^ * ^ 28533523^®* UNIVERSITY O F GHANA LIBRARY QP82.2 G35 G24blthr C. 1 The Balme Llbr mm G323774 University of Ghana http://ugspace.ug.edu.gh EFFECT OF GAMMA IRRADIATION ON AGRICULTURAL WASTE-DECOMPOSING AND FERMENTATION MICROORGANISMS IN GHANA A THESIS PRESENTED TO THE FACULTY OF SCIENCE UNIVERSITY OF GHANA LEGON BY CHARLES MAWULOM GBEDEMAH IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHILOSOPHY (BOTANY) DEPARTMENT OF BOTANY LEGON NOV. 1991. University of Ghana http://ugspace.ug.edu.gh Q 3 2 3 7 7 4 ~TLj2 00/0 / ^ W v v i University of Ghana http://ugspace.ug.edu.gh ABSTRACT Maize production in Ghana between 1984 and 1990 exceeded 500,000 metric tonnes per year except in 1985 where there was a shortfall to 395,000 metric tonnes. The unfavourable years of drought between 1981-1983 were attended by a sharp decline in maize production ( 140,000-333,200 metric tonnes ). The bulk of the national maize production was contributed by Ashanti, Brong-Ahafo, Eastern and Northern Regions. Maize husk available after removing cobs was commensurate with the total maize harvest for each Region. As a raw material for fungal protein production, maize husk has a potential for sustainable use. Gamma irradiation ( 0-200 Krad ) was used as a mutagen to evaluate its effect on vegetative growth, cellulolytic, pectinase and amylase activity of AspergiI1 us niger. Rhisopus oryzae, Trichoderma. viride and Lactobacillus plantarum. screened for use in the production of fungal protein on corn husk slurry by solid substrate fermentation. A dose of 20 Krad decreased vegetative growth of A. niger by 46.6 percent and further increases up to 50 Krads increased dry matter accumulation by the fungus. Vegetative growth of R. orysae was increased by about 30 percent by 50 Krad and remained nearly the same up to 200 Krad. There was no stastitical difference ( < 0.05, Student’s t-test ) between dry weight of mycelium obtained with 50, 100, 200 Krad of gamma irradiation. The best vegetative growth of T viride was obtained when 100 or 200 Krad of gamma irradiation was applied to spores prior to culturing. The best vegetative growth of the University of Ghana http://ugspace.ug.edu.gh iii bacterium ( L . plantarum ) was attained when the culture was exposed to 50 Krads prior to incubation at 28 JC for 5 days. Optimum cellulase, amylase and pectinase activity was induced by 50 Krad of gamma irradiation in A. nig&T and L. pi an tar urn ; on the other hand, optimal cellulase activity in T. viride and R. oryzae was induced by a dose of 100 Krad whilst 50 Krad was optimal for maximal production of amylase and pectinase enzymes by the same fungi ( T. viride and R. oryzae ) . Generally, the gamma irradiation dose that induced optimal vegetative growth was also attended by optimal cellulase activity.The pH of the medium containing spores of T viride treated with 100 krad was between pH 5.0 - 6.5 corresponding to the best pH for cellulase activity in T viride. The potential for use of gamma irradiation as a mutagen for enzyme production is promising. Gamma irradiation up to 200 Krad linearly increase acid production by A. nig&r as pH shifted from 2.9 to 2.2. The culture medium containing gamma-irradiated ( i 50 Krad. ) R. oryzae sporangiospores also became more acidic ( pH 4.5 - 3.8 ) presumably indicating accumulation of acids. Hydrolysis of corn husk into a slurry was achieved by using either one percent or five percent sodium hydroxide or hydrochloric acid and heating at 80° , 100° or 120°C for 1-3 hours. The best treatment combination for corn husk hydrolysis was heating at 100 C fot at least 1 hr. in either one or five percent sodium hydroxide or 1 percent Hydrochloric acid. This gave good University of Ghana http://ugspace.ug.edu.gh accessibility of cellulase for microbial enzyme attack leading to high crude fungal protein ( 20.0 percent ) produced by T. viride irradiated with 100 Krad of gamma irradiation prior to innoculation of the solid substrate. Irradiation caused morphological changes in T. viride and A. niger cultures. In T viride, as much as the conidiophores remained irregularly branched, the bright green colour development decreased progressively with increasing gamma irradiation dose ( eg. at 200 Krad there was no colour development ). Practical implications of these findings are discussed and future studies leading to commercial application of the technique suggested. University of Ghana http://ugspace.ug.edu.gh DECLARATION I, CHARLES MAWULOM GBEDEMAH, hereby declare that, except for references to other peoples work which have been duly cited, this work is the result of my own original research and that this dissertation had neither in whole nor in part been presented for another degree elsewhere. Student. J b DR. GEORGE T. ODAMTTEN Supervisor. University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES: 1. Cellulose, hemicellulose and lignin contents of lignocelluloses. 2. Major industrial fungal enzymes and their uses. o 3A. Vegetative growth of A. ni$er in liquid medium at 28 C for 5 days after exposure to indicated doses of gamma irradiation. 3B. Vegetative growth of T. viride in liquid medium at 28°C for 5 days after exposure to indicated doses of gamma irradiation. o 4. Vegetative growth of R. oryzae in liquid medium at 28 C for 5 days after exposure to indicated doses of gamma irradiation. 5. Influence of gamma irradiation on growth of L. plantarutTi in o nutrient broth at 28 C for 5 days. 6A-D. Influence of temperature and duration of heating on crude protein formation by non-irradiated T. viride on corn slurry after 5 days at 28°C. 7. Table of Analysis of variance. 8. Duncan’s Multiple Range Test of Chemical Treatment. 9. Duncan’s Multiple Range Test of Temperature of Digestion. 10. Duncan’s Multiple Range Test of Duration of Digestion. 11A-D. Influence of Temperature and Duration of heating on crude protein formation by irradiated spores of T. viride on corn slurry after 5 days at 28°C. 12 Summary of dose requirements for optimal inducement of indicated physiological action. University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES. 1. Maize Production in Ghana 1981-1990. 2. Maize Production by region. 3. Maize Husk Production Estimates by region. 4. Estimated annual cost of maize husk produced in Ghana 1987-90. 5. Duncan’s Multiple Range Test of the Combination of Chemical Treatment, Heating Temperature and Duration of Digestion on Crude Protein Formation. 6. Duncan’s Multiple Range Test of Combination of Chemical Treatment, Heating Temperature, Duration of Digestion and Irradiation Treatment of T. viride. 7A-C. Enzyme ( cellulase, pectinase and amylase ) activity of microorganisms after exposure to gamma irradiation shown as percent reduction in viscosity of substrate. University of Ghana http://ugspace.ug.edu.gh CONTENTS. Page I. INTRODUCTION 1 II. LITERATURE REVIEW 12 III. MATERIALS AND METHODS 18 a. Maintenance of Stock Culture 18 b. Gamma Irradiation 18 c. Methods of Innoculation 19 d. Assessment of Growth of Microorganisms 19 e. Determination of Enzyme Activity of Irradiated Microorganisms 19 f. pH Determination 22 g. Crude Protein Determination 22 h. Culture Media 22 i. Studies on the Morphology of T. viride before and after Exposure to Gamma Irradiation. 23 j. Methods of Sterilization 23 k. Experimental Precautions 24 IV. EXPERIMENTAL PROCEDURE 25 a. Data on Maize Production in Ghana 1981-90. 25 b. Effect of Gamma Irradiation on Vegetative Growth of A. nigei , R. oryzae and T. viride. 25 c. Effect of Gamma Irradiation on Growth of L. plantar ant.. 26 d. Amylase Activity of Gamma Irradiated Microorganisms 26 University of Ghana http://ugspace.ug.edu.gh e. Cellulase Activity of Gamma Irradiated Microorganisms f. Pectinase Activity of Gamma Irradiated Microorganisms g. Comparative pH Changes during Growth of Gamma Irradiated A. niger, R. oryzae T viride and L pi ant arum. h. Effect of Chemical Treatment, Heating Temperature and Duration of Treatment on Digestibility of Lignocellulose in Corn Husk for the Production of Crude Protein Nitrogen by T. viride. i. Production of Fungal Protein by Gamma Irradiated T. viride spores on Corn Husk Slurry combined- treated with Heat and Chemicals. j. Effect of Gamma Irradiation on the Morphology of T. viride. RESULTS a. Data on Maize Production in Ghana ( 1981-90 ) b. Effect of Gamma Irradiation on Vegetative Growth of A. niger, R. oryzae and T. viride. c. Effect of Gamma Irradiation on Growth of L. p I an t arum. d. Comparative Amylase Activity of Gamma Irradiated Microorganisms. e. Cellulase Activity of Gamma Irradiated Microorganisms University of Ghana http://ugspace.ug.edu.gh XI f. Pectinase Activity of Gamma Irradiated Microorganisms g. Comparative pH Changes during Growth of Gamma Irradiated A. niger, R. oryzae, T. viride and L. pi ant ax urn. h. Effect of Chemical Treatment, Heating Temperature and Duration of Treatment on Digestibility of Lignocellulose in Corn Husk for the Production of Crude Protein by T. viride. i. Production of Fungal Protein by Gamma Irradiated T viride Spores on Corn Husk Slurry combined-treated with Heat and Chemicals. j. Effect of Gamma Irradiation on the Morphology of T. viride. V I . GENERAL DISCUSSION VII. SUMMARY VIII. ACKNOWLEDGEMENT IX. APPENDICES 47 49 52 X. LITERATURE CITED 61 66 68 79 82 83 94 University of Ghana http://ugspace.ug.edu.gh 1. INTRODUCTION Biomass, composed of wood and crop residues and manures is the largest renewable source of organic carbon in the world.It has been estimated that 2.25 billion tonnes of cereal straws, 560 million tonnes of leguminous crop residues and 234 million tonnes of sugarcane bagasse are produced every year in the world (Anon, 1988). Lignocellulose are made up of structural polymers, cellulose, lignin and hemicelluloses; cellulose fibrils are embedded in an amorphous matrix of lignin and hemicelluloses.In the native state 1ignocelluloses are also associated with various non-structural components. The non-structural components make up a considerable percentage of the weight of certain lignocelluloses,such as agricultural residues and wood of certain tropical trees. These components, particularly the organic extracts (phenolics, terpenes, alkaloids etc.) can significantly influence biodegradative ability of microorganism ( Scheffer and Cowling, 1966 ).Table A shows cellulose, hemicellulose and lignin contents of representative lignocelluloses in nature. Plant cells are surrounded by a structural tissue,the cell wall (which is made up of a thin primary wall and secondary wall with middle lamella located between adjacent cells); Cellulose, hemicellulose, pectin and lignin are the main constituents of the plant cell wall.Of the structural components, cellulose is the most abundant, making up 35-40 percent of the dry weight of most tissues. The hemicelluloses and lignin make up 20-40 percent and 15-35 percent respectively. University of Ghana http://ugspace.ug.edu.gh Cellulose, hemicellulose and lignin contents of lignocelluloses. ( After Kirk, 1983 ) TABLE 1. COMPOSITION ( % dry w t .) Lignocellulose Cellulose Hemicellulose Lignin Reference Birch Wood 42 38 19 Timell,1967 Maple 4 5 29 24 Timell,1967 Spruce Wood 41 31 27 Timell,1967 Hemlock Wood 41 23 33 Timell,1967 Bagasse (Sugar cane)41 { Saccharwn. officinarurn Soybean stalk 3 5 C Glyci ne noax s Wheat straw 40 C Tri tic ura aes t i vum Rice straw C Oryzae saliva J Maize staw (husk) CZea mrxys'j 36 67 >20 >25 >28 >25 >30 20 20 Dunning & Lathrop,1945 Aronovsky Nelson & Lathrop,1943 17 12 10 University of Ghana http://ugspace.ug.edu.gh Below 18-20% lignin content, lignocelluloses are degraded to increasing extent with decreasing lignin content by cellulases and hemicellulases, and consequently by many bacteria and fungi that secrete these enzymes ( Kirk, 1983 ). Fungi belonging to the Aspergillus spp. produce pectinase enzyme ( Beldman et al, 1984) Table 2 shows major industrial fungal enzymes and their uses. Gilbertson (1980) estimated that there are about 1600-1700 North American species of wood and litter decomposing fungi. On a world-wide basis, there are certainly over 2,000 species. Some of these fungi use an apparently unique mechanisms for circumventing the lignin, pectin and other barriers to enzymatic cellulose degradation. CELLULOSE DEGRADATION; Degradation of crystalline cellulose by white-rot fungi, (eg. Sporotrichum pulverulentum. Nov. ) and various soil fungi result from the concerted, synergistic action of three types of hydrolyses (i) endo-1,4 - i-glucanases, (C enzyme) which cleave the x cellulose randomly; (ii) exo-1,4-';-glucanases, { cellobiohydrolases, C enzymes ) which release cellobiose { together with glucose in some cases ) from non-reducing ends of cellulose; and (iii) /?