Anaerobic Digestion Of Wheat Straw Research Articles

Potential of Wheat Straw for Biogas Production by Anaerobic Digestion in South Africa: A Review

Wheat straw (WS) is a promising substrate for biogas production by anaerobic digestion (AD) due to its high carbohydrate content. An estimated 0.603 million t yr−1 of WS are generated from wheat production systems in South Africa. This is equivalent to an energy potential of 11 PJ. Despite this, WS is still undervalued as a bioenergy resource in South Africa due to its structural complexity and low nitrogen content. WS disposal methods, such as use in livestock bedding, burning and burying into the soil, inter alia, are not sustainable and may contribute to global warming and climate change. The commercialization of the AD of WS needs to be further developed and promoted. Pre-treatment (i.e., physical, chemical, biological and hybrid methods) and anaerobic co-digestion (AcoD) are novel strategies that can support the conversion of WS into biogas and other value-added products. Current and future research should focus on optimizing pre-treatment and AcoD conditions towards industrialization of WS into valuable products. This paper focuses on the potential use of WS for biogas production in South Africa. The aim is to create information that will promote research and development, and encourage policy makers and stakeholders to participate and invest in WS biogas technology. Were WS biogas technology fully adopted, we believe that it would alleviate energy insecurity and environmental degradation, and sustain the livelihoods of citizens in South Africa.

Energy Integration of Thermal Pretreatment in Anaerobic Digestion of Wheat Straw

Cereal straw stands out as one of the most abundant and globally distributed agricultural residues. Traditional applications cope with a limited amount of production, leaving the remainder in the field for natural decomposition. Managing cereal straw through controlled biological transformation under anaerobic conditions holds the potential to generate added value in the form of bioenergy. However, the lignocellulosic composition of these substrates poses challenges for organic degradation, often requiring energy-intensive pretreatments. A detailed study with a comprehensive calculation of the overall energy balance of the integrated process is proposed, aiming to provide real added value and replicability. Three scenarios for wheat straw transformation were investigated, incorporating two preliminary pre-treatment stages—mechanical milling and physicochemical steam explosion. Three conditions of pretreatment were essayed, varying the time exposure of the steam explosion. The subsequent energy integration analysis revealed that the process was optimized by up to 15% in the final energy balance when the steam explosion was set to 10 min. The macromolecular composition determination revealed that the thermal pretreatment reduced the lag phase of the hydrolysis step through hemicellulose breakdown.

Cold isostatic pressing-study on methane production and microbial communities in anaerobic digestion of wheat straw with high-solids

In this study, the effect of cold isostatic pressing (CIP) pretreatment on the physicochemical properties and subsequent anaerobic digestion (AD) performance of wheat straw (WS) was explored, which did not have additional chemical reagent over the whole process. The WS was subjected to CIP pretreatment by pressures from 350 to 700 MPa, respectively, while AD was carried out at mesophilic temperature with total solids (TS) 7% and 14%. The mapping indicated that the CIP pretreatment promoted the hydrolysis of WS by destroying the dense structure. The CIP pretreatment changed pH and volatile fatty acids (VFAs) in the AD system. Compared with the control group, the cumulative methane production of CIP pretreatment group with TS 14% increased by 22.07%–56.18%. The acetic acid concentration peaked on 4th day and 14th day with 3943.23 mg/L and 3815.21 mg/L, respectively, in H600, and propionic acid concentration also reached two peaks at the same time with 2014.92 mg/L and 2405.20 mg/L, respectively. It was also observed that the CIP pretreatment maintained the pH in the range of 6.44–7.40, maintaining the stability of the overall system. It was also found that CIP pretreatment increased 9 species of hydrolysis and acidogenic bacteria, and methanogen Methanosarcina, thereby facilitating the hydrolysis and methanogenic stages. Predictive functional profiling comfirmed that CIP pretreatment could promote glycolysis/gluconeogenesis, fatty acid metabolism, and fatty acid degradation, whose relative abundance increased from 1.10% to 1.13%, 0.60%–0.62%, and 0.19%–0.23%. Methanogenic capacity of the system was improved because of CIP pretreatment enhancing the accessibility of microorganisms by disrupting the microstructure of WS. The energy conversion analysis showed that this method without chemical reagent addition is feasible for high-solids anaerobic digestion.

