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

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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 Brit ish Columbia May, 1978 Maheteme SelassieIWolde-Tsadick In presenting this thesis in partial fulf i l lment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the l ibrary 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 financial gain shall not be allowed without my written permission. Maheteme Selassie Wolde-Tsadick University of Brit ish Columbia Department of Animal Science Vancouver, B.C. ABSTRACT It has been indicated through a review of the l i terature that wood by-products have a potential as a dietary source of energy for the ruminant animal. However, l ignin constitutes a barrier to the proper u t i l i z -ation of cel lulose. Generally, any treatment to remove or a l ter l ignin makes the cellulose within 1ignocellulose materials more susceptible to the act iv i ty of the ce l lu lo l y t i c enzymes. Therefore, an e f f i c ient bio-logical treatment would require a system to solubi l ize or to remove l ignin from the 1ignin-carbohydrate complex. Cellulose within ruminant feeds forms an effective substrate for eventual conversion to body protein. There are several methods available for de l ign i f icat ion. This study was carried 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 ut i l i zed by the bacteria for ce l l function and mult ipl icat ion. The re-mainder of the available energy was released as heat energy. In this method the heat necessary to maintain the temperature in the thermophilic range was derived from both mechanical and from microbial ac t iv i ty . Thermophilic act iv i ty is considered to reduce the time required for organic waste digestion over that experienced by mesophilic digestion. The rate of the destruction of pathogenic bacteria, virus and other organisms is increased as a result of the high temperatures fermentation. Batch studies conducted to delignify alder sawdust by the use of the aerobic thermophilic oxidation method demonstrated that the l ignin content of sawdust can be reduced by as much as 74%, and crude bacterial protein was generated by approximately 17%. i i i 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 del ignif icat ion. i'Y TABLE OF CONTENTS Page I. INTRODUCTION • • • ix II. LITERATURE REVIEW 1 1. Basic Biological Processes 1 A. Biochemical Reactions 1 B. The Energy Relationships in the System 3 C. Biochemical Transformations 5 a) Carbon 6 b) Nitrogen 6 c) N i t r i f icat ion .7 (i) Dissolved oxygen . 8 ( i i ) Temperature 9 ( i i i ) pH 9 d) Denitr i f ication 10 e) Phosphorus 10 2. Thermophilic Microorganisms 10 A. Origin and Distribution 11 a) Division of Thermophiles 11 b) Growth at High Temperature . . 14 c) Growth at Low Temperature 15 B. Potential of Thermophilic Aerobic Digestion of Organic Solids 16 C. Nutritional Requirements . 18 D. The Effect 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. Wood and Its Potential In Ruminant Diet . 26 A. Lignin Distribution in Wood Tissues . 29 B. Lignin 30 C. Del ignif ication 31 III. MATERIALS AND METHODS 37 1. Materials 37 2. Methods of Culturing the Microorganisms-. . . . . . 41 A. Init ia l Stage of Procedure for Substrate Preparation . . 41 B. Culturing Parameters 42 a) Temperature 42 b) pH 43 c) Dissolved oxygen 43 C. Delignif ication 44 D. Harvesting • • 45 E. Drying • . 45 F. Nutritional Value . . • • 46 G. Analyses 46 IV. RESULTS AND DISCUSSION 48 1. Environmental Parameter 48 2. Effect of Aerobic Thermophilic Bacteria on Lignin Content of Alder 55 Relationship Between Lignin Removal and Removal of Cellulose 57 3. In Vitro Digest ib i l i ty Test 59 4. Crude Protein 67 V. SUMMARY AND CONCLUSION .' 80 BIBLIOGRAPHY . . . . . . . . . 81 VITA 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 5. Relevant change in environmental variables during treatment -Batch II 50 6. Relevant change in environmental variables during treatment -Batch III 51 7. Results of del ignif ication to cellulose content and in vitro d igest ib i l i ty in terms of treatment duration and amount of sawdust added - Batch I 63 8. Results of del ignif icat ion to cellulose content and in vitro d igest ib i l i ty in terms of treatment duration and amount of sawdust added - Batch II 64 9. Results of del ignif icat ion to cellulose content and in vitro d igest ib i l i ty in terms of treatment duration and amount of sawdust added - Batch III 65 10. The nitrogen cycle 72 11. Results of the rate of del ignif icat ion and environmental variables in terms of the amount of sawdust added and duration of treatment of three batches combined . . . . . . . 78 12. Results of crude protein value and environmental variables in terms of treatment of the three batches combined . 79 v i i LIST OF TABLES Page 1. The distribution of l ignin in black spruce earlywood 29 2. Chemical composition and in vitro d iges t ib i l i ty of supernatant and processed products of alder sawdust - Batch I 60 3. Chemical composition and in vitro d igest ib i l i ty of supernatant and processed products of alder sawdust - Batch II 61 4. Chemical composition and in vitro d igest ib i l i ty of supernatant and processed products of alder sawdust - Batch III 62 5. Nitrogen composition of the waste and/or wood substrate at i n i t i a l and final stage of processing 70 6. Results of crude protein value and environmental variables in terms of sawdust volume and treatment time - Batch I . . . 75 7. Results of crude protein value and environmental variables in terms of sawdust volume and treatment time - Batch II . . . 76 8. Results of crude protein value and environmental variables in terms of sawdust volume and treatment time - Batch III . . 77 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 wi 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 their 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. CR. Krishnamurti, Department of Animal Science. Sincere thanks is also expressed to Professor C. L. Coulthard, Dr. C. Cross, and P. William for their 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 efforts made this study possible. ix I. INTRODUCTION Lignin present in natural fibers provides both a physical and a chemical barrier to enzymes that are able to attack isolated fibers of cel lulose. Physically, penetration by enzyme molecules is suppressed; chemically, 1ignin-carbohydrate complexes form metabolic blocks that inhibit polysaccharide hydrolysis (21). Therefore, an ef f ic ient 'b io log ica l treatment may require a system to solubi l ize or remove l ignin thus dis-sociating the 1ignin-carbohydrate complex. In general, any treatment to depolymerize and solubi l ize l ignin makes the cellulose in 1ignocellulosics more susceptible to the act iv i ty by ce l lu lo ly 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-it ion of plants owing to maturity and species as well as the presence of natural inhibitors to cellulases a preliminary physical, chemical and biological method to alter the structure of plant materials appears to be necessary before ce l lu lo ly t i c enzymes can be effect ive. 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 plentiful renewable energy resource. Use of cellulose as ruminant feed affords an effective means by which the pro-duction of high quality protein can result (97). Incorporation of c e l -lulosic material as the energy source in ruminant rations wil 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 tabi l izat ion, pasteurization and deodorization of waste arising from many sources such as animal and plant production. The X bacteria that occur naturally in agricultural wastes (eg. swine manure) under aerobic conditions wil 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 is required. Such nutrients, present in the waste, include nitrogen, phosphorus, sulfur and minor trace elements a l l of which are essential for bacterial cel l production. Bacterial degradation of waste releases a part of the energy for ce 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 effect 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 result 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-fo ld: 1. to examine the apparent capacity of aerobic thermophilic bacteria to delignify alder sawdust (wood). 2. to subject fermentation samples collected at different periods of fermentation and levels of temperatures to in vitro assays designed to evaluate the d igest ib i l i ty of residual carbohydrates, and 3. to study the potentiality of this biological method in single cel l protein production. II. LITERATURE REVIEW 1. Basic Biological Processes A microbiological process may be defined as a sequence of biochemical events under the control and direction of microscopically v is ib le l iv ing ce l l s , in particular bacteria, fungi, and algae. 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 yielding useful energy to the microorganisms. Microbes that use oxygen as the ultimate electron acceptor are termed "aerobic micro-organisms" (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 ce l l s . Agitation of the l iquid medium keeps the microbial "masses" in suspension thereby maintaining a continuous contact with the substrate and removal of waste products (110 )• A) Biochemical Reactions Eff ic ient ut i l i zat ion of the substrate for microorganisms involves a maximum conversion of energy and carbon into an increase in cel l numbers. Thus the organism is concerned primarily in ce l l duplication (110). The - 1 -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 systems with "synthesis" 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 lustrated 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 ce l l s . The energy ut i l ized in the equation is obtained during the metabolism of the wastes. Synthesis or. growth is affected by the ab i l i ty of the microorganisms to metabolize and assimilate the food. In addition, the presence of toxic materials, the temperature, the ava i lab i l i ty 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 imit the extent of microbial act iv i ty . Whatever the nature of the substrate, i t must contain a suff ic ient amount of the major elements such as carbon, nitrogen, phosphorus and minerals to meet the nutritional 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 vessel, carbon may become a l imiting factor as the carbonaceous material is metabolized and lost from the systems as carbon dioxide. When growth becomes l imited, or increases in cel l mass many of the microorganisms may die and lyse thereby releasing the nutrients of their protoplasm which may be ut i l ized by the remaining or scavenging ce l l s . Further in his explanation pertaining to biochemical reactions Loehr (67) stated that in the presence of waste material and congenial l iv ing conditions, microbial metabolism wil l occur allowing new cel l s to be formed; energy and the microbial solids wi l l increase. In the absence of food, endogenous respiration wil l predominate and a reduction of the net microbial solids wil l result. The depleted cel l residue wil 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 ig) reported that a residue of about 20 to 25% of synthesized microbial mass wil l remain. When organic matter is metabolized and resynthesized into microbial ce l l s , the converted waste also being organic matter is part ia l ly stab-i l i z ed . As indicated ear l ier , the microbial ce l l s are capable of further degradation. Only when the biochemical oxygen demand of the waste degrad-ation has stopped does a stabil ized effluent result (67). B) The Energy Relationships in the System . Al l ce l l s , whether animal or plant, use similar fundamental mechanisms for their energy transforming act iv i t ies (108). 