  9  150  mesh  -  37  93  11  -  37  93  13  150 mesh  Vat temperature  (control)  Supernatant:  Processed  in  filtrate  Tal •(b)  Accumulated, amount o f Average of t r i p l i c a t e  (c)  Dry  matter  basis  and  sawdust sample  (kg.)  average of  triplicate  sample  1-^  77.  T a b l e 8.  R e s u l t s o f c r u d e p r o t e i n v a l u e and e n v i r o n m e n t a l v a r i a b l e s t e r m s o f sawdust v a l u e and t r e a t m e n t t i m e - - B a t c h III  Amout o f sawdust Treatment added time (kq.)  Raw s a w d u s t  (control)  F r e s h s w i n e manure Supernatant:  , ,Crude( ) Vat Dry m a t t e r '• v p r o t e i n t e m p e r a t u r e  (day)  (%)  150 mesh  la) (b)  filtrate  pH  (°C)  (50  .  (mq/1)  -  -  -  -  22  3  • 96  15  46  7.8  1.0-  -  9  S3  15  44  8.17  2.5  -  12  96  13  43  8.75  4.4  -  15  94  15  57  8.20  2.2  21  94  17  47  6.51  3.3  '88 97 •  0 8  6.9  •  -  -  150 mesh  Dissolved oxygen  b  a  Processed products: 30 mesh  in  21  --'  -  -  21  94  13  -  21  92  21  A v e r a g e o f t r i p l i c a t e sample Dry m a t t e r b a s i s and a v e r a g e o f t r i p l i c a t e  sample  FIG.  71  THE R A T E OF DELIGNIFICATION  AND E N V I R O N M E N T A L  (THREE BATCHES COMBINED) •  • - N  VARIAeL^ VARIABLES. 70  ^ m p e r o t u r e  UJ rr  4.5-^  FIG. 12. R E S U LT S  79.  OF CRUDE PROTEIN V A L U E AND ENVIRONMENTAL V A R I A B L E S  T R E A T M E N T OF THE T H R E E B A T C H E S  COMBINED.  TREATMENT  PERIOD  (day).  IN TERMS OF  80. V.  SUMMARY AND CONCLUSION  Aerobic thermophilic bacterial treatment Of sawdust material was studied employing a batch system.  Swine manure was used both as a source  of inoculum and culture media. The high temperature which was produced solely from microbial action promoted the sawdust d e l i g n i f i c a t i o n rate to a satisfactory degree.  The decreases in l i g n i n content of the sawdust was associated  with a substantial  increase in d i g e s t i b i l i t y .  Studies concerning the e f f e c t of longer periods of fermentation on percent protein in the biomass showed that the percentage of protein in the biomass increased with the longer period of treatment and with the decreasing d i l u t i o n (less addition of sawdust) rate.  81. BIBLIOGRAPHY 1.  Alexander, M.  Nitrification.  and F. E. Clark, eds). Madison, Wisconsin 2.  In "Soil Nitrogen" (W.V. Bartholomew  Publ. No. pp. 303-343. Agr. Soc. Agron.  (1965).  A l l e n , M. B. (a) The Dynamic Nature of Thermophil. J . Gen. Physiol. 33:205 (1950).  3.  A l l e n , M. B. (b) "The Thermophilic Aerobic Sporeforming B a c t e r i a . " B a c t e r i d . Rev., 17:125 (1953).  4.  Anthony, W. B., Cunningham, J r . J . B., and Harris, R. R. Sawdust as Feed for Ruminants, p. 135. Cellulose and Their Applications.  5.  In Robert F. Gould [ e d . ] ,  Adv. Chem. Ser. 95.  Chemical Society, Washington, D. C.  American  (1969).  Baker, H., Hunter, S. H., and Sobotka, H. Thermophily:  Hardwood  Nutritional Factors in  A Comparative Study of B a c i l l i and Euglena.  New York  Academy of Sciences, 62:351 (1952). 6.  Baker, H., Frank, 0., Pashr, I.,  Hunter, S., and Sobotka, H.  "Growth Requirements of 94 Strains of Thermophilic B a c i l l i , " Canadian Journal of M i c r o b i o l . , 6:557 7.  Barlow, B.  (1960).  A Spoilage of Canned Corn Due to a Thermophilic Bacterium.  Thesis, University of I l l i n o i s , Urbana (1912). 8.  Beckman, J . W.  Recovery of Vegetable O i l s and Fats by a Bacterial  Process. J . Ind. Eng. Chem. 22:117 (1930). 9.  Bender, F., Heaney, D. P., and Bowden, A.  Potential of Steam Wood  as a Feed for Ruminants. For. Prod. J . , 20:36 (1970). 10.  Berlyn, G. P. and Mark, R. E. Walls.  11.  Lignin Distribution In Wood Cell  Forest Products, J . 15:140 (1965).  Bhat, J . V. and B i l l i m o r i a , M. H. "Problems in Thermophily," J .  Indian Inst. S c i . , 37:113 (1955). . 12.  •  Bland, D. E. and Menshum, M. The Liberation of Lignin By Pretreatment of the Wood of Eucalyptus Regnans With A l k a l i , J . of the Australian and New Zealand Pulp and Paper Industry Technicial Association (Appita),. 21:17 (1967).  13.  Bragg, D. B., Coulthard, T. L., K i t t s , W. D., and Saben, H. S. Thermophilic Digestion of Swine Waste:  The Production of Feed  Grade Single Cell Protein for Use in Livestock Rations.  Department  of Poultry Science, Agricultural Engineering and Animal Science, University of B r i t i s h Columbia, Vancouver, B. C. 14.  Breed, R. S.,  Murray, R. G. and Smith, N. R., eds.  "Bergey's Manual  of Determinative Bacteriology," 7th ed. Williams and Wilkins, B a l t i more, Maryland 15.  (1957).  Brock, T. D. and Darland, G. K. Temperature and pH.  16.  Brownell, H. H.  Limits of Microbial Existence:  Science 169: 1316 (1970).  S t a b i l i t y of the Lignin-Carbohydrate Complex.  Technical Association of the Pulp and Paper Industry (TAPPI), 54: 66 (1971 ). 17.  Cambell, E. G.  A Thermophil N i t r i t e Former.  18.  Cambell, L., Leon, J r . and Williams, 0. B.  Science, 75:23 (1932).  The Effect of Temperature  on the Nutritional Requirements of Facultative and Obligative Thermop h i l i c Bacteria. 19.  Cameron, E. J . and Esty, J . R. Food.  The Examination of Spoiled Canned  2. C l a s s i f i c a t i o n of Flat Sour Spoilage Organisms from Non  Acid Foods. 20.  J . Bact., 65:141 (1953).  J . Infectious Diseases, 39:89 (1926).  Caspian, E. P. and Rettger, L. E.  Limitation of Bacterial Growth at  Higher Temperatures. J . Bact. 26:77 (1933).  83. 21.  Chian, E. S. K.  Symposium on Nove Approaches to Microbial  and Control of Waste.  Utilization  Biotechnology and Bioengineering. Vol. 18:  1177 (1976). 22.  Cleverdon, R. C., Pelczar, M. J . J r . and Doetsch, R. N. Requirements of Stearothermophilic Aerobic Sporogenous  The Vitamin Bacteria.  J . Bact. 58:113 and 58:523 (1949). 23.  Cooney, C. L. and Daniel, I.  C. Oxygen Transfer and Control in  Biological  Page 63, e d i t o r : Raymond P. Canale, 1971.  Waste Treatment.  Interscience Publishers - A Division of John Wiley and Sons - Toronto, New York. 24.  Coulthard, T. L. and Townsley, P. M. Swine Waste.  Thermophilic Processing of  Agricultural Engineering Department and Food Science  Department, University of B r i t i s h Columbia, Vancouver, B. C , No. 73-222 25.  (1973).  Coulthard, T. L. and Hendren, G. A.  P i l o t Plant Studies on the  Thermophilic Bacterial Treatment of Animal Wastes. Engineering B. C , 26.  paper  Agricultural  Department, University of B r i t i s h Columbia, Vancouver,  paper no. 73-504  (1973).  Crampton, E. W. and Maynard, L. A.  The Relation of Cellulose and  Lignin Content to the Nutritive Value of Animal Feeds.  J . Nutr.  15:383 (1937). 27.  Curran, H. R.,  Bronstetter, B. C. and Mayers, A. T.  Analysis of Vegetative Cells and Spores of Bacteria.  Spectrochemical J . Bact. 45:  485 (1943). 28.  Dehority, B. A. and Johnson, R. R.  Effect of P a r t i c l e Size Upon the  In V i t r o Cellulose D i g e s t i b i l i t y of Forages by Rumen Bacteria. J . Diary S c i . 44:2242 29.  Di Palma, Domenico.  (1961). Thermophilic Aerobic Microbial Processing of  Chicken Waste *• Batch #4.  Department of Agricultural Engineering,  University of B r i t i s h Columbia, Vancouver, B. C, (1974), 30.  Donefer, E., Proc. Symp. E f f e c t of Processing on Nutritient Value of Feeds.  In National Academy of Sciences, p. 211 Washington,  D. C. (1973). 31.  Donefer, E.  Effect of Processing on Nutrient Value of Roughages.  In National Academy of Sciences, p. 224, Washington, D. C. (1973). 32.  Douning, A. L., Painter, H. A., and Knowles, G. the Activated Sludge Process.  N i t r i f i c a t i o n in  Inst. Sewage P u r i f . , J . Proc. 130:  158 (1964). 33.  Eckenfelder, W. W., J r . H i l l Co. Inc.  Industrial  Water Pollution Control, McGraw-  (1966).  34.  Eckford, M. 0. Thermophilic Bacteria In Milk, Am. J . Hyg. 7:201  35.  Egorova, L. A.  Growth and Development of Extremely Thermophilic  Bacteria at 70°C. 36.  (1927).  Microbiol. 44:117 (1975).  Fischer, A., Volesungen i i b e r Bakterien 1897.  Jones, A. J . ,  t r a n s l a t i o n , Claredon, Oxford (1900). 37.  Foter, M. J . and Rahn, 0.  Growth and Fermentation of Bacteria  Near Their Maximum Temperature. 38.  Gaughran, E. R. L.  J . Bact. 32:485 (1936).  The Thermophilic Microorganisms.  Department of  Bacteriology, Rutgers University, New Brunswick, N. J . Bacteriological Reviews, 11:189 (1947). 39.  Gibbs, W.. M.  The Isolation and Study of N i t r i f y i n g Bacteria.  Soil S c i . Proc. 8:427 (1919). 40.  Goring, D. A. I., Chemical Pulping.  Microscopic Patterns of Lignin Removal During The Physics and Chemistry of Wood Pulp Fibers,  pp. 107-145 Technical Association of the Pulp and Paper Industry, 360 Lexington Avenue, New York 10017 (1970).  85.  41.  Hansen, D. A,, "The Growth of Thermophilic Bacteria" Arch. Mickrobiol. 4:23 (1933).  42.  Harrison, D. E. F.  "Physiological E f f e c t s . o f Dissolved Oxygen  Tension and Radox Potential on Growing Populations of Microorganisms," 5th International Symposium on the Continuous Culture of isms, edited by Dean A. C. R., Press 43.  P i r t , S. J . and Tempest, D. W., Academic  (1972).  Haug, R. T. and McCarty, P. L., N i t r i f i c a t i o n With the Submerged Filter.  44.  Microorgan-  J . Water P o l l u t . Contr. Fed., 44:2086 (1972).  