-glucosidases, which split cellobiose into glucose units or it may be oxidised to cellobionic acid and then cleaved. The endo- and exoglue a na s es which actually act synergistically - perhaps as a loose complex ( Wood and McCrae, 1977 ) are repressed by high concentrations of monosaccharides ( Eriksson, 1978 ). 3 University of Ghana http://ugspace.ug.edu.gh Ma.ior industrial fungal enzymes and their uses.____________________ Enzyme Source Catalytic action Examples of use TABLE 2. Glucose oxidase Catalase C!-Amylase Gluco- amylase /?-Glua nase Cellu - lase Pecti nase Lactase Fu r.gal re-nniri Lipase Fungal protease /Ispergi 1lus n i $ e r AspergiIlus niger AspergiIlus oryzae Aspergi I I'us n ig e r AspergiIlus rage r Trichodernto. AspergiIlus Fenic i I hum. AspergiIlus Aspergi11 us ai ger Mucor pus 1 11 us AspergiIlus Aspergi 11 u-: oryzae Oxi.di.zes glucose lo D-glucono-<5-lactone with consumption of oxygen. cata lyses the decom­ position of hydrogen peroxide to oxygyen and water Breaks Ot-1,4-links on the interior of the starch molecule g iv ing a mixture of m allo- dextnns. Acts from the end of the starch chains liberating glucose Hydrolyses f t - 1,3-glu cans which are not susceptible lo hydro­ ly s is by am ylase or gluco-am ylase S p Catal /ses the breaking S p c i / j-i,4 -links in s p ce llu lose and in some cases its specific ity permits lL to act on other /9-linked glucans S p A polygalacturonase effecting hydrolysis of a -i,4 -D -ga lacL o siduronic linkages in pectin A f?-qalactosi ia se that h\ dro lyses milk sugar ■lactose' to galactose and qlucose C l e a v e s c e r t a i n p e p t i d e bo t ' id i in c a s e i n Sp A ti i a c y l g l y c e r o l o c y l - olas ll>at I. iherases fa t t v a c i d s 11 -r-m ,_,l „■ 1 ..I i-JflS H a s >j w id e s p e d i urn o f p r o t e o l y t i c a c t i. v i l i e s Blood glucose an a lys is Egg de-sugaring H O rem oval 2 2 after milk sterilization Maltose syrup production Glucose syrup production Filtration of K<&£.r Di-peslive aids Extration and c larification of fruit juices and vines V he y p r oct'ssi nci O l‘i * e s en i al: i Ch eo s o f l a v o u r enhance j1 S o y s ou c e p rodu c t ion Biscuil d^ -ugh i mp v o v erne n t rungi. Vol. A, Fungal Tech, al. ( 1 983 ). University of Ghana http://ugspace.ug.edu.gh Fig. 1. A model for the degradation of native cellulose by the action of these enzymes is shown below. ENDOGLUCANASES + CELLOHYOflOLASES EXOGLUCOSIDASES University of Ghana http://ugspace.ug.edu.gh 6The cellulases from Trichoderni/x spp notably, Trichoderma. reeset, 7. koningii and T. viride have been studied. ( Beldman et al. 1984,1985; Mandels et et I . 1981; Tangu et al 1981; Shin et al 1978 ). Cellulases from fungal origin also known to be powerful in cellulose hydrolysis have been investigated in Sporotrichxm. pulverulent uni ( Eriksson, 1978; Eriksson and Petterson, 1975 ) Fusariwn solani, Penicilliutn fumiculosum and Talararnyces errosrsonii C Wood et al 1980, 1982 ) HEMICELLULOSE DEGRADATION Dekker and Richards ( 1976 ) reviewed microbial hemicellulases. Wood-rotting fungi produce enzymes capable of hydrolysing a variety of /?-(l->4) linked glycan ( mannan and xylan ) substrates as well as various glycosides ( Ahlgren and Eriksson, 1967; Keilich, Bailey and Liese, 1969; Highley, 1976 ). Endoglucanase from white-, brown- and soft-rot fungi all apparently act randomly, producing dimeric and higher oligomeric products ( Ishihara and Shimizu, 1980 ). Information regarding regulation of the synthesis of hemicellulose is somewhat contradictory ( Dekker and Richards, 1976 ). However, multiple hemicelluase activity is found in culture filtrates of many fungi including white-, brown- and soft rot fungi after growth on a variety of substrates including simple sugars ( Highley, 1976 ). University of Ghana http://ugspace.ug.edu.gh LIGNIN DEGRADATION The pertinent literature is replete with research on fungal degradation of lignin. Some of the reactions comprising degradation have been elucidated, and the unusual boichemical and physiological features are gradually begining to be described ( Amer and Drew, 1980; Crawford and Crawford, 1980; Kirk et al 1980; Kirk, 1980,1982 ). Lignin degradation is distinct from cellulose and hemicellulose degradation. Indeed, it differs from the biodegradation of all other studied biopolymers. Not a single enzyme involved in lignin degradation has been identified ( Kirk, 1971; 1983 ). Because the lignin polymer is attacked by an extracellular non-specific oxidising agent, it is possible that enzymes may not be directly involved. Hall ( 1980 ) suggested that "diffusible species" derived from molecular oxygen may be involved. Current evidence suggests that regulation of secondary metabolism, including lignin degradation is somehow connected with glutamate metabolism ( Kirk, 1981 ). LIGNOCELLULOSE DEGRADATION AND CONVERSION INTO FEED OR FOOD. Because of their abundance, 1ignocellulose plays a dominant role in the terrestrial carbon cycle. Lignin-degrading filamentous fungi play a prominent role, and probably predominant, role in the biodegradative part of this all-important cycle. Recent studies have aimed at feed production by controlled cultivation of lignolytic fungi on lignocelllosic substrates. Substantial increases in crude protein have been University of Ghana http://ugspace.ug.edu.gh reported for woods, barks and Iignocellulosic waste following cultivation of various fungi ( Daugalis and Bone, 1978; Ek and Eriksson, 1980; Matteau and Bone, 1980 ). Increases in vitro polysaccharide digestibility of lignocelluloses have accompanied solid substrate incubation with fungi ( Kirk and Moore, 1972; Detroy et a.I 1981; Zadrazil,1980; Zadrazil and Brunnert, 1980, 1981 ). Agricultural 1ignocellulosic waste disposal problems are the order of the day in Ghana. During the harvest season, considerable amount of maize plant debris is left to decompose in the fields as stubble or mulch. The removal of the maize cob from the husk is accompanied by the disposal of husk to waste. The local kenkey industry makes use of a small percentage of the maize husk as wrapping material for balls of kenkey during the manufacturing process. Kenkey consumers normally discard the husk after use. There is therefore a high potential for such waste being used as substrate in the production of microbial protein surjrleiiient biomass. The increased nutritive value of the husk could be used as animal feed supplement. One of the largest single factors affecting animal production in the developing countries is poor nutrition as farmers cannot afford to buy the recommended feed from the mills { McDowell, 1968 ). This is because the high cost of the ingredients of the feed contribute immensely to the exorbitant price of animal feeds used in livestock production. For example, in Ghana it is estimated that feed cost alone constitute 80 ,.p *i.- .-.-n cost of animal production ( Andah, 1974 ). 8 University of Ghana http://ugspace.ug.edu.gh Recent rapid increases in prices of ingredients of feedstuffs namely fish meal, wheat bran, maize, vitamin premixes etc. exacerbate the problem. If indeed microorganisms can provide, during their growth on solid waste, high levels of protein double their weight in a relatively smaller space ( Dunlop, 1973 ), it would be worthwhile to explore the possibility of exploiting their potential to produce feeds by solid substrate "fermentation". There are three technical problems in using lignocellulosic fungi to produce feeds by solid substrate "fermentation": 1. In scaling-up with the required careful control of humidity, aeration and temperature necessary for uniform treatment. 2. Preventing contamination of unwanted microbes. 3. The slowness of degradation of some plant residues. The development of a low-technology process, analogous to ensiling for use on a small scale from easy-to-treat lignocellulosics should be the rule rather than the exception. Conversion of lignocellulosic substances into economic products can be achieved through hydrolysis followed by fermentation ( Ladisch et a l , 3 983 ) or by direct fermentation ( Ng et al , 1981 ) . Hydrolysis of 1ignocellulose can be achieved with either chemical or enzymes or both. Because of certain disadvantages associated with acid hydrolysis, the enzymatic or biological method has received more attention ( Manonmami and Sreekantiah, 1987 ) _____Biomass in the form of maize husks, oat hulls and rice University of Ghana http://ugspace.ug.edu.gh husks are taken through three types of pretreatment so that they can suitably be fractionated into their major components: cellulose, hemicellulose and lignin. 1. ALKALINE TREATMENT: Substrate is treated with 5-10 percent ( w/w ) sodium hydroxide at 80-121 >C for a prescribed period depending on the nature of the substrate. 2. STEAM TREATMENT: The moist substrate is treated with steam at a o * temperature between 200-230 C for a prescribed period. 3. PARTIAL HYDROLYSIS: Substrates like maize husks, oat hulls and rice husks can also be partially hydrolysed by treatment with 3 o , percent HCl at a temperature of 90 C for four hours ( Anon, 1988 ). This partially maize husk could contain approximately 10 percent reducing sugar content and can serve as an excellent substrate on which to grow microoganisms. Cellulolytic, pectinolytic and amylolytic enzymes are produced by a large number of microorganisms including fungi. Highly active cellulases are often found in culture media supporting growth of Trichoderrria viride. T. viride is noted for its high cellulolytic activity and its ability to utilize various substrates ( oat hull, maize husk cellulose etc. ) with the attendant production of high protein into the medium ( Mandel and Weber, 1961 ). The fungus Aspergillus niger produces pectinase enzymes and there is a synergistic action of cellulases from T viride and pectinases from A. niger in plant biomass conversion. In this thesis, the effect of low gamma radiation doses on the cellulolytic, roectinase and amylase enzymes activity by T. viride 10 University of Ghana http://ugspace.ug.edu.gh and A niger were tested. Two other microorganisms Rhizopus oryzae and Lactobacillus plantaruin ( bacterium ) were included for purposes of comparison. Finally protein production by gamma-irradiated 7". viride on partially digested corn husk slurry was also investigated. University of Ghana http://ugspace.ug.edu.gh 11. LITERATURE REVIEW. The abundance of agricultural waste materials in the form of plant lignocellulose is well known. The attendant problem of utilization of lignocellulose for the purpose of producing fungal protein for direct human consumption ( Spicer, 1973 ) or as animal feedstuff ( Forss et al. 1972; Imrie, 1973 ) has engaged the attention of many researchers in the developed countries. Developing countries have only recently taken keen interest in plant biomass conversion. The plant cell wall which consists of middle lamella, primary wall and a secondary wall has pectic substances, present as polygalacturonides with non-uronide carbohydrates covalently bound to an unbranched chain of (1,4) a-D-galacturonic acid units. The walls of soft, non-differentiated tissues are of the primary type rich in cellulose, hemicellulose and pectin; the cellulose here has a lower degree of polymerisation and crystallization than in the secondary wall ( McNeil et al. 1984 ). The degradation of cellulosic materials by fungi has been well documented. Sandhu and Sidhu (1980) showed that fungal succession during compositing process of agricultural waste material involved fungi like Aspergillus funiigatus, A. niger, A. ter reus, A. / lavas, Hue or pusillus, Penic i I liwri spp. , Rhizopus rtdcrosporus, Trichodernva viride, T. longibrachiatum and members of the order Agaricales (Basidiomycotina). Prior to this, Siu ( 1951 ) summarised research findings on cellulolytic activity of fungi and he placed Aspergillus fum.iga.tus and Trichadertna viride among the strongly cellulolytic group, the moderately cellulolytic ones were 12 University of Ghana http://ugspace.ug.edu.gh Fxisariutn monili forme, F oxysporum. and F solani. The fungi Aspergillus japonicus and A. ochraceus were placed in the weak cellulolytic category whilst A. ccnxdidus, A. f lav us and Rhizopus oryzae were classified as non-cellulolytic. Basu (1948) reported that his strain of Aspergillus niger on jute fibre was non-cellulolytic but ,4. ustus, A. terreus and A. furnigatus were cellulolytic. Reese and Downing (1951) classified twelve Aspergillus spp according to their ability to degrade cellulose and he placed A. flavus, ,4. ochraceus and A. niger in