Constructing metabolic pathways for biogas production at low temperatures through metagenomic and metatranscriptomic analyses

BUDIANTO; Diah KUSMARDINI; Krysnha MURTY; NURFADHILAH; Gito Tri Putra ISWANDI

doi:10.55779/nsb17312374

Authors

BUDIANTO Institute Science and Technology Al-Kamal, Chemical Engineering Department, Jl. Raya Al-Kamal No. 2, Kedoya Selatan, Kebon Jeruk, Jakarta Barat 11520 (ID) https://orcid.org/0000-0002-8277-6202
Diah KUSMARDINI Institute Science and Technology Al-Kamal, Chemical Engineering Department, Jl. Raya Al-Kamal No. 2, Kedoya Selatan, Kebon Jeruk, Jakarta Barat 11520 (ID) https://orcid.org/0000-0002-5052-334X
Krysnha MURTY Institute Science and Technology Al-Kamal, Chemical Engineering Department, Jl. Raya Al-Kamal No. 2, Kedoya Selatan, Kebon Jeruk, Jakarta Barat 11520 (ID) https://orcid.org/0009-0009-5861-6987
NURFADHILAH Institute Science and Technology Al-Kamal, Chemical Engineering Department, Jl. Raya Al-Kamal No. 2, Kedoya Selatan, Kebon Jeruk, Jakarta Barat 11520 (ID) https://orcid.org/0009-0003-5277-6863
Gito Tri Putra ISWANDI Institute Science and Technology Al-Kamal, Chemical Engineering Department, Jl. Raya Al-Kamal No. 2, Kedoya Selatan, Kebon Jeruk, Jakarta Barat 11520 (ID) https://orcid.org/0009-0006-9209-3532

DOI:

https://doi.org/10.55779/nsb17312374

Keywords:

acidogenesis, acetogenesis, COGs, methanogenesis, 16S rDNA, 16S mRNA, 16S rRNA

Abstract

This study aimed to determine the metabolic pathways based on the abundance of microorganisms and the activity of genetic clusters of orthologous groups of proteins (COGs) involved in biogas production. Cow manure and tofu waste were used as substrates in a 1:1 ratio. The fermentation process was carried out in a glass reactor at 10-12 °C using long-term inoculum derived from active sludge. Metagenomic analysis based on 16S rDNA sequences and metatranscriptomic analysis (16S rRNA) were performed. The results showed that the average biogas production at low temperatures was only 0.73 L/day, with CH₄ levels exceeding 0.517 L/g VS. Clostridium cellulovorans dominated the microbial community with a relative abundance of 24%. COGs activities such as glucosidase and periplasmic β-galactosidase were predominant during the hydrolysis stage, while 3-hydroxyacyl-CoA dehydrogenase dominated acidogenesis, and acetate kinase was exclusively found in the acetogenesis stage. Biogas (CH₄) formation was primarily driven by the H₂/CO₂ pathway (49.2%), involving formylmethanofuran dehydrogenase and H₄MPT S-methyltransferase from archaeal genera Methanocorpusculum, Methanobrevibacter, and Methanobacterium. These findings provide insights into the microbial and enzymatic mechanisms underlying biogas production at low temperatures. Enhancing biogas yield under such conditions can be achieved by prioritizing the hydrogenotrophic pathway during methanogenesis and managing VFA accumulation during acidogenesis. Genetic engineering to enhance the expression of key enzymes and control specific microbes such as Syntrophomonas zehnderi may overcome enzymatic limitations at low temperatures, accelerate the conversion of CO₂ and H₂ into methane, and minimize inhibition of the methanogenesis process.

Metrics

Metrics Loading ...

References

Amato P, Christner BC (2009). Energy metabolism response to low-temperature and frozen conditions in Psychrobacter cryohalolentis. Applied and Environmental Microbiology 75(3):711-718. https://doi.org/10.1128/AEM.02193-08

Budianto B, Okta FZ, Yasin RE. The role of bacterial and archaea in determining the metabolic pathway of biogas fermentation at low temperatures (2024). Acta Biológica Colombiana 29(1):99-111. https://doi.org/10.15446/abc.v29n1.106266

DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, ... Andersen GL (2006). Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and Environmental Microbiology 72(7):5069-5072. https://doi.org/10.1128/AEM.03006-05

Farrell J, Rose A (1967). Temperature effects on microorganisms. Annual Reviews in Microbiology 21(1):101-120. https://doi.org/10.1146/annurev.mi.21.100167.000533

Gaboyer F, Burgaud G, Alain K (2015). Physiological and evolutionary potential of microorganisms from the Canterbury Basin subseafloor, a metagenomic approach. FEMS Microbiology Ecology 91(5):fiv029. https://doi.org/10.1093/femsec/fiv029

