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A thesis proposal Presented to Dr. Minda Formacion College of Arts and Sciences University of the Philippines in the Visayas Miag-ao, Iloilo

In partial fulfilment Of the requirements for the course BIO 296 - Seminar in Biology

Ervin Malones December 2011


Outline I.Status of the Philippine Swine Industry II.Anaerobic Digestion Process and Biogas digester III.Importance of Pre-treatment a. Physical Pre-treatment b. Biological Pre-treatment VI. Related Studies on Fungal Degradation VII. Potentials of Aspergillus niger and Aspergillus fumigatus VIII.The Process for Biogas Production

Enhancement of Biogas Production through Fungal Pre-treatment Using Pig Manure and Rice Hay


Objective of the Study

The main aim of this study is to investigate the effects of fungal pre-treatment from rice straws and pig manure and to measure the difference in methane yield.

Status of Philippine Swine Industry The Philippines is mainly an agricultural country. About 130, 000 km2 or 44% of its total area is devoted to agricultural crops like rice, corn, sugarcane and coconut. These crops generate residues during harvesting and processing and constitute a potentially important source of energy. Hog raising is the second largest commercial agri-food industry in the Philippines, after the banana industry. It generated about 16% of the Agriculture and Forestry Industry sales in 2006 (Census of the Philippine Business and Industry). As of 2009 there were 13,685,000 hog population in the Philippines (BAS, 2009) and it generates about 9,990 MT of manure based on the assumed specific manure yield (SMY) of 2 kg of manure per day per live head (2kg/day/head) for hog, 3grows/year, 120 days/ grow (Ulgado, 2009). About 71% of the swine population were raised in backyard farms while 29% were in commercial farms. Among all sorts of livestock, hog raising appears to have the most potential for a domestic biogas market. Hog raisers with > 6 hogs usually have pens that allow collection of manure, and apart from that, hog manure is a strong polluter and produces a large volume of gas per kg (Lapus, 2011). In the Philippines, piggery wastes were dumped in nearby rivers that lead to the increasing amounts of nutrients, frequent algal blooms, depletion of oxygen in the water followed by reduction in fish population and other negative environmental impacts. In general,

the main environmental problems caused by improper disposal of pig manure that needs attention: (1) Contamination soil and ground water (2) Contamination surface water (3) Emission of greenhouse gasses (carbon dioxide and methane) (4) Odour (5) Emission of ammonia gas (acidification). With agriculture including livestock production among the major drivers of the countys economic growth, the Philippines is a good example of an economy with significant resource potential for methane recovery from livestock wastes and wastes from the agro-industrial sector.

Anaerobic Digestion Process and Biogas digester

Anaerobic digestion is a biological process in which biodegradable organic material is decomposed in the absence of oxygen to produce biogas which is a mixture of CH 4 (55-75%), CO2 (25-45%), H2 (0-3%), N2 (1-5%), CO (0-0.3%), H2S (0.1-0.5), O2 and water vapors (traces) (Pauss et al., 1987; Gerardi, 2003)). Anaerobic digestion is a microbiological process of decomposition of organic matter, common to many natural environments and largely applied today to produce biogas in airproof reactor tanks, commonly named digesters. A wide range of micro-organisms are involved in the anaerobic process which has two main end products: biogas and digestate. Biogas is a combustible gas consisting of methane, carbon dioxide and small amounts of other gases and trace elements. Digestate is the decomposed substrate, rich in macro- and micro nutrients and therefore suitable to be used as plant fertiliser (Al Seadi et al., 2008). Anaerobic digestion has been developed worldwide to treat crop residues (corn stalks, rice straws, banana leaves peanut hulls, etc.), agricultural (cattle and pig manure and slurry, poultry manure, etc.), household and industrial residues (fish and meat wastes, vegetable and dairy processing residues (e.g. potatoes, cheese and yoghurt) and sewage sludge. These wastes can be used as feed stocks for anaerobic digestion. The quality of the feedstock used in the digester will determine how much gas is produced and its constituents. Production of biogas through anaerobic digestion of animal manure and slurries as well as of a wide range of digestible organic wastes, converts these substrates into renewable energy and offers a natural fertiliser for agriculture. In addition to the production of renewable energy,

