Unveiling microbial structures during raw microalgae digestion and co-digestion with primary sludge to produce biogas using semi-continuous AnMBR systems

[EN] Methane production from microalgae can be enhanced through anaerobic co-digestion with carbon-rich substrates and thus mitigate the inhibition risk associated with its low C:N ratio. Acclimated microbial communities for microalgae disruption can be used as a source of natural enzymes in bioenergy production. However, co-substrates with a certain microbial diversity such as primary sludge might shift the microbial structure. Substrates were generated in a Water Resource Recovery Facility (WRRF) and combined as follows: Scenedesmus or Chlorella digestion and microalgae co-digestion with primary sludge. The study was performed using two lab-scale Anaerobic Membrane Bioreactors (AnMBR). During three years, different feedstocks scenarios for methane production were evaluated with a special focus on the microbial diversity of the AnMBR. 57% of the population was shared between the different feedstock scenarios, revealing the importance of Anaerolineaceae members besides Smithella and Methanosaeta genera. The addition of primary sludge enhanced the microbial diversity of the system during both Chlorella and Scenedesmus co-digestion and promoted different microbial structures. Aceticlastic methanogen Methanosaeta was dominant in all the feedstock scenarios. A more remarkable role of syntrophic fatty acid degraders (Smithella, Syntrophobacteraceae) was observed during co-digestion when only microalgae were digested. However, no significant changes were observed in the microbial composition during anaerobic microalgae digestion when feeding only Chlorella or Scenedesmus. This is the first work revealing the composition of complex communities for semi-continuous bioenergy production from WRRF streams. The stability and maintenance of a microbial core over-time in semi-continuous AnMBRs is here shown supporting their future application in full-scale systems for raw microalgae digestion or codigestion.The Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF) are gratefully acknowledged for their support to this research work through CTM2011-28595-C02-02 and CTM2014-54980-C2-1-R projects. The authors are thankful to Ph.D. Silvia Greses and Ph.D. candidate Rebecca Serna-Garcia (Universitat de Valencia, Spain) for allowing the collection of digestate samples from their bioreactors and providing a brief data characterization of their performance. As well, authors thank the support of Maria Paches (IIAMA, Valencia, Spain) during phytoplankton monitoring in the photobioreactor plant. Finally, the sequencing service from FISABIO (Valencia, Spain) is also gratefully acknowledged for their technical support during the design stage of this work.Zamorano-López, N.; Borrás, L.; Seco, A.; Aguado García, D. (2020). Unveiling microbial structures during raw microalgae digestion and co-digestion with primary sludge to produce biogas using semi-continuous AnMBR systems. The Science of The Total Environment. 699:1-12. https://doi.org/10.1016/j.scitotenv.2019.134365S112699APHA, APHA/AWWA/WEF, 2012. In: Standard Methods for the Examination of Water and Wastewater. Stand. Methods, pp. 541 doi.org/ISBN 9780875532356.Astals, S., Musenze, R. S., Bai, X., Tannock, S., Tait, S., Pratt, S., & Jensen, P. D. (2015). Anaerobic co-digestion of pig manure and algae: Impact of intracellular algal products recovery on co-digestion performance. Bioresource Technology, 181, 97-104. doi:10.1016/j.biortech.2015.01.039Baudelet, P.