Bioaugmentation by enriched hydrogenotrophic methanogens into trickle bed reactors for H2/CO2 conversion.

Biomethanation represents a promising approach for biomethane production, with biofilm-based processes like trickle bed reactors (TBRs) being among the most efficient solutions. However, maintaining stable performance can be challenging, and both pure and mixed culture approaches have been applied to address this. In this study, inocula enriched with hydrogenotrophic methanogens were introduced to to TBRs as bioaugmentation strategy to assess their impacts on the process performance and microbial community dynamics. Metagenomic analysis revealed a metagenome-assembled genome belonging to the hydrogenotrophic genus Methanobacterium, which became dominant during enrichment and successfully colonized the TBR biofilm after bioaugmentation. The TBRs achieved a biogas production with > 96 % methane. The bioaugmented reactor consumed additional H2. This may be due to microbial species utilizing CO2 and H2 via various CO2 reduction pathways. Overall, implementing bioaugmentation in TBRs showed potential for establishing targeted species, although challenges remain in managing H2 consumption and optimizing microbial interactions.

Metaproteomic Analysis of Biogas Plants: A Complete Workflow from Lab to Bioinformatics.

Metaproteomics represents a promising and fast method to analyze the taxonomic and functional composition of biogas plant microbiomes. However, metaproteomics sample preparation and bioinformatics analysis is still challenging due to the sample complexity and contaminants. In this chapter, a tailored workflow including sampling, phenol extraction in a ball mill, amido black protein quantification, FASP digestion, LC-MS/MS measurement as well as bioinformatics and biostatistical data evaluation are here described for the metaproteomics advancements applied to biogas plant samples.

Pre-treatment with Trichoderma viride: Towards a better understanding of its consequences for anaerobic digestion.

Understanding and optimising biological pre-treatment strategies for enhanced bio-methane production is a central aspect in second-generation biofuel research. In this regard, the application of fungi for pre-treatment seems highly promising; however, understanding the mode of action is crucial. Here, we show how aerobic pre-treatment of crystalline cellulose with the cellulolytic Trichoderma viride affects substrate degradability during mesophilic, anaerobic digestion. It could be demonstrated that fungal pre-treatment resulted in a slightly reduced substrate mass. Nevertheless, no significant impact on the overall methane yield was found during batch fermentation. Short chain organic acids accumulation, thus, overall degradation dynamics including methane production kinetics were affected by the pre-treatment as shown by Gompertz modelling. Finally, 16S rRNA amplicon sequencing followed by ANCOM-BC resulted in up to 53 operative taxonomic units including fermentative, syntrophic and methanogenic taxa, whereby their relative abundances were significantly affected by fungal pre-treatment depending on the duration of the pre-treatment. The results demonstrated the impact of soft rot fungal pre-treatment of cellulose on subsequent anaerobic cellulose hydrolysis as well as on methanogenic activity. To the best of our knowledge, this is the first study to investigate the direct causal effects of pre-treatment with T. viride on basic but crucial anaerobic digestion parameters in a highly standardised approach.

High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform.

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.

Industrializing methanotrophs and other methylotrophic bacteria: from bioengineering to product recovery.

Microbes that use the single-carbon substrates methanol and methane offer great promise to bioindustry along with substantial environmental benefits. Methanotrophs and other methylotrophs can be engineered and optimized to produce a wide range of products, from biopolymers to biofuels and beyond. While significant limitations remain, including delivery of single-carbon feedstock to bioreactors, efficient growth, and scale-up, these challenges are being addressed and notable improvements have been rapid. Development of expression chassis, use of genome-scale and regulatory models based on omics data, improvements in bioreactor design and operation, and development of green product recovery schemes are enabling the rapid development of single-carbon bioconversion in the industrial space.

Chromosomal integration of the pSOL1 megaplasmid of Clostridium acetobutylicum for continuous and stable advanced biofuels production.