Algal Biomass Accumulation in Waste Digestate after Anaerobic Digestion of Wheat Straw

Cultivation of microalgae in waste digestate is a promising cost-effective and environmentally friendly strategy for algal biomass accumulation and valuable product production. Two different digestates obtained as by-products of the anaerobic fermentation at 35 °C and 55 °C of wheat straw as a renewable source for biogas production in laboratory-scale bioreactors were tested as cultivation media for microalgae after pretreatment with active carbon for clarification. The strains of microalgae involved were the red marine microalga Porphyridium cruentum, which reached 4.7 mg/mL dry matter when grown in thermophilic digestate and green freshwater microalga-Scenedesmus acutus, whose growth was the highest—7.3 mg/mL in the mesophilic digestate. During cultivation, algae reduced the available nutrient components in the liquid digestate at the expense of increasing their biomass. This biomass can find further applications in cosmetics, pharmacy, and feed. The nitrogen and phosphorus uptake from both digestates during algae cultivation was monitored and modeled. The results led to the idea of nonlinear dynamic approximations with an exponential character. The purpose was to develop relatively simple nonlinear dynamic models based on available experimental data, as knowing the mechanisms of the considered processes can permit creating protocols for industrial-scale algal production toward obtaining economically valuable products from microalgae grown in organic waste digestate.

Employing micro-aeration in anaerobic digestion of poultry litter and wheat straw: Batch kinetics and continuous performance

In this study, different micro-aeration (MA) strategies for anaerobic digestion (AD) of poultry litter (PL) and wheat straw (WS) were examined. MA at different stages (pretreatment, middle, pretreatment plus middle, and daily) in batch AD of WS showed that daily MA had the highest increase (16.5 %) of the cumulative methane yield (CMY) compared to the control. Batch co-digestion (Co-AD) of WS and PL with daily MA obtained a furtherly improved (15.1 %) CMY of 225.44 N mL CH4/g vS added. The modified Gompertz model and Cone model were good in fitting the methane yield kinetics of MA engaged AD process (R2 greater than 0.99). Daily MA shortened the lag phase of Co-AD by 3.4 %. The sequencing batch reactor for the Co-AD of WS and PL showed an increased (21.5 %) daily methane yield when 0.5-h/d MA was employed. The results provided support for the application of micro-aeration in the AD of agricultural wastes.

Synthetic design of methanotroph co-cultures and their immobilization within polymers containing magnetic nanoparticles to enhance methanol production from wheat straw-based biogas

In this study, various methanotroph co-cultures were designed to enhance methanol production from biogas produced through the anaerobic digestion of wheat straw (WS). Furthermore, whole-cell immobilization was performed using magnetic nanoparticle (MNP)-loaded polymers to develop an efficient bioprocess. The anaerobic digestion of WS by cattle dung yielded 219 L/kg of total solids reduced. Methanol produced was 5.08 and 6.39 mmol/L by pure- and co-cultures from biogas, respectively. The optimization of process parameters enhanced methanol production to 6.82 mmol/L by co-culturing Mithylosinus sporium and Methylocella tundrae. The immobilized co-culture within the MNP-doped polymers exhibited much higher cumulative methanol of up to 70.74 mmol/L than the production of 22.34 mmol/L by free cells after ten cycles of reuse. This study suggests that MNP-doped polymer-based immobilization of methanotrophs is a unique approach for producing renewable fuels from biomass-derived biogas, a greenhouse gas.

Targeted addition of CeO2 nanoparticles to promote methane production in anaerobic digestion systems of wheat straw

As a large agricultural country, China produces a large amount of wheat straw each year that needs to be treated harmlessly. Anaerobic digestion (AD) has a great potential to produce clean energy biogas while treating wheat straw. AD as a typical fermentation process, the regeneration of NAD+ is achieved by fermentation using NADH to reduce metabolic intermediates and therefore there is a lack of reduction capacity in the metabolic process. The low reducing power of the digestion system and the low methane yield of wheat straw in the AD process have limited the further development of this technology. Cerium dioxide nanoparticles (CDNs) are known for their excellent reducing powers. At the same time the large amount of cerium contained in the earth provides the conditions for its large-scale use. In this study, we evaluated the effects of CDNs on the AD of wheat straw. The CDNs were successfully prepared via hydrothermal synthesis. The particle size of CDNs was about 5 nm, the measured specific surface area was 168.02 m2/g, and the zeta potential was 10.70 mV. Experiments involving volatile fatty acids (VFAs), cyclic voltammetry curves, electrochemical impedance spectroscopy, oxidation-reduction potential, and NADH/NAD+ showed that CDNs effectively enhanced the reducing power and utilization of VFAs. Additionally, the cumulative methane production of the C10 group was 20.15% higher than CK group, reaching 367.78 mL CH4/g·VS. The cumulative methane production was well fitted using the modified Gompertz equation (R2 = 0.99677). Analysis of microbial community structure showed that CDNs increased bacterial abundance in hydrolytic acidification. The reduction ability of CDNs is very important for the improvement of the methane production efficiency of AD, which is of great interest for the study of nanoparticles that promote AD.