4. Loehr (67) indicated that knowledge of the energy relationships of microbial cel l s permits an understanding of energy available for syn-thesis and respiration, of production of microbial ce 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 ut i l i zed by the l iv ing cel l is in part conserved chemically in the compound known as adenosine t r i -phosphate (ATP). ATP is the carrier of chemical energy from the oxidation of foods to those processes of the cel 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 cel l s could be from animal wastes (swine) and sawdust (alder). Adenosine triphosphate is formed from adenosine diphosphate (ADP) during oxidation in the ce l l s . 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 cycle, receiving energy during the oxidation of foods and releasing energy during the performance of ce l lu lar 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 ce l lu lar reaction, i t is the common intermediate in the energy transformation in the cel l s (67). The ultimate electron acceptor is oxygen in aerobic organisms. However, electrons are not transferred direct ly from fuel molecules and their breakdown product to oxygen. Instead, these substrates transfer electrons to special carr iers, such as pyridine nucleotides or f lav ins. The reduced forms of these carriers then transfer their high-potential electrons to oxygen via an electron-transport chain located in the inner membrane of mitochondria. It was stated ear l ier 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 wil l produce a more oxidized end product or effluent than wil l anaerobic conditions and wi l l permit synthesis of a greater quantity of microbial ce l l s . These additional cel l s are an asset because i t is 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 for 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 cel ls synthesized per unit of food metabolized wi l l be s ignif icantly less than for aerobic organisms. C) Biochemical Transformations Several studies described in l iteratures (67) (109) ( i l l ) and (61) have ascertained the fact that to achieve satisfactory biological break-down of wastes, the material must contain suff ic ient 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 cel l synthesis. Loehr (67) br ief ly 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 systems. a) 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, CgH^N, and CO,,. Organic Carbon + 0 2 — • CgH^N + C0 2 The uptake of oxygen and formation of carbon dioxide represent the effects of respiration. 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 wil 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 te and nitrate nitrogen. Organic N * ammonium N « n i t r i te N * nitrate N. 7. The oxidation of ammonia to n i t r i te and nitrate is termed n i t r i -f ication and occurs under aerobic conditions. A more basic definit ion of n i t r i f i ca t ion 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 1s statement,, a residual dissolved oxygen concentration of about 2 mg/1 has been found necessary to have optimum n i t r i f i ca t ion . 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 te to nitrate, 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 wil l require 4.57 kg. of oxygen. c) N i tr i f icat ion N i t r i f i cat ion can be defined basically 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 act iv i ty . This suggestion was verif ied in sewage and soil studies which showed that oxygen was essential and that alkaline conditions favored n i t r i f i ca t ion . Several genera of n i tr i fy ing 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 te to nitrate. Of these genera, only Nitrosomonas and Nitrobacter 8. are generally encountered in aquatic and soil ecosystems and are the n i tr i fy ing autotrophs of importance. Two new genera of obligate auto-trophic nitrite-oxydizing bacteria, Nitrospira and Nitrococcus species have been reported (120). Factors l ike dissolved oxygen, temperature and pH play an important role in the n i t r i f i ca t ion process. (i) Dissolved Oxygen: Since the n i t r i fy ing organisms are aerobic, adequate dissolved oxygen (DO) is necessary to support n i t r i f i ca t ion 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 sensit iv ity of the Nitrobacter to low dissolved oxygen concentrations is one of the reasons complete n i t r i f i ca t ion is d i f f i c u l t to accomplish in heavily loaded systems where the oxygen demand is significant and where adequate oxygen does not exist in the microbial f loe. A c r i t i c a l dissolved oxygen concentration exists below which n i t r i f i ca t ion 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 imit is more dependant on the rate of oxygen diffusion to the microorganisms than on the oxygen concentration in the mixed l iquor. To assure that the dissolved oxygen concentration in a treatment unit is not l imiting n i t r i f i c a t i on , the dissolved oxygen in a treatment unit generally is kept above 1.0 mg/l. N i t r i f i cat ion proceeds at a rate independent of the dissolved oxygen above the 9. c r i t i c a l concentration (67). ( i i ) Temperature: N i t r i f i cat ion is affected by the temperature of the media. 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). One of the early studies (39) indicated that an exposure for 10 min. at 53°C - 55°C and at 56° - 58°C k i l led Nitrosomonas and Nitrobacter, respectively. Laboratory activated sludge studies indicated that the rate of n i t r i f i ca t ion increased throughout the range of 5° - 35°C (i23) -Information on n i t r i f i ca t ion at low temperatures indicates confl ict ing data. Different studies have indicated that n i t r i f i ca t ion did not develop below 10°C (96), that i t was possible to maintain n i t r i f i ca t ion at 8°C (82), that l i t t l e n i t r i f i ca t ion was achieved at temperatures below 6°C (32) and that n i t r i f i ca t ion was achieved at temperatures below 1°C (43). ( i i i ) 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 alkaline side (67). In a detailed study of n i t r i f i c a t i on , 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 fy ing organisms can adapt to low pH levels and achieve adequate n i t r i f i ca t i on . 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) Denitrif ication Microbial denitr i f icat ion takes place under anoxic conditions where n i tr i tes and nitrates are used as terminal electron acceptors in place of molecular oxygen (67). The nitrates and ni tr i tes 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. Nitrogen gas (N2) is the primary gaseous end product. Other gases produced include nitrous oxide (N^O), n i t r i c acid (NO), C0 2 and H 2 (67). Denitrif ication is brought about by facultative heterotrophic bacteria. Most of the active denitrifying organisms belong to the genera of Pseudomonas, Achromobacter, Bacillus and Micrococcus (67). e) Phosphorus Phosphorus is an important nutrient in biological processes. The phosphorus content of bacterial ce l l s is about two percent (1). The sources of phosphorus in waste processes include organic matter, phos-phates 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 wil 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 lass i f icat ion of microorganisms, thermophiles f a l l under bacteria. 11. A) Origin and Distribution The origin 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 br ief ly . 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 inal 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 poss ib i l i t ies -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-logical area seem to accept Imsenecki's ejt al_. (49) assumption. Along the same l ine Loginova and Tsaplina (70) assumed that most of the thermo-phi l ic organisms developed from mesophilic species close to them in such a way that the enzymes and other ce l l components of the thermophiles acquired heat resistance. a) Division of thermophiles According to Smith et al_. (104), thermophilic bacteria are c lass i f ied 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 suff ic ient data to make possible a comparison among organisms. The most recent designations reviewing the early descriptive terms of 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 ies between 55°C and 60°C. They may be divided into two groups: a) 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 slight to abundant growth at 28-30°C. Thermophiles constitute a very heterogenous group, i f a l l organ-isms are included with an optimum temperature for growth above 50°C. Their morphology and staining reactions are varied. Fundamental d i f fe r -ences appear in their nutritional requirments and metabolic act iv i t ies (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 solid material which in his case was 20% potato broth. The optimum growth temperature for thermophilic bacteria is usually somewhat lower in l iquid medium than on an agarized medium. . This has been con-firmed by Loginova (68). Based on this fact, Egorova (35) assumed that thermophilic bacteria are no exception in this respect. According to Egorova's studies, no differences in cel l morphology were observed during the 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 different stages of i ts 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 linear 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 volat i le acids liberated into the external medium (91). Pozmogova (93), in his ear l ier 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 Mil ler et al_. (79). The ab i l i ty of thermophiles to survive at higher temperatures s t i l l remains unanswered. Among poss ib i l i t ies 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, particularly 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 iv ing objects known, and vegetable cel ls (27). Regarding the growth limitation of thermophilic bacteria Allen (3) 14. reported that i t is due to exhaustion of the dissolved oxygen ava i lab i l i ty at elevated temperatures. This statement has been backed by results of Tanner et al_. ( I l l ) , but neither has mentioned nutrient exhaustion effect. 