Microbial communities involved in biogas production from wheat straw as the sole substrate within a two-phase solid-state anaerobic digestion

Microbial communities involved in biogas production from wheat straw as the sole substrate were investigated. Anaerobic digestion was carried out within an up-flow anaerobic solid-state (UASS) reactor connected to an anaerobic filter (AF) by liquor recirculation. Two lab-scale reactor systems were operated simultaneously at 37 °C and 55 °C. The UASS reactors were fed at a fixed organic loading rate of 2.5 g L⁻¹ d⁻¹, based on volatile solids. Molecular genetic analyses of the bacterial and archaeal communities within the UASS reactors (digestate and effluent liquor) and the AFs (biofilm carrier and effluent liquor) were conducted under steady-state conditions. The thermophilic UASS reactor had a considerably higher biogas and methane yield in comparison to the mesophilic UASS, while the mesophilic AF was slightly more productive than the thermophilic AF. When the thermophilic and mesophilic community structures were compared, the thermophilic system was characterized by a higher Firmicutes to Bacteroidetes ratio, as revealed by 16S rRNA gene (rrs) sequence analysis. The composition of the archaeal communities was phase-separated under thermophilic conditions, but rather stage-specific under mesophilic conditions. Family- and order-specific real-time PCR of methanogenic Archaea supported the taxonomic distribution obtained by rrs sequence analysis. The higher anaerobic digestion efficiency of the thermophilic compared to the mesophilic UASS reactor was accompanied by a high abundance of Firmicutes and Methanosarcina sp. in the thermophilic UASS biofilm.     

Discovery of Microorganisms and Enzymes Involved in High-Solids Decomposition of Rice Straw Using Metagenomic Analyses

High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production.     

Change in Microbial Numbers during Thermophilic Composting of Sewage Sludge with Reference to CO2 Evolution Rate

Dewatered sewage sludge was composted in a laboratory-scale autothermal reactor in which a constant temperature of 60°C was kept as long as possible by regulating the air feed rate. The change in CO₂ evolution rate was measured continuously from the start up through the cessation of compositing. The succession of mesophilic bacteria, thermophilic bacteria, and thermophilic actinomycetes was also observed during the composting. Specific CO₂ evolution rates of thermophilic bacteria and actinomycetes in the constant-temperature region of 60°C were assessed quantitatively. It was found that the CO₂ evolution rate was attributed to thermophilic bacteria at the initial stage of 60°C and to thermophilic actinomycetes at the later stage of 60°C.     

Effects of Temperature and Carbon-Nitrogen (C/N) Ratio on the Performance of Anaerobic Co-Digestion of Dairy Manure,Chicken Manure and Rice Straw: Focusing on Ammonia Inhibition

Anaerobic digestion is a promising alternative to disposal organic waste and co-digestion of mixed organic wastes has recently attracted more interest. This study investigated the effects of temperature and carbon-nitrogen (C/N) ratio on the performance of anaerobic co-digestion of dairy manure (DM), chicken manure (CM) and rice straw (RS). We found that increased temperature improved the methane potential, but the rate was reduced from mesophilic (30∼40°C) to thermophilic conditions (50∼60°C), due to the accumulation of ammonium nitrogen and free ammonia and the occurrence of ammonia inhibition. Significant ammonia inhibition was observed with a C/N ratio of 15 at 35°C and at a C/N ratio of 20 at 55°C. The increase of C/N ratios reduced the negative effects of ammonia and maximum methane potentials were achieved with C/N ratios of 25 and 30 at 35°C and 55°C, respectively. When temperature increased, an increase was required in the feed C/N ratio, in order to reduce the risk of ammonia inhibition. Our results revealed an interactive effect between temperature and C/N on digestion performance.     

Temperature-Driven Adaptation of the Bacterial Community in Peat Measured by Using Thymidine and Leucine Incorporation

The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25°C to 35, 45, or 55°C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55°C (thermophilic activity) and 25°C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25°C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log₁₀ ratio of TdR incorporation at 55 and 25°C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55°C 3 or 11 days before incubation at 25°C. Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.     

