Chain Elongation Using Native Soil Inocula: Exceptional <i>n</i>-Caproate Biosynthesis Performance and Microbial Mechanisms

Lin Deng; Yang Lv; Tian Lan; Qing-Lian Wu; Wei-Tong Ren; Hua-Zhe Wang; Bing-Jie Ni; Wan-Qian Guo

doi:10.1016/j.eng.2023.10.017

PDF(2769 KB)

Engineering ›› 2024, Vol. 39 ›› Issue (8) : 262-272. DOI: 10.1016/j.eng.2023.10.017

Research

Chain Elongation Using Native Soil Inocula: Exceptional n-Caproate Biosynthesis Performance and Microbial Mechanisms

Author information +

History +

Abstract

This study demonstrates the feasibility and effectiveness of utilizing native soils as a resource for inocula to produce $n$ -caproate through the chain elongation (CE) platform, offering new insights into anaerobic soil processes. The results reveal that all five of the tested soil types exhibit CE activity when supplied with high concentrations of ethanol and acetate, highlighting the suitability of soil as an ideal source for $n$ -caproate production. Compared with anaerobic sludge and pit mud, the native soil CE system exhibited higher selectivity (60.53%), specificity (82.32%), carbon distribution (60.00%), electron transfer efficiency $165.00 %$ , and conductivity $0.59 m s ⋅ c m - 1$ . Kinetic analysis further confirmed the superiority of soil in terms of a shorter lag time and higher yield. A microbial community analysis indicated a positive correlation between the relative abundances of Pseudomonas, Azotobacter, and Clostridium and $n$ -caproate production. Moreover, metagenomics analysis revealed a higher abundance of functional genes in key microbial species, providing direct insights into the pathways involved in $n$ -caproate formation, including in situ $C O 2$ utilization, ethanol oxidation, fatty acid biosynthesis (FAB), and reverse beta-oxidation (RBO). The numerous functions in FAB and RBO are primarily associated with Pseudomonas, Clostridium, Rhodococcus, Stenotrophomonas, and Geobacter, suggesting that these genera may play roles that are involved or associated with the CE process. Overall, this innovative inoculation strategy offers an efficient microbial source for $n$ -caproate production, underscoring the importance of considering CE activity in anaerobic soil microbial ecology and holding potential for significant economic and environmental benefits through soil consortia exploration.

Graphical abstract

Keywords

Soil / Chain elongation / $n$ -caproate / Reverse beta-oxidation / Fatty acid biosynthesis / Metagenomics

Cite this article

EndNote

Ris (Procite)

Bibtex

Download citation ▾

Lin Deng, Yang Lv, Tian Lan, Qing-Lian Wu, Wei-Tong Ren, Hua-Zhe Wang, Bing-Jie Ni, Wan-Qian Guo. Chain Elongation Using Native Soil Inocula: Exceptional n-Caproate Biosynthesis Performance and Microbial Mechanisms. Engineering, 2024, 39(8): 262‒272 https://doi.org/10.1016/j.eng.2023.10.017

References

Publishing order | Descend order by publishing year | Descend order by cited within

[1]	R. Zagrodnik, A. Duber, M. Lezyk, P. Oleskowicz-Popiel. Enrichment versus bioaugmentation-microbiological production of caproate from mixed carbon sources by mixed bacterial culture and Clostridium kluyveri. Environ Sci Technol, 54 (9) (2020), pp. 5864-5873.

[2]	J. Xu, J. Hao, J.J.L. Guzman, C.M. Spirito, L.A. Harroff, L.T. Angenent. Temperature-phased conversion of acid whey waste into medium-chain carboxylic acids via lactic acid: no external e-donor. Joule, 3 (3) (2019), pp. 885-888.

[3]	Y. Liu, P. He, L. Shao, H. Zhang, F. Lu. Significant enhancement by biochar of caproate production via chain elongation. Water Res, 119 (2017), pp. 150-159.

[4]	X. Huang, W. Dong, H. Wang, Y. Feng. Role of acid/alkali-treatment in primary sludge anaerobic fermentation: insights into microbial community structure, functional shifts and metabolic output by high-throughput sequencing. Bioresour Technol, 249 (2018), pp. 943-952.