-H., Ricochon, G., Linder, M., & Muniglia, L. (2017). A new insight into cell walls of Chlorophyta. Algal Research, 25, 333-371. doi:10.1016/j.algal.2017.04.008Bovio, P., Cabezas, A., & Etchebehere, C. (2018). Preliminary analysis ofChloroflexipopulations in full-scale UASB methanogenic reactors. Journal of Applied Microbiology, 126(2), 667-683. doi:10.1111/jam.14115Calusinska, M., Goux, X., Fossépré, M., Muller, E. E. L., Wilmes, P., & Delfosse, P. (2018). A year of monitoring 20 mesophilic full-scale bioreactors reveals the existence of stable but different core microbiomes in bio-waste and wastewater anaerobic digestion systems. Biotechnology for Biofuels, 11(1). doi:10.1186/s13068-018-1195-8Carrillo-Reyes, J., Barragán-Trinidad, M., & Buitrón, G. (2016). Biological pretreatments of microalgal biomass for gaseous biofuel production and the potential use of rumen microorganisms: A review. Algal Research, 18, 341-351. doi:10.1016/j.algal.2016.07.004Chen, C., Ming, J., Yoza, B. A., Liang, J., Li, Q. X., Guo, H., … Wang, Q. (2019). Characterization of aerobic granular sludge used for the treatment of petroleum wastewater. Bioresource Technology, 271, 353-359. doi:10.1016/j.biortech.2018.09.132Cheng, W., Chen, H., Yan, S., & Su, J. (2014). Illumina sequencing-based analyses of bacterial communities during short-chain fatty-acid production from food waste and sewage sludge fermentation at different pH values. World Journal of Microbiology and Biotechnology, 30(9), 2387-2395. doi:10.1007/s11274-014-1664-6Colzi Lopes, A., Valente, A., Iribarren, D., & González-Fernández, C. (2018). Energy balance and life cycle assessment of a microalgae-based wastewater treatment plant: A focus on alternative biogas uses. Bioresource Technology, 270, 138-146. doi:10.1016/j.biortech.2018.09.005Córdova, O., Chamy, R., Guerrero, L., & Sánchez-Rodríguez, A. (2018). Assessing the Effect of Pretreatments on the Structure and Functionality of Microbial Communities for the Bioconversion of Microalgae to Biogas. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.01388Correa, D. F., Beyer, H. L., Fargione, J. E., Hill, J. D., Possingham, H. P., Thomas-Hall, S. R., & Schenk, P. M. (2019). Towards the implementation of sustainable biofuel production systems. Renewable and Sustainable Energy Reviews, 107, 250-263. doi:10.1016/j.rser.2019.03.005Crutchik, D., Frison, N., Eusebi, A. L., & Fatone, F. (2018). Biorefinery of cellulosic primary sludge towards targeted Short Chain Fatty Acids, phosphorus and methane recovery. Water Research, 136, 112-119. doi:10.1016/j.watres.2018.02.047De Vrieze, J., Christiaens, M. E. R., & Verstraete, W. (2017). The microbiome as engineering tool: Manufacturing and trading between microorganisms. New Biotechnology, 39, 206-214. doi:10.1016/j.nbt.2017.07.001De Vrieze, J., Pinto, A. J., Sloan, W. T., & Ijaz, U. Z. (2018). The active microbial community more accurately reflects the anaerobic digestion process: 16S rRNA (gene) sequencing as a predictive tool. Microbiome, 6(1). doi:10.1186/s40168-018-0449-9Dodsworth, J. A., Blainey, P. C., Murugapiran, S. K., Swingley, W. D., Ross, C. A., Tringe, S. G., … Hedlund, B. P. (2013). Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage. Nature Communications, 4(1). doi:10.1038/ncomms2884Dojka, M. A., Harris, J. K., & Pace, N. R. (2000). Expanding the Known Diversity and Environmental Distribution of an Uncultured Phylogenetic Division of Bacteria. Applied and Environmental Microbiology, 66(4), 1617-1621. doi:10.1128/aem.66.4.1617-1621.2000Farag, I. F., Davis, J. P., Youssef, N. H., & Elshahed, M. S. (2014). Global Patterns of Abundance, Diversity and Community Structure of the Aminicenantes (Candidate Phylum OP8). PLoS ONE, 9(3), e92139. doi:10.1371/journal.pone.0092139Fontana, A., Kougias, P. G., Treu, L., Kovalovszki, A., Valle, G., Cappa, F., … Campanaro, S. (2018). Microbial activity response to