the non-cellulolytic group. On the other hand, Flannigen (1970) reported A. f urnigatus and A. niger isolated from barley kernel as cellulose decomposing. Presumably, cellulolytic activity of fungi might be species and strain specific as well as depending on the type of substrate which is being metabolised by the fungus ( Odamtten and Kampelmacher, 1985 ). Several members of the genus Aspergillus appear to be capable of hydrolysing the ft-(1-4)-glucosidic linkage in the cellulose chain ( Alexander, 1961; Raper and Fennel, 1972; Mazen, 1973; Steward and Walsh, 1972 ). But this does not itself determine whether they can attack cellulose or not since many non-cellulolytic microorganisms possess the C enzyme carrying out that reaction ( Reese et al. x 1950 ), ( Fig. 1 ). Weber (1969) found Trichoderma viride, T. reesii, and T honingii to have very high cellulolytic activity with the ability to utilize substrates with both reducing sugars in hydrolysed oat bull as well as cellulose. When grown on media containing maize or cotton fiber as sole carbon source, T viride synthesizes and releases into solution all enzymes that are University of Ghana http://ugspace.ug.edu.gh essential for the breakdown of native cellulose to glucose ( Mandels and Reese, 1964 ). Beldman et al. (1985), and Beldman, (1986) purified the cellulases of T. viride and characterised and compared all detectable endoglucanases, exoglucanases and f3-glucosidases. Synergism in cellulose hydrolysis by endoglucanases and exoglucanases purified from T viride were demonstrated by Beldman, (1986). Cellulolytic enzymes are also produced by other microorganisms including actinomycetes, gliding bacteria and bacteria ( Wood, 1985 ). However whilst bacterial cellulases are often cell wall bound, highly active cellulases of fungi are released into the culture media. It showed that only pre-treatment of cellulose involving either swelling or dissolution increases acessibility to attack by both cellulases and other reagents. Oat hulls contain 34 percent cellulose, and 30 percent pentosans giving a total carbohydrate content of 64 percent ( Caldwell and Pomeranz, 1973; Rosenberg et al. , 1978 ). Maize husks contain about 6 7.3 percent cellulose, 10.3 percent lignin, 1.8 percent ash and 1.1 percent protein. The amino acid composition of four T. viride strains ( QM 6 , QM 913, QM 94414 and NRRL 3653 ) tested on hydrolysed a oat hulls ammended with 12g/l glucose at 25°C for 10 days in shake culture flasks werp similar. Amino acids encountered include Aspartic acid, alanine,threonine, serine, glutamic acid, proline, glycine, cystine, valine, histidine, arginine and tryptophan. Some strains exhibited higher content of alanine, valine methionine and University of Ghana http://ugspace.ug.edu.gh histidine. The total crude protein of cultures of T viride QM6A on hydrolysed oat hull increased from 40.5g Kg ^ (10.02 percent crude protein) to a maximum of 71.Og Kg ^ (15.44 percent crude protein) in 8 days. This represented an overall 75.8 percent protein content enrichment of the oat hull.Growth of T viride on hydrolysed oat hulls therefore offer a potential for improved balanced animal feed for the livestock industry. The use of maize husk slurry as substrate for improving protein content of fungi eg. T. viride has not been tried to any great extent. There are limited references in the pertinent literature to the use of maize husk. Saah (1985) found an increase in the protein content of T vii'ide grown on maize husk slurry. Protein content of hydrolysed maize husk was enriched by 72.7 percent in 15 days by T viride. The pectinolytic enzymes of T. viride on the other hand has not been exhaustively investigated. The only reference to T. viride pectinase activity is the work of Rosenberg et al. ( 1978 ) who used a liquid medium of the following composition to culture T viride: glucose 12-18 gm/1; KH„PO , 0.0250g/l; KNO_ 5.00g/l; £j 4 O FeSO^.711^0, 0.013g/l; NH^Cl, 0.750g/l . The release of protein into the medium during growth was closely and positively correlated with the appearance of cellulase and pectinolytic enzymes. There is a synergistic action of cellulases from T. viride and pectinases from A. niger in plant biomass conversion. A combination of pectinolytic and cel1ulolytic enzyme was able to produce monomeric sugar solutions from beet pulp and potato fibre ( Beldman et al. 1984 ). The process is optimal at pH 3.5-4.0 and University of Ghana http://ugspace.ug.edu.gh is mainly caused by the polygalacturonase from the pectic enzyme which has its optimum at pH 6.0 ( Jones et al. 1976 ). In a second step, the extensive hydrolysis of the cell wall polysaccharides, a cellulose with high activity is needed ( Beldman et al. 1976 ). Mutation breeding is one important tool in developing new, better-performing variety of crops. Radiation and some chemicals ( mutagens ) induce mutations by altering genes and creating genetic variability. Plant breeders have derived from induced mutations additional genetic resources for improving crops ( Doninij 1983 ). Although sudden heritable changes in microorganisms have long been known to occur, it has only recently been possible for microbiologists to show that mutation in microorganisms can be induced by ultra violet light, mutagenic chemicals such as nitrogen mustard, X-rays and gamma rays. The lethality of ultra violet radiation is most easily expressed by the fraction of irradiated spores which fail to germinate. Doses that are not lethal delay germination or induce abnormalities ( Cochrane, 1958 ). In surveys of fungal susceptibility it is often noted that dark spores tend to be more resistant. Pigmentation alone, however, cannot explain specific differences in sensitivity ( Cochrane, 1958 ) since ultra violet, induced mutagenesis is also affected by several modifying enviromental factors. Ionising radiations are radiations whose passage through a material cause the production of ion-pairs. The process of food irradiation involves exposing the food or microrganisras therein to ionising radiation so that a prescribed quantity (dose) is absorbed 16 University of Ghana http://ugspace.ug.edu.gh Radiation sources used are the following: A. Gamma-rays from the nuclides Co-60 or Cs-137. B. X-rays generated from machine sources operated at or below an energy level of 5 MeV (millielectron Volts). C. Electron beams generated from machine sources ( eg. Van der Graaf generator ) operated at or below an energy level of 10 MeV. The way in which the radiation dose absorbed is measured differs according to the source of radiation. Most studies on fungi eg. Aspergillus spp, Penicillium. spp and Neurospora spp have employed X-rays or gamma rays as mutagens. The technique has been put into practice where the time of maximum synthesis of the desired product can be accurately judged and mutagenesis carried out during this time. In the case of Cephalosporiunx acremonium, the time of maximum cephalosporin C synthesis appears to correlate with differentiation into athrospores ( Nash and Haber, 1971 ), so that mutagenesis during the time of differentiation could increase specificity for antibiotic titre genes. Application of ionizing radiation for improvement of enzyme ( amylase, pectinase, cellulase, etc. ) production by A. niger, T. viride R, orysae and L plant arum on the other hand has not been tried. 17 University of Ghana http://ugspace.ug.edu.gh 111. MATERIALS AND GENERAL METHODS I.MATERIALS: FUNGI: Both Aspergillus niger Van Tiegh and R.hizop'‘is oryzae Went and Prinsen-Gerling were isolated from maize stored in humid chamber at : 80% R.H. for 10 days. Trichodernuy. viride was isolated from soil within the Botany Department, Univ. of Ghana, Legon. BACTERIUM: L actobaciIlus plant arum, was isolated from fermenting cassava pulp. Dry maize husks were obtained from the Research Farm of the Ghana Atomic Energy Commission, Kwabenya. II. GENERAL METHODS: A. MAINTENANCE OF STOCK CULTURE: Stock cultures of A.niger, R. oryzae and T viride were maintained on slants of oxytetracycline glucose yeast extract ( OGYE ) agar o at 10 C and subcultured every two weeks. L. plant arum was maintained on Tryptone glucose yeast o 1 extract agar at 35 C and was subcultured every two weeks. B. GAMMA IRRADIATION: Dry spores of the fungi were seperated by sieving after being cultured in lOOg of blended and autoclaved maize grains. The spores were then irradiated in air at pre-selected doses of 0, 20, 40, 50, 100, 200 ICrads in a Cobalt-60 Trradiator ( Gamma Cell 220, Atomic Energy of Canada Limited ). The source dose rate was 541.44 Gy/hr, For each dose applied there were four replicates. L. pi ant arum spores were cultured on Tryptone glucose Yeast Extract agar at 35 C for 3 days. The spores were then 5 ) peptone solution ( 10 spores/nil ). About 18 University of Ghana http://ugspace.ug.edu.gh 10 ml in MaCartney tubes were irradiated with the different indicated doses as above. C. METHODS OF INNOCULATION: Erlenmyer flasks containing 30 ml of V-8 broth amraended with 20g/l of glucose were inoculated with lml aliquot of spore suspension of either control or irradiated spores of .4. niger. . , 5 R.oryzae or T. viride. Each spore suspension contained about 10 spores/ml. About lml aliquot of L. plant.tarvm was used in inoculating 30 ml nutrient broth. D. ASSESSMENT OF GROWTH OF MICROORGANISMS: Growth of T. viride, A. niger and R. oryzae in static liquid medium was assessed by estimating the dry weight of harvested mycelium at the end of the 5 days incubation. at O , , , O 28-35 C. The A. niger and T. viride were incubated at 35 C ( Odamtten, 1977 ) and R. oryzae at 28°C ( Akushie, 1980 ). Mycelium collected on a previously weighed and dried Whatman No.2 filter paper was dried and weighed, after cooling in a dessicator. Growth of L. planiarwn was determined by measuring the optical density of the substrate at 660nm after 5 days incubation at 28°C. E. DETERMINATION OF ENZYME ACTIVITY OF IRRADIADED MICROORGANISMS: 1. AMYLASE: Amylase activity was determined by measuring the reduction in viscosity of 0.5 per cent starch solution with o Fenkse-Ostwald’s viscometer at 30 C. (Plate 1) The reaction mixture contained 5ml. of starch solution and 5ml, of culture filtrate of either .4. niger, R. oryzae or 7. viride. After­ mixing the solution was left for 2hrs to allow enzyme digestion before measurement were taken. The control reaction mixture was heated to 100 C for 5-10inin. to inactivate the ensyraes. -------------------------------------- i } University of Ghana http://ugspace.ug.edu.gh The reaction mixture for the determination of amylase activity for L. planta.rv.ffi contained 5ml of starch solution and 5ml of the culture solution ( ie. the solution containing the spores of L. plantarum II. CELLULASE: The cellulase activity of A. niger, R. oryzae and T. viride was determined by the same procedure as that of the amylase activity except that instead of starch solution, 0.5 per cent solution of carboxymethylcellulose ( CMC ) was used. In the determination of the cellulase activity of L. plantarum, the same procedure as in amylase determination for L. plantarum was adopted except that CMC solution was used instead of starch solution. III. PECTINASE: The pectinase activity of A. niger-, R. oryzae and T. viride was determined by the same procedure as that used in the determination of amylase activity except that instead of starch solution, 0.5 per cent pectin solution was employed. In the determination of pectinase activity of L. plantarum, the same procedure as in the amylase activity determination of L. plantarum was adopted but instead of starch solution, 0.5 per cent pectin solution was used. The reduction in viscosity expressed as the percentage loss in viscosity was calculated by the following mathematical expression: Percentage loss in viscosity = f T - T_ ] o 1 ---------- x 100 [ T T ] o - w T f l ow Um e of r ©action 0 mixture at Oriun. T 1 - f low t i iri* of r eaction rmxtui e ot o particular tune interval T = flow time of dis I it Led w - j ter. 20 University of Ghana http://ugspace.ug.edu.gh Plate 1, Photograph of set up used in determining changes in viscosity of different media caused by amylase, pectinase and celiulase enzymes produced by T, viride, R, oryzae, A. niger and L.plantarum. ( Note the hand holding the Fenske-Ostwald Viscometer) Plate 2. A close up of the Fenske-Ostwald Viscometer seen in Plate 1, University of Ghana http://ugspace.ug.edu.gh 22 F. pH DETERMINATION: The pH of the media before and after incubation was measured by using a pH Meter ( PYE Unicam Model 290 MK 2 ) G. CRUDE PROTEIN DETERMINATION: For crude protein determination, Kjeldahl’s method of estimation ( macroprocedure ) was employed as outlined by the Association of Official Analytical Chemists.