controlled anaerobic digestion of animal manures reduces emissions of greenhouse gases, nitrogen and odour from manure management, and intensifies the recycling of nutrients within agriculture (Amon et al., 2006; Clemens et al., 2006). The following are the benefits of Anaerobic Digestion: Environmental benefits, Energy balance, Reducing greenhouse gases, Displacing use of finite fossil fuels, Recycling nutrients, Reducing land and water pollution, Reducing odour, Reducing spread of weeds and disease. Biomass is one such renewable energy resource, but it must not compete with food production since the clearance of forest for farmland gives a significant contribution to the increase of CO2 in the atmosphere. Therefore, organic wastes and residues are preferred. Biogas technology is based on the phenomenon that when organic matter containing cellulose is fermented in the absence of air (anaerobically) a combustible gas (methane) is formed (Acharya, 1956). Biogas digesters are airtight fed with waste where it is fermented without oxygen to produce a methane-rich gas. This gas can be used to produce electricity, or for cooking and lighting. It is a safe and clean-burning gas, producing little carbon monoxide and no smoke. Biogas systems make clean energy for household use. Cooking on biogas is quicker and easier than cooking with firewood. Biogas systems kill the bacteria in livestock manure. A farm with a biogas system is a cleaner and safer place. Biogas systems produce excellent safe fertilizers for use on the farm.

Importance of pre-treatment

Pre-treatment prior to anaerobic digestion has been proven to be one of simple and effective methods to improve biodegradability and biogas production of lignocellulosic materials (Pavlostathis et al., 1985; Chen et al., 2005). The physical structures and chemical compositions of lignocellulosic materials could be altered through various pre-treatments, making the compositions in lignocellulosic materials more accessible and more readily biodegradable to anaerobic microorganisms, thus increasing digestion efficiency and biogas production. Pre-treatment by physical, chemical or biological means is a well-investigated process for ethanol production from lignocellulosic materials. The pre-treatment can enhance the biodigestibility of the wastes for ethanol and biogas production and increase accessibility of the

enzymes to the materials. It results in enrichment of the difficult biodegradable materials, and improves the yield of ethanol or biogas from the wastes (Taherzadeh and Karimi, 2008). Each pre-treatment has its own effect on the cellulose, hemicellulose and lignin; the three main components of lignocellulosic biomass. Commonly used pre-treatment alternatives are heat/thermal treatment, centrifugation, chemical- and ultrasonic treatment and grinding (Nordberg and Edstrm, 1997; Agrobiogas, 2006). However, the physical and chemical pre-treatments require high energy and corrosion resistant, high-pressure reactors, which increase the need of equipment and cost of pretreatment. Furthermore, the chemical pre-treatments can be detrimental to the methanogens apart from generating acidic or alkaline water, which needs pre-disposal treatment to ensure environment safety (Keller et al., 2003). Thus, an alternative approach is microbial pretreatment especially fungi to increase digestibility. Advantages of biological pre-treatment include inexpensive, low energy requirement and mild environmental conditions (Saratale et al., 2008). In biological pre-treatments either microbes and/or microbial enzymes are used for partial degradation of lignocellulose. These methods bear the advantage that they are usually simple and do not require major capital investments (Lissens et al., 2003). Biological degradation of lignocellulose is a complex process where many factors, as fungal strain, culture conditions, fungal enzymatic secretion and oxidative mechanisms, are implicated (Wan and Li, 2010). Therefore, analysis of the whole process is relevant to understand the mechanisms of fungal degradation and to get the best fungi and optimum growth conditions for obtaining the maximum amount of fermentable sugars. Biological treatments with microorganisms or

enzymes are also investigated to improve digestion in biogas production. The biological pretreatment might be used not only for lignin removal, but also for biological removal of specific components such as antimicrobial substances. Solid state fermentation of orange peels by fungal strains of Sporotrichum, Aspergillus, Fusarium and Penicillium enhanced the availability of feed constituents and reduced the level of antimicrobial substances (Srilatha et al., 1995). In a similar work, cultivation of white-rot fungi was used to detoxify olive mill wastewater and improve its digestion (Dhouib et al., 2006). Low energy requirement, no chemical treatment, and mild environmental conditions are the main advantages of biological pre-treatment.

