Автоселекция микроорганизмов избыточного активного ила, использованного в качестве инокулята при темновом получении биоводорода из субстратов с разным биополимерным составом

При использовании смешанных органических отходов в микробном сообществе реактора темновой ферментации происходит автоселекция микроорганизмов, наиболее приспособленных к данному биополимерному составу субстрата. В этом исследовании были использованы 6 субстратов, моделирующих разные биополимеры (белки, жиры, углеводы) и их смеси, для обогащения адаптированных к данным субстратам водород-продуцирующих бактерий из необработанного активного ила. С помощью высокопроизводительного секвенирования показано, что независимо от использованного субстрата, в микробном сообществе доминировал филум Firmicutes (67-100%). При использовании субстратов, богатых углеводами, микробное разнообразие было низким и в основном представлено родами Ruminococcus (26-90%) и Thermoanaerobacterium (6-67%). Темновая ферментация жиров и белков характеризовалась большим микробным разнообразием. При использовании жиров в наибольшей степени развивались Thermoanaerobacterium (21%), Thermobrachium (19%), Tepidiphilus (16%) и Acetomicrobium (14%), при использовании белков – Thermobrachium (34%), Acetomicrobium (16%) и Clostridium sensu stricto 7 (12%). Различные микробные сообщества и субстраты приводили к различиям в характеристиках процесса и метаболических путях. Максимальным был выход водорода из крахмала и составил 138 мл/г органического вещества с 60,4 % содержанием водорода в биогазе. Доминирование рода Ruminococcus, предположительно, внесло основной вклад в производство водорода. Согласно анализу стабильных изотопов ¹³С небольшие количества метана при темновой ферментации белков и жиров были образованы Methanothermobacter и Methanosarcina по преимущественно водородотрофному типу метаногенеза.

1. Kothari R., D. Buddhi, R.L. Sawhney, Comparison of environmental and economic aspects of various hydrogen production methods. Renew. Sust. Energ. Rev. 12 (2008) 781 553-563. https://doi.org/10.1016/j.rser.2006.07.012.

2. Abbasi T., Abbasi S.A. ‘Renewable’ hydrogen: Prospects and challenges, Renew. Sust. Energ. Rev. 15 (2011) 3034-3040. https://doi.org/10.1016/j.rser.2011.02.026.

3. Khan MA, Ngo HH, Guo W, Liu Y, Zhang X, Guo J, Chang SW, Nguyen DD, Wang J, Biohydrogen production from anaerobic digestion and its potential as renewable energy, Renewable Energy 2018 V. 129B. Pp. 754-768. https://doi.org/10.1016/j.renene.2017.04.029.

4. Wang JL, Yin Y. Fermentative hydrogen production using various biomass-based materials as feedstock. Renew Sustain Energy Rev 2018; 92:284-306. https://doi.org/10.1186/s12934-018-0871-5.

5. Łukajtis, R., Hołowacz, I., Kucharska, K., Glinka, M., Rybarczyk, P., Przyjazny, A., & Kamiński, M. (2018). Hydrogen production from biomass using dark fermentation. Renewable and Sustainable Energy Reviews, 91, 665–694. https://doi.org/10.1016/j.rser.2018.04.043.

6. Elbeshbishy E, Dhar BR, Nakhla G, Lee HS. A critical review on inhibition of dark biohydrogen fermentation. Renew Sustain Energy Rev 2017; 79:656-68. https://doi.org/10.1016/j.rser.2017.05.075.

7. Wang JL, Yin YN. Principle and application of different pretreatment methods for enriching hydrogenproducing bacteria from mixed cultures. Int J Hydrogen Energy 2017; 42:4804e23. https://doi.org/10.1016/j.ijhydene.2017.01.135.

8. Wong YM, Wu TY, Juan JC. A review of sustainable hydrogen production using seed sludge via dark fermentation. Renew Sustain Energy Rev 2014; 34:471–82. https://doi.org/10.1016/j.rser.2014.03.008.

9. Li C, Fang HHP. Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Environ Sci Technol 2007; 37:1–39. https://doi.org/10.1080/10643380600729071.

10. Valdez-Vazquez I, Ríos-Leal E, Esparza-García F, Cecchi F, Poggi-Varaldo HM. Semi-continuous solid substrate anaerobic reactors for H2 production from organic waste: mesophilic versus thermophilic regime. Int J Hydrog Energy 2005; 30:1383–91. https://doi.org/10.1016/j.ijhydene.2004.09.016.