- AOAC- ( 1970 ). H. CULTURE MEDIA: All chemicals used in the preparation of media were either of the "Analar", B.D.H. (British Drug House ) or Oxoid grade. The composition of medium varied with the experiment as stated in the appropriate places in the text. Erlenmeyer pyrex flasks ( 250ml-capacity ) each containing 30ml media were used for all the liquid cultures. Twenty milliliter of solid agar medium were used per 9cm diameter Petri dish. THE COMPOSITION OF THE MEDIA WAS AS FOLLOWS: I.LIQUID MEDIUM: V-8 (Cambell Soup Company U.S.A. ) Broth: 200ml of V-8 juice made up to 1,000 ml with distilled water (pH adjusted to 4.2 ) . RESENBERGH”S MEDIUM: ( Resenbergh el al. 1978 ) A modification of the medium used by Resenbergh et al ( 1978 ) was employed. The composition of the medium was as follows: 180 mg/1, glucose; KH.PO^ 25 mg; KNO .7H„0, 30 mg; FeSO . 7H O, 1.3 mg; Nil C l , 75 mg; 1,000 ml distilled water.I 4 11. PREPARATION OF MAIZE HUSK SLURRY: A slurry of maize husk was prepared using different ►">;e husk was hydrolysed with either 1% Sodium \ University of Ghana http://ugspace.ug.edu.gh hydroxide or 5% Sodium hydroxide at 80°C, 100°C or 120 C for varying periods ( 1, 2, 3, hours. ) In a parallel experiment, lOg of maize husk was hydrolysed with either 1 % Hydrochloric acid or 5% Hydrochloric acid at 80 C, 100 C or 120°C for varying periods ( 1, 2, or 3 hours. )• In all instances the hydrolysed corn husk was aseptically washed with several changes of distilled water before being transferred aseptically into the sterilized media. The number of replicates in each treatment was four. I. STUDIES ON THE MORPHOLOGY OF T. VIRIDE AND .4. NIGER BEFORE AND AFTER EXPOSURE TO GAMMA IRRADIATION. Dry conidia of T. viride and A. m g e r were exposed to 0-200 Krr.d of gamma irradiation and then used in inoculating petri plates containing 20 ml of V-8 Agar. The cultures were incubated o at 35 C for 5 days to allow sporulation. Mycelia from the cultures were mounted in lactophenol cotton blue and the morphology of the cultures compared. Photomicrographs were taken to facilitate this experiment. K. METHODS OF STERILIZATION: All media, conical flask, McCartney tubes, Fenkse-Ostwald’s Viscometer, etc. were sterilized by autoclaving at 121 C for 15 mins.( 15 psi ). Cotton wool plugs temporarily covered with grease proof paper were used to prevent the penetration of any condensed water during the autoclaving. University of Ghana http://ugspace.ug.edu.gh 2h Petri dishes ( 9.0 cm diameter ) were sterilized by heating at 165°C for 6-8 hours in an electrically heated oven (.Gallenkamp Hotbox Oven Size 1 ). R The Laminar flow cabinet ( Microflow ) was set going for at least 15-30 rain before using the inoculating chamber. The air conditioner was put on to augment the safety of the room for inoculat ion. J. EXPERIMENTAL PRECAUTIONS: 1. Glassware was kept scrupulously clean. Glassware which had already been cleaned with water and detergents was rinsed several times with tap water and three times with distilled water and then allowed to drain before use. 2. Oven-dried filter paper usually lost some weight because of heating. Filter paper used throughout these o investigations was therefore heated at 75 C for 24 hours prior to use. 3. Batches of filter paper with loads of dried mycelium were always conveyed to the balance room in a closed dessicator to avoid absorption of moisture. University of Ghana http://ugspace.ug.edu.gh IV. EXPERIMENTAL PROCEDURE A. DATA ON MAIZE PRODUCTION IN GHANA 1981 - 1990. The information was compiled from the 10 regions in Ghana in order to ascertain how much "waste" in terras of corn husk was available for use in supplementing feed production using microbial protein. Data from small scale farmers are not included This connotes that data obtained could be 5-10 percent higher in the overall picture throughout the country. Results are presented in Figs. 1-4 and in Appendices 1-4. B. EFFECT OF GAMMA IRRADIATION ON VEGETATIVE GROWTH OF A. NIGER R. ORYZAE AND T VIRIDE. In this Section, vegetative growth of the listed fungi after exposure to low gamma irradiation doses was assessed. About lml aliquot of irradiated spores of .4. niger, P.. oryzae and 7 viride suspended in 0.1 percent peptone solution was used to innoculate 30ml of V-8 broth amended with 20g/l of glucose in Erlenmeyer flasks. There were four replicates. Each mixture was swirled around to ensure thorough mixing of the spores in the o growth medium. The flasks were incubated at 28 C for 5 days. Growth of A. niger. R. orysae and T. viride was assessed by estimating the weight of harvested mycelium at the end of the incubation period following procedures out!ined under Section D of the General Methods. Results are presented in Fig. 5 and Table 3A, 3B, 4 . University of Ghana http://ugspace.ug.edu.gh 2 6 C. EFFECT OF GAMMA IRRADIATION ON GROWTH OF L. PL ANTARUM. Experiments in Section A were repeated using L. plant arxmi. About 1ml aliquot of irradiated spores of L. pi an Latum suspended in peptone solution was used in inoculating 30ml nutrient broth. The mixture was incubated at ambient o temperature of 28 C for 5 days. Vegetative growth of L. plantarum was determined by measuring the optical density of the substrate at 660 nm. Fig. 6 and Table 5 summarise the results obtained. D. AMYLASE ACTIVITY OF GAMMA IRRADIATED MICROORGANISMS. Amylase production after gamma irradiation of the test microorganisms was investigated following procedures outlined under General Methods. 'The results are presented in Fig. 7 and Appendix 7. E. CELLULASE ACTIVITY OF GAMMA IRRADIATED MICROORGANISMS. The determination of cellulase activity of A. niger. R. oryzae, T. viride and L. plantar urn followed the same procedure as outlined for the determination of amylase activity. The reaction mixture was made of 5 ml of culture filtrate of the appropriate microorganism and 5 ml of Carboxymethylcellulose (CMC) F. F’ECTINASE ACTIVITY OF GAMMA IRRADIATED MICROORGANISMS. Pectin degrading enzymes in microorganisms are essential for effective nutrient recycling of agricultural waste. It was University of Ghana http://ugspace.ug.edu.gh therefore necessary to ascertain the effect of gamma irradiation on the selected organisms. The determination of pectinase activity of .4. niger. R. oryzae, T. viride and L. plantar am. followed the same procedure as the determination of amylase activity of the microorganisms. The reaction mixture was made up of 5 ml of the cultural filtrate of the appropriate microorganism and 5 ml of pectin. The set up was exactly as in Section D and E ( Plate 1 ). G. COMPARATIVE pH CHANGES DURING GROWTH OF GAMMA IRRADIATED .4. NIGER, R. ORYZAE, T. VIRIDE AND L. PLANTARUM. Vegetative gowth of microorganisms is accompanied by production of secondary metabolites that cause drift in the pH of the medium. Thus a medium may become more acidic or may drift to the basic side depending on its initial pH. The changes in the pH during growth of the irradiated spores of the indicated organisms was investigated using a pH metre ( PYE Unicam Model 290 MK 2 ). The spores were incubated at their optimal temperatures for 5 days. H. EFFECT OF CHEMICAL TREATMENT, HEATING TEMPERATURE, AND DURATION OF TREATMENT ON DIGESTIBILITY OF LIGNOCELLULOSE IN CORN HUSKS FOR THE PRODUCTION OF CRUDE PROTEIN NITROGEN DY T . I-'/;■'! DE. Fully lignified tissues are degradable only by iui crobes that are able to degrade lignin, with the exception of the brown-rot fungi » Cell ullose arid hemicel lu I ose are closely associated with lignin in the pLant cell wall. 1 he structural resistance of these polymers £■( University of Ghana http://ugspace.ug.edu.gh 28 has made cellulosic materials generally non-nutritive and difficult to digest. For an increase in nutritive value, the material must be first converted into monomers or oligomers ( Kirk, 1983 ). Use of other non-1ignolitic microbes to process lignocellulose requires chemical or physical disruption of the lignin barrier, to fermentable products. Extant and potential use of fungi for lignocellulose conversion can be divided into five categories: a. conversion into feed or food; b. manufacture of mechanical pulp; c. production of microbial chemical products; d. production of chemicals from lignin; and e. treatment of lignocellulose-derived waste. In this Section, the best combination of treatments that would facilitate the utilization of corn husk by T. viride was investigated. T. viride was selected because of its versatility in degrading lignocellulose and it is not kwown to produce any harmful toxins to human. The procedure followed is as outlined under General Methods. The results are summarised in Table 3. I. PRODUCTION OF FUNGAL PROTEIN BY GAMMA IRRADIATED 7. VIRIDE SPORES ON CORN HUSK SLURRY COMBI NED-TREATED WITH HEAT AND CHEMICALS. Formation of crude protein by irradiated T. v ! i' i cie was studied. If irradiation enhanced crude University of Ghana http://ugspace.ug.edu.gh protein accumulation on hydrolysed corn husks, there would be an added advantage in using irradiation to stimulate enzymes required to effect the necessary chemical reactions. Results obtained are presented in Table 12 A-D. J. EFFECT OF GAMMA IRRADIATION ON THE MORPHOLOGY OF A. NIGER AND T. VIRIDE. Different strains of fungi respond differently to radiation treatment. Mutagenesis may be reflected not only in change in enzyme production patterns but also in the morphology of the test organism. In the concluding Section of this theses, the effect of low gamma irradiation on the morphology of ,4. niger and T. vii'ids- was studied. Temporary mounts of the mycelium were made and were stained with Lactophenol cotton blue. Plate 3 shows the results obtained. «-y University of Ghana http://ugspace.ug.edu.gh 30 RESULTS A. DATA ON MAIZE PRODUCION IN GHANA. C 1981-1990 ') Results are presented in Fig. 2-5. Maize production between 1984 and 1990 exceeded 500,000 metric tonnes except in 1985 where there was a decline to 395,000 metric tonnes ( Fig. 2 ). The unfavourable years of drought between 1981-1983 was attented by a sharp decline in maize production. The poorest maize production of the decade ( 198J-1990 ) was in 1983 ( 140,800 metric tonnes ) . The bulk of the national maize production was contributed by Ashanti, Brong Ahafo, Eastern and Northern Regions ( Fig. 3 ). Maize husk production was commensurate with the total maize harvest for each region ( Fig.4 ). Therefore as a raw material for fungal protein production, maize husk has a potential for sustainable use. The estimated cost of the maize husk ( Fig.5 ) shows that it can serve as a source of revenue for the farmer if other uses are found for it in the feed industry. University of Ghana http://ugspace.ug.edu.gh 31 Fig. 2. MAIZE PRODUCTION IN GHANA.19B1-90. (FIGURES IN .000 Ml.) YEARS S PRODUCTION (.000 MT.) MAIZE PRODUCTION University of Ghana http://ugspace.ug.edu.gh F H U O .I C T IO M B 'f I )' :f 3 l(j f JL ', 1 3 0 7 -1 9 9 0 I Fig. a. MAIZE PRODUCTION BY REGION 1387-1990, (FIGURES IN .000 MT) 160 140 120 SO sc 40 20 0 WESTERN GT,ACCRA VOLTA EkAHAFO UEAST CENTRAL FARTFRM ASHANTI NORTHERN U.W?ST REGIONS B 18fl7 I s 1W 8 hi 1888 □ 188(1 University of Ghana http://ugspace.ug.edu.gh Fig,4, MAIZE HUSK PRODUCTION BY REGION 1907-1990. (FIGURES IN .000 MT) 1367 0 WESTERN 0 CENTRAL [J GT.ACCRA [/J EASTERN □ VOLTA □ ASHANTI □ BAHAFO □ NORTHERN 1 U.EAST m U.WEST 1990 University of Ghana http://ugspace.ug.edu.gh 3b Fig. 5. ANNUAL COST OF MAIZE HUSK IN GHANA 1987-1990. 