hydrogen injection in thermophilic anaerobic digesters revealed by genome-centric metatranscriptomics. Microbiome, 6(1). doi:10.1186/s40168-018-0583-4Garrido-Cardenas, J. A., Manzano-Agugliaro, F., Acien-Fernandez, F. G., & Molina-Grima, E. (2018). Microalgae research worldwide. Algal Research, 35, 50-60. doi:10.1016/j.algal.2018.08.005González-Camejo, J., Jiménez-Benítez, A., Ruano, M. V., Robles, A., Barat, R., & Ferrer, J. (2019). Optimising an outdoor membrane photobioreactor for tertiary sewage treatment. Journal of Environmental Management, 245, 76-85. doi:10.1016/j.jenvman.2019.05.010Gonzalez-Fernandez, C., Sialve, B., & Molinuevo-Salces, B. (2015). Anaerobic digestion of microalgal biomass: Challenges, opportunities and research needs. Bioresource Technology, 198, 896-906. doi:10.1016/j.biortech.2015.09.095Gonzalez-Fernandez, C., Barreiro-Vescovo, S., de Godos, I., Fernandez, M., Zouhayr, A., & Ballesteros, M. (2018). Biochemical methane potential of microalgae biomass using different microbial inocula. Biotechnology for Biofuels, 11(1). doi:10.1186/s13068-018-1188-7González-González, L. M., Correa, D. F., Ryan, S., Jensen, P. D., Pratt, S., & Schenk, P. M. (2018). Integrated biodiesel and biogas production from microalgae: Towards a sustainable closed loop through nutrient recycling. Renewable and Sustainable Energy Reviews, 82, 1137-1148. doi:10.1016/j.rser.2017.09.091Greses, S., Gaby, J. C., Aguado, D., Ferrer, J., Seco, A., & Horn, S. J. (2017). Microbial community characterization during anaerobic digestion of Scenedesmus spp. under mesophilic and thermophilic conditions. Algal Research, 27, 121-130. doi:10.1016/j.algal.2017.09.002Greses, S., Zamorano-López, N., Borrás, L., Ferrer, J., Seco, A., & Aguado, D. (2018). Effect of long residence time and high temperature over anaerobic biodegradation of Scenedesmus microalgae grown in wastewater. Journal of Environmental Management, 218, 425-434. doi:10.1016/j.jenvman.2018.04.086Herrmann, C., Kalita, N., Wall, D., Xia, A., & Murphy, J. D. (2016). Optimised biogas production from microalgae through co-digestion with carbon-rich co-substrates. Bioresource Technology, 214, 328-337. doi:10.1016/j.biortech.2016.04.119Ju, F., Lau, F., & Zhang, T. (2017). Linking Microbial Community, Environmental Variables, and Methanogenesis in Anaerobic Biogas Digesters of Chemically Enhanced Primary Treatment Sludge. Environmental Science & Technology, 51(7), 3982-3992. doi:10.1021/acs.est.6b06344Kadnikov, V. V., Mardanov, A. V., Beletsky, A. V., Karnachuk, O. V., & Ravin, N. V. (2019). Genome of the candidate phylum Aminicenantes bacterium from a deep subsurface thermal aquifer revealed its fermentative saccharolytic lifestyle. Extremophiles, 23(2), 189-200. doi:10.1007/s00792-018-01073-5Klassen, V., Blifernez-Klassen, O., Wobbe, L., Schlüter, A., Kruse, O., & Mussgnug, J. H. (2016). Efficiency and biotechnological aspects of biogas production from microalgal substrates. Journal of Biotechnology, 234, 7-26. doi:10.1016/j.jbiotec.2016.07.015Klassen, V., Blifernez-Klassen, O., Wibberg, D., Winkler, A., Kalinowski, J., Posten, C., & Kruse, O. (2017). Highly efficient methane generation from untreated microalgae biomass. Biotechnology for Biofuels, 10(1). doi:10.1186/s13068-017-0871-4Leng, L., Yang, P., Singh, S., Zhuang, H., Xu, L., Chen, W.-H., … Lee, P.