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. At 55°C, Tanner and Wallace observed most rapid increase in cel l numbers and, after the period of active growth, a rapid death; and since cultures often became s ter 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 iquid medium at an elevated temper-ature, in accord with the observed relationship of oxygen tension and the capacity to sporulate among the aerobes, as well as facultative 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 invest i -gation, i t was mentioned that calcium carbonate was added in order to 1 8 neutralize acid production. The maximum viable cel l y ie ld (6 x 10 per ml) was obtained at 42°C, rather than at 55°C; with a second optimum cel l y ie ld at 20°C. A study of the rate of biochemical act iv i ty of a number of ther-mophiles indicated that the high reproductive rate is inadequate in explaining the intense biochemical act iv i t ies of the thermophilic bacteria. In some thermophilic cultures the progress of proteolysis may, of course, be related to reproduction rate, death and autolysis, 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 bio-chemical act iv i ty of thermophilic bacteria may be found in the very intense metabolic act iv i ty of these organisms, and not merely in their rapid proliferation (38). It has been suggested that rapidly metabolizing bacterial vegetatiye cel l s growing in a depleted medium with an accumulation of waste products of metabolism would l imit bacterial survival unless they were capable of existing into a dormant or resistant stage. The ab i l i ty 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 their optimum growth range, but experience a depression in this act iv i ty as the maximum growth temperature l imit is reached (38). c,) Growth at low temperature Thermophilic microorganisms, arb i t rar i ly characterized by an optimum temperature above 45 - 50°C exhibit considerable latitude in their overall temperature range for growth. A large number of thermo-phi l ic 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 imits fj.or growth. Morrison and Tanner (81) have suggested that during the growth of thermophilic bacteria at lower temperatures, the time of observation is of greatest significance. Foter and Rahn (37) in their analysis of the l imit ing 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 cel l are influenced differently by a change in temperature, with the result that the growth mechanism is upset. In their further comment, they mentioned that the accumulation of toxic metabolites within the cel l is not considered as a possible cause for a disturbance of the growth mechanism in the case of bacteria or other ce l l s with large surface area. Contrary to their 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 leve l , temperature and pH are also important, since these changes in the environment may be produced by the microorganisms themselves, thus l imiting the development of the microbial population. Brock and Darland (15), in their study pertaining to effect of temperature and pH on microbial existence, concluded that bacteria have the ab i l i t y to grow at either high temperature or high acidity, but not at both high temperature and high acidity. 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 required to maintain thermophilic temperatures. However, heat can be produced by microbial thermogenesis as evidenced by the autothermal nature of composting processes which usually operate in the thermophile range (51). 17. Simulation studies have shown that suff ic ient heat can be gen-erated 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 ted the possible advantages of ther-mophilic aerobic digestion over the corresponding mesophilic process: a) Increased reaction rate. According to the classic thermodynamic rule of Van't Hoff-Arrhenius, the bio-chemical reaction rates wil l double with each 10°C temperature increase (109). b) Increased eff ic iency. c) Improved solids - l iquid separation. d) Increased destruction of pathogenic organisms. e) Biomass harvested from thermophilic aerobic digestion process may have relat ively high protein and vitamin content ( 109) • f) The single ce 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 compos-it ion for nutritional purposes (73). Although many thermophiles had been isolated from canned food early in the history of the canning industry, the canned food spoilage effect did not receive suff ic ient attention until Barlow's (7) work was published. The significance of these bacteria l ies in their ab i l i t y to ferment lactose, or less commonly decompose proteins, and cause undes-irable flavours or odours (38). Apart from their undesirable activities,thermophiles do have some 18. beneficial aspects as well. They are considered as potential agents: a) in controlled fermentation of cellulose to useful products (38); b) in the recovery of vegetable o i l s and fats (8); c) in the degumming of s i lk (52); d) in enzyme application, i .e. amylase (38) and degummase (48) as well as in pharmaceutical products. Certain enzymes of thermophilic microorganism are being tested as drug preparation. A determination of the proteolytic act iv i ty in different species of the isolated spore-forming bacteria showed that the largest zones of casein hydrolysis are characteristic of B. Stearothermophilus, B. substitis  B. brevis and B. megaterium (69). The authors (Loginova, et al.) also refer to the role not only of the above l i s ted organisms but also to B. cereus in the decomposition of organic substance. C) Nutritional 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 nutritional requirements of 12 strains of B. stearothermophilus and they responded wel 1 with the addition of thiamine, nicotinic 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 their study. Similar experiments have been carried to a greater depth by Cambell and Williams (18) which 19. were based on essential amino acids and vitamins as a function of temper-ature (55°C). 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 ref lect the inabi l i ty of cel l to synthesize these vita l components ahead of their destruction. Furthermore, Baker ejt al_, (6), after studying strains of B. coagulans and 68 strains of B. stearothermophilus, reported that these organisms f e l l into four main nutritional patterns, i . e . : a) methionine; b) methionine plus other metabolites; c) methionine but clearly stimulated by other factors and, d) no methionine. These results are also in agreement with Cambell and Williams (18), and Bhat and Bil l imoria (11). Several investigators have indicated that the three vitamins: b iot in, niacin and thiamin are essential for thermophiles growth (3). Similarly, the same authors (3) reported that glucose, lactate and malate were the best carbon sources for growth at temperatures of 20 - 37°C. However, addition of 0.3% monosodium glutamate to the simple glucose medium permitted growth at 55°C for some thermophiles. 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 ce l l s , which in turn sug-gests that thermophiles may be halophilic (salt 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 bac i l l i are in real i ty quite simple and that they do not need extensive vitamin and amino acid supplement - and that past culture d i f f i cu l ty 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 relat ively high permeability and high osmotic tolerances of thermophiles permit the ef f ic ient provision of catalysts, fuel and building blocks to these systems (5). Based on their findings, Baker ejt aj_. (5) concluded that "high temperature" strains u t i l i ze a wider range of substrates, have a higher osmotic tolerance and also a heightened permeability than do "low temperature" strains. Various thermophilic organisms are unable to u t i l i ze the simple mono and disaccharides (121). Prickett (94) indicated that some of the thermophiles are also unable to u t i l i ze starch and cellulose. It has been pointed out that, although some thermophiles are proteolytic (8) and several are non-cel lulolyt ic, the act iv i ty of these organisms in symbiotic relationships in nature would suggest that they are, at most, feebly capable of attacking native proteins (38). This has been found to be true with pure cultures of food spoilage organisms, such as the hydrogen sulfide or sulfur producing stinker organisms in canned corn (122) and the thermophiles found in milk (34). Hydrogen sulfide and 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 sulfide were produced only by a limited number of organisms. Gaughran (38) in his review, cited types of thermophilic microorganisms according to their specific ac t i v i t i e s , - i .e. nitrogen f ix ing, n itros i fy ing, denitr i fy ing, 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 ibof lavin. Excellent growth also resulted, surprisingly, i f instead of the vitamins a large quantity of calcium ions (12 mg%) was added to the culture solution (3). D) The Effect of Temperature on the Nutritional Requirement Cleverdon et a_l_. (22) studied the vitamin requirements of Bacillus  coagulans at 37°C and 55°C. Employing a vitamin-free casein hydrolyzate medium they found that niacin, thiamin and biotin 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 workers (22 ) noted that biot in, 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 lass i f icat ion proposed by Cameron and Esty (19) in order to avoid confusion. Their c lass i f icat ion i s : 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 effect of temperature on nutritional requirements commented, in agreement with the 22. majority bf the investigators, that at higher