3b 30 25 20 15 10 0 1987 1988 COST (MILLIONS OF CEDIS) 1989 1990 University of Ghana http://ugspace.ug.edu.gh 8. EFFECT OF GAMMA IRRADIATION ON VEGETATIVE GROWTH OF A. NIGER, P.. ORYZAE AND T. VIRIDE. The application of gamma irradiation to dry spores of 4. niger, R, oryzae and 7 . viride prior to culturing in liquid medium had variable effects. Results are presented in Fig 6, Appendix 5, Tables 3a, 3b and 4. A dose of 20 Krad decreased vegetative growth of A. rtiger by 46.6 percent, and thereafter further increases in dose ( 20-50 Krad ) increased dry matter accumulation by the fungus to nearly the same level as in the control ( Fig. 6 ).The changes in pH of medium during growth were as follows ; control (pH 4.4-2.7 ); 20 Krad (pH 4.4-2.6); 40 Krad (pH 4.4-2.6); 50 Krad (pH 4.4- 2.5 ); 100 Krad (pH 4.4-2.2 ); 200 Krad (pH 4.4-2,2 ). Vegetative growth of P.. oryzae was increased by about 30 percent when spores were exposed to 50 Krad and remained nearly the same up to 200 Krads. ( Fig 6 ). There was no stastistical difference ( p = 0.05, Student t-test ) between d r y matter obtained at 50, 100, or 200 Krad ( Fig 6, Tables 3-4 ). The best vegetative growth ( 28-30mg ) for T.vizide was obtained when 100-200 Krad was applied to the spores. University of Ghana http://ugspace.ug.edu.gh DRY WE IGH T OF MY CE LIU M 36 Fig. 6 Effect of gamma irradiation on vegetative growth of A. niger, 3. Oryzae. and T. viride for 5 days. University of Ghana http://ugspace.ug.edu.gh Table 3a Vegetative growth of A. niger in liquid medium at 28°C for 5days after exposure to indicated doses of gamma irradiation Organism Dose Aoolied ( Krad) teplicaies pH of Medium Initial Final Mean Dry Weight c>f Mycelium (mg Mean S.E. 1 4.4 2.7 13 2 4.4 2.8 16 0 2.8 15+1 2 3 4.4 2.8 16 4 4.4 2.8 16 1 4,4 2.0 8 2 4.4 2.8 720 2.7 70+0 .8 3 4.4 2.6 6 4 4.4 2.6 6 1 4.4 2.8 12 2 4.4 2.7 10 40 2.7 10011.2 3 4.4 2.7 9 4 4.4 2.7 9 A nigsr 1 4.4 2.5 15 2 4.4 2.5 14 50 2.5 15 t0 .8 3 4.4 2.5 14 4 4.4 2.6 16 1 4.4 2.2 15 2 4.4 2.2 13 100 2.2 14 .0*0 .7 3 4.4 2.1 14 4 4.4 2.2 14 1 4.4 2.4 13 2 4.4 2.0 15 200 3 4.4 2.3 2.2 12 13+1.2 4 4.4 2.2 12 University of Ghana http://ugspace.ug.edu.gh Table 3B Vegetative growth of T. viride in liquid medium at 28°C for 5 days after exposure to indicated doses of gamma irradiation (Data provided Fig 5) Organsm Dose Applied (Krad) Replicates pH of Medium Initial Final Mean Dry Weight of Mycelium (m? Mean ± S.E. 0 1 2 3 4 4.4 4.4 4.4 4.4 5.4 5.4 5.5 5.3 5.4 26 28 24 25 26.0±1,5 1 4,4 5.5 28 2 4.4 5.5 5.6 28 20 27.0+1.2 3 4.4 5.8 25 4 4.4 5.6 27 1 4,4 5.8 27 2 4,4 5,7 27 Mi 27.0+0.7 3 4,4 5.7 5.7 26 T. viride 4 4,4 5,7 28 1 4.4 6,4 27 2 4.4 6,3 29 50 28.0+0.7 3 4.4 6.4 28 4 4.4 6.5 6.4 28 1 4.4 6,3 29 2 4.4 6,4 29 100 29,0+0,4 3 4.4 6.2 29 4 4,4 6.3 6.3 28 1 4,4 6.3 32 2 4,4 6,3 30 200 30.0+1.2 3 4.4 6.1 6.2 29 4 4.4 6.2 20 University of Ghana http://ugspace.ug.edu.gh 39 Table 4 Vegetative growth of R, oryzae in liq u id medium at 28°C for 5 days after exposure to indicated doses of gamma irradiation (Data provided Fsg.5) Organism Dose Applied (Krad) Replicates pH of Medium Initial Final Mean Dry Weight of Mycelium (mgj Mean ± S.E, 0 1 2 3 4 4.4 4.4 4.4 4.4 4.3 4.4 4.4 4.4 4,4 13 12 17 16 1 I 0+2.0 20 1 2 3 4 4 4 4.4 4.4 4.4 4.1 43 4.2 4.2 4.2 16 19 15 16 1 p n-t-o.4 1 4,4 4,0 4.1 17 2 4.4 4,2 19 40 180+0fl 3 4,4 4 I 18 4 4.4 4.0 17 R oryzae I 4.4 4,0 4.0 16 2 4.4 3.9 n 50 17.0±0.4 3 4.4 4 I 17 4 4.4 4.0 17 1 4,4 4.0 4.0 17 2 4.4 4.1 17 100 1 a,o±i .2 3 4.4 4.0 20 4 4.4 4.0 ia 1 4.4 4,1 4.0 16 2 4,4 4.0 16200 17.0+1,3 3 4.4 3,9 10 4 4.4 4.0 19 University of Ghana http://ugspace.ug.edu.gh kO C. EFFECT OF GAMMA IRRADIATION OH GROWTH OF L. PLANT ARUM: Figure 7 and Table 5 summarise results obtained. The best growth of the bacterium was obtained with a dose of 50 Krad and thereafter further increases in irradiation dose applied { 100-200 Krad ) depressed vegetative growth by nearly 50 percent ( Fig. 7 ). The pH of the medium drifted from 1.4 to 5.5 in the control. There was therefore a significant difference between the bacterium and the fungi in relation to their response to irradiation. University of Ghana http://ugspace.ug.edu.gh Op tic al De nsi ty (66 0 nm ) DOSE (K rad) Fig. 7 Influence of gamma irradiation on vegetative growth of L. plantarum incubated in nutrient broth at 28 for 5 days. University of Ghana http://ugspace.ug.edu.gh k2 Table 5 Influence of gamma irradiation on growth of L. plantarum in nutrient broth at 28°C for 5 days. (Data provided Fig. 6) Dose Applied (Krad) Replicates pH of culture medium Initial Final Mean Optical Density at 660 nm. Mean ± S.E 1 7.4 5.5 0.08 2 7.4 5.4 0.10 o 5.5 0.10+0 01 3 7.4 5.6 0.12 4 7.4 5.5 0.10 1 7.4 5.1 0.12 2 7.4 5,1 0.12 20 5.2 0.12+0.00 3 7.4 5.4 0.12 4 7.4 52 0.12 1 7.4 4.7 0.15 2 4.7 0.16 40 7.4 4.7 0.1610.01 3 7.4 4.8 0.16 4 7.4 4.6 0.17 1 4.6 0.247,4 2 7 4 4.6 0.24 50 4.6 0.25+0.01 3 7.4 4.5 0.27 4 7.4 4.6 0.25 1 7.4 4.7 0.18 2 7,4 4.6 0.16 100 4.7 0.1710.01 3 7.4 4.8 0.17 4 7.4 48 0,17 1 7.4 4.8 01 4 2 7.4 48 0,12200 3 7.4 48 4.8 0.16 014+0 .03 4 7.4 4.8 0.15 University of Ghana http://ugspace.ug.edu.gh D. COMPARATIVE ACTIVITY OF AMYLASE IN GAMMA-IRRADIATED MICROORGANISMS: Gamma irradiation doses up to 50 Krads increased amylase activity ( measured as reduction in viscosity of starch ) of all the microorganisms, namely ,4. niger.ii. oryzae, 7. viride and L. plaritarvm. ( Fig. 8 ). There was significant decline in enzyme activity ( p < 0.05 ) when higher doses ( 100, 200 Krad ) were applied. ( Fig. 8 ). Although the unirradiated bacteria L plantarvik did not show any amylase activity, its enzyme production and activity markedly increased with increasing doses ( 20, 40, 50 Krad ) and thereafter declined. E. CELLULASE ACTIVITY OF GAMMA-IRRADIATED MI CROORGANI SMS': Optimal cellulase activity ( measured as reduction in viscosity of carboxymethylcellulose CMC ) of all the organisms varied depending on the dose applied prior to incubation of spores. Fig. 9 summarises the results obtained. A dose of 100 Krads was the optima] for obtaining b3 University of Ghana http://ugspace.ug.edu.gh Re du cti on in Vis co sit y l* DOSE (Krad) Fig. 8 Effect of gT.mma irradiation on amylase activity of indicated microorganisms. ( Note the decline in enzyme activity after 50 Krad). University of Ghana http://ugspace.ug.edu.gh Fig. 9 Effect of gamma irradiation on cellulase activity of indicated microorganisms. ( Note the variation in response to dose applied). University of Ghana http://ugspace.ug.edu.gh maximum cellulase activity in T. viride and R. oryzae. A dose of 200 Krads was clearly unsuitable as it brought about significant ( p < 0.05 ) decline in the ability of the culture filtrate in reducing the viscosity of CMC. ( Fig. 9 ). On the other hand 50 Krads was optimal for .-4. niger and L pi ant arum as the highest activity of cellulase enzyme was obtained at this dose ( Fig. 9 ). b6 University of Ghana http://ugspace.ug.edu.gh .F. PECTINASE ACTIVITY OF GAMMA-IRRADIATED MICROORGANISMS: Fig. 10 shows results obtained when pectinase activity of the same microorganisms was tested after gamma irradiation. A dose of 50 Krad about doubled the pectinase enzyme activity in T. viride and increased pectinase activity of A. niger by about 10 percent but marginally increased the same activy in R. oryzae and L. plantarum.. Clearly in this instance higher doses beyond 50 krad depressed pectinase activity. University of Ghana http://ugspace.ug.edu.gh DOSE (K rad) Fig. 10 Comparative pectinase activity of garims irradiated spores of indicated microorganisms after 5 days incubation. University of Ghana http://ugspace.ug.edu.gh G. pH CHANGES DURING GROWTH OF GAMMA-IRRADIATED NIGER, P. ORYZAE, 2\ VIRIDE AND L . FLANTARUM. : The pH of a medium influences enzyme activity. In this Section pH of the gamma-irradiated spores of ,4. niger, P.. oryzae, 7. viride and L. plantar urn during incubation was examined. Results are presented in Fig. 11. Changes in the acidity of the media varied from one microorganism to another. For example, the pH of medium in which Pi. oryzae and L. pi ant arum were cultured after gamma irradiation progressively drifted from pH 4.4 to pH 4.0 and from 5.5 to 4.5 respectively when dose applied increased from 0 to 50 Krad. Thereafter the pH of the medium remained constant or nearly so when dose applied was increased up to 200 Krad ( Fig. 11 ). On the other hand, the pH of the medium inoculated with 7. viride drifted from pH 5.4 to pH 6.4 with increasing dose up to 50 Krad ( Fig. 11 ), and thereafter declined only marginally. Culture filtrate of .4. niger became increasingly acidic as gamma doses applied to spores prior to incubation increased from 20 to 200 Krad. Thus whilst unirradiated spores of 4. niger produced culture filtrate with pH 2.8, spores exposed to 100 and 200 Krad prior to incubation produced metabolites with a pH of 2.2. ^9 University of Ghana http://ugspace.ug.edu.gh pH of Me diu m 50 Fir;. 11 Figure showing pH changes in irradiated spores of the indicated microorganisms after incubation for 5 days. University of Ghana http://ugspace.ug.edu.gh 51 In summary one can say that: 1. A dose of 50 Krad applied to dry spores of P.. and L. plantarum was optimal for maximum acid production in the culture medium used. 2. A dose of 50 Krad applied to spores of T. vii ids prior to incubation drifted pH of the medium to the neutral side ie from pH 5.4 to 6.4, 3. .4. nif&r acid production was enhanced considerably by gamma irradiation of 100 -200 Krad. University of Ghana http://ugspace.ug.edu.gh 52 H. EFFECT OF CHEMICAL TREATMENT, HEATING TEMPERATURE. AND DURATION OF TREAmENT ON DIGESTIBILITY OF LIGNOCELLWLOSE IN CORN HUSK FOR THE PRODUCTION OF CRUDE PROTEIN NITROGEN BY 7. VIRIDE. Results are presented in Tables 6A-D. Analysis of variance ( Table 7 ) showed that the structural resistance of lignocellulose in corn husk is significantly ( p = 0 . 0 1 ) circumvented by chemical treatment with either 1 percent or 5 percent of Sodium Hydroxide or Hydrochloric acid. Duncan’s Multiple Range Test ( Table 8 ) however showed that there was no significant difference between treatments with 1 percent Sodium Hydroxides 5 percent Sodiuia Hydroxide or 1 percent Hydrochloric acid, ii. PHYSICAL TREATMENT WITH HEAT: Duncan’s Multiple Range Test of the statistical analysis showed that temperature applied at ( 80°, 100°, 120° C ) during digestion treatment significantly affect the degrative process prior to fermentation by 7. >jirid* for lignocellulose conversion. The optimum tcmpsrature for digestion was 100°C which eventually yielded a product with crude protein content of 19.09 percent ( Table 9 ). University of Ghana http://ugspace.ug.edu.gh Influence of tem pera tu re and duration of heating on crude protein formation by non-irradiated T, viride on corn slurry after 5 days at 28°C. TABLE 6 A i lemical Treatment Temperature of Digestion Duration of Digestion % Crude Protein formed Mean ± S.E With Temp. (°C) (hns) on slurry 80 i 168 1%NaOH 80 60 80 1 1 1 17.0 18.6 172 174+07 80 2 18.6 1 % NaOH 80 80 2 2 178 189 183+02 80 2 17.9 60 3 183 so 3 186 !%NaO^ 80 3 17 9 1S7+06 80 3 19.9 1 GO 1 18.7 1%NaOH 100 100 1 1 18.4 18.8 18.6+0.1 100 1 18.5 100 2 17.9 1%NaO- 100 100 2 2 19.8 18 6 18 8 ± 1.2 100 2 18.9 100 3 189 ICC 100 3 3 196 195 189+0.8 100 3 176 120 1 199 120 12" 120 1 1 1 189 179 189 18 S±0 7 ^"in oC. 19 6 1 'a IfctCn 120 120 2 n«L 19.5 19.8 19 4+0 4 120 2 187 120 3 195 ■ % iJaOh 120 12C 3 3 19 8 16.9 10911.5 120 3 19.2 University of Ghana http://ugspace.ug.edu.gh Influence of temperature and duration of heating on crude protein formation by non-irradiated T. viride on corn slurry after 5 days at 28°C. hemica! Treatment Temperature of Digestion Duration of Digestion % Crude Protein formed Mean ± S.E. With Temp. (°C) (hrs) on slurry 60 ! 18.8 80 1 19.2 5% NaOH 18.8+0 480 1 18.2 80 1 190 80 2 18 7 80 2 19.4 5% NaOH 80 2 19.2 18.9 ±0.4 80 2 18.3 80 3 18.9 80 3 19.6 5% NaOH 19.0±0.480 3 18.9 90 3 18.6 100 1 194 100 1 19.0 5% NaOH 100 1 19.8 19.2±0.4 100 1 18.6 100 2 19.6 100 2 192 5% NaOH 19.410-1'100 2 193 — 100 2 19.5 100 3 196 100 3 199 5% NaOH 19.7+1.0100 3 19 4 ICO 3 20 0 " i 120 1 19 6 '■i'j 1 18 4 i :jNaOH 188+06120 i 192 120 1 180 < 'in 2 185 120 2 189=0- .1 rv 18.6+0.31 '22 2 180 , 120 2 182.... .. ' c J 2 182 . . 3'«> NaOh 120 3 18.4 18.0 ±0.3 1 120 3 17.9 3 175 University of Ghana http://ugspace.ug.edu.gh T A B L E 6 C Influence of temperature and duration of heating on crude protein formation by non-irradiated T. viride on corn slurry after 5 days at 28°C. University of Ghana http://ugspace.ug.edu.gh 56 TABLE 6D Influence of temperature and duration of heating on crude protcsn formation by nest'Irradiated T. viride on corn slurry after & days at 28°C. Chemical 1 reatment With 'I emperature ot Digestion Temp (“C ) Duration ot Digestion ( t e ) % Crude Piotein formed on corn slurry Mean ± S.E. 16.2+0.91% HCl 60 00 GO 80 1 1 1 1 16.2 16.0 101 16.2 1% MCI 00 00 80 80 2 2 2 2 16.6 16.8 16.4 17.0 167 ±0.2 no 3 1(i 6 1 % HCl 00 3 17.0 17. 110.3 80 3 1/.2 80 3 17 A (00 1 |» y 1%M(| 100 i i { C' 17,6 ±0.9 100 1 17.8 100 1 17.9 100 ?. 17.fi 100 2 1?.0 1 % HCl 17.8 ±0.3 100 2 I7.D 100 ? 17.7 100 3 17,9 1 % HCl 100 3 18.2 ie.i-H>.9 100 3 18.0 100 3 10.4 1?G 1 18.6 120 •I 1% HCl 1?0 1 18.7 18.710.2 120 1 ia.4 120 2 18.7 120 p i0.9 1 % HOI 1ft, 8+0.1120 a 19.0 120 2 18.8 120 3 18.9 120 3 19.1 19.1 ±0.1 120 3 19 2 120 3 190 University of Ghana http://ugspace.ug.edu.gh 57 TABLE 7. ANALYSIS OF VARIANCE TABLES 6 A-D. Degrees of Sum of Mean F Code Source Freedom Squares Square Value Prob 1 Rep 3 0.52 0.174 1.12 .342 2 A 3 63.91 21.304 136.84 .000 4 B 2 37.55 18.773 120.59 .000 6 AB 6 52. 