11. De Gioannis, G., Muntoni, A., Polettini, A., Pomi, R., 2013. A review of dark fermentation hydrogen production from biodegradable municipal waste fractions. Waste Manage. 33, 1345–1361. https://doi.org/10.1016/j.wasman.2013.02.019.

12. Kobayashi, T., Xu, K.-Q., Li, Y.-Y., Inamori, Y., 2012. Evaluation of hydrogen and methane production from municipal solid wastes with different compositions of fat, protein, cellulosic materials and the other carbohydrates. Int. J. Hydrogen Energy 37, 15711–15718. https://doi.org/10.1016/j.ijhydene.2012.05.044.

13. Alibardi, L., & Cossu, R. (2016). Effects of carbohydrate, protein and lipid content of organic waste on hydrogen production and fermentation products. Waste Management, 47, 69–77.

14. Campuzano & González-Martínez, S. (2016). Characteristics of the organic fraction of municipal solid waste and methane production: A review. Waste Management, 54, 3–12. https://doi.org/10.1016/j.wasman.2016.05.016.

15. Wei S Z, Xiao B Y, Liu J X. Impact of alkali and heat pretreatment on the pathway of hydrogen production from sewage sludge. Chinese Sci Bull, 2010, 55: 777−786, https://doi.org/10.1007/s11434-009-0591-7.

16. Lokshina, L., Vavilin, V., Litti, Y., Glagolev, M., Sabrekov, A., Kotsyurbenko, O., & Kozlova, M. (2019). Methane Production in a West Siberian Eutrophic Fen Is Much Higher than Carbon Dioxide Production: Incubation of Peat Samples, Stoichiometry, Stable Isotope Dynamics, Modeling. Water Resources, 46(S1), S110–S125. https://doi.org/10.1134/S0097807819070133.

17. Litti Y., Nikitina A., Kovalev D., Ermoshin A., Mahajan R., Gunjan G., Nozhevnikova A. Influence of cationic polyacrilamide flocculant onhigh-solids’ anaerobic digestion of sewage sludge under thermophilic conditions, Environmental Technology. 2017. Dec 14:1-26 https://doi.org/10.1080/09593330.2017.1417492.

18. Conrad, R. Quantification of methanogenic pathways using stable carbon isotopic signatures: a review and a proposal. Org. Geochem. 36, 739–752 (2005). https://doi.org/10.1016/j.orggeochem.2004.09.006.

19. Lever M.A., Torti A., Eickenbusch P., Michaud A.B., Šantl-Temkiv T., Jørgensen B.B. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types // Front Microbiol. 2015. V. 6. P. 476. https://doi.org/10.3389/fmicb.2015.00476.

20. Fadrosh D.W., Ma B., Gajer P., Sengamalay N., Ott S., Brotman R.M., Ravel J. An improved dualindexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform // Microbiome. 2014. V. 2(1). P. 6. https://doi.org/10.1186/2049-2618-2-6.

21. Caporaso J.G., Lauber C.L., Walters W.A., Berg-Lyons D., Huntley J., Fierer N., Owens S.M., Betley J., Fraser L., Bauer M., Gormley N., Gilbert J.A., Smith G., Knight R. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms // ISME J. 2012. V. 6(8). P. 1621-1624. https://doi.org/10.1038/ismej.2012.8.

22. Caporaso J.G., Kuczynski J., Stombaugh J., Bittinger K., Bushman F.D., Costello E.K., Fierer N., Peña A.G., Goodrich J.K., Gordon J.I., Huttley G.A., Kelley S.T., Knights D., Koenig J.E., Ley R.E., Lozupone C.A., McDonald D., Muegge B.D., Pirrung M., Reeder J., Sevinsky J.R., Turnbaugh P.J., Walters W.A., Widmann J., Yatsunenko T., Zaneveld J., Knight R.. QIIME allows analysis of high-throughput community sequencing data // Nat. Methods. 2010 V. 7. P. 335-336. https://doi.org/10.1038/nmeth.f.303.

23. Quast C., Pruesse E., Yilmaz P., Gerken J., Schweer T., Yarza P., Peplies J., Glöckner F.O. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools // Nucleic Acids Res. 2013. V. 41. P. D590-D596. https://doi.org/10.1093/nar/gks1219.

24. Liu I.C., Whang L.-M., Ren W.-J., Lin P.-Y. The effect of pH on the production of biohydrogen by clostridia: Thermodynamic and metabolic considerations, Int. J. Hydrogen Energ. 36 (2011) 439-449 https://doi.org/10.1016/j.ijhydene.2010.10.045.