Methanosarcinaceae and Acetate-Oxidizing Pathways Dominate in High-Rate Thermophilic Anaerobic Digestion of Waste-Activated Sludge

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH₄/kg of VS_added. According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the Methanosarcinaceae, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.     

Adaptation of Phytoplankton-Degrading Microbial Communities to Thermal Reactor Effluent in a New Cooling Reservoir

In water column and sediment inocula from a nuclear reactor cooling reservoir, natural phytoplankton substrate labeled with ¹⁴C was used to determine aerobic and anaerobic mineralization rates for a range of temperatures (25, 40, 55, and 70°C) expected during reactor operation. For experiments that were begun during reactor shutdown, aerobic decomposition occurred at temperatures of <55°C. After 2 months of reactor operation, aerobic rates increased substantially at 55 and 70°C, although maximum rates were observed at temperatures of ≤40°C. The temperature range for which maximum anaerobic mineralization (i.e., the sum of CH₄ and CO₂) was observed was 25 to 40°C when the reactor was off, expanding to 25 to 55°C during reactor operation. Increased rates at 55°C, but not 70°C, correlated with an increase in the ratio of cumulative methane to carbon dioxide produced over 21 days. When reduced reactor power lowered the maximum temperature of the reservoir to 42°C, aerobic decomposition at 70°C was negligible, but remained substantial at 55°C. Selection for thermophilic decomposers occurred rapidly in this system in both aerobic and anaerobic communities and did not require prolonged exposure to elevated temperatures.     

Isolation and Characterization of a Fast-Growing, Thermophilic Methanobacterium Species

A thermophilic, autotrophic methanogen (strain CB12, DSM 3664) was isolated from a mesophilic biogas digestor. This bacterium used H₂-CO₂ or formate as a substrate and grew as short rods, sometimes in pairs and in crooked filaments. Motility was not observed. Its optimum temperature (56°C) was lower than that of other thermophilic members of the genus Methanobacterium. The maximum observed specific growth rate was 0.564 h⁻¹ (74-min doubling time).     

Mesophilic versus thermophilic anaerobic digestion of cattle manure: methane productivity and microbial ecology

In this study, productivity and physicochemical and microbiological (454 sequencing) parameters, as well as environmental criteria, were investigated in anaerobic reactors to contribute to the ongoing debate about the optimal temperature range for treating animal manure, and expand the general knowledge on the relation between microbiological and physicochemical process indicators. For this purpose, two reactor sizes were used (10 m³ and 16 l), in which two temperature conditions (35°C and 50°C) were tested. In addition, the effect of the hydraulic retention time was evaluated (16 versus 20 days).Thermophilic anaerobic digestion showed higher organic matter degradation (especially fiber), higher pH and higher methane (CH₄) yield, as well as better percentage of ultimate CH₄ yield retrieved and lower residual CH₄ emission, when compared with mesophilic conditions. In addition, lower microbial diversity was found in the thermophilic reactors, especially for Bacteria, where a clear intensification towards Clostridia class members was evident.Independent of temperature, some similarities were found in digestates when comparing with animal manure, including low volatile fatty acids concentrations and a high fraction of Euryarchaeota in the total microbial community, in which members of Methanosarcinales dominated for both temperature conditions; these indicators could be considered a sign of process stability.     

Characterization of wheat straw-degrading anaerobic alkali-tolerant mixed cultures from soda lake sediments by molecular and cultivation techniques

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion.     

Phylogenetic Analysis of Bacterial Communities in Mesophilic and Thermophilic Bioreactors Treating Pharmaceutical Wastewater

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32°C) and thermophilic (50 to 58°C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the β subdivision of the division Proteobacteria (β-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (α-proteobacteria, β-proteobacteria, γ-proteobacteria and δ-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity—a function of both the total number of species present (richness) and their relative distribution (evenness).     