-H. (2018). A review on the bioenergetics of anaerobic microbial metabolism close to the thermodynamic limits and its implications for digestion applications. Bioresource Technology, 247, 1095-1106. doi:10.1016/j.biortech.2017.09.103Li, R., Duan, N., Zhang, Y., Liu, Z., Li, B., Zhang, D., & Dong, T. (2017). Anaerobic co-digestion of chicken manure and microalgae Chlorella sp.: Methane potential, microbial diversity and synergistic impact evaluation. Waste Management, 68, 120-127. doi:10.1016/j.wasman.2017.06.028Li, R., Duan, N., Zhang, Y., Liu, Z., Li, B., Zhang, D., … Dong, T. (2017). Co-digestion of chicken manure and microalgae Chlorella 1067 grown in the recycled digestate: Nutrients reuse and biogas enhancement. Waste Management, 70, 247-254. doi:10.1016/j.wasman.2017.09.016Mahdy, A., Mendez, L., Ballesteros, M., & González-Fernández, C. (2015). Algaculture integration in conventional wastewater treatment plants: Anaerobic digestion comparison of primary and secondary sludge with microalgae biomass. Bioresource Technology, 184, 236-244. doi:10.1016/j.biortech.2014.09.145Mansfeldt, C., Achermann, S., Men, Y., Walser, J.-C., Villez, K., Joss, A., … Fenner, K. (2019). Microbial residence time is a controlling parameter of the taxonomic composition and functional profile of microbial communities. The ISME Journal, 13(6), 1589-1601. doi:10.1038/s41396-019-0371-6McIlroy, S. J., Kirkegaard, R. H., Dueholm, M. S., Fernando, E., Karst, S. M., Albertsen, M., & Nielsen, P. H. (2017). Culture-Independent Analyses Reveal Novel Anaerolineaceae as Abundant Primary Fermenters in Anaerobic Digesters Treating Waste Activated Sludge. Frontiers in Microbiology, 8. doi:10.3389/fmicb.2017.01134Nakamura, K., Iizuka, R., Nishi, S., Yoshida, T., Hatada, Y., Takaki, Y., … Funatsu, T. (2016). Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform. Scientific Reports, 6(1). doi:10.1038/srep22259Pachés, M., Romero, I., Hermosilla, Z., & Martinez-Guijarro, R. (2012). PHYMED: An ecological classification system for the Water Framework Directive based on phytoplankton community composition. Ecological Indicators, 19, 15-23. doi:10.1016/j.ecolind.2011.07.003Peces, M., Astals, S., Jensen, P. D., & Clarke, W. P. (2018). Deterministic mechanisms define the long-term anaerobic digestion microbiome and its functionality regardless of the initial microbial community. Water Research, 141, 366-376. doi:10.1016/j.watres.2018.05.028Qiao, J.-T., Qiu, Y.-L., Yuan, X.-Z., Shi, X.-S., Xu, X.-H., & Guo, R.-B. (2013). Molecular characterization of bacterial and archaeal communities in a full-scale anaerobic reactor treating corn straw. Bioresource Technology, 143, 512-518. doi:10.1016/j.biortech.2013.06.014Rinke, C. (2018). Single-Cell Genomics of Microbial Dark Matter. Microbiome Analysis, 99-111. doi:10.1007/978-1-4939-8728-3_7Rivière, D., Desvignes, V., Pelletier, E., Chaussonnerie, S., Guermazi, S., Weissenbach, J., … Sghir, A. (2009). Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge. The ISME Journal, 3(6), 700-714. doi:10.1038/ismej.2009.2Robles, Á., Ruano, M. V., Charfi, A., Lesage, G., Heran, M., Harmand, J., … Ferrer, J. (2018). A review on anaerobic membrane bioreactors (AnMBRs) focused on modelling and control aspects. Bioresource Technology, 270, 612-626. doi:10.1016/j.biortech.2018.09.049Sanz, J. L., Rojas, P., Morato, A., Mendez, L., Ballesteros, M., & González-Fernández, C. (2017). Microbial communities of biomethanization digesters fed with raw and heat pre-treated microalgae biomasses. Chemosphere, 168, 1013-1021. doi:10.1016/j.chemosphere.2016.10.109Seco, A., Aparicio, S., González-Camejo, J., Jiménez-Benítez, A., Mateo, O., Mora, J. F., … Ferrer, J. (2018). Resource recovery from sulphate-rich sewage through an innovative anaerobic-based water resource recovery facility (WRRF). Water Science and Technology, 78(9), 1925-1936. doi:10.2166/wst.2018.492Sialve, B., Bernet, N., & Bernard, O. (2009). Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnology Advances, 27(4), 409-416. doi:10.1016/j.biotechadv.2009.03.001Skouteris, G., Hermosilla, D., López, P., Negro, C., & Blanco, Á. (2012). Anaerobic membrane bioreactors for wastewater treatment: A review. Chemical Engineering Journal, 198-199, 138-148. doi:10.1016/j.cej.2012.05.070Solden, L., Lloyd, K., & Wrighton, K. (2016). The bright side of microbial dark matter: lessons learned from the uncultivated majority. Current Opinion in Microbiology, 31, 217-226. doi:10.1016/j.mib.2016.04.020Solé-Bundó, M., Salvadó, H., Passos, F., Garfí, M., & Ferrer, I. (2018). Strategies to Optimize Microalgae Conversion to Biogas: Co-Digestion, Pretreatment and Hydraulic Retention Time. Molecules, 23(9), 2096. doi:10.3390/molecules23092096Solé-Bundó, M., Garfí, M., Matamoros, V., & Ferrer, I. (2019). Co-digestion of microalgae and primary sludge: Effect on biogas production and microcontaminants removal. Science of The Total Environment, 660, 974-981. doi:10.1016/j.scitotenv.2019.01.011Stämmler, F., Gläsner, J., Hiergeist, A., Holler, E., Weber, D., Oefner, P. J., … Spang, R. (2016). Adjusting microbiome profiles for differences in microbial load by spike-in bacteria. Microbiome, 4(1). doi:10.1186/s40168-016-0175-0Vanwonterghem, I., Jensen, P. D., Dennis, P. G., Hugenholtz, P., Rabaey, K., & Tyson, G. W. (2014). Deterministic processes guide long-term synchronised population dynamics in replicate anaerobic digesters. The ISME Journal, 8(10), 2015-2028. doi:10.1038/ismej.2014.50Wang, Y., Hammes, F., De Roy, K., Verstraete, W., & Boon, N. (2010). Past, present and future applications of flow cytometry in aquatic microbiology. Trends in Biotechnology, 28(8), 416-424. doi:10.1016/j.tibtech.2010.04.006Weinrich, S., Koch, S., Bonk, F., Popp, D., Benndorf, D., Klamt, S., & Centler, F. (2019). Augmenting Biogas Process Modeling by Resolving Intracellular Metabolic Activity. Frontiers in Microbiology, 10. doi:10.3389/fmicb.2019.01095Widder, S., Allen, R. J., Pfeiffer, T., Curtis, T. P., Wiuf, C., … Soyer, O. S. (2016). Challenges in microbial ecology: building predictive understanding of community function and dynamics. The ISME Journal, 10(11), 2557-2568. doi:10.1038/ismej.2016.45Xie, B., Gong, W., Tian, Y., Qu, F., Luo, Y., Du, X., … Liang, H. (2018). Biodiesel production with the simultaneous removal of nitrogen, phosphorus and COD in microalgal-bacterial communities for the treatment of anaerobic digestion effluent in photobioreactors. Chemical Engineering Journal, 350, 1092-1102. doi:10.1016/j.cej.2018.06.032Zamalloa, C., De Vrieze, J., Boon, N., & Verstraete, W. (2011). Anaerobic digestibility of marine microalgae Phaeodactylum tricornutum in a lab-scale anaerobic membrane bioreactor. Applied Microbiology and Biotechnology, 93(2), 859-869. doi:10.1007/s00253-011-3624-5Zamorano-López, N., Borrás, L., Giménez, J. B., Seco, A., & Aguado, D. (2019). Acclimatised rumen culture for raw microalgae conversion into biogas: Linking microbial community structure and operational parameters in anaerobic membrane bioreactors (AnMBR). Bioresource Technology, 290, 121787. doi:10.1016/j.biortech.2019.121787Zamorano-López, N., Greses, S., Aguado, D., Seco, A., & Borrás, L. (2019). Thermophilic anaerobic conversion of raw microalgae: Microbial community diversity in high solids retention systems. Algal Research, 41, 101533. doi:10.1016/j.algal.2019.101533Zou, Y., Xu, X., Li, L., Yang, F., & Zhang, S. (2018). Enhancing methane production from U. lactuca using combined anaerobically digested sludge (ADS) and rumen fluid pre-treatment and the effect on the solubilization of microbial community structures. Bioresource Technology, 254, 83-90. doi:10.1016/j.biortech.2017.12.054Lv, Z., Chen, Z., Chen, X., Liang, J., Jiang, J., & Loake, G. J. (2019). Effects of various feedstocks on isotope fractionation of biogas and microbial community structure during anaerobic digestion. Waste Management, 84, 211-219. doi:10.1016/j.wasman.2018.11.04

