Utilization of cellulose and hemicellulose of pig faeces by Trichoderma viride

The purpose of this investigation was to study the microbiological degradation of the cellulose-hemicellulose-lignin complexes of the faeces of pigs. Cellulose, hemicellulose and lignin are components of the cell wall of plants and residues of plant material occur in large quantities in faeces and other organic waste material. The development of the intensive livestock farming is leading to the production of large quantities of manure that cannot always be disposed of in the usual way by lack of agricultural land. To prevent deterioration of the environment, other ways of handling the manure should be sought. Priority should be given to those procedures according to which the insoluble organic components of the faeces are decomposed by microorganisms and the energy produced utilized by man. Examples of such procedures are the aerobic breakdown of the organic residues with production of large amounts of biomass that can be utilized as feed additive (single-cell protein). Anaerobic treatment of manure may lead to the production of methane. In the present investigation attention was centred on the aerobic break-down of the undigested organic residues of pig faeces.

Chapter 1 contains a general introduction and gives a review of literature pertaining to waste material with cellulose, hemicellulose and lignin as main components. In the Netherlands most of the organic waste material occurs as manure, sewage sludge and domestic wastes. The chemistry and structure of the plant cell wall were discussed. The presence of lignin strengthens the structure of plant tissue but seriously hampers the break-down of other cell wall components like cellulose and hemicellulose which are closely linked to the lignin molecules. Fungi play an important part in the degradation of cellulose-hemicellulose-lignin complexes of plant residues. Some fungi are able to decompose lignin, others utilize cellulose and hemicellulose, leaving lignin untouched. Cellulolytic bacteria occur in considerable numbers in nature, but break-down of lignin by bacteria is hardly reported in the literature. Extra-cellular, substrate-specific enzymes produced by microorganisms are involved in the break-down of plant residues. Several enzymes play a part in the degradation of the cellulose-hemicellulose-lignin complexes. Those involved in the decomposition of cellulose are known under the collective name of cellulase. Hemicellulose is a group name for several plant cell wall heteropolysaccharides which are degraded by substrate-specific enzymes. Little is known of enzymes involved in lignin break-down.

In Chapter 2 a survey is given of materials and methods used in this investigation. A method was described for the collection of insoluble residues of plant material from pig faeces. For practical reasons, this preparation was used in the experiments to be described. Chapter 3 gives the results of microscopic and chemical analyses of undigested plant residues of pig faeces. These residues were derived from plant material. mainly ground seeds and fruits, supplied as mixed feed to the animals. Various types of plant tissue (parenchyma, collenchyma and sclerenchyma) were observed showing that considerable amounts of cellulose-, hemicellulose-, and lignin-containing cell walls had passed more or less undamaged the digestive track of the pigs (Figs. 3.1 - 3.11). The chemical analysis of pig faeces showed that 30-40 % of the dried matter consisted of insoluble plant residues containing 65 - 70 % polysaccharides (cellulose and hemicellulose) and nearly 30 % lignin. In order to use these plant residues as carbon source for fungi, pretreatment of the material is necessary as the high lignin content hampers the ready utilization of the cellulose and hemicellulose by the cellulolytic fungi.

Chapter 4 described the isolation of cellulolytic microorganisms. Mainly fungi were isolated and a number of them together with some fungi obtained from culture collections were tested for their ability to utilize as carbon source: (a) monosaccharides that occur in the polysaccharides of plants (b) some model polysaccharides related to plant cell walls (c) lignin and (d) undigested plant residues from pig faeces. The results of these experiments are shown in Tables 4.1, 4.2 and 4.3. None of the fungi tested was able to decompose lignin. When the undigested plant residues of pig faeces had been supplied as carbon source only 30-40 % of the material was utilized. This was mainly due to the protection of the polysaccharides against fungal attack by the lignin. In order to improve the availability of the polysaccharides to the fungi, breaking or removal of the lignin matrix of the plant tissue is required. Of the fungi tested for growth on residues of faeces, Penicillium nigricans, Myrothecium verrucaria and Trichoderma viride QM 9419 gave the best results. As the Trichoderma strain produced the highest amounts of cellulolytic enzymes, this organism was chosen for further research.

In Chapter 5 experiments were described concerning the growth of Trichoderma viride on (a) crystalline cellulose (Avicel), (b) undigested plant residues from pig faeces, and (c) plant residues from faeces treated with alkali to dissolve and remove lignin. Crystalline cellulose was the best carbon source. It was almost completely utilized by T.viride giving about 250 mg of protein per g of cellulose consumed. The undigested carbon compounds of pig faeces were utilized for about 49 % and the alkali-treated residues for 60 %. The amount of cellulolytic enzymes in the culture liquid of T.viride, calculated per unit of soluble protein, was with cellulose as carbon source three times as high, and with alkali- treated residues twice as high as with untreated plant residues from pig faeces. Spent culture solutions of T. viride supplied with pure, cellulose or undigested residues of pig faeces contained relatively large amounts of soluble protein. An unknown proportion of this protein represented enzymes, presumably mainly cellulolytic enzymes. The production of biomass from the undigested residues of pig faeces by use of Trichoderma viride ('single-cell protein') to prepare mixed feed is for the following reasons no appropriate procedure: (a) T.viride only partly utilizes the polymers of the undigested residues of pig faeces, (b) the fungal protein is mixed with part of the faeces constituents which could not be metabolized by the fungus and which are also resistant to digestion by the animal owing to the protection of cellulose and hemicellulose against break-down by the presence of lignin, (c) the soluble protein of the culture solution of the fungus is difficult to collect.

Chapter 6. A procedure was proposed (Fig 6.1) according to which the insoluble residues of pig faeces are submitted to a solution of cellulolytic enzymes. The hydrolysis of the polysaccharides (at 50°C and pH 4.8) proceeds in a relatively short time giving a solution of sugars which can be used for further purposes. A suitable enzyme solution can be obtained by growing T.viride on residues of faeces. The enzyme solution of the spent nutrient solution of the fungus can be easily concentrated by acetone precipitation or by vacuum evaporation. The substrate for the enzymic procedure, the undigested residues of faeces, are adequately degraded when a pretreatment has been applied (grinding to a particle size of 0.08 mm). When the proposed procedure is applied, 150 g of insoluble residues per 1 of enzyme solution can be hydrolysed; 70 % of the residues is converted-into soluble sugars within 4 h. The cellulolytic enzymes are initially adsorbed to the insoluble substrate but are released when hydrolysis proceeds; they can be separated from the undissolved material together with the dissolved sugars. Separation of sugars and enzymes can be achieved by mixing with new substrate which adsorbs the enzymes. After separation of the sugar solution the substrate with enzymes are transferred to the reactor. In this way part of the enzymes can be re-used. In Chapter 6 also the results are given of the purification and separation of a number of enzymes involved in the degradation of the cellulose-, hemicellulose-, lignin complex (Fig. 6.8). The mixture of enzymes, excreted by T. viride when the organism was growing on undigested plant residues of pig faeces, was separated into several protein fractions each with its own enzyme activity with respect to different substrates, i.c. endoglucanase, exoglucanase and hemicellulase activity (Figs. 6.9-6.12). Degradation of undigested components of pig faeces in view of recycling of the faeces is no simple procedure. Microbial (enzymic) break-down is possible if a suitable pretreatment is applied. The economical applicability of the results obtained in this investigation will depend on: (a) the occurrence of excessive amounts of manure, (b) disposal problems, particularly in connection with the environment, (c) alternative methods for processing excessive amounts of manure.