Herbert, D. "Continuous Culture of Microorganism, Some Theoretical Aspects," Continuous Cultivation of Microorganisms. Symp. p. 45, Prague (1958).  45.  Hudson, J . M. Factors Affecting Hydrolyzed Sawdust as a Feed for Animals.  Ph.D. Thesis University of Nebraska, L i n c o l n , Nebraska  (1971). 46.  Hungate, R. E.  The Rumen and Its Microbes.  New. York, Academic,  p. 175 (1966). 47.  Imhoff, K., Muller, W. J . and Thistlethwayte, D.  K. B.  Disposal  of Sewage and Other Water-Borne Wastes, pp. 30, 31 and 150.  Ann  Arbor Science Publishers Inc. Ann Arbor (1971). 48.  Imsenecki, A. A. and Solnzeva, L. I. Cultures of Thermophilic Bacteria.  49.  Imsenecki, A. A. and Solnzeva, L. I.  Production of Amylase from Chem. Abstracts, 39:3807 (.1945). The Growth of Aerobic  Thermophilic Bacteria. J . Bact. 49:539 (1945). 50.  Jaworski, N., Lawton, G. N. and Rohlich, G. G. "Aerobic Sludge Digestion," I n t l . Journ. Air and Water P o l l . ( B r i t ) . , 4:106 (1961).  51.  Kambhu, K. and Andrews, J . F.  Aerobic Thermophilic Process for the  86. Biological Treatment of Wastes - Simulation Studies. Water and. Pollution Control Federation, 41:Ri27 52.  K a t a g i r i , M. and Nakahama, T.  Journal of  (1969).  Useful Thermophilic Bacteria for  Fermentation Degumming, J . Agr, Chem Soc. Japan 15;1042, B u l l . Agr. Chem. Soc. Japan, 15:144 (1939). 53.  Kellerman, K. and McBeth, G. "The Fermentation of C e l l u l o s e , " Cent. Baht. Parasitenk. II.  54.  34:485 (1912).  Kirk, T. K. and Moore, W. E.  Removing Lignin From Wood With White-  Rot Fungi and D i g e s t i b i l i t y of Resulting Wood.  Wood Fiber, 4:72  (1972). 55.  K i t t s , W. D., Krishnamurti, C. R.,  Shelford, J . A. and Huffman,  J . G. Use of Wood and Wood Byproducts as a Source of Energy in Beef Cattle Rations, p. 279. Their Applications.  In Robert F. Gould (ed.) Cellulases and  Adv. Chem. Ser. 95, American Chemical Society,  Washington, D. C. (1969). 56.  K i t t s , W. D., Bragg, D. B., Coulthard, T. L., and Saben, H. S. Symposium - Uses of Agricultural Wastes.  In Canadian Plains Proce-  edings - 2, p. 76-79 (1974). 57.  K i t t s , W.. D. and Krishnamurti, C. R. Livestock Feed. Agricultural Institute  58.  The P o s s i b i l i t i e s of Wood as Review, p. 18, Nov.-Dec.  (1970).  K i t t s , W. D. and Krishnamurti, C. R. "Wood Wastes as Livestock Feed," Symposium held at University of B r i t i s h Columbia, Vancouver, B. C. (1976).  59.  K i t t s , W. D. and Underkrofler, L. A. "Digestion By Rumen Microorganisms" Hydrolytic Products of Cellulose and the C e l l u l o y t i c Enzymes.  J.  Agr. Food Chem. 2:639 (1954). 60.  Klopfenstein, T. J . , Krause, V. E., Jones, M. J . and Woods, W. Chemical Treatment of Low Qua!ity Roughages. (1972).  J . Anim. S c i . 35:418  61.  Kountz, R. R. and Forney, C. Oxidation Activated  Metabolic Energy Balances in a Total  Sludge System.  Sewage Ind. Wastes, 31:810  (1972). 62.  K r u i j , E.  Cent. Bakt. Parasitenk, II.  63.  Larsen, R. E. and Jones, G. M.  Abt. 26, page 26 (.1910).  A Modified Method for the In Vitro  Determination of Dry Matter and Organic Matter D i g e s t i b i l i t y .  Can.  J . Anim. S c i . 53:251 (1973). 64.  