Galperin MY, Kristensen D M, Makarova KS, Wolf YI, Koonin EV (2019). Microbial genome analysis: the COG approach. Briefings in Bioinformatics 20(4):1063-1070. https://doi.org/10.1093/bib/bbx117

Harirchi S, Wainaina S, Sar T, Nojoumi SA, Parchami M, Parchami M, Varjani S, Khanal SK, Wong J, Awasthi MK (2022). Microbiological insights into anaerobic digestion for biogas, hydrogen or volatile fatty acids (VFAs): a review. Bioengineered 13(3):6521-6557. https://doi.org/10.1080/21655979.2022.2035986

Hassa J, Wibberg D, Maus I, Pühler A, Schlüter A (2019). Genome analyses and genome-centered metatranscriptomics of Methanothermobacter wolfeii strain SIV6, isolated from a thermophilic production-scale biogas fermenter. Microorganisms 8(1):13. https://doi.org/10.3390/microorganisms8010013

He Y, Caporaso J G, Jiang XT, Sheng HF, Huse SM, Rideout JR, Edgar R C, Kopylova E, Walters WA, Knight R (2015). Stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity. Microbiome 3:20. https://doi.org/10.1186/s40168-015-0081-x

Kataki S, Chatterjee S, Vairale MG, Sharma S, Dwivedi SK, Gupta DK (2021). Constructed wetland, an eco-technology for wastewater treatment: A review on various aspects of microbial fuel cell integration, low temperature strategies and life cycle impact of the technology. Renewable and Sustainable Energy Reviews 148:111261. https://doi.org/10.1016/j.rser.2021.111261

Li A, Chu Y, Wang X, Ren L, Yu J, Liu X, Yan J, Zhang L, Wu S, Li S (2013). A pyrosequencing-based metagenomic study of methane-producing microbial community in solid-state biogas reactor. Biotechnology for Biofuels 6:3. https://doi.org/10.1186/1754-6834-6-3

Li Y, Meng Z, Xu Y, Shi Q, Ma Y, Aung M, Cheng Y, Zhu W (2021). Interactions between anaerobic fungi and methanogens in the rumen and their biotechnological potential in biogas production from lignocellulosic materials. Microorganisms 9(1):190. https://doi.org/10.3390/microorganisms9010190

Liu C, Wachemo AC, Tong H, Shi S, Zhang L, Yuan, H, Li X (2018). Biogas production and microbial community properties during anaerobic digestion of corn stover at different temperatures. Bioresource Technology 261:93-103. https://doi.org/10.1016/j.biortech.2017.12.076

Liu FH, Wang SB, Zhang JS, Zhang J, Yan X, Zhou HK, Zhao GP, Zhou ZH (2009). The structure of the bacterial and archaeal community in a biogas digester as revealed by denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis. Journal of Applied Microbiology 106(3):952-966. https://doi.org/10.1111/j.1365-2672.2008.04064.x

Markowski M, Białobrzewski I, Zieliński M, Dębowski M, Krzemieniewski M (2014). Optimizing low-temperature biogas production from biomass by anaerobic digestion. Renewable Energy 69:219-225. https://doi.org/10.1016/j.renene.2014.03.039

Mukherjee A, Reddy MS (2020). Metatranscriptomics: an approach for retrieving novel eukaryotic genes from polluted and related environments. 3 Biotech 10(2):71. https://doi.org/10.1007/s13205-020-2057-1

Murugan N, Appavu P (2020). Investigation on low temperature biogas generation. International Journal of Ambient Energy 41(1):5-7. https://doi.org/10.1080/01430750.2018.1443283

Mutungwazi A, Ijoma GN, Matambo TS (2021). The significance of microbial community functions and symbiosis in enhancing methane production during anaerobic digestion: A review. Symbiosis 83(1):1-24. https://doi.org/10.1007/s13199-020-00734-4

Popovic M (2019). Thermodynamic properties of microorganisms: determination and analysis of enthalpy, entropy, and Gibbs free energy of biomass, cells and colonies of 32 microorganism species. Heliyon 5(6):e01950. https://doi.org/10.1016/j.heliyon.2019.e01950

Price PB, Sowers T (2004). Temperature dependence of metabolic rates for microbial growth, maintenance, and survival. Proceedings of the National Academy of Sciences 101(13):4631-4636. https://doi.org/10.1073/pnas.0400522101

Rademacher A, Zakrzewski M, Schlüter A, Schönberg M, Szczepanowski R, Goesmann A, Pühler A, Klocke M (2012). Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing. FEMS Microbiology Ecology 79(3):785-799. https://doi.org/10.1111/j.1574-6941.2011.01265.x