Mathematical Model of a Thermophilic Anaerobic Digestion for Methane Production of Wheat Straw

This paper presents a newly created mathematical model of thermophilic anaerobic digestion of wheat straw carried out in a 2 dm3 bioreactor for methane production. Two batch processes, with 30 mL/dm3 and 35 mL/dm3 organic load, are carried out—one set for parameter identification and one set for model verification. The identification of model parameter values is based on dynamical experiments. It is fulfilled using two different techniques: deterministic sequential quadratic programming algorithm and metaheuristic genetic algorithm. Verification of the developed mathematical models is conducted based on the different data sets of the process. Both models predict the set of the experimental data for all considered process variables well. Genetic algorithm visually fits the data with a higher degree of accuracy, as confirmed by the numerical results for the objective function value.

Mesophilic and Thermophilic Anaerobic Digestion of Wheat Straw in a CSTR System with 'Synthetic Manure': Impact of Nickel and Tungsten on Methane Yields, Cell Count, and Microbiome.

Lignocellulosic residues, such as straw, are currently considered as candidates for biogas production. Therefore, straw fermentations were performed to quantitatively estimate methane yields and cell counts, as well as to qualitatively determine the microbiome. Six fully automated, continuously stirred biogas reactors were used: three mesophilic (41 °C) and three thermophilic (58 °C). They were fed every 8 h with milled wheat straw suspension in a defined, buffered salt solution, called ‘synthetic manure’. Total reflection X-ray fluorescence spectrometry analyses showed nickel and tungsten deficiency in the straw suspension. Supplementation of nickel and subsequently tungsten, or with an increasing combined dosage of both elements, resulted in a final concentration of approximately 0.1 mg/L active, dissolved tungsten ions, which caused an increase of the specific methane production, up to 63% under mesophilic and 31% under thermophilic conditions. That is the same optimal range for pure cultures of methanogens or bacteria found in literature. A simultaneous decrease of volatile fatty acids occurred. The Ni/W effect occurred with all three organic loading rates, being 4.5, 7.5, and 9.0 g volatile solids per litre and day, with a concomitant hydraulic retention time of 18, 10, or 8 days, respectively. A maximum specific methane production of 0.254 m3 CH4, under standard temperature and pressure per kg volatile solids (almost 90% degradation), was obtained. After the final supplementation of tungsten, the cell counts of methanogens increased by 300%, while the total microbial cell counts increased by only 3–62%. The mesophilic methanogenic microflora was shifted from the acetotrophic Methanosaeta to the hydrogenotrophic Methanoculleus (85%) by tungsten, whereas the H2-CO2-converter, Methanothermobacter, always dominated in the thermophilic fermenters.

Effect of Nano-Magnetite and Ethanol on Methane Production in the Anaerobic Digestion of Wheat Straw

HighlightsStudied mesophilic and thermophilic anaerobic fermentation of wheat straw with nano-magnetite (Fe3O4) and ethanol.The maximum biogas yield of 421.5 mL/g in mesophilic AD was 60.8% higher than control.Revealed the mechanism of nano-Fe3O4 and ethanol addition to promote biogas production.Abstract. This study investigated the mesophilic and thermophilic anaerobic fermentation of wheat straw with nano-magnetite (Fe3O4) and ethanol addition. The effects of ethanol addition on the biogas yield, the degradation of lignocellulose, and the bacterial community were explored. The results showed that the biogas yield and methane content increased as the ethanol concentration increased. The ethanol concentration of 4 g/L resulted in highest biogas yield in mesophilic anaerobic fermentation at 421.5 mL/g, which was a 60.8% improvement compared to the control. The corresponding methane content was 66.2%. For the lignocellulose degradation, the cellulose and hemicellulose contents in mesophilic anaerobic fermentation were reduced from 55.42% and 17.19% of the pretreated wheat straw to between 22.88% and 25.98% and 11.60% and 13.05%, respectively. The microbial community results showed that as the ethanol content increased, the relative abundance of Firmicutes initially increased before it decreased. Among the microbial community results, the relative abundances of Firmicutes and Bacteroides in the biogas residue of the mesophilic anaerobic fermentation with the addition of 3 g/L of ethanol were the highest, at 31.26% and 30.94%, respectively. Keywords: Anaerobic Digestion, Biogas, Ethanol, Nano-Fe3O4, Wheat Straw.

