Toward industrial C8 production: Oxygen intrusion drives renewable n-caprylate production from ethanol and acetate via intermediate metabolite production

bioRxiv Pub Date : 2024-07-16 DOI:10.1101/2024.07.12.603245
Kurt Gemeinhardt, Byoung Seung Jeon, J. Ntihuga, Han Wang, Caroline Schlaiß, Timo N. Lucas, I. Bessarab, Nicolas Nalpas, Nanqing Zhou, J. Usack, Daniel H. Huson, Rohan B. H. Williams, Boris Maček, Ludmilla Aristilde, L. T. Angenent
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Abstract

Previous bioreactor studies achieved high volumetric n-caprylate (i.e., n-octanoate) production rates and selectivities from ethanol and acetate with chain-elongating microbiomes. However, the metabolic pathways from the substrates to n-caprylate synthesis were unclear. We operated two n-caprylate-producing upflow bioreactors with a synthetic medium to study the underlying metabolic pathways. The operating period exceeded 2.5 years, with a peak volumetric n-caprylate production rate of 190 ± 8.4 mmol C L-1 d-1 (0.14 g L-1 h-1). We identified oxygen availability as a critical performance parameter, facilitating intermediate metabolite production from ethanol. Bottle experiments in the presence and absence of oxygen with 13C-labeled ethanol suggest acetyl-coenzyme A-based derived production of n-butyrate (i.e., n-butanoate), n-caproate (i.e., n-hexanoate), and n-caprylate. Here, we postulate a trophic hierarchy within the bioreactor microbiomes based on metagenomics, metaproteomics, and metabolomics data, as well as experiments with a Clostridium kluyveri isolate. First, the aerobic bacterium Pseudoclavibacter caeni and the facultative anaerobic fungus Cyberlindnera jadinii converted part of the ethanol pool into the intermediate metabolites succinate, lactate, and pyroglutamate. Second, the strict anaerobic C. kluyveri elongated acetate with the residual ethanol to n-butyrate. Third, Caproicibacter fermentans and Oscillibacter valericigenes elongated n-butyrate with the intermediate metabolites to n-caproate and then to n-caprylate. Among the carbon chain-elongating pathways of carboxylates, the tricarboxylic acid cycle and the reverse ß-oxidation pathways showed a positive correlation with n-caprylate production. The results of this study inspire the realization of a chain-elongating production platform with separately controlled aerobic and anaerobic stages to produce n-caprylate renewably as an attractive chemical from ethanol and acetate as substrates. Broader context Next to renewable electric energy, carbon-based chemicals have to be produced sustainably and independently from fossil sources. To meet this goal, we must expand the portfolio of bio-based conversion technologies on an industrial scale to cover as many target chemicals as possible. We explore the bioprocess of chain elongation to provide medium-chain carboxylates that can function as future platform chemicals in the circular economy. The most valuable medium-chain carboxylate produced with chain elongation is n-caprylate (i.e., n-octanoate). This molecule with eight carbon atoms in a row (C8) is challenging to produce renewably for the chemical industry. Previous reports elucidated that elevated ethanol-to-acetate ratios, which are found in syngas-fermentation effluent, stimulated n-caprylate production. Until now, studies have suggested that chain elongation from high concentrations of ethanol and acetate is a fully anaerobic process. We refine this view by showing a trophic hierarchy of aerobic and anaerobic microbes capable of facilitating this process. Appropriate oxygen supplementation enables the synthesis of succinate, lactate, and pyroglutamate that permit high-rate chain elongation to n-caprylate under anaerobic conditions. Given these results, future research should focus on the segregated study of aerobic and anaerobic microbes to further enhance the process performance to produce n-caprylate renewably at an industrial scale.
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工业化生产 C8:氧气侵入通过中间代谢产物的产生推动乙醇和乙酸酯生产可再生的正辛酸酯
之前的生物反应器研究利用链延伸微生物群从乙醇和乙酸酯中获得了较高的正辛酸酯(即正辛酸)生产率和选择性。然而,从底物到正辛酸酯合成的代谢途径尚不清楚。我们使用合成培养基运行了两个生产正辛酸酯的上流式生物反应器,以研究基本代谢途径。运行时间超过 2.5 年,正辛酸酯的峰值体积生产率为 190 ± 8.4 mmol C L-1 d-1 (0.14 g L-1 h-1)。我们发现氧气供应是一个关键的性能参数,有利于乙醇中间代谢产物的生产。用 13C 标记的乙醇在有氧和无氧条件下进行的瓶内实验表明,乙酰辅酶 A 可产生正丁酸盐(即正丁酸盐)、正己酸盐(即正己酸盐)和正辛酸盐。在此,我们根据元基因组学、元蛋白组学和代谢组学数据,以及 kluyveri梭菌分离物的实验,推测了生物反应器微生物组内的营养层次结构。首先,好氧菌 Pseudoclavibacter caeni 和兼性厌氧真菌 Cyberlindnera jadinii 将部分乙醇池转化为中间代谢产物琥珀酸、乳酸和焦谷氨酸。其次,严格厌氧的 C. kluyveri 将乙酸与残余乙醇拉长为正丁酸。第三,Caproicibacter fermentans 和 Oscillibacter valericigenes 将正丁酸与中间代谢物一起延长为正己酸,然后再延长为正辛酸。在羧酸盐的碳链延长途径中,三羧酸循环和反ß-氧化途径与正辛酸酯的生成呈正相关。这项研究的结果启发人们建立一个链延伸生产平台,分别控制好氧和厌氧阶段,以乙醇和乙酸酯为底物,可再生地生产正辛酸酯这种有吸引力的化学品。更广泛的背景 除了可再生电力能源之外,碳基化学品的生产也必须以可持续的方式独立于化石资源。为了实现这一目标,我们必须扩大工业规模的生物基转化技术组合,以涵盖尽可能多的目标化学品。我们探索了生物链拉长过程,以提供可作为未来循环经济平台化学品的中链羧酸盐。利用链延长法生产的最有价值的中链羧酸盐是正辛酸酯(即正辛酸)。这种分子中一排有八个碳原子(C8),对于化学工业来说,以可再生方式生产具有挑战性。以前的报告阐明,合成气发酵废水中乙醇与乙酸的比率升高会刺激正辛酸酯的生产。到目前为止,研究一直认为高浓度乙醇和乙酸酯的链延伸是一个完全厌氧的过程。我们通过展示能够促进这一过程的需氧和厌氧微生物的营养层次,完善了这一观点。适当的氧气补充可使琥珀酸、乳酸和焦谷氨酸得以合成,从而允许在厌氧条件下高速链延长至正辛酸。鉴于这些结果,未来的研究应侧重于好氧和厌氧微生物的分离研究,以进一步提高工艺性能,从而在工业规模上可再生地生产正辛酸酯。
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