[5]	M.V. Reddy, S. Hayashi, D. Choi, H. Cho, Y.C. Chang. Short chain and medium chain fatty acids production using food waste under non-augmented and bio-augmented conditions. J Clean Prod, 176 (2018), pp. 645-653.

[6]	L.A. Kucek, J. Xu, M. Nguyen, L.T. Angenent. Waste conversion into n-caprylate and n-caproate: resource recovery from wine lees using anaerobic reactor microbiomes and in-line extraction. Front Microbiol, 7 (2016), p. 1892.

[7]	Q. Wu, X. Bao, W. Guo, B. Wang, Y. Li, H. Luo, et al. Medium chain carboxylic acids production from waste biomass: current advances and perspectives. Biotechnol Adv, 37 (5) (2019), pp. 599-615.

[8]	X. Shi, L. Wu, W. Wei, B. Ni. Insights into the microbiomes for medium-chain carboxylic acids production from biowastes through chain elongation. Crit Rev Environ Sci Technol, 52 (21) (2022), pp. 3787-3812.

[9]	S.L. Wu, J. Sun, X. Chen, W. Wei, L. Song, X. Dai, et al. Unveiling the mechanisms of medium-chain fatty acid production from waste activated sludge alkaline fermentation liquor through physiological, thermodynamic and metagenomic investigations. Water Res, 169 (2020), p. 115218.

[10]	S. Ge, J.G. Usack, C.M. Spirito, L.T. Angenent. Long-term n-caproic acid production from yeast-fermentation beer in an anaerobic bioreactor with continuous product extraction. Environ Sci Technol, 49 (13) (2015), pp. 8012-8021.

[11]	M.T. Agler, C.M. Spirito, J.G. Usack, J.J. Werner, L.T. Angenent. Chain elongation with reactor microbiomes: upgrading dilute ethanol to medium-chain carboxylates. Energy Environ Sci, 5 (8) (2012), pp. 8189-8192.

[12]	T.I.M. Grootscholten, F. Kinsky dal Borgo, H.V.M. Hamelers, C.J.N. Buisman. Promoting chain elongation in mixed culture acidification reactors by addition of ethanol. Biomass Bioenergy, 48 (2013), pp. 10-16.

[13]	P. Yang, L. Leng, G.Y.A. Tan, C. Dong, S.Y. Leu, W.H. Chen, et al. Upgrading lignocellulosic ethanol for caproate production via chain elongation fermentation. Int Biodeterior Biodegradation, 135 (2018), pp. 103-109.

[14]	X. Hu, H. Wang, Q. Wu, Y. Xu. Development, validation and application of specific primers for analyzing the clostridial diversity in dark fermentation pit mud by pcr-dgge. Bioresour Technol, 163 (2014), pp. 40-47.

[15]	S. Joshi, A. Robles, S. Aguiar, A.G. Delgado. The occurrence and ecology of microbial chain elongation of carboxylates in soils. ISME J, 15 (7) (2021), pp. 1907-1918.

[16]	M. Béchamp. Lettre de m. A. Béchamp a m. Dumas. Ann Chim Phys, 4 (13) (1868), pp. 103-111.

[17]	H.A. Barker, S.M. Taha. Clostridium kluyverii, an organism concerned in the formation of caproic acid from ethyl alcohol. J Bacteriol, 43 (3) (1942), pp. 347-363.

[18]	H.A. Barker, M.D. Kamen, B.T. Bornstein. The synthesis of butyric and caproic acids from ethanol and acetic acid by Clostridium kluyveri. Proc Natl Acad Sci USA, 31 (12) (1945), pp. 373-381.

[19]	J. He, Z. Shi, T. Luo, S. Zhang, Y. Liu, G. Luo. Phenol promoted caproate production via two-stage batch anaerobic fermentation of organic substance with ethanol as electron donor for chain elongation. Water Res, 204 (2021), p. 117601.

[20]	L.T. Angenent, H. Richter, W. Buckel, C.M. Spirito, K.J.J. Steinbusch, C.M. Plugge, et al. Chain elongation with reactor microbiomes: open-culture biotechnology to produce biochemicals. Environ Sci Technol, 50 (6) (2016), pp. 2796-2810.