temperature the enzymes responsible for the synthesis of a particular 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 poss ib i l i t ies 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 exter-nal source of the substance which i t can no longer produce. One of the bacterial strains Cambell (18) used in his studies fai led to synthesize histidine at culture incubation temperatures of 20 - 37 - and 55°C. Histidine therefore was required for growth. In interpreting this particular finding, Cambell (18) assumed that the gene necessary for the production of the enzyme responsible for the synthesis of hist idine, i f present, has been inactivated; thus this strain can no longer make histidine 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 cel l biomass at f i r s t increases with increasing concentration of the carbon source; however, after 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 effect 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 nitrate 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 tab i l i ty and the increase in the content of isoleucine, arginine and tryptophan, and a decrease in the content of phenylalanine and aspartic acid. The authors suggest that there are stronger hydrophob bonds between the subunits in the enzyme molecules of the thermophiles, which is responsible for their thermal s tab i l i ty . It has been reported by Heinen (70) that the enzyme amylase act iv i ty of the extreme thermo-phi l ic bacterium (70°C optimum for growth), plays an important role in thermal s tab i l i ty whereas Ca and Mg ions are essential for ensuring the thermal s tab i l i ty of amylase and i ts high act iv i ty . F) Oxidative Metabolism It has been reported by Allen (3) that the d i f f i cu l ty 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 ut i l ized remain completely unknown. However, Allen (3) managed to extract some general conclusions on the oxidative ab i l i t i e s of the thermophiles from the l iterature 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 denitr i f icat ion (3). Oxidation of ammonia to n i t r i te by cultures of thermophilic spore-formers has been reported by Cambell (17). Allen in his review (3), stated that the nitrogen fixation 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 zed; however, the mechanism is not well understood. "N i t r i f i cat ion" and "nitrogen f ixat ion" by cultures of thermo-phi l ic organisms, although reported by few investigators (78, 72), have not been extensively studied. H) Decomposition of Natural High Polymeric Materials It has been noted as early as 1894 (71) that the thermophilic aerobic sporeforming bacteria have the ab i l i ty 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 ra tes of thermophilic sporeformers; however, the properties of these enzymes have not been studies in deta i l . Viljoen et a l . (117) have isolated the organisms responsible for the primary attack on cellulose and have reached the conclusion that the thermophilic bacteria which actually decompose cellulose are obligate anaerobes. Tetrault (112) has reported that the aerobic and facultatively anaerobic sporeformers associated with the anaerobes play a secondary role. McBee (75) in his study using pure cultures also obtained a similar result. Claims have also been made, however, for cellulose decomposition by 26. aerobic thermophiles. The f i r s t of these claims came from Kellerman and McBeth (53). Allen (3) in his review reported that results of several studies have evidence for aerobic thermophilic bacteria's ab i l i ty to decompose cellulose. Linder natural conditions, anaerobic cellulose breakdown may be considered to be more prevalent (107) (3). Hydrogen, carbon dioxide, ethyl alcohol, acetic acid and occasionally butyric acid have been found as products of high temperature cellulose breakdown (100). Murray (85) reported on the aerobic growth of thermophilic cellulose 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 to 90 percent or less. He concluded that the many fai lures to obtain pure cultures of thermophilic aerobic cellulose decomposers may not be due to the anaerobic nature of the ce l lu lo ly t i c organisms, but due to insuf-f i c ient water vapor in the a i r . S t i l l less is known regarding the decomposition of other constituents of plant material by thermophilic bacteria. There is almost no l i terature on the breakdown by thermophiles of such materials as hemicellulose, pectin, l ignin and ch i t in. 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 ut i l i zed by rumen microflora without some form of pretreatment (99). The high l ignin and .the low cel l contents are believed to be the controlling 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 nutrit ive value. These are due to changes in the chemical composition of the plants owing to age and species and the presence of natural inhibitors of cel luloses. 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 wil 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 con-vert fibrous material containing large amounts of cellulose into food that is acceptable to man. Since wood and wood by-products contain mainl free cel lulose, cellulose chemically associated with l ignin 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 ze these sub-stances and in turn have the end-products of this fermentation trans-formed 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 iges t ib i l i ty of the feed's energy. These factors are inter-related and depend to a large extent on the chemical composition of the herbage. The relative amounts of solubles, cel lulose, hemicellulose and l ignin 28. are a l l aspects of composition that influence d iges t ib i l i ty . Intake is related to these composition factors as well as to the rate of digestion and rate of passage of the fiber mass (118). Smith et aK (105) reported that there is a significant correlation between the rate constant and percent l ignin in the plant tissue of several grass and legume forages. Lignin cellulose ratio 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 effect of plant composition parameters such as intake, d i ges t ib i l i t y , or digestion rate. Because of the fact that plant materials that di f fer only in the component of interest, i .e . differences in l ignin percentages are generally con-founded with maturity, or differences in the content of other fibrous components. They (64) also indicated that occasionally genetic mutants offer an opportunity to obtain samples of plant tissue which d i f fer appreciably in only one chemical component. In order to substantiate their statement, they referred to Muller's et al_. (83,84) work which ver i f ied the fact that plant material which has less l ignin content has greater cel l wall d igest ib i l i ty and intake. The results of Riquelme et al_. in lamb fattening rations (97) indicated that the cellulose fibers (bleached hardwood kraft) supported animal weight gain essentially equal to the control ration (70% wheat + 24% a l fa l fa hay + 4% molasses + 2% premix) and did so e f f i c ient ly . Further-more, the carcasses of the f iber fed lambs exhibited a conformation and quality comparable to the control group, and because of the reduction in backfat thickness, kidney and pelvic fat, the calculated y ie ld grade was improved, indicating more salable meat cuts per carcass. 29. A. Lignin Distribution in Wood Tissues Ultraviolet microscopy has been used by Goring et_ al_. (40) to measure the distribution of l ignin in spruce and birch wood. The spruce xylem was considered to consist predominantly of three dist inct morpho-logical regions: 1. Secondary wall (S) of the f iber (tracheid); 2. The compound middle lamella (ML) between fibers 3. Middle lamella (ML ) at the cel l corners. cc Table 1. The distribution of l ignin in black spruce earlywood Tissue Tissue Lignin Lignin type volume {%) % total concn. ,g/g S 87 72 0.22 ML 9 16 0.50 ML_ 4 12 0.85 As shown in Table 1 most of the l ignin in spruce earlywood exists in the secondary wall of the f iber even though the concentration of l ignin in the middle lamella is high. It was also mentioned in their report that similar results have been obtained in latewood studies. The d i s t r i -bution of l ignin found in these three areas supports the prediction made by Berlyn and Mark (10). Goring et a l . (40) concern in this particular study was to remove l ignin and record the rate of removal from various regions in the wood tissue during chemical pulping (Kraft and su l f i te pulping). According to their results the secondary wall loses l ignin f a i r l y rapidly in the 30. early stages of the work while the middle lamella shows strong u l t ra -violet (UV) absorption until late in the cook. They also reported that similar trends were found in acid su l f i te pulping. Both in kraft and su l f i te 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 later in the cook. This extraction sequence of spruce lignins and the microscopic pattern of l ignin removal have been interpreted in terms of a theory of del ignif icat ion based on the degrad-ation of a polymer gel. B) Lignin Lignin is located in the woody parts of plants, such as bobs, hulls and the fibrous portions of roots, stems and leaves. Its chemical structure remains uncertain. 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 differing as regards attached units. 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 different products isolated. Methoxy groups have been reported to occur in percentages ranging from 5 to 15 or more. The percentage increases as the plant matures. The nucleus is a polyhydrate aromatic compound. Thus, l ignin 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 cellulose and is included with the carbohydrates in the conventional methods of feed analysis. Its recognition as a separate entity is important because of its dominant influence on the degree of d igest ib i l i ty of many feeds (74). 