Biofuel production by Clostridium acetobutylicum is compromised by strain degeneration due to loss of its pSOL1 megaplasmid. Here we used engineering biology to stably integrate pSOL1 into the chromosome together with a synthetic isopropanol pathway. In a membrane bioreactor continuously fed with glucose mineral medium, the final strain produced advanced biofuels, n-butanol and isopropanol, at high yield (0.31 g g-1), titre (15.4 g l-1) and productivity (15.5 g l-1 h-1) without degeneration.

The potential of Bacillus species isolated from Cinnamomum camphora for biofuel production.

BACKGROUND: Increasing concerns about climate change and global petroleum supply draw attention to the urgent need for the development of alternative methods to produce fuels. Consequently, the scientific community must devise novel ways to obtain fuels that are both sustainable and eco-friendly. Bacterial alkanes have numerous potential applications in the industry sector. One significant application is biofuel production, where bacterial alkanes can serve as a sustainable eco-friendly alternative to fossil fuels. This study represents the first report on the production of alkanes by endophytic bacteria. RESULTS: In this study, three Bacillus species, namely Bacillus atrophaeus Camph.1 (OR343176.1), Bacillus spizizenii Camph.2 (OR343177.1), and Bacillus aerophilus Camph.3 (OR343178.1), were isolated from the leaves of C. camphora. The isolates were then screened to determine their ability to produce alkanes in different culture media including nutrient broth (NB), Luria-Bertani (LB) broth, and tryptic soy broth (TSB). Depending on the bacterial isolate and the culture media used, different profiles of alkanes ranging from C8 to C31 were detected. CONCLUSIONS: The endophytic B. atrophaeus Camph.1 (OR343176.1), B. spizizenii Camph.2 (OR343177.1), and B. aerophilus Camph.3 (OR343178.1), associated with C. camphora leaves, represent new eco-friendly approaches for biofuel production, aiming towards a sustainable future. Further research is needed to optimize the fermentation process and scale up alkane production by these bacterial isolates.

Construction of Xylose-Utilizing Cyanobacterial Chassis for Bioproduction Under Photomixotrophic Conditions.

Xylose is a major component of lignocellulose and the second most abundant sugar present in nature after glucose; it, therefore, has been considered to be a promising renewable resource for the production of biofuels and chemicals. However, no natural cyanobacterial strain is known capable of utilizing xylose. Here, we take the fast-growing cyanobacteria Synechococcus elongatus UTEX 2973 as an example to develop the synthetic biology-based methodology of constructing a new xylose-utilizing cyanobacterial chassis with increased acetyl-CoA for bioproduction.

A comparative assessment of microbial biodiesel and its life cycle analysis.

Biodiesel is a type of sustainable, biodegradable energy made from natural sources like vegetable oils, animal fat, and from microbes. Unlike traditional diesel, it has a lower carbon footprint and produces fewer harmful emissions when burned. Biodiesel has gained popularity as a more sustainable substitute for hydrocarbon-based diesel and may be utilized in diesel engines without any modification. In this review, biodiesel from microorganisms such as algae, yeast, and fungi and advantages over another feedstock were discussed. The life cycle evaluation of biodiesel is a thorough assessment of the ecological and economic effects of biodiesel production and use, from the extraction of raw ingredients to the waste disposal process. The life cycle analysis considers the entire process, including the production of feedstocks, the production of biodiesel, and the use of biodiesel in vehicles and other applications. Life cycle analysis of biodiesel produced from microorganisms takes into consideration the environmental impact and sustainability of each step in the production process, including the impact on land use, water use, greenhouse gas emissions, and the availability of resources. In this section, biodiesel produced from microorganisms and other raw materials, its comparisons, and also steps involved in the life cycle such as the cultivation of microorganisms, harvesting of biomass, and conversion to biodiesel were discussed. The processes like extraction and purification, hydrothermal liquefaction, and their environmental impacts were examined by using various LCA software from the previously mentioned process.

Appraising the phycoremediation potential of cyanobacterial strains Phormidium and Oscillatoria for nutrient removal from textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass.