Nutrient composition of spent wash and its impact on sugarcane growth and biochemical attributes

Nutrient composition of crude and digested spent wash and effect of their application on sugarcane growth and biochemical attributes were studied. Higher concentrations of essential nutrients (P, S, Fe, Mn, Zn, Cu) and heavy metals (Cd, Cr, Ni and Pb) were present in crude spent wash (CSW) as compared to the digested spent wash (DSW); sulphur content was the highest (765 μg ml⁻¹ in DSW and 1,609 μg ml⁻¹ in CSW) among all nutrients analyzed. Sugarcane (Saccharum spp. hybrid cultivar CoLk 8102) setts grown in soil pot culture conditions with different rates of crude spent wash (5, 10, 20 and 100 ml kg⁻¹ soil) along with digested spent wash (100 ml kg⁻¹ soil) showed improvement in bud sprouting (10.5 %), settling height (40 %), root number (9.4 %), root length (13.2 %), chlorophyll a (52.9 %) and b (55.3 %) contents and activity of catalase (98 %) enzyme over control at low rate of crude spent wash (5 ml kg⁻¹ soil). Whereas, higher doses of spent wash (20 and 100 ml kg⁻¹ soil) decreased these parameters markedly except peroxidase which was found higher at all the levels of both CSW and DSW. Findings indicated stimulatory effect of low rate of crude spent wash (5 ml kg⁻¹ soil) on root and shoot growth and inhibitory effect of higher dose (100 ml kg⁻¹ soil) of both crude and digested spent wash, therefore, judicious application of spent wash will improve crop productivity and alleviate environmental pollution problems.     

Metabolic Activity of Fatty Acid-Oxidizing Bacteria and the Contribution of Acetate, Propionate, Butyrate, and CO2 to Methanogenesis in Cattle Waste at 40 and 60°C

The quantitative contribution of fatty acids and CO₂ to methanogenesis was studied by using stirred, 3-liter bench-top digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60°C under identical loading conditions (6 g of volatile solids per liter of reactor volume per day, 10-day retention time). In the thermophilic digestor, acetate turnover increased from a prefeeding level of 16 μM/min to a peak (49 μM/min) 1 h after feeding and then gradually decreased. Acetate turnover in the mesophilic digestor increased from 15 to 40 μM/min. Propionate turnover ranged from 2 to 5.2 and 1.5 to 4.5 μM/min in the thermophilic and mesophilic digestors, respectively. Butyrate turnover (0.7 to 1.2 μM/min) was similar in both digestors. The proportion of CH₄ produced via the methyl group of acetate varied with time after feeding and ranged from 72 to 75% in the mesophilic digestor and 75 to 86% in the thermophilic digestor. The contribution from CO₂ reduction was 24 to 29% and 19 to 27%, respectively. Propionate and butyrate turnover accounted for 20% of the total CH₄ produced. Acetate synthesis from CO₂ was greatest shortly after feeding and was higher in the thermophilic digestor (0.5 to 2.4 μM/min) than the mesophilic digestor (0.3 to 0.5 μM/min). Counts of fatty acid-degrading bacteria were related to their turnover activity.     

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

The incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T_m) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K_m and k_cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.     

Anaerobic Co-digestion of Swine Manure and Microalgae <Emphasis Type="Italic">Chlorella</Emphasis> sp.: Experimental Studies and Energy Analysis

Integration of algae production with livestock waste management has the potential to recover energy and nutrients from animal manure, while reducing discharges of organic matter, pathogens, and nutrients to the environment. In this study, microalgae Chlorella sp. were grown on centrate from anaerobically digested swine manure. The algae were harvested for mesophilic anaerobic digestion (AD) with swine manure for bioenergy production. Low biogas yields were observed in batch AD studies with algae alone, or when algae were co-digested with swine manure at ≥43 % algae (based on volatile solids [VS]). However, co-digestion of 6–16 % algae with swine manure produced similar biogas yields as digestion of swine manure alone. An average methane yield of 190 mL/g VS_fed was achieved in long-term semi-continuous co-digestion studies with 10?±?3 % algae with swine manure. Data from the experimental studies were used in an energy analysis assuming the process was scaled up to a concentrated animal feeding operation (CAFO) with 7000 pigs with integrated algae-based treatment of centrate and co-digestion of manure and the harvested algae. The average net energy production for the system was estimated at 1027 kWh per day. A mass balance indicated that 58 % of nitrogen (N) and 98 % of phosphorus (P) in the system were removed in the biosolids. A major advantage of the proposed process is the reduction in nutrient discharges compared with AD of swine waste without algae production.     