25. Bundhoo M.A.Z., Mohee R. Inhibition of dark fermentative bio-hydrogen production: A review, Int. J. Hydrogen Energ. 41 (2016) 6713-6733. https://doi.org/10.1016/j.ijhydene.2016.03.057.

26. Carrillo-Reyes J, Buitr ´on G, Moreno-Andrade I, Tapia-Rodr´ıguez AC, Palomo-Briones R, Razo-Flores E, Ju´arez OA, Arreola-Vargas J, Bernet N, Braga AFM, Braga L, Castell ´o E, Chatellard L, Etchebehere C, Fuentes L, Le´on-Becerril E, M´endez-Acosta HO, Ruiz-Filippi G, Venegas ET, Trably E, Wenzel J, Zaiat M, Standardized protocol for determination of biohydrogen potential, MethodsX (2019). https://doi.org/10.1016/j.mex.2019.11.027.

27. Goud, R.K., Venkata Mohan, S., 2012. Acidic and alkaline shock pretreatment to enrich acidogenic biohydrogen producing mixed culture: Long term synergetic evaluation of microbial inventory, dehydrogenase activity and bioelectro kinetics. RSC Adv. 2, 6336–6353. https://doi.org/10.1039/C2RA20526B.

28. Calusinska, M., Happe, T., Joris, B., Wilmotte, A., 2010. The surprising diversity of clostridial hydrogenases: A comparative genomic perspective. Microbiology 156, 1575–1588. https://doi.org/10.1099/mic.0.032771-0.

29. Meyer, J., 2007. (FeFe) hydrogenases and their evolution: A genomic perspective. Cell. Mol. Life Sci. 64 (9), 1063–1084. https://doi.org/10.1007/s00018-007-6477-4.

30. Laothanachareon T, Kanchanasuta S, Mhuanthong W, Phalakornkule C, Pisutpaisal N, Champreda V. Analysis of microbial community adaptation in mesophilic hydrogen fermentation from food waste by tagged 16S rRNA gene pyrosequencing. J Environ Manag 2014; 144:143-51. https://doi.org/10.1016/j.jenvman.2014.05.019.

31. Jumas-Bilak, E.; Roudiere, L.; Marchandin, H. (2009). "Description of 'Synergistetes' phyl. nov. and emended description of the phylum 'Deferribacteres' and of the family Syntrophomonadaceae, phylum 'Firmicutes'". Int. J. Syst. Evol. Microbiol. 59: 1028–1035. https://doi.org/10.1099/ijs.0.006718-0.

32. Riviere, D., Desvignes, V., Pelletier, E., Chaussonnerie, S., Guermazi, S., Weissenbach, J., Li, T., Camacho, P., and Sghir, A. (2009). Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge. ISME. J. 3, 700–714. https://doi.org/10.1038/ismej.2009.2.

33. Dahle H, Birkeland N-K (2006) Thermovirga lienii gen. Nov., sp. nov., a novel moderately thermophilic, anaerobic, amino-aciddegrading bacterium isolated from a North Sea oil well. Int J Syst EvolMicrobiol 56:1539–1545. https://doi.org/10.1099/ijs.0.63894-0.

34. Honda T, Fujita T, Tonouchi A (2013) Aminivibrio pyruvatiphilus gen. Nov., sp. nov., an anaerobic, amino-acid-degrading bacterium from soil of a Japanese rice field. Int J Syst Evol Microbiol 63: 3679–3686. https://doi.org/10.1099/ijs.0.052225-0.

35. Meng X, Yuan X, Ren J, Wang X, Zhu W, Cui Z (2017) Methane production and characteristics of themicrobial community in a two stage fixed-bed anaerobic reactor using molasses. Bioresour Technol 241:1050–1059. https://doi.org/10.1016/j.biortech.2017.05.181.

36. Yi, Y., Wang, H., Chen, Y., Gou, M., Xia, Z., Hu, B., … Tang, Y. Identification of Novel Butyrateand Acetate-Oxidizing Bacteria in Butyrate-Fed Mesophilic Anaerobic Chemostats by DNA-Based Stable Isotope Probing. Microb Ecol 79, 285–298 (2020) https://doi.org/10.1007/s00248-019-01400-z.

37. Bernard M. Ollivier, Robert A. Mah, Thomas J. Ferguson, David R. Boone, J.L. Garcia, and Ralph Robinson. Emendation of the Genus Thermobacteroides: Thermobacteroides proteolyticus sp. nov., a Proteolytic Acetogen from a Methanogenic Enrichment. International Journal of Systematic Bacteriology. October 1985 vol. 35 no. 4 425-428 https://doi.org/10.1099/00207713-35-4-425.