31. Lignin occurs in plants ch i e f l y as 1ignocel lulose. There i s sup-port to the be 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 ce l lu lo se are f u l l y understood. Its behavior in nut r i t i on i s l ikewise unsett led, d i f fe rent feeding tests y i e ld ing c on f l i c t i n g re su l t s . Proof fo 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 tab l i sh , for un t i l i t s molecular structure i s known, no c r i t e r i o n of the accuracy of a quant itat ive test for l i g n i n i s possible (26). Crampton and Maynard (26) have shown in the 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 issues apparently are not attacked by alimentary bacter ia . According to t he i r assumption, th i s might be due to a certa in degree of ant i sept ic action of the l i g n i n resu l t ing from i t s phenolic nucleus. C) De l i gn i f i ca t i on In one of the i r studies, K i t t s e_t al_. (58) reported that unpro-cessed sawdust in ruminant d ie t has been able to provide energy as high as 244 kcal/kg d igest ib le energy, and 22.7% of d iges t ib le protein. In a further 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 pa r t i cu l a r l y benef ic ia l to u t i l i z e wood wastes without further 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 non-nutr it ive bulk in rations containing otherwise high levels of energy feeds (4,55). One l i q u i d by-product,, a hemicellulose extract prepared from hardboard 32. manufacture, has proved commercially successful as a feedstuff of high nutritive value (89). Sodium hydroxide has been one of the more popular agents for del ignif icat ion and has been applied to a variety of materials to improve their d igest ib i l i ty for use as a ruminant feed. Sodium hydroxide would appear to be the most effective and economical, particularly 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 physio-logical stress on animals consuming large quantities of treated feed-stuffs (31). Smith et aJL (103) compared a variety of agents for improving d igest ib i l i ty of ruminant fecal material. Sodium hydroxide and sodium peroxide substantially improved the ava i lab i l i ty of carbohydrate in the material. The increase resulted from the improvement in the ava i l -ab i l i ty of insoluble residual cel l wall as well as the formation of so l -uble matter. Brownell (16) found that when 20 mesh wood was subjected to various chemical pretreatments, the amount of l ignin obtainable after subsequent ball mil l ing was greatly increased above that obtained without mi l l ing. The most striking increase occurred after treatments with sodium hydroxide or ammonium hydroxide. Pew and Weyna (88) in their study of enzymatic l iberation of l i gn in, 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 ie ld of milled wood l ignin was increased by a mild alkal i treatment before mi l l ing. This suggested that l ignin was attached to the carbohydrate by alkal i sensitive bonds. Further in their report (12) they mentioned that prolonged standing of milled wood solutions of sodium or ammonium hydroxide at poor temperature fa i led to increase the y ie ld of l ignin. The y ie ld , however, did i n -crease with increasing temperature of alkal i treatments between 48° and 86°C. Final ly, they (12) concluded that the l ignin carbohydrate bond, i f such bond does exist, is stable to alkal i treatment below approximately 100°C and that the breakup of the complex is due to a lkal i peeling of the hemicellulose. Above 100°C hydrolysis of l ignin begins and compli-cates further measurements of l ignin recovery. It would appear,however, that the 1ignin-carbohydrate bond cannot be more a lka l i - l ab i le than bonds within the l ignin. The objective of treating 1ignocellulosic materials is to remove or dis^ rupt l igninso that their energy potential as a ruminant feedstuff is enhanced severalfold compared with their untreated state. As i t is mentioned in several references, ce l lu los ic 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 ce l lu los ic 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 ingle-cel l protein; 3. Feeding to ruminants to produce meat and milk. 34. Ear l ier, i t was mentioned that ruminants have the ab i l i ty to u t i l i ze ce l lu los ic materials through the rumen fermentation. The nature of fermentation is anaerobic. The anaerobic microorganisms convert carbohydrate to microbial proteins, various volat i le fatty acids, carbon dioxide and methane (46). Microbial proteins, microbial soluble matter and volat i le fatty acids are ut i l ized by the ruminant, while methane is a caloric loss. For adequate fermentation the nitrogen requirement of the microbes must be met. Since cel lu los ic wastes are usually very low in nitrogen, these feedstuffs must be supplemented with nitrogen. Digestion of cellulose is a function of the time the material resides in the rumen. Thus, feeding cel lu los ic materials in rations containing high levels of concentrates may decrease cellulose ut i l i zat ion because of the faster rates of passage associated with high concentrate rations. Fine grinding of the cel lu los ic material might obtain a similar result. It has also been postulated that high levels of soluble carbohydrate might reduce the digestion of c e l -lulosic materials (46). Kitts and Underkrofler (59) investigated the mechanism of break-down of cellulose to develop methods that would increase the ava i lab i l i ty of cellulose in low quality roughages dealing with ce l lu lo ly t i c enzymes from mixed rumen microorganisms by grinding the bacterial cel l s from strained rumen f lu id with alumina. They could not detect ce l lu lo ly t i c act iv i ty in the centrifuged and f i l tered rumen f l u i d , suggesting that the ce l lu lo ly t i c enzymes were not present as in the rumen f lu id but were associated with the bacterial ce l l s . Being aware of the fact that glucose was the only major product of hydrolysis of carboxymethyl cellulose (CMC) 35. they hypothesized that the cellulose degrading enzyme of the rumen microorganisms was a "celloglucosidase." The rumen microorganisms are limited by the composition of the cel lu los ic matter. They are unable to degrade l i gn in, which exerts a protective action on the cellulose with which i t is combined. This imposes a l imit of d igest ib i l i ty on the structural matter of a l l plant materials fed to ruminants, d igest ib i l i ty being negatively related to l ignin content (117). Therefore, removing l ignin by the cheapest means possible is a vital concern for ruminant nutr i t ionists. Various methods for del ignif icat ion 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 l ignin (54) and cel lulose. d) Chlorite treatment (98). e) Sodium peroxide treatment (103). f ) Fermentation (54). g) Steaming under different combinations of time and pres-sure [temperature] (9). h) Irradiation treatment (55). i) Ball mill ing (28). j ) Acid hydrolysis (45), etc. Kirk and Moore (54) have examined nine white-rot fungi for their ab i l i t y to remove lignin from bigtooth aspen (Populus grandidentata Michx) and yellow birch (Betula allghaniensis). During decay most of the fungi removed a larger percentage of the l ignin than the polysaccharides. Lignin removal was always accompanied by removal of polysaccharides, but l ignin removal did not corrolate with removal of any particular component of the polysaccharides. During decay l ignin was usually more selectively removed in the f i r s t few percentages of weight loss than were the polysaccharides. The decayed wood with less l ignin were more digestible by a mixture of polysaccharidases and by rumen f lu id than were the control samples. It was known from this type of study that in general the more lignin removed from wood, the more digestible is the residual material. Although some white-rot fungi are effective in removing l ignin faster than polysaccharides from wood, i t was mentioned by the authors that the process was relat ively slow. For practical use of white-rot fungi i t would be desirable to speed up the process. Therefore, they suggested meeting the following cr i ter ia which are believed to be opt-imizing factors: 1. Aeration, moisture, temperature and source and amount of nutrient nitrogen should enhance the rate of decay. 2. To find conditions that would improve the select iv i ty of l ignin removal. 3. To find better fungus species or obtain desirable mutants, as is common in industrial microbiology. 37. III. MATERIALS AND METHODS 1. Materials Treatment tank: A f iber glass fermentation tank (Figure 1) with a total capacity of 1690 1 itres(operating volume of 1362 l i t res ) 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 ut i l i zed in the study. Aerator: Aeration was accomplished with a commercial compressor. A four inch diameter rotor driven by a 1/2 H.P. e lectr ic 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 factor in thermophilic waste treatment processes (25). Mixer: Active agitation of the substrate was considered to be essential and was achieved by a 1 HP 1725 RPM motor directly connected to 7.62 cm - 3 blade propellor mounted on a 20.32 cm. diameter stainless steel draft tube (Figure 1). 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 showed that the two units supplied a total of over 1.81 kg per day of oxygen to the substrate (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 res per minute. b) Transfer Pump - The transfer pump is a smaller rever-sible 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. 0 0 CT) CO 39 FIGURE:'^ MIXING A C T I O N SHEPHERD , 1977. 40, 41. Transfer l ines: Al l transfer lines were of 5.08 cm. diameter (minimum) rubber tubing, equipped with quick coupling f i t t ings . Screening: Processed mixed liquor and suspended solids (MLSS) were screened into three major fractions by a "Sweco" vibrating screen which was driven by a 1/4 HP e lectr ic motor. a) The coarse sized material was retained on a 30-mesh sieve (30 holes/2.54 sq. cm.). b) The f iner sized material was collected on a 150-mesh sieve (150 holes/2.54 sq. cm.). c) The l iquid f i l t r a t e was collected and subjected to a "basket-type" centrifugation for further solids recovery(56). Drying: Process MLSS was subjected to drying using two different sources of energy depending on the weather condition. a) Solar energy was ut i l ized when weather permitted. A thin plastic sheet was spread over a dry ground surface on which the processed sawdust was evenly spread for drying. b) An electr ic 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 Microorganisms A) Init ia l Stage Procedure for Substrate Preparation a) 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 ita l importance for a successful aerobic.fermentation, a constant flow of fine air bubbles at the overhead of the sparger (Figure 1) 42. was maintained. c) One hundred and thirty to one hundred and forty l i te r s of fresh and part ia l ly 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 after 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 after 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 settleablesolids or particles of the substrate are between 50 and 75%. B) Culturing Parameters a) Temperature - temperature is one of the most important environmental parameters affecting the growth, act iv i ty and evolution of organisms (3). Surucu (110) stated that growth rate of microorganisms increases with temperature up to certain points and after that point, i t begins to decrease. This would indicate that temperature can be expressed as the resultant of two opposing act iv i t ies on microorganisms which are: i) rate of enzymatic act iv i ty is low at low temperatures and accelerates as the temperatures r i se , and 43. i i ) denaturation of cel l proteins (or enzymes) is not signif icant until moderate temperatures are attained. Protein inactivation increases rapidly with further increase in temperature. b) pH - the [H] ion concentration effect is one of the factors which has a very pronounced effect on enzyme reactions (77). A change in pH of the substrate or medium frequently occurs as the result of microbial growth (66). One of the conditions which l imits microbial growth is the hydro-gen ion concentration of the medium. At low pH denaturation of the key enzyme proteins wi l l occur. 