Effect of different charged Fe3O4 nanoparticles on methane production for anaerobic digestion of wheat straw

Anaerobic digestion (AD) is an effective measure to deal with organic waste, and biogas can be recovered from waste as clean energy. China is mainly an agricultural country, and a large amount of wheat straw is produced every year, which needs to be treated innocuously and resourcefully. In this context, the purpose of this study was to evaluate the effect of exogenous conductive material Fe3O4 nanoparticles on AD of wheat straw and to further evaluate the effect of the charge of nanoparticles on AD. The effects of different charged Fe3O4 nanoparticles on AD of wheat straw were studied. Fe3O4 nanoparticles with positive, negative, and no charge were successfully prepared and used them in the AD of wheat straw. The experiments were carried out in a 500-mL glass bottle with an effective volume of 350 mL and a total solid (TS) concentration of 6%. The results showed that Fe3O4 nanoparticles with positive, negative, and no charge could increase the cumulative methane production of AD, and the pure ligands used to modify Fe3O4 nanoparticles had no considerable effect on AD of wheat straw. The negatively charged nanoparticles (NNPs) had the greatest positive effects on the AD of wheat straw, which was 51.33% higher than that of the blank control group and reached 333.14 mL CH4/g·TS. The peak concentration of volatile fatty acids (VFAs) in the negative experiment was 31.65% higher than that of the blank control group at the beginning of the experiment and 30.57% lower than that of the blank control group at the end of the experiment. The cyclic voltammetry curve showed that the NNPs improved the redox performance in the AD process, and the maximum value was reflected in the methane production. NNPs can increase the metabolic rate of VFAs and the content of active substances catalyzing biochemical reactions. The charge properties of Fe3O4 nanoparticles play an important role in improving the efficiency of AD, which is of great significance for nano-electron transfer mediators to improve the efficiency of AD. These results show that NNPs have the most significant effect on AD. The addition of this material is helpful in regulating the indices in the process of AD and can be applied to the AD of more substrates.

Anaerobic Digestion of Wheat Straw Pretreated with Soaking in Water and Alkali Medium

Anaerobic Digestion of Wheat Straw Pretreated with Soaking in Water and Alkali Medium

Lignocellulose degradation, biogas production and characteristics of the microbial community in solid-state anaerobic digestion of wheat straw waste

Waste recycling is pivotal for deep space exploration or space habitation in life support systems (LSS) to enhance the material closure. This study investigated the enzymatic pretreatment and solid-state anaerobic digestion (SS-AD) of wheat straw as the major component of biomass waste in LSS for resource reclamation. Wheat straw compounds, such as cellulose, hemicellulose and lignin were significantly degraded after pretreatment with degradation at 37.47%, 46.96%, and 14.05%, respectively. SS-AD with the C/N ratio of 25 resulted in more intense lignocellulose degradation (74.20%) and more biogas yield (77.59 L/kg volatile solid) with 30 days digestion. The microbial community variation and diversity were analyzed that common fiber-degrading bacteria including Firmicutes, Bacteroidetes and Proteobacteria were dominant while the composition of the microbial genera shifted along with the digestion time. Moreover, a potential feasible strategy for biomass waste management in LSS by SS-AD was proposed.