In this study, phycoremediation of textile wastewater (TWW) by freshwater cyanobacterial strains such as sp., Oscillatoria sp. F01 and Oscillatoria sp. F02 was evaluated, and lipids were simultaneously extracted from biomass for biodiesel production. Onset of the study, Phormidium sp. and Oscillatoria sp. F01 has better growth rates, increased biomass production, high chlorophyll content, and efficient nutrient utilization in TWW compared to Oscillatoria sp. F02. Phormidium sp. showed 1.41 g/L dry weight, followed by Oscillatoria sp. F01 with 1.39 g/L and Oscillatoria sp. F02 with 1.02 g/L biomass. Both strains demonstrated their capability to elevate the pH level while reducing TDS and eliminating/reducing several nutrients such as nitrates, nitrites, phosphates, sulphates, sulphides, chlorides, calcium, sodium, and magnesium. Further, the total lipids extracted from the TWW-grown Phormidium sp., Oscillatoria sp. F01 and Oscillatoria sp. F02 was estimated to be 8.20, 13.70 and 11.20 %, respectively, on day 21, which was higher than the lipid content obtained from control cultures. Further, biodiesel produced from the lipids of all strains showed higher levels of C12:0, C16:0, C16:1, C18:1, C18:2, and C18:3 among all the fatty acids. Therefore, they can potentially offer a valuable source of lipids and diverse fatty acids for high-quality biodiesel production. This integrated system not only offers a solution for TWW treatment but also provides a feedstock for renewable fuel production simultaneously.

Enhancing biogas generation from lignocellulosic biomass through biological pretreatment: Exploring the role of ruminant microbes and anaerobic fungi.

Biogas production from Lignocellulosic Biomass (LB) via anaerobic digestion (AD) has gained attention for its potential in self-sustainability. However, the recalcitrance of LB cell walls pose a challenge to its degradability and biogas generation. Therefore, pretreatment of LB is necessary to enhance lignin removal and increase degradability. Among the different approaches, environmentally friendly biological pretreatment ispromising as it avoids the production of inhibitors. The ruminal microbial community, including anaerobic fungi, bacteria, and protozoa, has shown an ability to effectively degrade LB through biomechanical and microbial penetration of refractory cell structures. In this review, we provide an overview of ruminant microbes dominating LB's AD, their degradation mechanism, and the bioaugmentation of the rumen. We also explore the potential cultivation of anaerobic fungi from the rumen, their enzyme potential, and their role in AD. The rumen ecosystem, comprising both bacteria and fungi, plays a crucial role in enhancing AD. This comprehensive review delves into the intricacies of ruminant microorganisms' adhesion to plant cells, elucidates degradation mechanisms, and explores integrated pretreatment approaches for the effective utilization of LB, minimizing the impact of inhibitors. The discussion underscores the considerable potential of ruminant microbes in pretreating LB, paving the way for sustainable biogas production. Optimizing fungal colonization and ligninolytic enzyme production, such as manganese peroxidase and laccase, significantly enhances the efficiency of fungal pretreatment. Integrating anaerobic fungi through bioaugmentation during mainstream processing demonstrably increases methane production. This study opens promising avenues for further research and development of these microorganisms for bioenergy production.

Nitrogen and phosphorus stress as a tool to induce lipid production in microalgae.

Microalgae, capable of accumulating large amounts of lipids, are of great value for biodiesel production. The high cost of such production stimulates the search for cultivation conditions that ensure their highest productivity. Reducing the content of nitrogen and phosphorus in the culture medium is widely used to change the content and productivity of lipids in microalgae. Achieving the right balance between maximum growth and maximum lipid content and productivity is the primary goal of many experimental works to ensure cost-effective biodiesel production from microalgae. The content of nitrogen and phosphorus in nutrient media for algal cultivation after converted to nitrogen (-N) and phosphorus (-P) lies in an extensive range: from 0.007 g L- 1 to 0.417 g L- 1 and from 0.0003 g L- 1 to 0.227 g L- 1 and N:P ratio from 0.12:1 to 823.33:1. When studying nutritional stress in microalgae, no single approach is used to determine the experimental concentrations of nitrogen and phosphorus. This precludes the possibility of correct interpretation of the data and may lead to erroneous conclusions. This work results from the systematisation of information on using nitrogen and phosphorus restriction to increase the lipid productivity of microalgae of different taxonomic and ecological groups to identify future research directions. The results of 301 experiments were included in the analysis using the principal components method. The investigation considered various divisions and classes: Cyanobacteria, Rhodophyta, Dinophyta, Haptophyta, Cryptophyta, Heterokontophyta/Ochrophyta (Bacillariophyceae, Eustigmatophyceae, Xanthophyceae), Chlorophyta, and also the ratio N:P, the time of the experiment, the light intensity during cultivation. Based on the concentrations of nitrogen and phosphorus existing in various nutrient media, a general scheme for designating the supply of nutrient media for nitrogen (as NO3- or NH4+, N g L- 1) and phosphorus (as РO4-, P g L- 1) has been proposed: replete -N (˃0.4 g L- 1), moderate -N (0.4-0.2), moderate N-limitation (0.19-0.1), strong N-limitation (˂0.1), without nitrogen (0), replete -Р (˃0.2), moderate -P (0.2-0.02), moderate P-limitation (0.019-0.01), strong P-limitation (˂0.01), without phosphorus (0).