73 8.789 56.45 .000 8 C 2 4.67 2.334 14.99 .000 10 AC 6 3.92 0.653 4.19 .000 12 BC 4 6.48 1.620 10.40 .000 14 ABC 12 3.49 0.291 1.87 .039 16 D 1 25.03 25.028 160.77 .000 18 AD 3 4.96 1.655 10.63 .000 20 BD 2 0.37 0.184 1.18 .308 22 ABD 6 2.39 0.398 2.56 .020 24 CD 2 0.01 0.005 0.03 26 ACD 6 0.42 0.070 0.45 28 BCD 4 0.22 0.055 0.35 30 ABCD 12 1.13 0.094 0.60 -31 Error 213 33.16 0.156 Coefficient of Variation = 2.10 X A = Chemical Treatment ( 1% NaOH, 5% NaOH, IX HC1, 5X HC1 ) B = Temperature of Digestion ( 80, 100 120 °C ) C = Duration of Digestion ( 1, 2, 3, hours ) D = Non-irradiated spores. AB, AC, BC, ABC, AD, BD, ABD, C D , ACD, BCD, ABCD are combination treatments. all University of Ghana http://ugspace.ug.edu.gh 58 Duncan’s Multiple Range Test of Chemical Treatment. TABLE 8 S- = 4.654747-02 at alpha = 0.01 x LSD value = .1710941 Original Order Mean 1 = 19.00 A (IX NaOH) Mean 2 = 19.13 A (5% NaOH) Mean 3 = 17.96 B (IX HC1 ) Mean 4 = 18 97 A (5X HC1 ) Ranked Order Mean 2 = 19.13 A Mean 1 = 19.00 A Mean 4 = 18.97 A Mean 3 = 17.96 B Means are the percent ( X ) Crude Protein produced. TABLE 9. Duncan’s Multiple Range Test of Temperature of Digestion S- = 4.031129E-02 at alpha = 0.01 x LSD value = 0.1481719 Original Order Ranked Order Mean 1 = 18.26 C ( 80°C ) Mean 2 = 19.09 A Mean 2 = 19.09 A ( 100°C ) Mean 3 = 18.94 B Mean 3 = 18.94 B ( 120°C ) Mean 1 = 18.26 C Figures with the same letters are not significantly different. University of Ghana http://ugspace.ug.edu.gh TABLE 10. Duncan’s Multiple Range Test- Duration of Digestion. S- = 4.Q31129E-02 at alpha = 0.01 x LSD value = .1481719 Original Order Ranked Order Mean 1 = 18.59 B (1 hour) Mean 3 = 18.88 A Mean 2 = 18.82 A (2 hour) Mean 2 = 18.82 A Mean 3 = 18.88 A (3 hour) Mean 1 = 18.59 B Means are the percent ( % ) Crude Protein produced. University of Ghana http://ugspace.ug.edu.gh 60 iii. DURATION OF DIGESTION OF CORN HUSK ON PROTEN NITROGEN FORMATION BY T. VIRIDE. There was no statistical difference between crude protein formed by T viride in corn husk slurry digested for 2 and 3 hours. ( 18.82 and 18.88 percent respectively ). Duncan’s Multiple Range Test ( Table 10 ) shows that digestion of corn husk for 1 hour prior to inoculation by T. viride resulted in a protein content of the fungus ( 18.59% ) which was inferior to what obtained after 2 and 3 hours of digestion. iv. COMBINED EFFECT OF CHEMICAL TREATMENT, HEATING TEMPERATURE AND DURATION OF DIGESTION OF CORN HUSK ON CRUDE PROTEIN PRODUCTION BY T. VIRIDE. Combination of the three parameters namely, chemical treatment, heating teperature and duration of corn husk showed variable effects ( Appendix 5 ). The best combination for optimal production of crude protein by unirradiated T. viride spores on digested corn husk was treatment with 5% NaOH solution at 100°C for 2 or 3 hours. In this instance nitrogen content of the product was between 19.4-19.7 percent. This was significantly ( p 5 0.05 ) different from the other treatment combinations ( Appendix 5 ), The treatment combination which gave the lowest yield of crude protein ( 16.2 percent Nitrogen ) was a digestion in 1 percent Hydrochloric acid solution at 80°C for 1 hour. University of Ghana http://ugspace.ug.edu.gh I. PRODUCTION OF FUNGAL PROTEIN BY GAMMA-IRRADIATED T. VIRIDE SPORES ON CORN HUSK SLURRY COMBINED-TREATED WITH HEAT AND CHEMICALS. The experiments in Section H were repeated. This time, spores of T viride were exposed to 100 Krad of gamma radiation prior to inoculation of the combined-treated corn husk slurry. Results obtained are presented in Tables 11A-D. Analysis of variance of the data ( Table 7 ) showed that irradiation significantly ( p £ 0.05 ) increased crude protein ( X Nitrogen ) formation by T. viride on corn husk slurry. Crude protein ( Nitrogen Content ) of the resultant culture from irradiated spores had a mean nitrogen content of 20.0 percent. University of Ghana http://ugspace.ug.edu.gh TABLE 11 A. Influence of temperature and duration of heating on crude protein formation by irradiated spores T. viride on corn slurry after 5 days at 28°C. Chemical Treatment Temperature of Digestion Duration of Digestion % Crude Protein formed Mean±S.E. With CQ) (hra) on slurry 80 1 18.4 80 1 18.4 IttNtOH 18J10.2 80 1 18.3 80 1 18.9 80 2 18.8 1%N*OH 80 2 1«.3 18.811.0 80 2 18.7 80 2 18.9 80 3 18.0 WNtOH 80 3 18.0 18.2x0.3 80 3 18.1 80 3 18.7 100 1 18.8 100 1 18.8 1%NlOH 18.8*0.2 100 1 18.3 100 1 18.7 100 2 18.8 100 2 20.0 1«NtOH 18.8±0.1 100 2 18.7 100 2 18.8 100 3 18.8 100 3 202 1%N»OH 18.810.2 100 3 18.8 100 3 18.8 120 1 18.8 120 1 m 1«N&OH 18.210.4 120 1 18.4 120 1 18.8 120 2 20 i 12a 2 19.81%N«OH 18.810.4 120 2 18.4 120 2 18.2 120 3 18.6 120 3 18.0 1%N«OH 18.4lQ.ii120 3 ia.a 120 3 20.0 University of Ghana http://ugspace.ug.edu.gh TABLE 11B. Influence of temperature and duration of heating on crude protein formation by irradiated spores T. viride on corn slurry after 5 days at 286C. University of Ghana http://ugspace.ug.edu.gh 6k TABLE 11C. Influence of temperature and duration of heating on crude protein formation by irradiated spores T. viride on corn slurry after 5 days at 28°C. Chamlcal Treatment Temperature of Dlgeetlon Duration of Dlgeetlon % Crude Protein formed MeantS.E. With c o (hra) on alurry 60 1 16.8 60 1 16.6 1*HCt 16.710.1 60 1 16.7 80 1 16.5 «a 2 16.6 60 2 16.9 WHO 16.8*0.1 60 2 16.6 60 2 16J 60 a 17.4 60 a n .a 17.510.1 1%HCI 60 a 17.5 60 3 17.6 100 1 175 100 1 17,6 1*HCI 100 1 17.6 17.610.1 100 1 17.6 100 2 16.2 100 2 16.4 1HHCI 16.310.1 100 2 16.2 100 2 16.3 100 3 16.6 100 a 16.6 1*HB 16.610.1 100 a 16.7 100 a 16.6 120 1 16.9 120 1 16,8 18.810.1 1%HO 120 1 161 120 1 16.6 120 2 18,3 120 2 18.8 1HHCI 18.110.1 120 2 18.1 120 2 1 8 i 120 3 18.3 1HHCI 120 3 18.4 18.510.1 120 3 18.5 120 a 18.6 University of Ghana http://ugspace.ug.edu.gh TABLE 11D. Influence of temperature and duration of heating on crude protein formation by irradiated spores T. viride on corn slurry after 5 days at 28°C. Chemical Treatment Temperature of Digestion Duration of Digestion % Crude Protein formed MeantS.E. With CO (h rs ) on slurry 80 1 ia,9 ao 1 m 5*NiOH ia .9±o .i ao 1 ia.7 so 1 18.9 ao 2 18.9 m uoH so 2 18.1 19.0*0.1 ao 2 19.1 ao 2 19.0 ao a 19.3 m uoH ao 3 19.2 19.2*0.1 ao 3 19.3 80 a 19.1 100 1 19.6 100 1 19.7 5%NlDH 19.7*0.1 100 1 19.6 100 1 19.7 100 i 19.8 100 2 19.8 !%NiOH 19.8* 0.1 100 2 19.7 100 2 i9.a 100 3 19.8 100 3 19.9 S%N*OH 19.8*0.1 100 3 19.7 100 3 19.8 120 1 19.8 120 1 19.8 SttNtOH 19.7*0.1 120 1 19.7 120 1 19.8 120 2 19.3 120 2 19.6 5%N«OH 19.610.1 120 2 19.7 120 2 19.6 120 3 19.6 120 3 19.4l«N*OH is.5±a.i120 3 19.S 120 3 19.3 University of Ghana http://ugspace.ug.edu.gh 66 J. EFFECT OF GAMMA IRRADIATION ON THE MORPHOLOGY AND SPORULATION OF A. NIGER AND T. VIRIDE. Gamma irradiation did not cause any significant changes in the morphology and sporulation of T. viride. In T. viride as much as the conidiophores remain irregularly branched, the bright green colouor development decreased progressively with incresing irradiation dose ( eg. at 200 Krad there was no colour development after 8 day of incubation ). Plate 3. University of Ghana http://ugspace.ug.edu.gh 67 Plate 3, A close up of 8 day old gamma irradiated T. viride grown on V-8 agar. ( Note the decrease in green colour formation with increasing irradiation) University of Ghana http://ugspace.ug.edu.gh 68 GENERAL DISCUSSION The pertinent literature is replete with information on increasing interest in the conversion of agricultural waste, lignocellulose, effluent from chemical industries etc. by microbiological process into food and animal feed ( Imrie, 1973; Schellart, 1975; Beldman et al. 1985 ). One of the resulting products, the microbial or fungal biomass itself can be used as a protein source in animal and human nutrition. It is reported that 2.3 billion metric tonnes of cereal straw, 560 million metric tonnes of leguminous crop residue and 234 million metric tonnes of sugar cane bagasse are produced throughout the year ( Anon, 1988 ). These "waste" materials can be used as substrates for microbial protein production. In Section A data collected showed that Ghana’s annual maize production between 1984 and 1990 exceeded 500,000 metric tonnes except in 1985 when it declined to 395,000 metric tonnes ( Fig. 2 ). The bulk of the production was contributed by Ashanti, Brong Ahafo, Eastern and Northern Regions ( Fig. 3 ). During the harvest season, maize plant debris is left to decompose in the field as stubble. After removal of the cob, the corn husk is used as wrapping in the kenkey industry; some are discarded. The kenkey consumer normally discards the husk after eating. In economic terms, money is being poured down the drain because maize husk from harvested maize cost 24.0 - 31.6 million cedis between 1987 - 1990. ( Fig. 5 ). Each region has maize husk stock commensurate with its annual maize production figures ( University of Ghana http://ugspace.ug.edu.gh Therefore, as a raw material for fungal protein production, maize husk has potential for sustained feedstuff production. In many developed countries, cellulose and lignocellulose in forestry and, agricultural waste is hydrolysed to prepare substrates for production of food and feedstuff ( Bunker, 1963; Hospova, 1966 ) using microorganisms. Bacteria, yeast and fungi have been investigated. Bacteria employed include: Bacillus, Hydrogenomonas, Methcmonas, Methylmonas, Lactobacillxis; yeasts: Candida, Rhodoturola, Sacch.arom.yces; Filamentous fungi: Aspergi I lust Fusarium, Trichoderma, Rhizopxis, Chaetomivm., Penicillxtm. . ( Kihlberg, 1972 ). The microorganisms of choice should satisfy the following conditions: (a) Optimum utilization of waste components and simple nutritional demands; any additive required may influence the process costs adversely. (b) High specific growth rate, and "competitive ability". This makes a non-sterile process possible. (c) Simple and cheap separation. (d) Non-toxic; safety in animal and human diet. In the studies reported in this thesis, Aspergi I lus niger was chosen because Pringsheim and Lichtenstein ( 1920 ) reported the feeding of animals with this fungus and Aspergillus fv.migat'us, grown on straw supplemented with inorganic fertilizer during World War 1. During the World War 11, several industrially produced oy University of Ghana http://ugspace.ug.edu.gh 70 fungi ( Fxisariwn, Rhizopus, Candida J were reported to have been incorporated into human diets with satisfactory results ( Thatcher, 1954 ). T. viride is known to produce antibiotics and mycotoxins ( Weindling and Emerson, 1936; Wright, 1956; Ooka et. al. 1966; Meyer, 1966; Pyke and Dietz, 1966 ) but one has to keep in mind that the production of such substances is dependent on medium composition and conditions of growth, whereas many of these substances may be destroyed by special treatments. Such feeding trials by T. viride gave satisfactory results ( Church et. al. , 1972; Peitersen, 1975 ). On the whole T. viride seem to possess favourable properties for use in bioconversion of agricultural waste into animal feedstuff. Good vegetative growth is invariably attended by high enzyme activity ( Mandel and Reese, 1957 ). Cellulolytic, pectinolytic and amylolytic enzymes are produced during vegetative growth by a large number of microorganisms. In Section B gamma irradiation was used as a mutagen to ascertain its efficacy in improving vegetative growth of the test organisms, namely: A. niger, R. oryzae, T. viride and L. plantarvm. Results obtained showed that effect of low gamma irradiation on vegetative growth is variable. A dose of 20 Krad decreased vegetative growth of A. niger by 46.6 percent and thereafter further increase of radiation dose up to 50 Krad increased dry natter accumulation to the same level as in the control. The general mutational effect of irradiation evidently causes damage to genetic and biochemical University of Ghana http://ugspace.ug.edu.gh mechanisms, creating impairement of function and biological demands where non existed before ( Ingram and Farkas, 1977 ). A dose of 20 Krad applied to A. niger spores presumably led to partial damage of DNA helix that altered biochemical synthetic pathways prerequisite for normal growth. However further doses up to 50 Krad was enough to unfurl the DNA helix and reform new strands to restore vegetative growth. Differences in radiosensitivity of fungal spores is well known and the best vegetative growth of T. viride and R. oryzae obtained with a dose of 100 Krad ( Fig. 1 ) can be explained on the basis of mutations resulting from exposure to radiation. Indeed, Sadi et. al. ( 1983 ) stated that radiation can increase radial growth of microorganisms by up to 50 percent. However, above a certain threshold ( varying from one species to another ) irradiation may destroy genetic material and decrease germination capacity and metabolism of the spores ( Siagian, 1983 ). This partly explains why the bacterium L. plantarwn declined in vegetative growth beyond a dose of 50 Krad ( Fig 2 ). Gamma irradiation doses up to 50 Krad ( 0.5 KGy. ) improved or caused a significant { p < 0.05 ) increase in amylase activity of A. niger , R. oryzae , T. viride and L. plantarum ( Fig. 3 ). Many Phycoraycetes ( Phycomacotina ) and Ascomycetes ( Ascomycotina ) can convert starch to sugar. Amylase activity of some Lactobacillus have been shown in fermentation of vegetative waste ( Sibir et. al. 1984 ). Rhizopus, and especially Aspergillus 71 University of Ghana http://ugspace.ug.edu.gh 72 have found practical applications in using stareh for amylase, pectinase and biomass ^poductiana ( Moo^Yotfli^ et. al. 1983.). In this-thesis, low gamma irradiation dose of 50 Krad enhanced amylase production. This is the first record of induced enzyme activity of A. niger, R. oryzae, T. viride and L. plantarxun following gamma irradiation. Although many microorganisms produce cellulases, very few can break down lignocellulose and use it efficiently for biomass or other end product formation. The best cellulose converters include Trichoderma reesei, T. lignorvm, T. koningii, T. viride, Ch.aetom.iv.rn. cellulolyticxm. and Sporotrichxan pulverulentxm. ( Moo-young et. al. 1983 ). In recent times, much attention has been paid to the enzymatic hydrolysis of cellulose because it is a renewable carbon source and available in large quantities. Some microorganisms produce a multicomponent cellulase enzyme system, including the 1,4-/?