[21]	D. Luo, X. Meng, N. Zheng, Y. Li, H. Yao, S.J. Chapman. The anaerobic oxidation of methane in paddy soil by ferric iron and nitrate, and the microbial communities involved. Sci Total Environ, 788 (2021), p. 147773.

[22]	J. Yang, L. Zou, L. Zheng, Z. Yuan, K. Huang, W. Gustave, et al. Iron-based passivator mitigates the coupling process of anaerobic methane oxidation and arsenate reduction in paddy soils. Environ Pollut, 313 (2022), p. 120182.

[23]	L. Fan, D. Schneider, M.A. Dippold, A. Poehlein, W. Wu, H. Gui, et al. Active metabolic pathways of anaerobic methane oxidation in paddy soils. Soil Biol Biochem, 156 (2021), p. 108215.

[24]	K. Tsutsuki, F.N. Ponnamperuma. Behavior of anaerobic decomposition products in submerged soils—effects of organic material amendment, soil properties, and temperature. Soil Sci Plant Nutr, 33 (1) (1987), pp. 13-33.

[25]	R. Conrad. Importance of hydrogenotrophic, aceticlastic and methylotrophic methanogenesis for methane production in terrestrial, aquatic and other anoxic environments: a mini review. Pedosphere, 30 (1) (2020), pp. 25-39.

[26]	O. Sivan, M. Adler, A. Pearson, F. Gelman, I. Bar-Or, S.G. John, et al. Geochemical evidence for iron-mediated anaerobic oxidation of methane. Limnol Oceanogr, 56 (4) (2011), pp. 1536-1544.

[27]	J.M. Saquing, Y.H. Yu, P.C. Chiu. Wood-derived black carbon (biochar) as a microbial electron donor and acceptor. Environ Sci Technol Lett, 3 (2) (2016), pp. 62-66.

[28]	M. Keiluweit, P.S. Nico, M.G. Johnson, M. Kleber. Dynamic molecular structure of plant biomass-derived black carbon (biochar). Environ Sci Technol, 44 (4) (2010), pp. 1247-1253.

[29]	J. Cui, N.M. Holden. The relationship between soil microbial activity and microbial biomass, soil structure and grassland management. Soil Tillage Res, 146 (2015), pp. 32-38.

[30]	Q. Wu, W. Guo, X. Bao, X. Meng, R. Yin, J. Du, et al. Upgrading liquor-making wastewater into medium chain fatty acid: insights into co-electron donors, key microflora, and energy harvest. Water Res, 145 (2018), pp. 650-659.

[31]	Q. Wu, X. Feng, Y. Chen, M. Liu, X. Bao. Continuous medium chain carboxylic acids production from excess sludge by granular chain-elongation process. J Hazard Mater, 402 (2021), p. 123471.

[32]	T. Luo, Q. Xu, W. Wei, J. Sun, X. Dai, B.J. Ni. Performance and mechanism of Fe₃O₄ improving biotransformation of waste activated sludge into liquid high-value products. Environ Sci Technol, 56 (6) (2022), pp. 3658-3668.

[33]	G. Chen, X. Wu, D. Wang, J. Qin, S. Wu, Q. Zhou, et al. Cluster analysis of 12 Chinese native chicken populations using microsatellite markers. Asian-Australas J Anim Sci, 17 (8) (2004), pp. 1047-1052.

[34]	S. Bao, G. Zhang, P. Zhang, Q. Wang, Y. Zhou, X. Tao, et al. Valorization of mixed volatile fatty acids by chain elongation: performances, kinetics and microbial community. Int J Agric Biol, 22 (6) (2019), pp. 1613-1622.

[35]	Y. Liu, P. He, W. Han, L. Shao, F. Lü. Outstanding reinforcement on chain elongation through five-micrometer-sized biochar. Renew Energy, 161 (2020), pp. 230-239.

[36]	H.B. Ding, G.Y.A. Tan, J.Y. Wang. Caproate formation in mixed-culture fermentative hydrogen production. Bioresour Technol, 101 (24) (2010), pp. 9550-9559.

[37]	M. Coma, R. Vilchez-Vargas, H. Roume, R. Jauregui, D.H. Pieper, K. Rabaey. Product diversity linked to substrate usage in chain elongation by mixed-culture fermentation. Environ Sci Technol, 50 (12) (2016), pp. 6467-6476.