Overview of microalgae and cyanobacteria-based biostimulants produced from wastewater and CO2 streams towards sustainable agriculture: A review.

For a long time, marine macroalgae (seaweeds) have been used to produce commercial biostimulants in order to ensure both productivity and quality of agricultural crops under abiotic stress. With similar biological properties, microalgae have slowly attracted the scientific community and the biostimulant industry, in particular because of their ability to be cultivated on non-arable lands with high biomass productivity all year long. Moreover, the recent strategies of culturing these photosynthetic microorganisms using wastewater and CO2 opens the possibility to produce large quantity of biomass at moderate costs while integrating local and circular economy approaches. This paper aims to provide a state of the art review on the development of microalgae and cyanobacteria based biostimulants, focusing on the different cultivation, extraction and application techniques available in the literature. Emphasis will be placed on microalgae and cyanobacteria cultivation using liquid and gaseous effluents as well as emerging green-extraction approaches, taking in consideration the actual European regulatory framework.

Rhizopus stolonifer biomass catalytic transesterification capability: optimization of cultivation conditions.

BACKGROUND: Using fungal biomass for biocatalysis is a potential solution for the expensive cost of the use o enzymes. Production of fungal biomass with effective activity requires optimizing the cultivation conditions. RESULTS: Rhizopus stolonifer biomass was optimized for transesterification and hydrolysis of waste frying oil (WFO). Growth and biomass lipolytic activities of R. stolonifer improved under shaking conditions compared to static conditions, and 200 rpm was optimum. As biomass lipase and transesterification activities inducer, olive oil was superior to soybean, rapeseed, and waste frying oils. Biomass produced in culture media containing fishmeal as an N-source feedstock had higher lipolytic capabilities than corn-steep liquor and urea. Plackett Burman screening of 9 factors showed that pH (5-9), fishmeal (0.25-1.7%, w/v), and KH2PO4 (0.1-0.9%, w/v) were significant factors with the highest main effect estimates 11.46, 10.42, 14.90, respectively. These factors were selected for response surface methodology (RSM) optimization using central composite design (CCD). CCD models for growth, biomass lipase activity, and transesterification capability were significant. The optimum conditions for growth and lipid modification catalytic activities were pH 7.4, fishmeal (2.62%, w/v), and KH2PO4 (2.99%, w/v). CONCLUSION: Optimized culture conditions improved the whole cell transesterification capability of Rhizopus stolonifer biomass in terms of fatty acid methyl ester (FAME) concentration by 67.65% to a final FAME concentration of 85.5%, w/w.

Quantification of indicator and pathogenic bacteria in manures and digestates from three agricultural biogas plants over a one-year period.