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 neutrality. As pH rises over 9.5, the hydroxyl-ipn begins to exert a toxic effect. Few, i f any, microorganisms can survive above pH H.O. Control of pH at either a high or low range can be used to prevent or retard bacterial decomposition of potentially degradable materials (77). c) Dissolved oxygen - aeration in biological processes serves two important functions: i) provides adequate oxygen to meet the demand of the microorganisms and i i ) provides adequate means to ensure mixing in order to prevent settl ing of the solids (23). Aeration and subsequent dissolved oxygen in the substrate is a c r i t i c a l 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 oxidative act iv i ty (24). d) Imhoff cone measurement (Amount of Settleable Solids)-the settleable solids most frequently encountered in waste treatment practice are of such particle sizes that they tend to settle through water at constant rates depending on the effective sizes and relative densities of the individual particles (47). While a certain ratio 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). Coulthard (25) indicated that 50 to 75% settleable solids is a desirable quantity in the slurry for the thermo-phi l ic treatment process. Large volumes of water to solids ratio reduces the level of heat energy derived from oxidation while an excessively high solid content reduces the efficiency of mixing and aeration. C) Del ignif ication As stated above, the digestor was f i l l e d to three-fourths of i ts capacity. 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 after 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 del ignif icat ion of the sawdust. The declining phase of the microbial cel 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 stabil ized sludge. A sample was collected from the vat before adding new unproces-sed sawdust. Excessive foam formation and low Imhoff cone percentage were the basic guides for sample col lect ion. Collected samples were stored in a deep-freezer until analyzed. Unprocessed raw sawdust was used as a control for analysis. The collected samples and unprocessed (control) sawdust were analyzed or tested for: a) acid detergent f iber b) cellulose c) l ignin d) crude protein and e) d iges t ib i l i ty (in vitro digestion) D) Harvesting 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 fac i l i t a te the storage and handling of the processed product. Regarding the odour and f ly problem, the processed product had a negligible effect once i t has reached this dry stage. A moisture content in the 10-15% range wil l inhibit 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 nutritive value of the product. 1. Dry matter 2. Crude protein 3. Bomb calorimetry gross energy 4. Ash G) Analyses Frozen processed sawdust (supernatant) samples were thawed and dried in an oven at 65°C. Similar drying treatment was carried out on unprocessed sawdust (control). Dried samples were ground to pass a 40-mesh screen and analyzed for: a) dry matter b) acid detergent f iber c) cellulose d) l ignin e) crude protein f) gross energy and g) ash In vitro digestion was also conducted to determine the.effect of such microbial treatment towards dry matter d igest ib i l i ty . 47. Procedures as described in the Association Off ic ia l s of Agricul-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 itro digestion was a development of the rumen liquor and pepsin-acid procedures from Larsen and Jones (63) with slight modification. The source of inoculum was from a rumen of f istulated steer which was fed a diet of a l fa l fa hay with premix mineral salt-free choice, and water available at a l l times. 48. IV. RESULTS AND DISCUSSION 1. Environmental Parameter Studies were conducted in order to examine the poss ib i l i ty of alder sawdust del ignif icat ion by aerobic thermophilic fermentation. The purpose of the studies using the batch technique was to obtain in -formation relating to the environmental variables thought necessary to ensure the growth of microorganisms and to their effectiveness in del ignif icantion. 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, II and III are shown in Figures 4, 5, and 6 respectively. 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 terature on the growth of thermophilic organisms in pure cultures contains suff ic ient information to indicate that many thermophilic organ-isms grow best when the pH of the medium is between 6.5 to 7.5 (67). Regarding mixed liquor and suspended solids, Bragg, et al_. (13) reported that for the best result the aerobic thermophilic processing a lka l in i ty should be in the range of 7.5 to 8.5 pH value. Brock and Darland (15) have shown in their work that the bacteria have the ab 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 specif ic as to the genus or species of FIG. 4. R E L E V A N T - C H A N G E JN ENVIRONMENTAL VARIABLES DURING TREATMENT. B A T C H X . I 45 o o U J rr ZD h-< or LL! 0_ I i i I-7 0 56 42 28 14 Oj ambient t e m p . 5 10 15 2 0 2 5 PERIOD OF T R E A T M E N T (day). 35 5 X ° a. 0 FIG. 5. 5 0 -R t L E V A N T ' CHANGE IN ENVIRONMENTAL VARIABLES DURING TREATMENT B A T C H TX H 7.5 15 22.5 30 37.5 4 5 5 2 . 5 60) PERIOD OF TREATMENT (day). 51 52. bacteria studied. Nevertheless, these results suggest that there are physico-chemical limitations of the environment beyond which l i f e is impossible. 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 neutrality. Temperature changes in Batch I, II, and III are shown in Figures 4, 5, and 6 respectively. These results show a relationship between high rate of del ignif ication and high temperature (Tables 2, 3, and 4). Surucu (110) reported that 55°C is the temperature which the highest observed net ce l l mass production occurred while the maximum growth and substrate ut i l izat ion rates were noted at 58°C. However, these results did not d i f fer s ignif icantly between 55 to 58°C (110). Samples collected within this range of temperature are low in l ignin and acid detergent f iber (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 del ignif icat ion. The foam produced as a result of the biochemical act iv i ty 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 te 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 act iv i ty . Regarding the effect 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 particular 53. organism. Further in their comment, they stated that at a higher temperature the enzyme(s) responsible for the synthesis of a particular 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 ut i l ized 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 stabi l izat ion of the waste material (24). The result of Popel's (90) work has indicated that 3.55 kcal of heat is produced per gram of organic solids. Observations of these studies and several other "batch" process t r ia l s showed that the more heat produced, the greater the degradation of organic solids by the aerobic bacteria. Therefore, the maintenance of high temperature during processing is of v ita l importance for increasing the rate of biodegradation. To achieve this, the following factors must be closely observed: a) Increasing oxygen transfer eff iciency (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 iquid 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 this 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 imit ing. It has been pointed out by Coulthard (24) that the oxygen transfer coefficient is affected by the physical and chemical characteristics of the system under aeration. Along the same point Eckenfelder (7) l i s t s the variable characteristics as temperature, turbulent mixing, l iquid depth and waste composition. This observation can be explained by the fact that the concentration and the biological act iv i ty of mixed liquor suspended solids decreased with increasing treatment period. This has been demonstrated also by Surucu (110) that at higher solid retention time values, autoxidation of biological solids becomes very important and increases the apparent amount of oxygen ut i l ized per quantity of organic matter removed. As another poss ib i l i ty , the increase in oxygen ut i l i zat ion 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 terature. For example, a ir 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 solid retention time is longer (78). The oxygen input and output were expressed as functions of the air flow rate and oxygen transfer eff iciency 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 result they found was negligible. Therefore, according to these studies reported in this thesis, 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) sawdust1 was obtained from the lower Fraser Valley, Brit ish Columbia and was c lass i f ied as matured wood. The size of the sawdust particle was approximately 2.5 x 3 x 10.15 millimeters. The chemical composition and d igest ib i l i ty of the untreated alder sawdust (control) the fresh swine manure and the processed samples of a l l 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. Batch III Purchased from Spruce Specialties Ltd. 56. was designed to investigate: a) Whether del ignif icat ion was taking place before vat temperature reaches 55°C or not; b) Whether the stage of processing would y ie ld maximum del ignif icat