Lechtenberg, V. L., Colenbrander, V. F., Bauman, L. F. and Rhyberd, C. L.  Effect of Lignin on Rate of In V i t r o Cell Wall and Cellulose  Disappearance in Corn. J . Anim. S c i . 39:1.165 (1974). 65.  Levy, J . , Cambell, J . J . R. and Blackburn, T. H. Microbiology, page 185.  66.  Linday, E., Margery.  Introductory  John Wiley and Sons, Inc. Toronto (1973).  In Practical Introduction to Microbiology,  page 58-59, E. and F. N. Spon Ltd.  Northumberland Press Limited,  Gateshead on Tyne (1962). 67.  Loehr, R. C.  Agricultural Waste Management.  Academic Press, New  York and London (1974). 68.  Loginova, L. G., Golovacheva, R. S. and Egorova, L. A.. The L i f e of Microorganisms at High Temperature, Nauka, Moscow (1966).  69.  Loginova, L. G., Khraptsova, G. I.,  Golvina, I.  Yakovleva, M. B. and Bogdanova, T. I., 70.  Loginova, L. G. and T s p l i n a , I. of Thermophilic Microorganisms.  71.  72.  A.  G., Tsaplina, I.  A.,  Microbiology 45:927 (1977).  Symposium on Enzymes and Proteins  Microbiology, 45:494 (1976).  MacFadyen, A. and B l a x a l l , F. R.  Thermophilic Bacteria, J . Path.  Bact., 3:87; B r i t . Med. J . 2:644  (1896).  Mandelstam, J . and McQuillen, K., Biochemistry of Bacterial Growth, p. 183, John Wiley and Sons, Inc.,  N. Y.  (1968).  88. 73.  Mateles, R. I.,  Baruah, J . N. and Tannenbaum.  p h i l i c Bacterium on Hydrocarbons:  Growth of Thermo-  A New Source of Single-Cell  -Protein, Science 157:1322 (1967). 74.  Maynard, L. A. and L o o s l i , J . K. page 76.  75.  Animal N u t r i t i o n , Sixth e d i t i o n ,  McGraw-Hill Book Company, Toronto (1969).  McBee, R. H.  The Culture and Physiology of a Thermophilic C e l l u l o s e -  Fermenting Bacterium. J . Bact. 56:653 (1948). 76.  Mcdonald, P., Edwards,  R. A. and Greenhalgh.  pp. 17 and 19, O l i v e r , and Boyd, Edinburgh. L t d . , Edinburgh, Great B r i t a i n 77.  McKinney, R. E.  Animal N u t r i t i o n , T. and A. Constable  (1971).  In Microbiology for Sanitary Engineers, pp. 58,  135, 185 and 186. McGraw-Hill Book Company, Inc. Toronto, N. Y. (1972). 78.  Metcalf and Eddy, Inc., Wastewater Engineering. McGraw H i l l , N. Y. (1972).  79.  M i l l e r , Yu. M., Pozmogova, I.  N. and Loginova, L. G., Mikrobiologiya,  41:479 (1972). 80.  Mischustin, E. N. and Shilnicova.  Biological  Fixation of Atmospheric  page 198, The MacMillan Press L t d . , London/Madras 81.  Morrison, L. E. and Tanner, F. W. Bacteria.  82.  Studies on the Thermophilic  Botan. Gaz. 77:171 (1924).  Mulbarger, M. C. Sludge Systems.  83.  N i t r i f i c a t i o n and D e n i t r i f i c a t i o n in Activated J . Water P o l l u t . Contr. Fed. 43:2054 (1971).  Muller, L. D., Barnes, R. F., Bauman, L. F. andColenbrander, V. F. Variation in  L i g n i n and Other Structural Components of Brown  Midrib Mutants of Maize (Zea mays L.) 84.  (1971).  Crop. S c i . 11:413 (1971).  Muller, L. D., Lechtenberg, V. L., Bauman, L, F,, Barnes, R,' F. and Rhykerd, C. L. In Vivo Evaluation of a Brown Midrib Mutant of (Zea  mays L.), J . Anim. S c i . 35:885. (1972). 85.  Murray, H.  Aerobic Decomposition of Cellulose by Thermophilic  Organisms. J . Bact, 47:117 (1944). 