38. Etchebehere, C; Pavan, M. E.; Zorzopulos, J.; Soubes, M.; Muxi, L. (1998). "Coprothermobacter platensis sp. nov., a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge". International Journal of Systematic and Evolutionary Microbiology. 48 (4): 1297–1304. https://doi.org/10.1099/00207713-48-4-1297.

39. Sasaki K, Morita M, Sasaki D, Nagaoka J, Matsumoto N et al. Syntrophic degradation of proteinaceous materials by the thermophilic strains Coprothermobacter proteolyticus and Methanothermobacter thermautotrophicus. J Biosci Bioeng 2011; 112:469–472. https://doi.org/10.1016/j.jbiosc.2011.07.003.

40. Sivarajan, A., Shanmugasundaram, T., Thirumalairaj, J., & Balagurunathan, R. (2016). Production and optimization of biohydrogen from saccharolytic actinobacterium, Streptomyces rubiginosus (SM16), using sugarcane molasses. Biofuels, 8(6), 717–723. https://doi.org/10.1080/17597269.2016.1257317.

41. Ntaikou, I., Gavala, H. N., Kornaros, M., & Lyberatos, G. (2008). Hydrogen production from sugars and sweet sorghum biomass using Ruminococcus albus. International Journal of Hydrogen Energy, 33(4), 1153–1163. https://doi.org/10.1016/j.ijhydene.2007.10.053.

42. Sheng T, Gao L, Zhao L, Liu W, Wang A. Direct hydrogen production from lignocellulose by the newly isolated Thermoanaerobacterium thermosaccharolyticum strain DD32. RSC Adv. 2015; 5:99781–8. https://doi.org/10.1186/s12934-017-0692-y.

43. Cao G, Ren N, Wang A, Lee D-J, Guo W, Liu B, et al. Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16. Int J Hydrogen Energy.2009; 34:7182–8. https://doi.org/10.1016/j.ijhydene.2009.07.009.

44. Mamimin C, Jehlee A, Saelor S, Prasertsan P, Sompong O. Thermophilic hydrogen production from co-fermentation of palm oil mill effluent and decanter cake by Thermoanaerobacterium thermosaccharolyticum PSU-2. Int J Hydrogen Energy. 2016. https://doi.org/10.1016/j.ijhydene.2016.07.152.

45. Zhao Lei, Chen Chuan, Ren Hong-Yu, Wang Zi-Han, Wu Kai-Kai, Meng Jia, Cao Guang-Li, Ren Nan-Qi, Ho Shih-Hsin Unraveling hydrogen production potential by glucose and xylose co-fermentation of Thermoanaerobacterium thermosaccharolyticum W16 and its metabolisms through transcriptomic sequencing // Int. J. Energy Res.2020. https://doi.org/10.1002/er.5468.

46. Qin, Y., Li, L., Wu, J., Xiao, B., Hojo, T., Kubota, K., Li, Y.-Y. (2019). Co-production of biohydrogen and biomethane from food waste and paper waste via recirculated two-phase anaerobic digestion process: Bioenergy yields and metabolic distribution. Bioresource Technology. https://doi.org/10.1016/j.biortech.2019.01.004.

47. Sinha, P., & Pandey, A. (2014). Biohydrogen production from various feedstocks by Bacillus firmus NMBL-03. International Journal of Hydrogen Energy, 39(14), 7518–7525.

48. Shah, A. T., Favaro, L., Alibardi, L., Cagnin, L., Sandon, A., Cossu, R., … Basaglia, M. (2016). Bacillus sp. strains to produce bio-hydrogen from the organic fraction of municipal solid waste. Applied Energy, 176, 116–124. https://doi.org/10.1016/j.apenergy.2016.05.054.

49. Alves, J. I., van Gelder, A. H., Alves, M. M., Sousa, D. Z., & Plugge, C. M. (2013). Moorella stamsii sp. nov., a new anaerobic thermophilic hydrogenogenic carboxydotroph isolated from digester sludge. Int J Syst Evol Micr, 63(Pt 11), 4072–4076. https://doi.org/10.1099/ijs.0.050369-0.

50. Slobodkin, A., Reysenbach, A.-L., Mayer, F., & Wiegel, J. (1997). Isolation and Characterization of the Homoacetogenic Thermophilic Bacterium Moorella glycerini sp. nov. International Journal of Systematic Bacteriology, 47(4), 969–974. https://doi.org/10.1099/00207713-47-4-969.