Male-Specific Coliphages as Indicators of Thermal Inactivation of Pathogens in Biosolids

Male-specific (F⁺) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F⁺ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F⁺ DNA and F⁺ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F⁺ RNA phages. Separate experiments on individual isolates at 53°C confirmed the apparent heat resistance of group I F⁺ RNA coliphages as well as the susceptibility of group III F⁺ RNA coliphages. Although few F⁺ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F⁺ RNA phages. Hence, F⁺ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F⁺ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment.     

Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.     

A comparison study on the high-rate co-digestion of sewage sludge and food waste using a temperature-phased anaerobic sequencing batch reactor system

Assessing contemporary anaerobic biotechnologies requires proofs on reliable performance in terms of renewable bioenergy recovery such as methane (CH₄) production rate, CH₄ yield while removing volatile solid (VS) effectively. This study, therefore, aims to evaluate temperature-phased anaerobic sequencing batch reactor (TPASBR) system that is a promising approach for the sustainable treatment of organic fraction of municipal solid wastes (OFMSW). TPASBR system is compared with a conventional system, mesophilic two-stage anaerobic sequencing batch reactor system, which differs in operating temperature of 1st-stage. Results demonstrate that TPASBR system can obtain 44% VS removal from co-substrate of sewage sludge and food waste while producing 1.2 m³CH₄/m³_system/d (0.2 m³CH₄/kgVS_added) at organic loading rate of 6.1 gVS/L/d through the synergy of sequencing-batch operation, co-digestion, and temperature-phasing. Consequently, the rapid and balanced anaerobic metabolism at thermophilic stage makes TPASBR system to afford high organic loading rate showing superior performance on OFMSW stabilization.     

Characteristics of Mesophilic Bacteria Isolated during Thermophilic Composting of Sewage Sludge

The degree of inactivation by UV irradiation was different between vegetative cells and spores of bacteria isolated from sewage sludge composting at 60°C. By using this property, a method to estimate the spore ratio of a mixture of vegetative cells and spores was presented. This UV irradiation method was applied to the estimation of the spore ratio of sewage sludge compost samples collected at several stages of composting. The spore ratio of mesophilic bacteria in the samples obtained at the thermophilic stage of 60°C was 40% at most. The vegetative form of mesophilic bacteria showed a thermotolerance property at 60°C by forming colonies but showed no respiratory activity at that temperature.     

Comparison of Seven Chemical Pretreatments of Corn Straw for Improving Methane Yield by Anaerobic Digestion

Agriculture straw is considered a renewable resource that has the potential to contribute greatly to bioenergy supplies. Chemical pretreatment prior to anaerobic digestion can increase the anaerobic digestibility of agriculture straw. The present study investigated the effects of seven chemical pretreatments on the composition and methane yield of corn straw to assess their effectiveness of digestibility. Four acid reagents (H₂SO₄, HCl, H₂O₂, and CH₃COOH) at concentrations of 1%, 2%, 3%, and 4% (w/w) and three alkaline reagents (NaOH, Ca(OH)₂, and NH₃·H₂O) at concentrations of 4%, 6%, 8%, and 10% (w/w) were used for the pretreatments. All pretreatments were effective in the biodegradation of the lignocellulosic straw structure. The straw, pretreated with 3% H₂O₂ and 8% Ca(OH)₂, acquired the highest methane yield of 216.7 and 206.6 mL CH₄ g VS ⁻¹ in the acid and alkaline pretreatments, which are 115.4% and 105.3% greater than the untreated straw. H₂O₂ and Ca(OH)₂ can be considered as the most favorable pretreatment methods for improving the methane yield of straw because of their effectiveness and low cost.