Optimization of the Biomethane Production Process by Anaerobic Digestion of Wheat Straw Using Chemical Pretreatments Coupled with Ultrasonic Disintegration

Biomass is an attractive energy source that can be used for production of heat, power, and transport fuels and when produced and used on a sustainable basis, can make a large contribution to reducing greenhouse gas emissions. Anaerobic digestion (AD) is a suitable technology for reducing organic matter and generating bioenergy in the form of biogas. This study investigated the factors allowing the optimization of the process of biogas production from the digestion of wheat straw (WS). The statistical analysis of the experiments carried out showed that ultrasonic processing plays a fundamental role with the sonication density and solids concentration leading to improved characteristics of WS, reducing particle size, and increasing concentration of soluble chemical oxygen demand. The higher the sonicating power used, the more the waste particles are disrupted. The optimality obtained under mesophilic conditions for WS pretreated with 4% w/w (weight by weight) H2O2 at temperature 36 °C under 10 min of ultrasonication at 24 kHz with a power of 200 W improves the methane yield by 64%.

Insights of enhancing methane production under high-solid anaerobic digestion of wheat straw by calcium peroxide pretreatment and zero valent iron addition

Recent investigations verified CaO2 accelerated hydrolysis of lignocellulose, but its residue caused a harmful oxidizing environment. This study therefore proposed the combined CaO2 pretreatment and zero-valent iron (ZVI) addition to enhance high-solid anaerobic digestion of wheat straw, trying to eliminate negative effect of residual oxides. Effects of CaO2 pretreatment without/with ZVI addition on digestion performance were investigated. Results showed that in non-ZVI added digesters, as CaO2 dosage increased, total production of SCOD and VFAs increased linearly, while activities of coenzyme F420 and electron transport system were negatively correlated. Pretreatment using appropriate CaO2 dosage indirectly increased biogas production by promoting hydrolysis and acidification. Comparably, initial ORP in ZVI-added digesters sharply declined below −350 mV, which was suitable for methanogen growth. ZVI significantly improved activities of cellulase, hemicellulase, acetic kinase and coenzyme F420, with the highest improvement percentages of 138%, 48%, 35% and 58%, respectively. Since ZVI could provide electrons for conversion of CO2 to CH4, compared with non-ZVI added digesters (R0-R4), methane content in ZVI-added digesters (RZ0-RZ4) increased by 8.1%, 9.0%, 11.2%, 17.0% and 18.0% successively. Consequently, 0.6 g/g VS CaO2 pretreatment combined with 0.4 g/g VS ZVI addition achieved the maximum methane production, increasing by 35.6% compared to control digester.

ANAEROBIC DIGESTION OF WHEAT STRAW AND MICOBIOLOGICAL ASSESMENT OF THE RESULTED DIGESTATE

A study on the anaerobic digestion of wheat straw in a pilot scale bioreactor with an organic load of 2, 5, 7, 10 and 20 g/l and a process duration of 18 to 80 days was performed. The pilot bioreactor used has a computerized system for control and monitoring of various operational parameters – temperature, pH, biogas composition, etc. Total solids, total organics and volatile fatty acids were measured by standard methods and gas chromatography. Daily biogas yield and its main components (CH4, CO2, H2S) were analysed too. During the anaerobic digestion, different species of microorganisms have been isolated from the genera Bacillus, Pseudomonas, Enterococcus and Aeromonas, as well as the species Terribacillus halophilus. With a known pathogenic potential are described Pseudomonas sp., Enterococcus sp. and Aeromonas sp. Studies on the antimicrobial resistance of all isolated strains show resistance to ampicillin, amoxicillin, bacitracin, ceftriaxone, gentamicin and vancomycin. The cellulose degrading activity of some of the bacterial isolates, their pathogenic potential and antimicrobial resistance are discussed in detail in the light of the data on the mechanisms of proven resistance.

Effects of different pretreatment methods on biogas production and microbial community in anaerobic digestion of wheat straw.

The pretreatment of wheat straw has been recognized to be an essential step prior to anaerobic digestion, owing to the high abundance of lignocellulosic materials. In order to choose economical and effective techniques for the disposal of wheat straw, effects of five pretreatment methods including acid, alkali, co-pretreatment of acid and alkali, CaO2, and liquid digestate of municipal sewage sludge on anaerobic digestion of wheat straw were investigated by analyzing biogas production and organic matter degradation in the study. The results showed that among these pretreatment methods, the methane yield was highest in the liquid digestate pretreated-wheat straw with 112.6mL gTS-1, followed by the acid, alkali, and CaO2 pretreatments, and the lowest was observed in the co-pretreatment of acid and alkali. Illumina MiSeq sequencing of the microbial communities in the anaerobic digesters revealed that the genera Ruminiclostridium including Ruminiclostridium and Ruminiclostridium 1, Hydrogenispora, and Capriciproducens were the main hydrolytic bacteria, acidogenic bacteria, and acetogenic bacteria, respectively, in the anaerobic digesters. Capriciproducens and Hydrogenispora dominated in the first and the later stages, respectively, in the anaerobic digesters, which could work as indicators of the anaerobic co-digestion stage of sludge and wheat straw. The total solid and SO42--S contents of the solid digestate and the NH4+-N concentration of the liquid digestate had a significant influence on the microbial community in the digesters. These findings indicated that liquid digestate pretreatment was a potential option to improve the anaerobic digestion of wheat straw, due to the low cost without additional chemical agents.