Related Studies on Fungal Degradation

Microorganisms can also be used to treat the lignocelluloses and enhance enzymatic hydrolysis. The applied microorganisms usually degrade lignin and hemicellulose but very little part of cellulose, since cellulose is more resistant to biological attack. Several fungi, e.g. brown-, white- and soft-rot fungi, have been used for this purpose. Lignin degradation occurs through the action of lignindegrading enzymes such as peroxidises and lactase. White-rot fungi are among the most effective microorganisms for biological pretreatment of lignocelluloses (Sun and Cheng, 2002). White-rot fungi are the only known living organisms capable of complete lignin degradation, and their application has been suggested for partial delignification to increase digestibility (Mller & Trsch, 1986; Ghosh & Bhattacharyya, 1999). Most of the white-rot fungi degrade lignin and cellulose simultaneously. A selective white-rot fungus, Ceriporiopsis subvermispora is known to selectively degrade lignin in softwood and hardwood (Okano et al., 2005). The white rot fungi treatment (21 d at 21C) and short-term composting (7 d) resulted in high losses of organic matter due to biological activity, as a result of which, the increase in methane potential was low or even negative (Lehtomki et al., 2004). The suitable fungi for biological pre-treatment should have higher affinity for lignin and degrade it faster than the carbohydrate components. Currently, enzymes derived from the aerobic fungal genera Trichoderma and Aspergillus are most widely used in industrial settings (Nichols et al., 2008). Nevertheless, several microorganisms are known to completely depolymerize hemicellulases (mainly xylans) to xylose. These include the fungi Penicillum capsulatum and Talaromyces emersonii (Filho et al., 1991), the thermophilic actinomycete Thermomonospor fusca (Bachmann and McCarthy, 1991), the hyper- thermophile Caldicellulosiruptor saccharolyticus (Van De Werken et al., 2008). On a study by Abdel-Sater and El-Said (2001), the fungal flora decomposing rice straw were investigated all over the soil of Sharkia Province in Egypt, appeared during this study was; Aspergillus, and the next frequent fungal genera were ranked by Penicillium, Chaetomium and Fusarium, Acremonium, Alternaria, Cladosporium, Cochliobolus, Emericella, Humicola,

Mucor, Myrothecium, Stachybotrys and Trichoderma (Helal, 2005). Members of Aspergillus, Penicillium and Trichoderma were also found to be the most prevalent fungi isolated from rice straw as xylan decomposing fungi (Abdel-Sater and El-Said, 2001).

Among microorganisms, fungi are of great interest for enzyme production because they excrete their enzymes extracellularly (Bollok, 2005). In the fungal family, Trichoderma reesei is regarded as the most efficient producer of cellucellulase (Miettinen-Oinnonen, 2002). However, the fungus does not excrete sufficient amount of -glucosidase for efficient enzymatic hydrolysis (Bollok, 2005), for which Aspergillus strains are known to be good producers (Juhasz et al., 2003).

Potential of Aspergillus niger and Aspergillus fumigatus

The representative fungi that will be used for the study will be Aspergillus niger and Aspergillus fumigatus. These two fungi were chosen for their availability in the surroundings and their unique ability in degradation process. According to Malik and Rajoka (1973), Aspergillus species is plentiful in rice fields. High recovery of the organism from rice field may not be unconnected with the fact that cellulose of rice straw has a medium lignin content (12 to 16%) when compared with other cereal stalks (17%) so that the cellulose is easily metabolized (Youngquist, 1993; Qingxiang, 2002). This makes Aspergillus species (even the least in cellulose expression) to easily colonize soil where stalks of rice have decayed and probably mineralized the soil. The degraded cellulose is utilized as source of carbon and energy. The black Aspergilli have a number of characteristics which make them ideal organisms for industrial applications, such as good fermentation capabilities, and high levels of protein secretion (De-Vries and Visser, 2001); ability to assimilate various organic substrates and suppress development of other micro-organisms and high sporulation capacity (Millner, 1994). Aspergillus strains are known for their ability to produce glucosidase with significantly higher yields than Trichoderma species (Kang, 1999). Numerous fungal strains were used for lignocellulosic hydrolysis such as Aspergillus niger, A. terreus, Fusarium moniliforme and Chaetomium celluloyticum (Kim et al., 1985). Aspergillus niger and T. viride are known to be effective in the composting of lignocellulosic materials by producing oxidative and hydrolytic enzymes (Zayed and Abdel-Motaal, 2005). A. niger is a thermo-tolerant (Sanchez et al., 2000) ligninolytic microbe. Aspergillus niger isolated from different soil sources with cellulolytic ability were tested for the production of cellulase on