-D-glucan glucanohydrolase ( endoglucanase ), l-4-/?-D-glucan cellobiose hydrolase ( exoglucanase ) and /?-D-glucoside glucohydrolase f?-glucosidase. T. viride produces six endoglucanases ( Endo 1; 11; 111; IV; V; VI ), three exoglucanases ( Exo 1; 11; and 111 ) and a /?-glucosidase ( /?-gluc. 1 ). A conbination of these three types of enzymes is necessary for the complete hydrolysis of crystalline cellulose ( Beldman et. al. 1985 ). Endoglucanase and exoglucanase are known to act synergistically in cellulose hydrolysis, while /?-glucosidase is needed for removal of cellobiose ( a strong University of Ghana http://ugspace.ug.edu.gh inhibitor of both endoglucanase and exoglucanase ). Although the endoglucanases and exoglucanases of gamma-irradiated spores of T. viride and R. oryzae were not determined in Section E f it is conjected that gamma irradiation at 100 Krad was presumably optimal in inducing the synergistic action in cellulose hydrolysis. Optimal cellulolytic activity of A. niger and L. plantarwri was however obtained at a lower dose of 50 krad ( Fig. 9 ) indicating differences in radiosensitivity of fungal spores used for same purpose. Beldman et. al. ( 1985 ) stated that the chemical composition of plant cell wall necessitates a combination of cellulase, pectinase and hemicellulase enzyme action for efficient utilization of biomass. Results from Section F show that pectinase activity of A. niger, R. oryzae T. viride and L. plantarvm was variably increased by a dose of 50 Krad and that dosebeyond 50 Krad depressed pectinase activity. Thus the overall effect of gamma irradiation on enzyme production by the test microorganisms can be summarised as follows: 73 TABLE 12. Summary of dose requirements for optimal inducement of indicated physiological action Type of Dose (Krad) activity A. nieer requirement T. viride for optimal R.oryzae improvement L.plantarum Vegetative Growth 50 100 100 50 Amylase production 50 50 50 50 Cellulase " 50 100 100 50 Ppr.tinase " 50 50 50 50 University of Ghana http://ugspace.ug.edu.gh ?k Generallyt a dose which enhanced vegetative growth of the microorganism was also optimal for cellulase activity. However the same dose ( 50 Krad ) was optimal for inducement of amylase and pectinase activity in all the test microorganisms In Section G, gamma irradiation of up to 200 Krad gave a linear increase in acid production by A. niger ( Fig. 11 ). This fungus is used in commercial production of some acids including citric acid, oxalic acid and D-gluconic acid. It will be interesting to quantify, in future, which organic acids are formed in abundance by gamma-irradiated A. niger spores. Fumaric and lactic acids are produced by Rhizopus orygite ( Blain,^,t975 ). The culfctrre medium containing R. ofyzde treated with a dose of > 50 Krad became more acidic ( pH 4.5 - 3.8 ) presumably indicating accumulation of acids. Future studies will aim at identifying the types of acids formed by this fungus after gamma irradiation treatment. The nature of the substrate ( substrate composition ) may also influence acid production. In subsequent experiments the medium used in culturing the microorganism will be varied to find out their effect on the quantity and type of acids formed after gamma irradiation of the spores. Cellulase activity in T. viride is kwown to be optimal between pH 5.0 - 6.5. This corresponds to the pH obtained by spores exposed to 100 Krad of ganuna irradiation. Some fungi are sources of industrial production of enzymes ( Table 2 ). These include species used in these investigations namely A. niger ( o( amylase, amyloglucosidase, University of Ghana http://ugspace.ug.edu.gh 75 pectic enzymes, proteases, glucose oxidase, naringinase ) Rhisopus oryzae ( Amyloglucosidase, lipase, rennet proteases, pectic enzymes ), Trichoderma. viride ( Cellulose ). Results from this study show that gamma irradiation could improve yield of these enzymes. This information provides a springboard for future studies on quantities and qualities of enzymes produced after irradiation treatment. Structural resistance of cellulose and hemicellulose closely associated with lignin in the plant cell wall makes ce llul osic materials generally non-nutritive and difficult to digest. For an increase in nutritive value, the material must first be converted into monomers or oligomers ( Kirk, 1982 ). This is achieved by pre-treatment hydrolysis process using either chemical or enzymes or both ( Dunlap et. al. 1976 ). In Section H of this thesis , hydrolysis of corn husk into a slurry was achieved by using either sodium hydroxide ( IX and 5% ) or Hydrochloric acid ( IX and 5X ), heating at 80°, 100° and 120° C for 1 - 3 hours ( Tables 6 - 1 1 ). The best treatment o combination was heating at 100 C for at least 1 hour in either 1%, 5% NaOH or 1% HC1. Alkaline treatment of cellulosic material is probably the oldest and best kwown method of enhancement of microbial degradation of cellulose. The effects of the alkaline treatment are multiple. In general, the application of alkaline results in removal of lignin, an increase in surface area by swelling, and an alteration of crystalline and amorphous University of Ghana http://ugspace.ug.edu.gh 76 structure. The effectiveness of this treatment was demonstrated with an increase in enzymatic digestibility ( Detroy et. al. 1980 ) Acid treatment also decreased lignin content of variuos woods ( More et. al. 1972 ) and pre-treatment of cellulosic waste by steam at 100-220° C for 15 min to 2 hours provides a high yield of soluble xylo-oligomers and xylose and good accessibility of cellulose for microbial and enzymatic attack ( Buckholz et. al. 1981 ). Gamma irradiation ( 100 Erad ) applied to T. viride spores prior to inoculation of hydrolysed corn slurry improved crude protein formation ( % N x 6.25 ). Crude protein content of the resultant culture from irradiated local isolate of T. viride spore was 20.0 percent. Rosenberg et. al. 1978 showed that total crude protein of cultures of T. viride QM6 on hydrolysed oathull a was 15.4 %. ( Tables 12 - 13 ). Rhodes et. al. ( 1961 ) and Gray et. al. ( 1964 ) screened 175 species of Imperfect fungi and determined the crude and extratable protein produced in shake flasks after 4 days. They found the highest crude protein ( 9 % N ) in T. viride. Irradiation of T. viride spores increased considerably crude protein of cultures on hydrolysed maize husk slurry from 9 % to 20 %. (ie by 122 percent). Several wastes have been proposed in the pertinent literature as substrate for production of fungal protein such as molasses ( Gray and Abou-El—Seoud, 1966 ), soya beans whey ( University of Ghana http://ugspace.ug.edu.gh I ( Church et. a.1. 1972 ), corn waste ( Church et. al. 1972 ), waste from the coffee and rum distilling industries ( Updegraff et. ctl. 1973 ) etc. Results from this theses show that at least under laboratory conditions, the use of corn husk slurry to produce fungal protein food for animal feed is feasible. However, a number of other ancillary studies are required before practical application. Modern fermentations require that the industrial scale of the process takes place in a fermenter which provides an environment suitable for the growth of a pure culture on a substrate free from contamination and under controlled conditions. The design must incorporate a device for mixing the contents and air supply for aerobic processes, probes to monitor the enviroment and regulators to control it. There must also be provision for inoculation and sampling, as well as for charging and discharging the vessel. In continuous culture, ( as compared to the batch culture ), it is necesssary to monitor and control the flow rate of the medium as well as the culture volume and mass. These should be considered in designing an appropriate fermentor for fermentation of corn husk slurry by irradiated spores of T. viride. A cost-benefit analysis for the process is also important to justify any commercial application of the technique. Only a few studies have reported the nutritional value of fungal protein for animals ( Litchfield, 1968 ). Conclusions as to the value of fungal mycelium produced by T viride on corn husk University of Ghana http://ugspace.ug.edu.gh 78 slurry as food or feed, can only be drawn after extensive feeding studies with both domestic animals and pigs, to establish nutritional value and safety. Time limitation did not allow such studies to be carried out. However it is anticipated that future studies will examine the above-mentioned suggestions. Many morphological as well as physiological characteristics of fungi are generated by enviromental conditions. For example, Waterhouse ( 1974 ) reported that the size of Phylophthora palmivora ( Butl ) Butl sporangia vary widely according to medium, host, age of culture, moisture and light. The way in which each influenced the sporangia was, however, not stated. Nyerges-Rogrun ( 1975 ) showed that there are morphological changes of the conidial head of Penicillivm purpurogenxm following gamma irradiation. In the concluding Section of this thesis, the effect of gamma irradiation on sporulation and morphology of T. viride was examined. There was no significant change in the morphology. The development of colour was delayed for 8 days when 200 Krad was applied prior to culturing. Presumably the genetic coding for colour formation is somehow impaired at this dose. The physiological basis for this cannot be established by present data. What is interesting is the colourless mutant of T. viride produced by 200 Krad of gamma irradiation. Future studies will elucidate the significance of the colourless mutant and its implication in the ecological niche of the fungus. University of Ghana http://ugspace.ug.edu.gh 79 SUMMARY 1. Microorganisms namely, Aspergillus niger, Rhizopus oryzae, Trichoderma viride and L. plantarum have been used to study the effect of gamma irradiation doses on enzyme activity ( amylase, cellulase, pectinase ) and on vegetative growth in liquid culture. 2. A dose of 20 Krad decreased vegetative growth of A. niger by 46.6 percent; further increases up to 50 Krad increased dry matter accumulation of the fungus. 3. Vegetative growth of R. oryzae was increased by about 30 percent by 50 Krad and remained nearly the same up to 200 Krad. 4. There was no statistical difference ( p i 0.05, Student’s t-test ) between dry weight of mycelium of R. oryzae obtained in culture medium containing mycelium exposed to 50, 100 and 200 Krad of gamma irradiation. 5. The best vegetative growth of T. viride ( 28-30 ) was obtained in flasks containing spores irradiated with 100 or 200 Krad prior to incubation. 6 . The best vegetative growth of L. plantarxan was obtained in flasks containing vegetative cells exposed to 50 Krad prior to incubation at 28°C for 5 days. 7. Optimal cellulase, amylase and pectinase activity was induced by 50 Krad of gamma irradiation in A. niger and L. plantarwn. 