Enhanced Anaerobic Biogas Production From Wheat Straw by Herbal-Extraction Process Residues Supplementation.

Trace metals are essential constituents of cofactors and enzymes and that their addition to anaerobic digesters increases methane production. Many trace elements are contained in herbal-extraction process residues (HPR). The present study concerns the effect of six kinds of HPR [Danshen root (Dr), Astragalus membranaceus (Am), Isatis root (Ir), Angelica sinensis (As), and Pseudo-ginseng (Pg)] that were used as additives, respectively, in the anaerobic digestion of wheat straw on biogas and methane production. The ratios of HPR residues/wheat straw [based on total solids (TS), of wheat straw] were 3, 5, and 10%, respectively. The digesters were at 37 ± 1°C of water bath during 30 days of anaerobic digestion. The results showed that HPR had significant effects on the anaerobic co-digestion. The highest biogas productivity was achieved when treated with 10% Pseudo-ginseng residues (PGR), which yielded 337 ml/g TS of biogas and 178 ml/g TS of methane. Cumulative production of biogas and methane increased by 28 and 37% compared to the production achieved in the control. These results suggest that PGR is an effective HPR to enhance the production of methane.

Effects of freezing–thawing pretreatment on anaerobic digestion of wheat straw and its kinetics analysis

In this study, freezing–thawing (FT) pretreatment of different freezing time and freezing temperatures was investigated to find the effect on anaerobic digestion (AD) of wheat straw (WS). The freezing temperature gradient is − 10 °C, − 20 °C, − 40 °C and − 80 °C, and the freezing time gradient is 12 h, 24 h, 48 h and 96 h. Total methane production exhibited a mere distance among all samples. Morphology change sculptured by SEM showed this method broke the structure of WS leaving fragments and pores in varying degrees. Three kinetic models were performed on WS to represent the behavior of experimental data. Kinetic model parameters of total methane production and lag phase time showed that logistic function model had the best fit, followed by modified Gompertz model, yet transfer function model lost efficacy in this experiment. Logistic function model was then used to reveal the influence on lag phase caused by freezing time and freezing temperature, and the results implied that FT pretreatment can shorten the lag phase time of AD, providing a 21.39% improvement under the optimal conditions of − 20 °C 96 h. The analysis of response surface regression shows that the freezing temperature has more effect on the lag phase time of anaerobic digestion than freezing time. Warmer freezing temperature of − 20 °C do better than − 80 °C on lag time, which can be achieved in most cold regions, so this treatment can occur naturally in such area without additional energy input.Graphical abstract

Methane production from wheat straw pretreated with CaO2/cellulase.

There are various lignocellulosic biomass pretreatments that act as attractive strategies to improve anaerobic digestion for methane (CH4) generation. This study proposes an effective technique to obtain more CH4via the hydrothermal coupled calcium peroxide (CaO2) co-cellulase pretreatment of lignocellulosic biomass. The total organic carbon in the hydrolysate of samples treated with 6% CaO2 and 15 mg enzyme per g-cellulose was 7330 mg L−1, which represented an increase of 92.39% over the total organic carbon value of samples hydrolyzed with the enzyme alone. The promotion of the anaerobic digestion of wheat straw followed this order: hydrothermal coupled CaO2 co-cellulase pretreatment > hydrothermal coupled CaO2 pretreatment > enzymatic pretreatment alone > control group. The sample treated with 6% CaO2 and 15 mg enzyme per g-cellulose gave the highest CH4 production with a CH4 yield of 214 mL g−1 total solids, which represented an increase of 64.81% compared to the control group. The CH4 yield decreased slightly when the amount of added cellulase exceeded 15 mg enzyme per g-cellulose.