[38]	Y. Liu, F. Lu, L. Shao, P. He. Alcohol-to-acid ratio and substrate concentration affect product structure in chain elongation reactions initiated by unacclimatized inoculum. Bioresour Technol, 218 (2016), pp. 1140-1150.

[39]	D. Vasudevan, H. Richter, L. Angenent. Upgrading dilute ethanol from syngas fermentation to n-caproate with reactor microbiomes. Bioresour Technol, 151 (2014), pp. 378-382.

[40]	P.J. Weimer, M. Nerdahl, D.J. Brandl. Production of medium-chain volatile fatty acids by mixed ruminal microorganisms is enhanced by ethanol in co-culture with Clostridium kluyveri. Bioresour Technol, 175 (2015), pp. 97-101.

[41]	S. Villegas-Rodríguez, G. Buitrón. Performance of native open cultures (winery effluents, ruminal fluid, anaerobic sludge and digestate) for medium-chain carboxylic acid production using ethanol and acetate. J Water Process Eng, 40 (2021), p. 101784.

[42]	C. Zhang, L. Yang, P. Tsapekos, Y. Zhang, I. Angelidaki. Immobilization of Clostridium kluyveri on wheat straw to alleviate ammonia inhibition during chain elongation for n-caproate production. Environ Int, 127 (2019), pp. 134-141.

[43]	Y. Yin, Y. Zhang, D. Karakashev, J. Wang, I. Angelidaki. Biological caproate production by Clostridium kluyveri from ethanol and acetate as carbon sources. Bioresour Technol, 241 (2017), pp. 638-644.

[44]	P. Weimer, D. Stevenson. Isolation, characterization, and quantification of Clostridium kluyveri from the bovine rumen. Appl Microbiol Biotechnol, 94 (2012), pp. 461-466.

[45]	X. Hu, H. Du, Y. Xu. Identification and quantification of the caproic acid-producing bacterium Clostridium kluyveri in the fermentation of pit mud used for Chinese strong-aroma type liquor production. Int J Food Microbiol, 214 (2015), pp. 116-122.

[46]	S. Wu, W. Wei, Q. Xu, X. Huang, J. Sun, X. Dai, et al. Revealing the mechanism of biochar enhancing the production of medium chain fatty acids from waste activated sludge alkaline fermentation liquor. Acs Es&T Water, 1 (4) (2021), pp. 1014-1024.

[47]	W. Han, P. He, L. Shao, F. Lu. Road to full bioconversion of biowaste to biochemicals centering on chain elongation: a mini review. J Environ Sci (China), 86 (2019), pp. 50-64.

[48]	L.A. Kucek, C.M. Spirito, L.T. Angenent. High n-caprylate productivities and specificities from dilute ethanol and acetate: chain elongation with microbiomes to upgrade products from syngas fermentation. Energy Environ Sci, 9 (11) (2016), pp. 3482-3494.

[49]	C.M. Spirito, A.M. Marzilli, L.T. Angenent. Higher substrate ratios of ethanol to acetate steered chain elongation toward n-caprylate in a bioreactor with product extraction. Environ Sci Technol, 52 (22) (2018), pp. 13438-13447.

[50]	G.H. Dinesh, R.S. Murugan, K. Mohanrasu, N. Arumugam, M. Basu, A. Arun. Anaerobic process for biohydrogen production using keratin degraded effluent. J Pure Appl Microbiol, 13 (2) (2019), pp. 1135-1143.

[51]	Y. Gao, L. Guo, C. Jin, Y. Zhao, M. Gao, Z. She, et al. Metagenomics and network analysis elucidating the coordination between fermentative bacteria and microalgae in a novel bacterial-algal coupling reactor (BACR) for mariculture wastewater treatment. Water Res, 215 (2022), p. 118256.

[52]	W. Buckel, R.K. Thauer. Flavin-based electron bifurcation, ferredoxin, flavodoxin, and anaerobic respiration with protons (Ech) or NAD+ (Rnf) as electron acceptors: a historical review. Front Microbiol, 9 (2018), p. 401.

[53]	G. Pagliano, V. Ventorino, A. Panico, O. Pepe. Integrated systems for biopolymers and bioenergy production from organic waste and by-products: a review of microbial processes. Biotechnol Biofuels, 10 (2017), p. 113.

