Xiang Jinfeng, Zhou Zihan, Liu Zhihao, Ren Cong, Xu Yan
Lab of Brewing Microbiology and Applied Enzymology, Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi, China.
Lab of Brewing Microbiology and Applied Enzymology, Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi, China; China Key Laboratory of microbiomics and Eco-brewing Technology for Light Industry, Wuxi 214122, Jiangsu, China.
Water Res. 2025 Sep 15;284:123973. doi: 10.1016/j.watres.2025.123973. Epub 2025 Jun 7.
Converting biodegradable carbon in wastewater into medium-chain fatty acids (MCFAs) through stable microbiota is highly attractive. In this study, we utilized a top-down approach for constructing MCFA-producing microbial consortia. Specifically, an enrichment and plating-screening strategy employing lactic acid and ethanol as selective carbon sources was applied to isolate simplified caproic acid-producing microbial consortia from liquor-making pit mud. The representative microbial consortium SimpCom3 demonstrated high level of caproic acid production (14.62 ± 0.48 g/L) in a semi-synthetic medium, significantly outperforming consortium SimpCom1 (5.96 ± 0.11 g/L) and consortium SimpCom2 (9.63 ± 0.16 g/L). This performance of microbial consortium SimpCom3 was attributed to its ability to co-utilize lactic acid and ethanol, produce fewer odd-chain fatty acids byproducts, and maintain pH self-regulation between 6.45 and 8.29. Metagenomic analyses revealed the dominance of Clostridium kluyveri (30.69 %-50.46 %), C. butyricum (6.71 %-13.98 %) and C. tyrobutyricum (37.11 %-58.07 %) in consortium SimpCom3, which synergistically converted lactic acid and ethanol to caproic acid via reverse β-oxidation. Stable performance over 56 days of cyclic-batch fermentation processes confirmed the robustness of consortium SimpCom3. When applying consortium SimpCom3 to unsterilized liquor-making wastewater in a fermenter with a fed-batch approach, 22.13 g/L caproic acid was produced with 66.38 % selectivity, and microbial dynamics analysis demonstrated the consortium's high adaptability to real wastewater. Metabolic analysis based on high-quality assembly metagenomes (HQ-MAGs) revealed a novel cooperative metabolism: cross-feeding between Clostridium kluyveri (which utilizes ethanol and produces caproic acid) and lactate-utilizing butyrate producers maintained consortium stability and enhanced caproic acid production. Crucially, the decarboxylation of lactic acid counteracted acidification caused by ethanol-driven caproic acid synthesis, enabling self-regulated pH stability within the simplified microbiome system. Together, this study presents a simplified microbial consortium construction method for caproic acid production from liquor-making wastewater, overcoming the limitations of synthetic co-cultures and enhancing the viability of chain-elongation biorefineries in wastewater treatment.
通过稳定的微生物群将废水中的可生物降解碳转化为中链脂肪酸（MCFAs）极具吸引力。在本研究中，我们采用了一种自上而下的方法来构建产MCFAs的微生物群落。具体而言，采用以乳酸和乙醇为选择性碳源的富集和平板筛选策略，从酿酒窖泥中分离出简化的产己酸微生物群落。代表性微生物群落SimpCom3在半合成培养基中表现出高水平的己酸产量（14.62±0.48 g/L），显著优于群落SimpCom1（5.96±0.11 g/L）和群落SimpCom2（9.63±0.16 g/L）。微生物群落SimpCom3的这种性能归因于其能够共同利用乳酸和乙醇，产生较少的奇数链脂肪酸副产物，并将pH值自我调节维持在6.45至8.29之间。宏基因组分析揭示了在群落SimpCom3中，克氏梭菌（30.69%-50.46%）、丁酸梭菌（6.71%-13.98%）和酪丁酸梭菌（37.11%-58.07%）占主导地位，它们通过反向β-氧化协同将乳酸和乙醇转化为己酸。在56天的循环分批发酵过程中稳定的性能证实了群落SimpCom3的稳健性。当将群落SimpCom3以分批补料的方式应用于未灭菌的酿酒废水中时，可产生22.13 g/L的己酸，选择性为66.38%，微生物动力学分析表明该群落对实际废水具有高度适应性。基于高质量组装宏基因组（HQ-MAGs）的代谢分析揭示了一种新的合作代谢：利用乙醇并产生己酸的克氏梭菌与利用乳酸的丁酸产生菌之间的交叉喂养维持了群落稳定性并提高了己酸产量。至关重要的是，乳酸的脱羧作用抵消了由乙醇驱动的己酸合成引起的酸化，从而在简化的微生物系统内实现了自我调节的pH稳定性。总之，本研究提出了一种从酿酒废水中生产己酸的简化微生物群落构建方法，克服了合成共培养的局限性，并提高了废水处理中链延长生物精炼厂的可行性。