ion. However, the major system of processing was not different 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 del ignif icat ion with a 24 hour period (Table 2). This was with a limited sawdust load of 22 kg. On the other hand, 59% of del ignif ication was achieved within 22 days of processing having a total load of 99 kg. of sawdust (Table 2). The result of Batch II showed s l ight ly over 56% of del ignif icat ion within 13-17 days of processing with 44 kg of sawdust. There was an indication of poor del ignif ication as the vat temperature drops (Table 3). This can be explained by the poss ib i l i ty that aerobic thermophilic bacteria and temperature play an important role in del igni f icat ion. With Batch III, the highest del ignif icat ion rate (74%) was achieved within 15 days of processing (Table 4). As i t was mentioned ear l ier , this batch was treated only with 22 kg of sawdust throughout the processing period. Again, as the vat temperature dropped there was a lower del ignif icat ion rate (Table 4). In most cases the extended rate of del ignif icat ion was not as ef f ic ient as it 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 particular stage, the microorganisms are within their optimum growth range, accounting for the high chemical act iv i ty and measurable degree of degradation. As the period of processing progressed, there was an 57. increase of sawdust load periodically and exhaustion of the nutrients from the medium resulting in poorer del igni f icat ion. Under conditions described in these studies 10 to 15 days of processing appears to be suff ic ient time to delignify 20 to 25 kg of alder sawdust. Relationship between l ignin removal and removal of cellulose Due to the partial ut i l i zat ion of cellulose by microorganisms, i t was d i f f i c u l t to correlate the rate of del ignif icat ion with the amount of cellulose release. As the amount of raw sawdust increased within the fermenter, the amount of free cellulose in the medium and rate of del ignif icat ion decreased (Figures 7 and 8). Low level of total cellulose corresponded with high vat temperature (Table 2). Since the higher vat temperature promoted a general increase in chemical act iv i ty and growth, these data might indicate that the released cellulose was being ut i l ized by microorganisms as a nutrient source. In most experiments, low analytical values for cellulose 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 after sawdust addition, percent of cellulose remained relat ively high (Table 2). The high content of cellulose at an early stage of processing may be due to readily available nutrients from swine manure which is preferred to the cellulose by microorganisms. However, as the period of processing advanced, this would be followed by exhaustion of preferred nutrients, and thus cellulose would become the predominate medium. Consequently, more cellulose ut i l i zat ion took place resulting in reduced cellulose content. Most workers who attempted to isolate the organisms responsible 58. for the primary attack on cellulose have reached the conclusion that the thermophilic bacteria which actually decompose cellulose to carbon dioxide and short chain organic compounds are obligate anaerobes (38). Claims have also been made, however, for cellulose decomposition by aerobic thermophilic bacteria. The f i r s t of these claims came from Kellerman and McBeth (53). Cellulose content decreased as processing time increased. This decrease was accompanied by .an increase in l ignin especially when the vat temperature was high (approximately 66°C) as shown in Figure 2. A relat ively 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 pro-cessing seems to be a reasonable period of time to remove 50-7$ of l ignin from a 20 to 25 kg load of alder sawdust. Percent of ash was taken to represent the inorganic (mineral) constituents of the samples. It has been indicated by Macdonald, et a l . (76) that the ash may, however, contain material of organic origin such as sulfur and phosphorus from proteins and some loss of volat i le material in the form of sodium, chloride, potassium, phosphorus, and sulfur wil l take place during ignit ion. The ash content is thus not truly representative of the inorganic material in a given sample quan-t i ta t i ve ly or qual itat ively. Results of percent of ash of Batches I, II, and III are given in Tables 2, 3, and 4 respectively. In most cases the results 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 super-natant 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 ignin by complete oxidation of unit weight of a sample and does not occur (complete oxidation of l ignin) in. the animal's body. The y ie ld of processed product obviously depends on the volume of sawdust added and to a certain extent to the amount of swine waste used in the process. 3. In Vitro Digest ib i l i ty 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 ignin renders most wood practical ly indigestible. It is known from this and other types of study that in general the more l ignin removed from wood, the more digestible is the residual material by domestic animals. The chemical analyses and in vitro dry matter d iges t ib i l i ty results for alder sawdust are given in Tables 2, 3, and 4 for Batch I, II, and III, respectively. Values for d igest ib i l i ty are averages of duplicates. As the volume of sawdust added to the treatment vat increased, the percent of d igest ib i l i ty increased to a certain point and declined o 60. Table 2.- Chemical composition and in v i t r o digestibility of supernatant and processed product of alder s a w d u s t M - - Batch j " p e r n a t a n t sawdust added(b) In vitro dry matter Vat temper-(kg.) (day) (%) {%) (%) (cal/q) (%) digestibility ature Raw sawdust (control) - - 66 34 52 6298 \ JO j 1 : \ Io 1 19 ( C) Fresh swine manure - 52 19 (44) 44 5989 12 41 Supernatant: 22 1 49 20 (41) 43 5970 9 43 59 4 46 20 (41) 26 6161 10 - 45 49 44 (66) 9 45 19 (44) 14 6309 8 47 60 - n 49 . 17 (50) 14 6044 10 53 66 22 (88) 14 49 16 (53) 22 6096 10 50 67 11 (99) 22 44 14 (59) 16 4338 10 55 41 Processed products: 30 mesh 22 48 • 20 (41) 13 6214 6 35 150 mesh 22 42 33 (.3) 27 5584 13 29 150 mesh filtrate 22 39 • 28 (18) 20 5538 18 24 Yield of tt) processed product (kg) 144.0 17.0 2.0 T a b l e 3. Chemical compos i t i on and i n v i t r o d i g e s t i b i l i t y o f s upe rna t an t and processed product o f a l d e r s a w d u s t ( a ) — Batch II. Amount o f Treatment A c i d d e t . sawdust t ime f i b e r Added ( b ) (kg.) (day) (%) L i g n i n ^ con ten t (%) C e l l u l o s e con ten t (%) Gross energy ( c a l / g ) Ash {%) In v i t r o d r y m a t t e r d i g e s t i b i l i t y (%) Vat Y i e l d ofidl temper- p roces sed a t u r e p roduc t ( ° C ) (kg.) Raw sawdust ( c o n t r o l ) - 71 32 48 6129 1 19 Fresh swine manure - 60 22 (31) 40 6285 8 48 -Superna tan t : 44 13 49 14 (56) 31 5872 12 61 55 - 17 53 14 (56) 34 5790 12 63 56 11 (55) 26 55 17 (47) 35 5852 n 60 43 .11 (55) 37 55 19 (41) 32 5919 13 58 25 Proces sed p r o d u c t s : 30 mesh 37 60 17 (47) 38 5883 8 37 49.0 150 mesh 37 52 26 (19) 30 5531 17 28 11.0 150 mesh f i l t r a t e 37 53 30 (6) 25 5505 23 24 2.0 So l ka ( p u r i f i e d c e l l u l o s e ) 71 -(a) Dry ba s i s (b) Accumulated amount o f sawdust (kg.) (c) F i g u r e s i n parentheses r e p r e s e n t percent o f d e l i g n i f i c a t i o n (d) A i r dry b a s i s CM 62. Table 4. Chemical composition and in vitro digestibility of supernatant and processed product of alder sawdust(a)- - Batch III. Amoung of Treatment Acid det. L i g n i n ^ Cellulose Gross In vitro Vat Yield of (d) sawdust. . Added ( b) (kg.) time • (day) fiber (%) content (*) content (%) energy (cal/g) Ash (%) dry matter temper-digestibil ity ature (*) rc) processed product (kg.) Raw sawdust (control) - - 73 35 53 6278 3 16 Fresh swine manure - - 52 23 . 43 6023 8 50 Supernatant: 20 3 ' 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 Processed products: 30 mesh 21 - - - - - - -150 mesh 21 45 25 (29) 28 5235 17 30. 43.0 150 mesh f i l trate 21 37 28 (20) 23 4767 30 25 5.0 Solka (purified cellulose) 73 (a) Dry basis (b) Accumulated amount of sawdust (kg.) .(c) Figures in parentheses represent percent of delignification (d) Air dry basis 63. to 5 5 \v in vitro d.m. d iges t ib i l i t y FIG. 7 R E S U L T S OF DELIGNIFICATION TO C E L L U L O S E C O N T E N T AND in vitro DIGESTIBILITY IN T E R M S OF T R E A T M E N T DURATION. B A T C H I . c o n t r o l cellulose' content y-'~>-,~l* lignin content Ol 1 0 15 • 20" T R E A T M E N T P E R I O D (doy). 22 FIG. 8 64. R E S U L - T S OF DELIGNIFICATION TO C E L L U L O S E C O N T E N T in vitro DIGESTIBIL ITY IN TERMS OF TREATMENT* B A T C H H . 55 4 4 33 2 2 II c ontrol in vitro d.m. digestibility control cellulose content control l ignin content 0 8 16 24 T R E A T M E N T (day) 32 ' 40 R E S U L T S OF DELIGNIFICATION TO C E L L U L O S E C O N T E N T in vitro DIGESTIBILITY IN T E R M S OF T R E A T M E N T DURATION^. B A T C H HL• 0 5 10 15 2 0 T R E A T M E N T PERIOD (day). 25 as t n 66. as the load of sawdust further increased (Figures 7 and 8). A parallel relationship was detected between high del ignif icat ion and high d igest i -b i l i t y . However, low rate of del ignif icat ion and in v itro d iges t ib i l i ty was experienced along with low vat temperature after 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 igest ib i l i ty as well as del ignif icat ion (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, clearly indicate the effectiveness of microorganisms in del ignif icat ion. Progressive in vitro digestion corresponding with the improvement of del ignif icat ion was observed (Figure 9). The f i r s t 10 to 15 days of processing appears to have the major influence on d igest ib i l i ty . High d iges t ib i l i ty (%) was attained beyond which additional processing is of no value mostly in terms of del ignif icat ion In vitro d igest ib i l i ty test showed that processed sawdust is 55% digest-ible which is equivalent to high quality a l fa l fa hay in terms of animal feeding value. As a result of aerobic thermophilic bacteria treatment almost threefold increase of alder sawdust dry matter d iges t ib i l i ty has been achieved compared to the control (19%). Some of the samples which showed low l ignin content fa i led to show high d iges t ib i l i ty (Tabes 2, 3, and 4). It seems reasonable to expect that the rate constant for digestion of a forage is related to the structural composition of the material; however, factors other than l ignin content were involved to lower the d igest ib i l i ty 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 ta t i s t i ca l l y analyzed, there was an impressive trend which indicates that aerobic thermophilic bacteria treatment was more effective in increasing the in vitro dry matter d igest ib i l i ty of alder sawdust. Particle size in a l l batch studies reduced [150 mesh, 150 mesh f i l t r a te (centrifuged product)] the in vitro d iges t ib i l i ty 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 particular samples. Based on these in vitro d iges t ib i l i ty observations, i t can be concluded that aerobic thermophilic treatment appears to be very effective in making the nutrients in alder sawdust available to rumen microorganisms. 