carboxymethylcellulose. The maximum cellulase expression was at day 4 (0.07162 IU/ml/min). High recovery of the organism from rice field coupled with high cellulose productivity level may be adduced to the cellulose content of rice straw which has a medium lignin content that supports the growth of the organism. In the soil samples studied, Aspergillus niger was isolated in all instances. This confirms the ubiquity of Aspergillus niger in all soil environments. Elite strain of A. niger with improved rate of cellulase production and holds great potential as a candidate strain for commercial production of cellulose (Damisa et al., 2011). Finally, a consortium of Aspergillus niger and Trichoderma viride was tested for in-vitro biodegradation of rice straw. The fungal consortium was able to decompose cellulose, hemicelluloses, lignin, and total carbon significantly (p _ 0.05) over the control. The C/N ratio was reduced to 19.5 from an initial value of 29.3 in three weeks of the biodegradation process, thus showing the potential of this method for use in large-scale composting of rice straw (Kausar et al., 2010). Coronel et al. (1991) found that Aspergillus fumigatus was the most active cellulose producer on rice straw substrate among 144 tested strains of thermophilic lignocellulose-degrading fungi. Jin et al. (2007) studied the fifty strains of fungi from deadwood, rotted leaves, rice straw and wheat straw were tested for their ability to degrade rice straw, ten potential strains were further screened, and strain XC6 exhibited excellent ability in degrading rice straw. Identification of morphology and 18S rDNA gene sequences showed the strain was Aspergillus fumigatus. The initial pH and inoculation size were the most important factors affecting rice straw degradation, and an inoculation size of 10%, culture temperature 30 , (NH4)2SO4 as nitrogen source, inoculation age of 36 h and initial pH of 4.0 were optimum for rice straw degradation by the strain XC6. Under the optimal conditions, the rice straw degradation rate reached 100%0.2%, and biomass was 22.91.1 g/L. Sherief et al. (2010) showed that Aspergillus fumigatus, A. terreus and Emericella niveus were the most active cellulolytic species based upon their high yield of exoglucanase, endoglucanase, CMCase, -glucosidase and xylanase activities. However, the results also indicate that no significant relation between enzyme activities and both amounts of total protein and released free sugars in all tested fungi. However, maximum yield of CMCase (9.65 IU g-1), exoglucanase (0.54 IU g-1) and xylanase (25.36 IU g-1) activities were obtained after 4 days.

However, maximum endoglucanase (0.33 IU g-1) and -glucosidase (8.71 IU g-1) activities were shown after 2-4 days.

Related Studies on Rice Hays Around the world, different crops have been screened for their methane potential on various occasions (Gunaseelan, 1997). In this study rice straw will be used for the co-digestion with pig manure because of its availability. Rice straw contains a high percentage of polysaccharides and lignin. The main components of rice straw is 36.2 (wt%), cellulose 19.0 (wt%), hemicellulose 9.9 (wt%) lignin (Nigam, 2009). Rice (Oryza sativa L.) is a major crop grown worldwide with the annual productivity around 800 million metric tonnes that also generates large production of rice (paddy) straw. The management of rice residue through direct incorporation of straw in soil is associated with certain problems such as immobilization of plant nutrients particularly nitrogen and reduces germination of subsequent crops. Moreover, rice straw waste contains high C:N ratio about 80:1 and is rich in silica and lignin which make it difficult to be degraded (Kumar et al., 2006). Rice straw is an attractive lignocellulosic material for biofuel production since it is one of the most abundant renewable resources. It has several characteristics, such as high cellulose and hemicelluloses content that can be readily hydrolyzed into fermentable sugars (Binod et al., 2009). Though, Hashimoto (1983) and Fischer et al. (1983) reported lower specific methane yields in co-digestion of manure with straw compared with digestion of manure alone. In the study by Zhou (2011), an anaerobic dry fermentation system was designed and erected to validate the feasibility and test its performance by using the mixtures of rice straw and fresh pig manures under the near middle temperature. Experimental results show that after 6 days CH4 content in biogas can be above than 45%. In addition, the total amount of biogas produced is about 16.55 m3 from 6-day to 22-day, and the maximum volume gas production rate can be about 1.2 m biogas (dm3 fermentation tank). Furthermore the CH4 content in the biogas after the start-up time can be maintained at about 50%~70%. These useful results imply that the anaerobic dry fermentation using rice straw and fresh pig manures is viable. Also, on the study by Phutela (2011), the chopped and moist paddy straw was pre-treated with, Trichoderma reesei