Interest in the conversion of manure in biogas via anaerobic digestion (AD) is growing, but questions remain about the biosafety of digestates. For a period of one year, we monitored the impact of three mesophilic agricultural biogas plants (BPs) mainly fed with pig manure (BP1, BP3) or bovine manure (BP2) on the physicochemical parameters, the composition of the microbial community and the concentration of bacteria (E. coli, enterococci, Salmonella, Campylobacter, Listeria monocytogenes, Clostridium perfringens, Clostridium botulinum and Clostridioides difficile). The BP2 digestate differed from those of the two other BPs with a higher nitrogen content, more total solids and greater abundance of Clostridia MBA03 and Disgonomonadacea. Persistence during digestion ranked from least to most, was: Campylobacter (1.6 to >2.9 log10 reduction, according to the BP) < E. coli (1.8 to 2.2 log10) < Salmonella (1.1 to 1.4 log10) < enterococci (0.2 to 1.2 log10) and C. perfringens (0.2 to 1 log10) < L. monocytogenes (-1.2 to 1.6 log10) < C. difficile and C. botulinum (≤0.5 log10). No statistical link was found between the reduction in the concentration of the targeted bacteria and the physicochemical and operational parameters likely to have an effect (NH3, volatile fatty acids and total solids contents, hydraulic retention time, presence of co-substrates), underlining the fact that the fate of the bacteria during mesophilic digestion depends on many interacting factors. The reduction in concentrations varied significantly over the sampling period, underlining the need for longitudinal studies to estimate the impact of AD on pathogenic microorganisms.

Performance of co-culture strategy on nutrient removal and biogas upgrading by strigolactone induction.

In this study, we investigated the performance and elucidated the synergistic effects of microalgae-fungi symbionts co-cultured with 10-7 and 10-9  mol L-1 of GR24 and supplemented with endophytic bacteria, multi-walled carbon nanotubes (MWCNTs) or vitamin B12 (VB12), on nutrient removal and biogas upgrading. The results showed that the microalgae-fungi-bacteria symbiotic system co-cultured with 10-9  mol L-1 GR24 presented the optimal growth performance of 0.368 ± 0.04 d-1 , chlorophyll a of 249.36 ± 22.31 µg L-1 , and extracellular carbonic anhydrase activity of 42.55 ± 3.755 enzyme units. In this co-culture system, the organic matter, nutrients, and CO2 purification obtained the highest removal efficiency, with 81.35 ± 7.96% for chemical oxygen demand, 83.56 ± 7.91% total nitrogen, 84.17 ± 7.95% total phosphorus, and 63.72 ± 6.06% CO2 . The symbiont system also greatly increased the methane content in the biogas by 30.67%. The remarkable performance of the microalgae-fungi-bacteria symbiotic system shows its ability to be broadly applied in simultaneous biogas upgrading and wastewater treatment. PRACTITIONER POINTS: The optimal GR24 concentration for microalgae-fungi consortia was 10-9  M. Endophytic bacteria were superior to MWCNTs and VB12. Fungi-algae-bacteria consortia presented excellent growth and removal performance. Removal efficiencies of COD, TN, and TP were about 81% under optimum treatment.

Inter-kingdom interactions and stability of methanogens revealed by machine-learning guided multi-omics analysis of industrial-scale biogas plants.

Multi-omics analysis is a powerful tool for the detection and study of inter-kingdom interactions, such as those between bacterial and archaeal members of complex biogas-producing microbial communities. In the present study, the microbiomes of three industrial-scale biogas digesters, each fed with different substrates, were analysed using a machine-learning guided genome-centric metagenomics framework complemented with metatranscriptome data. This data permitted us to elucidate the relationship between abundant core methanogenic communities and their syntrophic bacterial partners. In total, we detected 297 high-quality, non-redundant metagenome-assembled genomes (nrMAGs). Moreover, the assembled 16 S rRNA gene profiles of these nrMAGs showed that the phylum Firmicutes possessed the highest copy number, while the representatives of the archaeal domain had the lowest. Further investigation of the three anaerobic microbial communities showed characteristic alterations over time but remained specific to each industrial-scale biogas plant. The relative abundance of various microorganisms as revealed by metagenome data was independent from corresponding metatranscriptome activity data. Archaea showed considerably higher activity than was expected from their abundance. We detected 51 nrMAGs that were present in all three biogas plant microbiomes with different abundances. The core microbiome correlated with the main chemical fermentation parameters, and no individual parameter emerged as a predominant shaper of community composition. Various interspecies H2/electron transfer mechanisms were assigned to hydrogenotrophic methanogens in the biogas plants that ran on agricultural biomass and wastewater. Analysis of metatranscriptome data revealed that methanogenesis pathways were the most active of all main metabolic pathways.