51. Balk, M., Weijma, J., Friedrich, M. W., & Stams, A. J. M. (2003). Methanol utilization by a novel thermophilic homoacetogenic bacterium, Moorella mulderi sp. nov., isolated from a bioreactor. Archives of Microbiology, 179(5), 315–320 https://doi.org/10.1007/s00203-003-0523-x.

52. Engle M, Li Y, Rainey F, DeBlois S, Mai V, Reichert A, Mayer F, Messner P, Wiegel J. Thermobrachium celere gen. nov., sp. nov., a rapidly growing thermophilic, alkalitolerant, and proteolytic obligate anaerobe. Int J Syst Bacteriol 1996; 46:1025- https://doi.org/1033.10.1099/00207713-46-4-1025.

53. Ciranna, A., Santala, V., & Karp, M. (2011). Biohydrogen production in alkalithermophilic conditions: Thermobrachium celere as a case study. Bioresource Technology, 102(18), 8714–8722. https://doi.org/10.1016/j.biortech.2011.01.028.

54. Maune MW, Tanner RS Description of Anaerobaculum hydrogeniformans sp. nov., an anaerobe that produces hydrogen from glucose, and emended description of the genus Anaerobaculum. // Int J Syst Evol Microbiol. 2012 Apr; 62(Pt 4):832-8. https://doi.org/10.1099/ijs.0.024349-0.

55. Gerritsen J, Fuentes S, Grievink W, van Niftrik L, Tindall BJ, Timmerman HM, Rijkers GT, Smidt H. Characterization of Romboutsia ilealis gen. nov., sp. nov., isolated from the gastro-intestinal tract of a rat, and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Romboutsia gen. nov., Intestinibacter gen. nov., Terrisporobacter gen. nov. and Asaccharospora gen. nov. Int J Syst Evol Microbiol 2014; 64:1600-1616. https://doi.org/10.1099/ijs.0.059543-0.

56. Černý, M., Vítězová, M., Vítěz, T., Bartoš, M., & Kushkevych, I. (2018). Variation in the Distribution of Hydrogen Producers from the Clostridiales Order in Biogas Reactors Depending on Different Input Substrates. Energies, 11(12), 3270. https://doi.org/10.3390/en11123270.

57. Rainey, F.A. Clostridiales. In Bergey’s Manual of Systematics of Archaea and Bacteria; JohnWiley & Sons, Inc.:Hoboken, NJ, USA, 2015; pp. 1–5.

58. Lee M, Hidaka T, Tsuno H. Two-phased hyperthermophilic anaerobic co-digestion of waste activated sludge with kitchen garbage. J Biosci Bioeng 2009; 108:408-13. https://doi.org/10.1016/j.jbiosc.2009.05.011.

59. Manaia, C. M. (2003). Tepidiphilus margaritifer gen. nov., sp. nov., isolated from a thermophilic aerobic digester. Int J Syst Evol Microbiol, 53(5), 1405–1410. https://doi.org/10.1099/ijs.0.02538-0.

60. Zhao X, Li D, Xu S, Guo Z, Zhang Y, Man L, Jiang B, Hu X. Clostridium guangxiense sp. nov. and Clostridium neuense sp. nov., two phylogenetically closely related hydrogen-producing species isolated from lake sediment // Int J Syst Evol Microbiol. 2017. 67(3). P. 710-715. https://doi.org/10.1099/ijsem.0.001702.

61. Yang, G., Wang, J. (2019). Changes in microbial community structure during dark fermentative hydrogen production. International Journal of Hydrogen Energy. https://doi.org/10.1016/j.ijhydene.2019.08.039.

62. Yang, G., Yin, Y., & Wang, J. (2019). Microbial community diversity during fermentative hydrogen production inoculating various pretreated cultures. International Journal of Hydrogen Energy. https://doi.org/10.1016/j.ijhydene.2019.03.216.

63. De Vrieze, J., Hennebel, T., Boon, N. & Verstraete, W. Methanosarcina: The rediscovered methanogen for heavy duty biomethanation. Bioresour. Technol. 112, 1–9 (2012). https://doi.org/10.1016/j.biortech.2012.02.079.

64. Dolfing, J. and W. G. B. M. Bloeman. (1985). Activity measurements as a tool to characterize the microbial composition of methanogenic environments. Journal of Microbiological Methods 4 (1):1–12. https://doi.org/10.1016/0167-7012(85)90002-8.