(cellulolytic fungus) and Coriolus versicolor ligno-cellulolytic fungus) and their effect on change in chemical composition of paddy straw was determined. The correlation between change in various contents of paddy straw and biogas production was developed. Taniguchi et al. (2005) had evaluated biological pre-treatment of rice straw using four white-rot fungi (Phanerochaete chrysosporium, Trametes versicolor, Ceriporiopsis subvermispora and Pleurotus ostreatus) on the basis of quantitative and structural changes in the components of the pre-treated rice straw as well as susceptibility to enzymatic hydrolysis. Pre-treatment with P. ostreatus resulted in selective degradation of the lignin rather than the holocellulose component, and increased the susceptibility of rice straw to enzymatic hydrolysis.

The composting process and its effects to the environment

Generally agricultural, industrial or household wastes not used for the process are being turned to compost as soil fertilizer. Composting is a process based on the management of microorganisms naturally present in waste materials. In addition to the positive beneficial effects of this process on the environment, there are a number of potential negative impacts. The waste material may contain within its microbial population organisms which are potentially pathogenic to humans, animals and plants. This is currently a major concern (Gale, 2002). On a study by Ghazifard on compost, the thermophilic and mesophilic microbiota in compost produced from Esfahan municipal solid waste (in Iran) were examined at different stages of composting process from day zero to 28 days and was conducted in four different seasons. Some of the mesophilic bacteria observed in initial stages of composting process were gram negative Escherichia, Klebsiella, Aeromonas and Alcaligenes, and gram positive Enterococcus and Bacillus species. After 20 days of the composting process lower species diversity of mesophiles (only Bacillus species) were isolatedlikely due to the high temperature (60-68C) condition. Among the mesophilic fungi, at the initial stages of composting process some types of yeasts and molds were isolated, but after day 20 due to high temperature condition (60-68C), no mesophilic fungi were obtained. On the 15th day of composting the highest diversity of thermotolerant fungi such as Cladosporium, Aspergillus, Mucor, Rhizopus, and Absidiae spp. were observed.

Fungal diversity during composting was investigated by culture-independent rDNA sequence analysis. Composting was carried out with pig manure and mushroom cultural waste using a field-scale composter (Hazaka system), and samples were collected at various stages. Based on partial sequence analysis of large subunit (LSU) ribosomal RNA (rRNA) and sequence identity values, a total of 12 different fungal species were found at six sampling sites; Geotrichum sp., Debaryomyces hansenii, Monographella nivalis, Acremonium strictum, Acremonium alternatum, Cladosporium sphaerospermum, Myriangium durosai, Pleurotus eryngii, Malassezia globosa, Malassezia restricta, Rhodotorula glutinis, and Fusarium sporotrichioides. Geotrichum sp. of the class Saccharomycetes was the most predominant fungal species throughout the composting process (185 out of a total of 236 identified clones, or 78.4%), followed by Acremonium strictum (7.6%), Monographella nivalis (5.1%), and Pleurotus eryngii (3.8%). The prevalence of Geotrichum sp. was the lowest (61.1%) at the beginning of composting, and then gradually increased to 92.5% after 10 days of composting (Cho et al., 2009). The composting process may generate by-products which have potentially negative impacts on the environment, in particular gaseous by-products. Ammonia may be volatilized as ammonium released from N-rich materials by microbial activity accumulates in the compost. Depending upon the redox conditions, microbial decomposition of organic materials will lead to the production of CO2, N2O, or CH4 (Hellmann et al., 1997). All of these three gases contribute to the greenhouse effect, but their net contributions to global climate change differ. According to Hellmann et al., (1997), CO2 is one of the products of aerobic respiration, but in contrast to CO2 produced by fossil fuel combustion, CO2 derived from degradation of plant materials does not contribute to global warming because it had been previously removed from the atmosphere by photosynthesis. N2O is a product mainly of the microbial reduction of NO3- under anaerobic conditions (denitrification) but also to some extent via nitrification. CH4 is a product of the microbial reduction of CO2 under anaerobic conditions. The release of N2O and CH4 to the atmosphere contributes to the enhancement of the greenhouse effect). The flux of CH4 to the atmosphere can be avoided if the material is subject to anaerobic digestion in which the CH4 is collected and used as a source of energy.