86.  Novick, A. "Growth of Bacteria," Ann. Rev. M i c r o b i o l . 9:97 (1955),  87.  O'Brian, R. J .  Symposium on Enzymes and Proteins of Thermophilic  Microorganisms. 88.  Micro. B i o l . 45:494 (1976).  Pew, J . C, and Weyna, P.  Fine Grinding, Enzyme Digestion, and the  Lignin-Cellulose Bond in Wood.  Technical Association of the Pulp  and Paper Industry [TAPPI,(Technical 89.  Section)], 45:247 (1962).  Pfonder, W. H., Grebing, S. E., Hajny, G. and Tyree. Wood Drived Products in Ruminant N u t r i t i o n , p. 298. Gould (ed.), Cellulases and Their Applications. American Chemical Society, Washington, D. C.  90.  Popel, F. and Ohnmacht, C. M.  The Value of In Robert F.  Av. Chem. Ser. 95.  (1969).  Thermophilic Bacterial Oxidation  of Highly Concentrated Substrates.  Water Research Program Press,  6:807 (1972). 91.  Pozmogova, I. Organisms.  92.  N. Nature of the Metabolism of the Thermophilic  Microbiol. 44:436 (1975).  Pozmogova, I.  N. Growth Dynamics of Mesophilic and Thermophilic  Microflora From a Thermal Spring. 93.  Pozmogova, I.  Microbiol. 44:276 (1975).  N. and Malyan, A. N. Physiology of Thermophilic  and Methophilic B a c i l l i During Cultivation at Optimal and Submaximal Temperature. 94.  Microbiol. 45:249 (1976).  P r i c k e t t , P. S.  Thermophilic and Thermoduric Micro-organisms  Special Reference to Species Isolated from Milk. Spore Forming Types.  with  V. Description of  N. Y. State Agr, Expt. Sta. Tech. B u l l . 147.  95.  Pringheim, H. "Uberden Fermentation Abbauder Cellulose" Z. Physiol. Chem. 78:266 (1912).  96.  Rimer, A. and Woodward, R. L.  Two Stage Activated Sludge P i l o t  Plant Operations - Fitchburg, Massachusetts.  J . Water P o l l u t .  Contro. Fed. 44:101 (1972). 97.  Riquelme, E., Dyer, I.  A., Baribo, L. E. and Couch, B. Y., Wood  Cellulose as an Energy Source in Lamb Fattening Rations.  J . Anim.  S c i . 40:977 (1975). 98.  Saarinen, P., Jensen, W. and Shojarvi.  Act. Agra, Fennica 94:41  (1958). 99.  Scott, R. W. Feeding.  100.  M i l l e t , M. A. and Hajny, G. J . Wood Wastes for Animal  Forest Prod. J . 19:14 (1969).  Scott, S. W., Fred, E. B. and Peterson, W. H., Products of the Thermophilic Fermentation of Cellulose.  Ind. Eng. Chem. 22:731  (1930). 101.  Shepherd, D. W. M.Sc. Thesis.  Waste Recycling By Thermophilic Fermentation. Department of Bio Resource Engineering, U . B . C ,  Vancouver, B. C. 102.  (1977).  Sherrard, J . M. and Schroeder, E. 0. "Variation of Cell Yield and Growth Rate in the Completely Mixed Activated Sludge Process" Presented at the 27th Annual Purdue Ind. Waste Conf. W. Lafayette, Ind.  103.  (1972).  Smith, L. W., Goering, H. K. and Gordon, C. H. Proc. Conf. Anim. Waste Management Conf., page 88, Cornell Univ., Ithaca, New York (1969).  104.  Smith, N. R., Bacteria.  Gordon, R. E. and Clark, F, E.  U.S.  Aerobic Sporeforming  Dept. Agr., Agr. Monograph No. 16:148 (1952).  105.  Smith, L. W., Goering, M. K. and Gordon L.H.  Relationships of  Forage Consumption With Rates of Cell Wall Digestion and  Indigestible  Contents of Dairy Science, 55:1140 (1972). 106.  Sobel, A. T. "Moisture Removal," Proc. 1971.  