Rama H, Ndaba B, Dhlamini MS, Cochrane N, Maaza M, Roopnarain A (2024). Elucidating Key microbial drivers for methane production during cold adaptation and psychrophilic anaerobic digestion of cattle manure and food waste. Fermentation 10(7):370. https://doi.org/10.3390/fermentation10070370

Ravindra R, Winter R (2003). On the temperature–pressure free‐energy landscape of proteins. ChemPhysChem 4(4):359-365. https://doi.org/10.1002/cphc.200390062

Rowland I., Gibson G, Heinken A, Scott K, Swann J, Thiele I, Tuohy K (2018). Gut microbiota functions: metabolism of nutrients and other food components. European Journal of Nutrition 57:1–24. https://doi.org/10.1007/s00394-017-1445-8

Shakya M, Lo CC, Chain PSG (2019). Advances and challenges in metatranscriptomic analysis. Frontiers in Genetics 10:472554. https://doi.org/10.3389/fgene.2019.00904

Sheridan PP, Panasik N, Coombs JM, Brenchley JE (2000). Approaches for deciphering the structural basis of low temperature enzyme activity. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1543(2):417-433. https://doi.org/10.1016/S0167-4838(00)00237-5

Struvay C, Feller G (2012). Optimization to low temperature activity in psychrophilic enzymes. International Journal of Molecular Sciences 13(9):11643-11665. https://doi.org/10.3390/ijms130911643

Varghese VK, Poddar BJ, Shah MP, Purohit HJ, Khardenavis AA (2022). A comprehensive review on current status and future perspectives of microbial volatile fatty acids production as platform chemicals. Science of the Total Environment 815:152500. https://doi.org/10.1016/j.scitotenv.2021.152500

Wang S, Bi X, Wang S (2015). Thermodynamic analysis of biomass gasification for biomethane production. Energy 90:1207-1218. https://doi.org/10.1016/j.energy.2015.06.073

Wang S, Ma F, Ma W, Wang P, Zhao G, Lu X (2019). Influence of temperature on biogas production efficiency and microbial community in a two-phase anaerobic digestion system. Water 11(1):133. https://doi.org/10.3390/w11010133

Westerholm M, Isaksson S, Lindsjö OK, Schnürer A (2018). Microbial community adaptability to altered temperature conditions determines the potential for process optimisation in biogas production. Applied Energy 226:838-848. https://doi.org/10.1016/j.apenergy.2018.06.045

Wolters B, Ding GC, Kreuzig R, Smalla K (2016). Full-scale mesophilic biogas plants using manure as C-source: bacterial community shifts along the process cause changes in the abundance of resistance genes and mobile genetic elements. FEMS Microbiology Ecology 92(2):fiv163. https://doi.org/10.1093/femsec/fiv163

Xia Y, Wang Y, Fang HHP, Jin T, Zhong H, Zhang T (2014). Thermophilic microbial cellulose decomposition and methanogenesis pathways recharacterized by metatranscriptomic and metagenomic analysis. Scientific Reports 4(1):6708. https://doi.org/10.1038/srep06708

Yang Z, Sun H, Zhao Q, Kurbonova M, Zhang R, Liu, G, Wang W (2020). Long-term evaluation of bioaugmentation to alleviate ammonia inhibition during anaerobic digestion: Process monitoring, microbial community response, and methanogenic pathway modeling. Chemical Engineering Journal 399:125765. https://doi.org/10.1016/j.cej.2020.125765

Yuan Y, Hu X, Chen H, Zhou Y, Zhou Y, Wang D (2019). Advances in enhanced volatile fatty acid production from anaerobic fermentation of waste activated sludge. Science of the Total Environment 694:133741. https://doi.org/10.1016/j.scitotenv.2019.133741

Zhang L, Loh KC, Lim JW, Zhang J (2019). Bioinformatics analysis of metagenomics data of biogas-producing microbial communities in anaerobic digesters: A review. Renewable and Sustainable Energy Reviews 100:110-126. https://doi.org/10.1016/j.rser.2018.10.021

Zhang M, Tan Z, Wang X, Cui M, Wang Y, Jiao Z (2018). Impact inoculum dosage of lactic acid bacteria on oat and wheat silage fermentation at ambient and low temperatures. Crop and Pasture Science 69(12):1225-1236. https://doi.org/10.1071/CP17250

Zhou X, Liu T, Zhang S, Kang B, Duan X, Yan Y, Feng L, Chen Y (2023). Metagenomic insight of fluorene-boosted sludge acidogenic fermentation: Metabolic transformation of amino acids and monosaccharides. Science of the Total Environment 865:161122. https://doi.org/10.1016/j.scitotenv.2022.161122