4. Crude Protein Results of crude protein value of Batch I,. II and III are shown .in Tables 6, 7, and 8, respectively. 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 and III. Before Batch II was conducted, i t was assumed that some loss of nitrogen might have taken place during the drying process. To confirm th is , samples were freeze dried and analyzed for crude protein value. However, no difference was observed. Therefore, Batch II was designed to determine whether long term (37 days) was a major contributing factor for the improvement of the crude protein percent or not. The result of long processing (Table 7) has shown a substantial 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 i t may be affected by environmental variables. In most cases, high crude protein value paral lels the sharp temperature rise and the high bacterial act iv i ty at the same time. Comparatively, 150 mesh solid material and 150 mesh-filtrate have shown high crude protein value from supernatant samples. The crude prote value of Batch III was relat ively high (Table 8) compared to other batches; this was mainly due to the addition of about 160 l i te r s of part ia l ly pre-processed substrate (after the effect of 22 kg of raw saw-dust towards del ignif ication 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 te 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 fraction 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 solid material that remains in the supernatant after centrifugation. This was found to be only 0.7%. However, on a large scale basis this could become a substantial 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 ear l ier , the main contributing factor for high crude protein value in this particular 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 identical 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 wil l increase di lution rates apart from creating a disfavourable environment to the microorganisms. Thus, less cel l y ie ld 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 di lution rate. Regarding the quality of the single ce l l (S.C.) protein, Mateles, et alk (73) reported that the amino acid composition of thermophilic organisms protein is of relat ively high quality. Surucu (110) commented that the nutritional value of S.C. protein is d irect ly 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 di lution rate (44), i t would seem desirable to operate the system accordingly. There-fore, operation of the system at a lower di lution rate is preferable from 70. the standpoint of the protein and nucleic acid content in the biomass, i .e . tradeoffs of di lution 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, nitrate, n i t r i t e , and ammonium compounds. There are, however, some species which can u t i l i ze atmospheric nitrogen; v iz . 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 inal stage of processing Total Nitrogen Composition (%) g a t c h Substrate at i n i t i a l Processed product stage 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 Figures in parentheses implies total nitrogen in percent L = Lowest Results of Batches II and III indicated an increase of 65% to 72% (Table 5) respectively of total nitrogen in the f inal processed product. The organic nitrogen contribution from the swine waste was only 35% for Batch II and 28% for Batch III (Table 5). But in the case of Batch I, 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. denitrifying organisms might have been involved in the fermentation process compared to n i tr i fy ing 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 negligible. The data of this study (Batch II and III) indicate that micro-organisms particularly those capable of fixing atmospheric nitrogen have contributed a substantial amount of nitrogen to the total nitrogen content of the processed product. Linday (66) stated that atmospheric nitrogen fixation by microorganisms together with electr ical discharge in the atmosphere, by which molecular nitrogen can under normal circum-stances, be made available for the majority of l iv ing 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 nitrate by bacteria (lithotrophic) Levy, et al_. (65). Nitrogen fixation is accomplished both by aerobes and anaerobes. Levy et al_. (65) stated that a key nitrogenous compound in microbial nutrition is inorganic ammonium, for i t combines with organic acids (d-keto acids) to form amino acids. The principle product of this event is glutamic acid. Further in their statement regarding the mechanism of n i t r i f i c a t i on , they also mentioned that many bacteria possess this ab i l i t y and have the additional enzymes to form a l l of their nitrogen-containing ce l lu lar constituents from glutamic acid. Microorganisms, therefore, can use inorganic ammonium salts as their sole source of nitrogen. The ut i l i zat ion of nitrate by microorganisms as a source of nitrogen for ce l lu lar synthesis entails the reduction of nitrate to the oxidation level of ammonia; at which i t can be incorporated into amino acids and other nitrogen-72. CN F ig . 10. T H E N I T R O G E N C Y C L E _ L E V Y * i al. I 9 7 3 N fixation ( Nitrification ) N H . Nitrosomonas Dissimilatory reduction I De ni t ri fication ) - > N 0 2 — Nitrobacter \ \ \ \ Immobilization , v \ . . . Microbial \ V degradation \ 2^. 0 r g a n i c Assimilatory reduction (1mm o bi Ii z a ti on ) _ Reduction Ox ida t i on No valency' change 73. containing cel l constituents. As shown in the nitrogen cycle (Figure 10),nitrogen fixation is a reduction process and a supply of potential reducing agents is required. 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 essentially anaerobic microenvironment. The nitrogenase enzyme system that performs the reduction is in a l l cases oxygen sensitive. It seems l ike ly that the nitrogenase is protected from oxygen by a very high rate of respir-ation in Azotobacter, which l imits the access of oxygen to the site of nitrogen f ixat ion. As i t was mentioned ear l ier , the fixed nitrogen f ina 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. Organic nitrogen compounds y ie ld ammonia when they decompose. This. is primarily a hydrolytic reaction involving no valency (65). Mischustin and Shilnicova (80) have reported that the nitrogen f ixing bacteria (Azotobacter chrooccum) is effective 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 denitrifying organisms which reverse this process, releasing atmospheric nitrogen. Thus some organisms f ix nitrogen for themselves, and this becomes available for others after cel l death and breakdown. Whilst others direct ly convert i t to a form more available for other organisms (66). Regarding deni t r i f i cat ion, Levy (65) stated 74. that denitr i f icat ion reaction characterist ical ly carried out by aerobes that find themselves in an anaerobic environment. If there are oxidizable carbon compounds present these wil l be oxidized and nitrate is ut i l ized 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 f inal 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 protein value and environmental variables in terms of sawdust volume and treatment time - - Batch I Amount of sav/dust added (kq.) Treatment time (day) Dry m a t t e r ( b ) (%) Crude (GT protein (X) Vat temperature (°C) pH Dissolved oxygen (mq/1) Raw sawdust (control) 86 0 - - -Fresh swine manure - - 97 8 - - -Supernatant: 22 1 97 3 59 • 8.2 2.4 _ 4 . 96 4 c 49 8.4 3.0 4A (66) 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-Processed products: 30 mesh 22 93 1 150 mesh - 22 89 2 150 mesh f i l t r a t e - 22 93 7 fa] Accumulated amount of sawdust (kg.) (b) Average of t r i p l i c a t e sample. . (c) Dry matter basis and average of t r i p l i c a t e sample 7 6 . e 7 . 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 a n d e n v i r o n m e n t a l v a r i a b l e s i n t e r m s o f s a w d u s t v o l u m e a n d t r e a t m e n t t i m e - - B a t c h I I Amount o f s a w d u s t T r e a t m e n t a d d e d (a) t i m e ( k g . ) ( d a v ) ( b ) D r y m a t t e r ' (%) C r u d e ^ p r o t e i n (%) V a t t e m p e r a t u r e (°0 PH D i s s o l v e d o x y g e n (ma/1) Raw s a w d u s t ( c o n t r o l ) - - 86 0 F r e s h s w i n e m a n u r e - - 95 7 S u p e r n a t a n t : 4 4 13 93 9 55 8 . 3 4 0 . 7 - 17 93 9 56 8 . 4 7 3 . 9 1 1 C55) 26 92 ' 11 43 8 . 2 9 5 .5 " (66) .37 93 12 25 7 . 3 6 5 . 0 P r o c e s s e d p r o d u c t s : 30 mesh - 37 93 9 150 mesh - 37 93 11 150 mesh f i l t r a t e - 37 93 13 Tal A c c u m u l a t e d , a m o u n t o f s a w d u s t ( k g . ) •(b) A v e r a g e o f t r i p l i c a t e s a m p l e ( c ) D r y m a t t e r b a s i s a n d a v e r a g e o f t r i p l i c a t e s a m p l e 1-^ T a b l e 8. R e s u l t s o f c rude p r o t e i n v a l u e and env i r onmen ta l v a r i a b l e s i n terms o f sawdust v a l u e and t r e a t m e n t t ime - - Ba tch I I I 77. Amout o f sawdust Treatment added t ime Dry m a t t e r , , C r u d e ( b ) '• av p r o t e i n Vat t empe ra tu r e pH D i s s o l v e d oxygen ( k q . ) (day) (%) (50 (°C) . (mq/1) Raw sawdust ( c o n t r o l ) - - '88 0 F r e sh swine manure - - 97 • 8 6.9 Supe rna tan t : 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 5 7 8.20 2.2 21 94 17 • 47 6.51 3.3 P roce s sed p r o d u c t s : -30 mesh - 21 - - ' -150 mesh - 21 94 13 150 mesh f i l t r a t e - 21 92 21 la) (b) Average 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 average o f t r i p l i c a t e sample FIG. 71 THE RATE OF DELIGNIFICATION AND E N V I R O N M E N T A L V A R I A e L ^ (THREE BATCHES COMBINED) V A R I A B L E S . • • - N 70 ^ m p e r o t u r e UJ rr 4.5-^ FIG. 12. 79. 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