Impact of carbon monoxide on performance and microbial community of extreme-thermophilic hydrogenotrophic methanation in horizontal rotary bioreactor.

A novel horizontal rotary bioreactor was developed for upgrading biogas from coke oven gas at extreme-thermophilic condition. The introduction of CO decreased the outlet methane content from 80% to 50% due to insufficient H2. This hindrance was overcome by increasing the proportion of incoming hydrogen, coupled with a prolonged gas retention time from 24 to 72 h, leading to a restoration of methane content to 91.6%. Notably, CO and CO2 exhibited a competitive relationship to hydrogen, which was determined by their contents. The substitution of Methanothermobacter for Methanobacterium as the dominant genus was observed at 70 °C, with relative abundance exceeding 98%. Incorporation of CO increased bacteria diversity and fostered a syntrophic relationship between the bacterial community and M. thermautotrophicus. This study provides both theoretical basis and practical support for biogas upgrading from coke oven gas using a biofilm reactor, thus aiding its future industrialization prospects.

Unraveling prevalence of homoacetogenesis and methanogenesis pathways due to inhibitors addition.

Three inhibitors targeting different microorganisms, both from Archaea and Bacteria domains, were evaluated for their effect on CO2 biomethanation: sodium ionophore III (ETH2120), carbon monoxide (CO), and sodium 2-bromoethanesulfonate (BES). This study examines how these compounds affect the anaerobic digestion microbiome in a biogas upgrading process. While archaea were observed in all experiments, methane was produced only when adding ETH2120 or CO, not when adding BES, suggesting archaea were in an inactivated state. Methane was produced mainly via methylotrophic methanogenesis from methylamines. Acetate was produced at all conditions, but a slight reduction on acetate production (along with an enhancement on CH4 production) was observed when applying 20 kPa of CO. Effects on CO2 biomethanation were difficult to observe since the inoculum used was from a real biogas upgrading reactor, being this a complex environmental sample. Nevertheless, it must be mentioned that all compounds had effects on the microbial community composition.

Taxonomic and enzymatic basis of the cellulolytic microbial consortium KKU-MC1 and its application in enhancing biomethane production.

Lignocellulosic biomass is a promising substrate for biogas production. However, its recalcitrant structure limits conversion efficiency. This study aims to design a microbial consortium (MC) capable of producing the cellulolytic enzyme and exploring the taxonomic and genetic aspects of lignocellulose degradation. A diverse range of lignocellulolytic bacteria and degrading enzymes from various habitats were enriched for a known KKU-MC1. The KKU-MC1 was found to be abundant in Bacteroidetes (51%), Proteobacteria (29%), Firmicutes (10%), and other phyla (8% unknown, 0.4% unclassified, 0.6% archaea, and the remaining 1% other bacteria with low predominance). Carbohydrate-active enzyme (CAZyme) annotation revealed that the genera Bacteroides, Ruminiclostridium, Enterococcus, and Parabacteroides encoded a diverse set of cellulose and hemicellulose degradation enzymes. Furthermore, the gene families associated with lignin deconstruction were more abundant in the Pseudomonas genera. Subsequently, the effects of MC on methane production from various biomasses were studied in two ways: bioaugmentation and pre-hydrolysis. Methane yield (MY) of pre-hydrolysis cassava bagasse (CB), Napier grass (NG), and sugarcane bagasse (SB) with KKU-MC1 for 5 days improved by 38-56% compared to non-prehydrolysis substrates, while MY of prehydrolysed filter cake (FC) for 15 days improved by 56% compared to raw FC. The MY of CB, NG, and SB (at 4% initial volatile solid concentration (IVC)) with KKU-MC1 augmentation improved by 29-42% compared to the non-augmentation treatment. FC (1% IVC) had 17% higher MY than the non-augmentation treatment. These findings demonstrated that KKU-MC1 released the cellulolytic enzyme capable of decomposing various lignocellulosic biomasses, resulting in increased biogas production.