[54]	W. Han, P. He, L. Shao, F. Lu. Metabolic interactions of a chain elongation microbiome. Appl Environ Microbiol, 84 (22) (2018), pp. e01614-e1618.

[55]	Y. Song, J. Bae, J. Shin, S. Jin, J.K. Lee, S.C. Kim, et al. Transcriptome and translatome of CO₂ fixing acetogens under heterotrophic and autotrophic conditions. Sci Data, 8 (1) (2021), p. 51.

[56]	C.W. de Araújo, R.C. Leitao, T.A. Gehring, L.T. Angenent, S.T. Santaella. Anaerobic fermentation for n-caproic acid production: a review. Process Biochem, 54 (2017), pp. 106-119.

[57]	A.J.M. Stams, F.A.M. De Bok, C.M. Plugge, M.H.A. van Eekert, J. Dolfing, G. Schraa. Exocellular electron transfer in anaerobic microbial communities. Environ Microbiol, 8 (3) (2006), pp. 371-382.

[58]	X. Zhang, J. Xia, J. Pu, C. Cai, G.W. Tyson, Z. Yuan, et al. Biochar-mediated anaerobic oxidation of methane. Environ Sci Technol, 53 (12) (2019), pp. 6660-6668.

[59]	L. Feng, S. He, Z. Gao, W. Zhao, J. Jiang, Q. Zhao, et al. Mechanisms, performance, and the impact on microbial structure of direct interspecies electron transfer for enhancing anaerobic digestion—a review. Sci Total Environ, 862 (2023), Article 160813.

[60]	Z. Zhao, C. Sun, Y. Li, H. Peng, Y. Zhang. Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: establishing direct interspecies electron transfer via ethanol-type fermentation. Renew Energy, 148 (2020), pp. 523-533.

[61]	Z. Zhao, J. Wang, Y. Li, T. Zhu, Q. Yu, T. Wang, et al. Why do DIETers like drinking: metagenomic analysis for methane and energy metabolism during anaerobic digestion with ethanol. Water Res, 171 (2020), p. 115425.

[62]	Z.M. Summers, H.E. Fogarty, C. Leang, A.E. Franks, N.S. Malvankar, D.R. Lovley. Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria. Science, 330 (6009) (2010), p. 14135.

[63]	X. Liu, S. Zhuo, X. Jing, Y. Yuan, C. Rensing, S. Zhou. Flagella act as geobacter biofilm scaffolds to stabilize biofilm and facilitate extracellular electron transfer. Biosens Bioelectron, 146 (2019), p. 111748.

[64]	X. Liu, X. Jing, Y. Ye, J. Zhan, J. Ye, S. Zhou. Bacterial vesicles mediate extracellular electron transfer. Environ Sci Technol Lett, 7 (1) (2020), pp. 27-34.

[65]	G. Wang, Q. Li, X. Gao, X. Wang. Synergetic promotion of syntrophic methane production from anaerobic digestion of complex organic wastes by biochar: performance and associated mechanisms. Bioresour Technol, 250 (2018), pp. 812-820.

[66]	A.J.M. Stams, D.Z. Sousa, R. Kleerebezem, C.M. Plugge. Role of syntrophic microbial communities in high-rate methanogenic bioreactors. Water Sci Technol, 66 (2) (2012), pp. 352-362.

[67]	S. Ishii, T. Kosaka, Y. Hotta, K. Watanabe. Simulating the contribution of coaggregation to interspecies hydrogen fluxes in syntrophic methanogenic consortia. Appl Environ Microbiol, 72 (7) (2006), pp. 5093-5096.

[68]	Y. Wang, J. Hou, H. Guo, T. Zhu, Y. Zhang, Y. Liu. New insight into mechanisms of ferroferric oxide enhancing medium-chain fatty acids production from waste activated sludge through anaerobic fermentation. Bioresour Technol, 360 (2022), p. 127629.

[69]	X. Liu, J. Zhan, L. Liu, F. Gan, J. Ye, K.H. Nealson, et al. In situ spectroelectrochemical characterization reveals cytochrome-mediated electric syntrophy in geobacter coculture. Environ Sci Technol, 55 (14) (2021), pp. 10142-10151.