8 . Optimal cellulase activity in T. viride and R. oryzae was induced by a dose of 100 Krad whilst 50 Krad was optimal for maximum production of amylase and pectinase enzyme. University of Ghana http://ugspace.ug.edu.gh 80 9. Gamma irradiation dose that induced optimal vegetative growth was also attended by optimal cellulase activity by the fungi. 10. pH of the medium containing T. viride treated with 100 Krad of gamma irradiation was between pH 5.0 - pH 6.5 corresponding to the best pH for cellulase activity in the fungus. 11. Gamma irradiation doses up to 200 Krad linearly increase acid production by A. niger and shifted pH of medium from initial 2.9 to final pH of 2.2. 12. Culture medium containing gamma-irradiated ( 50 Krad ) R. oryzae sporangiophores also became more acidic ( pH 4.5 - 3.8 ) presumably indicating accumulation of acids. 13. Hydrolysis of corn husk for use in solid-substrate fermentation by T. viride was achieved by using either 1 . o o percent or 5 percent NaOH or HCl and heating at 80 , 100 or 120°C for 1-3 hours. 14. The best treatment for corn husk hydrolysis prior to enzyme o digestion of cellulose was heating at 100 C for at least 1 hr. in either 1 percent NaOH, 5 percent NaOH or 1 percent HCl. 15. The treatment gave good accessibility of cellulose for microbial enzyme attack leading to high crude protein formation by T. viride on the maize husk slurry. 16. Irradiation of T. viride spores prior to inoculation of the solid substrate produced crude fungal protein which was 2 0 . 0 percent higher than that formed by the unirradiated T. viride spores a highly significant difference. University of Ghana http://ugspace.ug.edu.gh 81 17- National maize production figures between 1981 to 1990 show a shortfall in 1983 ( 395,000 metric tonnes ) as compared to the remaining years ( in excess of 500,000 metric tonnes ). Regional contribution to the national maize production showed that Ashanti, Brong Ahafo, Eastern and Northern Regions contributed the bulk of the national production figures between 1981 to 1990. 18. Amount of maize husk available after removing the cobs was commensurate with the total maize harvest for each region and cost about 23.0 - 32.0 million cedis. 19. As a raw material substrate for fungal protein production by T vil'ide , maize husks have a potential for sustainable use. University of Ghana http://ugspace.ug.edu.gh 82 ACKNOWLEDGEMENT I am Indebted to Dr. George T. Odamiten, my supervisor, for his guidance, patience and assistance during the preparation of this thesis. I express my sincere appreciation to the Ghana Atomic Energy Commission (GAEC) for Its support during my course. Finally, I am grateful to my numerous colleagues at GAEC for making me "computer literate" In the preparation of this thesis. University of Ghana http://ugspace.ug.edu.gh O j APPENDIX 1. --------------- 11 * i x Ltu_i m .. (FTGTTRKS 1 JLV/ji_a_L3— v ^ y * ------- —-— IN THOUSANDS METRIC TONNES) - YEAR PRODUCTION 1981 334.3 1982 284.3 1983 140.8 1984 574.0 1985 395.0 1986 559.1 1987 587. 7 1988 600.0 1989 715.0 1990 553.0 Source:PPMED (Statistics Division) Min. Of Agric. MAY. 1991. University of Ghana http://ugspace.ug.edu.gh APPENDIX 2. MAIZE PRODUCTION BY REGION (THOUSANDS OF METRIC TONNES). REGION 1987 1988 1989 1990 WESTERN 43.5 36.5 49.0 49.7 CENTRAL 60.3 67.0 105.4 42.2 G T .ACCRA 9.2 15.7 25.2 14.3 EASTERN 97.2 70.1 139.5 99.4 VOLTA 53.4 33.6 55.7 21.6 ASHANTI 83.1 106.6 83.0 108.3 B .AHAFO 122. 7 94.6 135.6 86.5 NORTHERN 102.1 134.6 87.0 106.7 U .EAST 3.0 37.9 2.7 0.9 U .WEST 23.2 3.4 31. 5 23.1 Source:PPMED (Statistics Division) Min. of Agric. MAY. 1991. University of Ghana http://ugspace.ug.edu.gh MAIZE HUSK PRODUCTION ESTIMATES BY REGION IN THOUSAND METRIC TONNE APPENDIX 3. REGION_________ 1987_________ 1988_________ 1989_________ 1990 WESTERN 6.5 5.5 7.4 7.5 CENTRAL 9.0 1 0 . 1 15.8 6.3 G T .ACCRA 1 . 4 2.4 3.8 2 . 1 EASTERN 14.6 10.5 2 1 . 0 14.9 VOLTA 8 . 0 5.0 8.4 3.2 ASHANTI 12.5 16.0 12.5 16 . 2 B .AHAFO 18 . 4 14.2 20.3 13.0 NORTHERN 15.3 2 0 . 2 13 .1 16.0 U .EAST 0.5 5.7 0.4 0 . 1 U .WEST 3.5 0.5 4.7 3.5 Source: PPMED (Statistics Division). Min. of Agric. MAY 1991. University of Ghana http://ugspace.ug.edu.gh 86 ESTIMATED ANNUAL COST OF MAIZE HUSK PRODUCED IN GHANA 1987-90. (CALCULATION BASED ON HUSK BEING 15% OF MAIZE PRODUCED AND AVERAGE ANNUAL COST OF HUSK BEING 290 CEDIS PER KILO.) APPENDIX 4. YEAR TONNAGE (METRIC) 1987 89.7 1988 90.1 1989 107.4 1990 82.8 COST (MILLIONS OF CEDIS) 26.0 26.1 31.2 24.0 University of Ghana http://ugspace.ug.edu.gh Duncan's M u ltip le J-ange Test com bination Of T reatm en t,H eating Temperature And Duration Of D ig es tion . APPENDIX 5. S - Cl. 13P6424 at a lpha - 0 .0 1 LSD v a lu e = O. 5132B24 Original Order Ranked Order Mean 1 17. P5 KL lKNaOH, 80°C,, lh r ; Mean 15 A IP . 86 Mean 2 IB. <51 FOHI l«N aO H , BO C, 2hr ; Mean 14 AB IP . 65 Mean 3 - IB. P4 CDEFG l«N aO H , 80°C , 3hr ; Mean 33 ABC IP . 51 Mean 4 IP . i o BCDEFG lXNaQH ,100°C , lh r ; Mean 8 ABC IP . 50 Mean 5 - IP . 30 ABCDE HKNa0H,100°C, 2hr ; Mean 32 ABC IP . 4P Mean <5 IP . 40 ABCD IMNaOH.lOO C,, 3 hr ; Mean 13 ABCD IP . 40 Mean 7 = IP . 05 BCDEFO lKNa0H ,120°C , lh r ; Mean 6 ABCD IP . 40 Mean 8 = IP . 50 ABC lKNa0H ,120°C , 2hr ; Mean 12 ABCD IP . 38 Mean p IP . 15 BCDEF l«N a0H ,1 20 C, 3hr ; Mean 5 ABCDE IP . 30 MeanlO IP . OO CDEFO SKNaOH, 80 C,, lh r ; Mean 27 BCDE IP . 25 M ean ll IP . 20 BCDEF 5KNaOH, BO°C, 2hr ; Mean 31 BCDE IP . 25 MeanlZ = IP . 36 ABCD 5*NaOH , 80 C, 3hr ; Mean 11 BCDEF IP . 20 Meanl3 IP . 40 ABCD 5KNaoh,100 C, lh r ; Mean 34 BCDEF IP . 16 Meanl4 = IP . 65 AB 5%NaDH,100 C,, 2 hr ; Mean 9> BCDEF IP . 15 Meanl5 - IP . 86 A 5KNaOH,100°C,, 3hr ; Mean 4 BCDEFO IP . IO MeanlcS = 18. PO CDEFC3H 5KNaOH,120 C, lh r ; Mean 7 BCDEFO IP . 05 Meant? = 18. 85 DEFOHI 5%NaOH,120 C, 2hr ; Mean :IO CDEFO IP . OO MeanlB = 17. PO KL 5KNa0H,120 C, 3hr ; Mean 26 CDEFO 18. PP M ean lP = 1(S. 44 O IKHC l, 80 C, lh r ; Mean 3 CDEFO IB. P4 Mean20 = 16. 77 NO lKHC l, 80 C, 2hr ; Mean 35 CDEFO 18. P4 MeanZl 17. 25 MN l*H C l, 80 C, 3hr ; Mean 16 CDEFGH IB. PO Mean22 = 17. 75 LM lXHC l, lOO C, lh r ; Mean 17 DEFOHI 18. 85 Mean23 = 18. 05 JKL lKHC l, lOO C, 2hr ; Mean 36 DEFOHI IB. 83 Mean24 18. 33 HIJK 1KHCL, lOO C, 3hr ; Mean 25 !DEFOHI 18. 7P Mean25 = 18. 7P DEFOHI lKHCl, 120 C, lh r ; Mean 30 EFOHI 18. 75 Mean26 = 18. PP CDEFO lHHCl, 120 C, 2hr ; Mean 2 FOHI IB. 61 Mean27 = IP . 25 BCDE lKHC l, 120 C, 3hr ; Mean ;2P OHIJ IB. 54 Mean2B = 18. 27 IJK L 5KHCI, BO C, lh r ; Mean ;24 H IJK 18. 33 Mean2P = 18. 54 OHIJ 5 «H C l, BO C, 2hr ; Mean 28 IJK L IB. 27 MeanSO - 18. 75 EFOHI SKHCl, 80 C 3hr ; Mean 23 JKL 18. 05 Mean31 = IP . 25 BCDE SJtfHCl, lOO C lh r ; Mean 1 KL 17. P5 Mean32 IP . 4P ABC 59NaOH,100°C,lhr,*; Mean 24= BCD IP . 75 Mean 8= IP . <50 DE o 196Na0H,100 C ,lh r,# ; Mean 2P= CD IP . 73 Mean P= 18. 80 MNO o lft>Na0H,100 C,2hr,*; Mean <52= CD IP . 70 Mean 10= IP . 80 BC ol&Na0H ,100 C,2hr,#; Mean <58= CD IP . <57 Mean 11= 18. PO JKLM o 19tNaOH,100 C,3hr,*; Mean 1Na0H,120 C,3hr,#; Mean 15= F IP . 40 Mean 19= 18. 80 MNO o 59«NaOH, 80 C .lh r,* ; Mean 27= F IP . 40 Mean 20= IP . 20 OH o , 5SKNaOH, 80 C ,lh r,# ; Mean 18= F IP . 40 Mean 21= 18. PO JKLM o 5*SNaOH, 80 C ,2hr,*; Mean <55= a IP . 23 Mean 22= IP . 50 EF o 596NaOH, 80 C,2hr,#; Mean <50= a IP . 23 Mean 23= IP . OO IJK L o SKNaOH, 80 C ,3hr,»; Mean 25= OH IP . 20 Mean 24= IP . 75 BCD SKNaOH, BO°C,3hr,#; Mean 14= OH IP . 20 Mean 25= IP . 20 GH 596NaOH, 8 0 °C ,lh r ,* ; Mean <5= OH[ IP . 20 Mean 2<5= IP . <50 DE 5S*Na0H,100°C,lhr,#; Mean 20= OH IP . 20 Mean 27= IP . 40 F 5%Na0H,100°C,2hr,*; Mean <53= OH IP . 20 Mean 28= IP . PO AB oSXNaOH.lOO C,2hr,#; Mean 52= GHI IP . 13 s University of Ghana http://ugspace.ug.edu.gh APPENDIX 6 Ccont’cD Mean 2P= IP . 73 CD o 5 «N a 0 H ,1 0 0 C ,3h r,* ; M ean 34= GHI IP . IO M ean 30= 20. OO A o 55tfNaOH,lQO C ,3hr,# ; M ean 53= H IJ IP . 0 5 M ean 31= IB. 80 MNO o 5%NaOH,12G C ,±h r,* ; M ean 58= IJ K IP . 02 M ean 32= IP . OO IJ K L o , 596Na0H,120 C ,lh r ,# ; M ean 32= IJ K L IP . OO Mean 33= 18. 60 PQR o 5 «N aO H ,120 C ,2h r,* ; M ean 23= IJ K L IP . OO -Mean 34= IP . i o GHI - -• >0 * - 596Na0H,120 C ,2hr,# ; M ean 4= JKLM 18. P2 Mean 35= 18. OO V o 596NaOH,120 C ,3h r,* ; M ean 21= JKLM 18. PO Mean 36= 17. 80 V o 5%NaOH,12G C ,3hr,# ; M ean 17= JKLM 18. PO Mean 37= 16. 23 V o ifeH C l, BO C , lh r ,* ; M ean 11= JKLM 18. PO Mean 38= 16. 65 [ o 1KHCI, 80 C ,lh r ,# ; M ean ;13= JKLM 18. PO Mean 3P= 16. 70 c o lK H C l, 80 C ,2h r,* ; M ean 50= KLM 18. 88 Mean 40= 16. 05 z lH HC l, 8 0 °C ,2h r ,# ; M ean 56= LMN 18. 85 Mean 41= 17. 05 Y lK H C l, 8 0 °C ,3 h r ,* ; M ean 51= LMN 18. 85 M ean 42= 17. 45 X lK H C l, 8 0 °C ,3h r ,# ; M ean 19= MNO 10. 80 Mean 43= 17. 73 V o lK H C l, iOO C , lh r ,* ; M ean 31= MNO 18. 80 Mean 44 = 17. 77 V o lK H C l, IOO C ,lh r #; M ean P= MNO 18. 80 Mean 45= 17,83 V 196HCI, 1 0 0 °C ,2 h r ,* ; M ean ■61= MNO 18. 80 Mean 46= 18. 27 ST o l «H C l , IOO C ,2hr,# ; M ean 4P= NOP 18. 70 Mean 47= 18. 13 TUV o i «H C l , IOO c ,3 h r ,* ; M ean 5= OPQ 18. 67 Mean 48= 18. 53 CtR o lK H C l, IOO C ,3hr,# ; M ean 67= OPOR 18. 65 Mean 4P= 18. 70 NOP o lH H C l, 120 C , lh r ,* ; M ean 33= PQR 18. 60 Mean 50= 18. 88 KLM o IK H C l, 120 C ,lh r ,# ; M ean 7= PQR 18. 60 Mean 51= 18. 85 LMN o l «H C l , 120 C ,2h r,* ; M ean 48= QR 18. 53 Mean 52= IP . 13 GHI O 196HCI, 120 C ,2hr,# ; M ean 2= R 18. 50 Mean 53= IP . 05 H I J o IK H C l, 120 C ,3h r,* ; M ean 3= S 18. 30 Mean 54= IP . 45 EF o IK H C l, 120 C ,3h r,# ; M ean 46= ST 18. 27 Mean 55= 17. 70 V o 5XHCI, 80 C , lh r , * ' , : M ean 5P= ST 18. 27 Mean 56= 18. 05 LMN o 55HSHCI, 80 C ,lh r ,ff; M ean 6P= ST 18. 27 Mean 57= 10. 05 UV o 5J6HCI, 80 C ,2hr,*.; M ean 71= STU 18. 20 M ean 58= IP . 02 IJ K o 596HCI, 80 C ,Zhr,# ; M ean 47= TUV 18. 13 M ean 50= 18- 27 ST , o 5 *H C l, 80 C ,3h r,* ; M ean 57= UV 18. 0 5 M ean 60= IP . 23 G SJCHCl, BO °C ,3hr,# ; M ean 35= V 18. OO M ean 61 = 18. 80 MNO 5%HCl, 1 0 0 ° c , lh r , * ; M ean 45= V 17. 03 Mean 62= IP . 70 CD 5 «H C l, 100 °G ,lh r,# -; M ean 36= V 17. 80 University of Ghana http://ugspace.ug.edu.gh 90 APPENDIX 6. Mean <53= IP . 20 OH o 5 «H C l, lOO C,2hr,*; Mean 44= W 17. 77 Mean <54= IP . 77 BC o SKHCl, lOO C,2hr,#; Mean 43= W 17. 73 Mean <55= IP . 23 G o 5 «H C l, lOO c ,3h r,* ; Mean 55= V 17. 70 Mean <5<5= IP . 80 BO o , 5HCL, lOO C,3hr,#; Mean 42= X 17. 45 Mean <57= 18. <55 OPQR 596HCI, 120°C ,lh r,*; Mean 1= X 17. 40 Mean <58= IP . <57 CD o 596HCI, 120 C ,lh r,# ; Mean 41= Y 17. 05 Mean. <5P= IB. 27 ST o 59CHCI, 120 C,2hr,*; Mean 40= Z 1<5. 85 Mean 70= IP . <50 DE o 5 «H C l, 120 C,2hr,#; Mean 3P= C 1<5. 70 Mean 71= 18. 20 STV o 596HCI, 120 C ,3hr,*; Mean 38= [ 1<5. <55 Mean 72= IP . 45 EF o 5%HCl, 120 C,3hr,#; Mean 37= \ 1<5. 23 * = N on -ir rad ia ted T. viride. M = Irra d ia ted T. viride. A lphabet used to show d if fe r e n t ia t io n s in the means. University of Ghana http://ugspace.ug.edu.gh 91 Appendix 7A. exposure to gamma irradiation shown as percent reduction in viscosity of starch. Amylase activity of microorganisms after Dose Applied Amylase activity (% reduction In viscosity of starch) (Krad) T. viride R.oryzae A. niger Lplantarum 0 60 53 65 40 20 65 60 68 45 40 68 62 70 50 50 75 64 72 65 100 70 60 68 60 200 45 60 66 55 University of Ghana http://ugspace.ug.edu.gh 92 Appendix 7B. exposure to gamma irradiation shown as percent reduction in viscosity of cellulose. Cellulase activity of microorganisms after Dose Applied Cellulase activity (% reduction In viscosity of cellulose) (Krad) T. viride R.oryzae A. niger L. plantar um 0 40 50 65 30 20 60 48 66 40 40 68 45 68 45 50 72 55 64 48 100 79 66 60 43 200 58 50 61 32 University of Ghana http://ugspace.ug.edu.gh Appendix 7C. exposure to gamma irradiation shown as percent reduction in viscosity of pectin. Pectinase activity of microorganisms after Dose Applied Pectinase activity (% reduction in viscosity of pectin) (Krad) T. viride R.oryzae A. niger Lplantarum 0 30 40 59 34 20 40 44 60 38 40 48 44 61 40 50 64 48 63 42 100 50 42 62 39 200 48 42 62 33 I University of Ghana http://ugspace.ug.edu.gh REFERENCES: AHLGREN, E and ERIKSSON, K.E, 1967. Characterisation of cellulases and related enzymes by isoelectric focussing, gel filtration and zone electrophoresis II. Studieson Sterextffi sanguinoleturn, Fom&s annosus and Chrysos por iuiti lignoruffi enzymes. Acta Chemica Scandinavicca 2J., 1193-1200. AKUSHIE, P-L. N. A. 1980. Studies on the physiology of Rhizopus oryzae Went & Prinsen Gerling causing deterioration of groundnut Arachis hypogeae in storage. PhD. Thesis, Dept. of Botany, Univ. of Ghana, Legon. ALEXANDER, M 1961. Introduction to Soil Microbiology. John Wiley and Sons, New York. AMER, G. I. and DREW, S. W. , 1980. Microbiology of lignin degradation. 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