Compendium of 4,941 rumen metagenome-assembled genomes for rumen microbiome biology and enzyme discovery

The Rowett Institute and SRUC are core funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. The Roslin Institute forms part of the Royal (Dick) School of Veterinary Studies, University of Edinburgh. This project was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/N016742/1, BB/N01720X/1), including institute strategic programme and national capability awards to The Roslin Institute (BBSRC: BB/P013759/1, BB/P013732/1, BB/J004235/1, BB/J004243/1); and by the Scottish Government as part of the 2016–2021 commission.Peer reviewedPublisher PD

“Candidatus Paraporphyromonas polyenzymogenes” encodes multi-modular cellulases linked to the type IX secretion system

Abstract Background In nature, obligate herbivorous ruminants have a close symbiotic relationship with their gastrointestinal microbiome, which proficiently deconstructs plant biomass. Despite decades of research, lignocellulose degradation in the rumen has thus far been attributed to a limited number of culturable microorganisms. Here, we combine meta-omics and enzymology to identify and describe a novel Bacteroidetes family (“Candidatus MH11”) composed entirely of uncultivated strains that are predominant in ruminants and only distantly related to previously characterized taxa. Results The first metabolic reconstruction of Ca. MH11-affiliated genome bins, with a particular focus on the provisionally named “Candidatus Paraporphyromonas polyenzymogenes”, illustrated their capacity to degrade various lignocellulosic substrates via comprehensive inventories of singular and multi-modular carbohydrate active enzymes (CAZymes). Closer examination revealed an absence of archetypical polysaccharide utilization loci found in human gut microbiota. Instead, we identified many multi-modular CAZymes putatively secreted via the Bacteroidetes-specific type IX secretion system (T9SS). This included cellulases with two or more catalytic domains, which are modular arrangements that are unique to Bacteroidetes species studied to date. Core metabolic proteins from Ca. P. polyenzymogenes were detected in metaproteomic data and were enriched in rumen-incubated plant biomass, indicating that active saccharification and fermentation of complex carbohydrates could be assigned to members of this novel family. Biochemical analysis of selected Ca. P. polyenzymogenes CAZymes further iterated the cellulolytic activity of this hitherto uncultured bacterium towards linear polymers, such as amorphous and crystalline cellulose as well as mixed linkage β-glucans. Conclusion We propose that Ca. P. polyenzymogene genotypes and other Ca. MH11 members actively degrade plant biomass in the rumen of cows, sheep and most likely other ruminants, utilizing singular and multi-domain catalytic CAZymes secreted through the T9SS. The discovery of a prominent role of multi-modular cellulases in the Gram-negative Bacteroidetes, together with similar findings for Gram-positive cellulosomal bacteria (Ruminococcus flavefaciens) and anaerobic fungi (Orpinomyces sp.), suggests that complex enzymes are essential and have evolved within all major cellulolytic dominions inherent to the rumen

Additional file 9: of “Candidatus Paraporphyromonas polyenzymogenes” encodes multi-modular cellulases linked to the type IX secretion system

Figure S4. Multiple alignment of GH5 domains. (DOCX 39 kb

Additional file 17: of “Candidatus Paraporphyromonas polyenzymogenes” encodes multi-modular cellulases linked to the type IX secretion system

Table S6. Data collection and refinement statistics for the crystal structure of Cel5C_N. (DOCX 14 kb