The Process for Biogas Production (Wolf, 1979; Ehrig and Dallos, 2009). The science underlying anaerobic digestion can be complex and the process is best understood if split into four main stages: hydrolysis, acidogenesis, acetogenesis and methanogenesis. Hydrolysis. One of the main objectives of pre-treatment methods is to increase solubility of the substrate and to accelerate the hydrolysis process. The waste processed material consists mainly of carbohydrates, lipids, proteins and inorganic materials. In the first processing step, the hydrolysis, the complex compounds of the input material are decomposed into elementary organic compounds like amino acids, sugar and fatty acids. The bacteria taking active part in this process therefore release extracellular enzymes. This process is known as polymer breakdown stage. These enzymes decompose the material in a biochemical process. For example, the cellulose consisting of polymerized glucose is broken down to dimeric, and then to monomeric sugar molecules (glucose) by cellulolytic bacteria. The hydrolytic activity is of significant importance in wastes with high organic content and may become rate limiting. Chemicals can be added during this step in order to decrease the digestion time and provide a higher methane yield. Acidogenic phase. After hydrolysis the formed intermediate products such as monomeric glucose are used in the acidogenic stage. Through acid-forming bacteria and under anaerobic condition the intermediate products with molecules of six atoms of carbon (glucose) are broken down into low fatty acids (acetic acid, propionic acid and butyric acid) and carbohydrates. Besides, lower amounts of alcohols (ethanol) and lactic acid are formed. Acetogenic phase. In the second stage, acetogenic bacteria, also known as acid formers, convert the products from the first stage into simple organic acids, carbon dioxide and hydrogen. The principal acids produced are acetic acid, butyric acid, propionic acid and ethanol. Too high contents of hydrogen are harmful for the acetic acid forming bacteria. Therefore the creators of acetic acid have to constitute a symbiotic community with the bacteria of the methanogenic phase. The latter consume hydrogen when creating methane and thus provide acceptable living conditions for the acetogenic bacteria.


Methanogenic phase. Finally, methane is produced during methanogenesis by bacteria called methane formers in two ways: by means of cleavage of two acetic acid molecules to generate carbon dioxide and methane, or by reduction of carbon dioxide with hydrogen. The acetate reaction is the primary producer of methane because of the limited amount of hydrogen available. Methane production from organic substrates mainly depends on their content of substances that can be degraded to CH4 and CO2. Composition and biodegradability are key factors for the methane yield from energy crops and animal manures (Balsari et al., 1983). As methane is produced spontaneously from the organic sediments in natural water bodies (Nozhevnikova et al., 1997), it is not surprising to find similar production from artificial water bodies such as manure storage tanks and lagoons (Park et al., 2006; Safley and Westerman, 1988; Sharpe et al., 2002). The more soluble and easily degradable the substrate, the easier is the design and operation. In particular, pig wastes are considered to be relatively easy to handle. According to Moeller et al. (2006), using swine manure that was allowed to ferment for 15 days at 50C has a methane yield of 230 l/kg and for 30 days at 30C of 300 l/kg. An estimation of the potential to produce methane of animal manures is essential. Parameters such as nutrients content, pH, temperature, organic loadings, solid content, mixture ratio and hydraulic retention time (HRT) are detrimental to biogas quality and quantity. These parameters would also determine the size of digester, auxiliary equipment needed, as well as the sizes and specifications of biogas treatment facilities. Maximum methane yield requires adequate and efficient nutrient supply for micro-organisms in the digester. According to Oosterkamp (2011), information is given on the effects of retention time, temperature, addition of trace elements, addition of surface active elements, addition of enzymes, addition of bacteria, co-digestion of manure with straw and co-digestion of manure with straw pre-treated with fungi. Methane yields of 380 l/kg volatile solids (75 % energy recovery) can be obtained with mixtures of manure and straw and long retention times (120 days). High solids digestion of cattle manure with long retention times in family-size digesters gave methane yields of 230 l/kg volatile solids (45 % energy recovery). Thus, the need for the study is to investigate


the effects of fungal pre-treatment of rice hays and pig manure, as well as to measure the difference in methane yield.


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