Cornell Agr. Waste  Management Conf. pp. 107-114, Cornell University, Ithaca, New York (1971). 107.  Stainer, R. Y., Doudoroff, M. and Adelbert, E. A. "The Microbial World'. Prentice H a l l , N.Y. 1  108.  Stryer, L.  (1970).  Biochemistry, pp. 42, 167, 263, and 266.  Freeman,  W. H. and Company, San Francisco (1975). 109.  Surucu, G. A., Chian, E. S. K. and Engelbrecht. p h i l i c Treatment of High Strength Wastewaters.  Aerobic ThermoJ . Water P o l l u t .  Cont. Fed. 48:669 (1976). 110.  Surucu, G. A. Thermophilic Aerobic Microbiological Treatment of High Strength Wastewaters and Recovery of Protein, Ph.D. Thesis, 1975. University of I l l i n o i s At Urbana - Champaign.  111.  Tanner, F. W. and Wallace, G. I. Growth of Thermophilic Bacteria.  112.  T e t r a u l t , P. A.  Relation  of Temperature to the  J . Bact. 10:421 (1925).  The Growth of Thermophilic Cellulose Decomposing  Organisms on Agar. J . Bact. 19:15 (1930). 113.  Townsley, P. M. Personal Communication, Department of Food Science, University of B r i t i s h Columbia, Vancouver (1978).  114.  Van Goest, P. J .  Symposium bn Nutrition and Forage and Pastures:  New Chemical Procedures for Evaluating Forages.  J . Anim. S c i . 23:  838 (1964). 115.  Van Goest, P. J . Development. of Comprehensive System of Food Analysis and Its Application to Forages.  J . Anim. S c i . 26:119 (1967).  116.  Van Goest, P. J , and Mertens, D, R,  Composition and Nutritive  Characteristics of Low Quality C e l l u l o s i c Wastes. Fed. Proc. 33: 1942 (1974). 117.  V i l j o e n , J . A., Fred, E. B. and Peterson, W. H. of Cellulose by Thermophilic Bacteria.  118.  Waldo, D. R.,  119.  J . Agr. S c i . 16:1  Smith, L. W. and Cox, E. L.  Disappearance from the Rumen.  The Fermentation (1926).  Model of Cellulose  J . Dairy S c i . 55:125 (1972).  Washington, D. R. and Symons, J . M. V o l a t i l e Sludge Accumulated in Activated Sludge Systems.  J . Water P o l l u t . Contr. Fed. 34:767  (1962). 120.  Waterbury, J . B., Remsen, C. C. and Watson, S. W.  Nitrospira  sp.  and Nitrococcus sp. - Two New Genera of Obligate Autotrophic N i t r i t e Oxidizing Bacteria. 121.  Weinzirl, J .  B a c t e r i d . Proc. 45 (1968).  The Bacteriology of Canned Food.  J . Med. Research,  39:349 (1919). 122.  Werkman, C. H. Bacteriological Vegetables.  Studies of Sulfide Spoilage of Canned  Iowa Agr. Expt. Sta. Research B u l l . 117, pp. 163-180  (1929). 123.  Wild, H. E. , Sawyer, C. N. and MacMahon, T. C. N i t r i f i c a t i o n Kinetics.  124.  Factors Affecting  J . Water P o l l u t . Contr. Fed. 43:1845 (1971).  Wilson, R. K. and Pigden, W. J .  Effect of a Sodium Hydroxide  Treatment on the U t i l i z a t i o n of Wheat Straw and Poplar Wood by Rumen Microorganisms. 125.  Can. J . Anim. S c i . 44:122  Woodman, H. E. and Stewart, J . in the Ruminant Organism.  The Mechanism of Cellulose  Digestion  The Action of C e l l u l o s e - S p l i t t i n g Bacteria  on the Fiber of Certain Typical Feeding Stuffs, (1932).  (1964).  J . Agr. Sci'. 22:527  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094543/manifest

Comment

Related Items