综述：微生物相互作用解析：中链脂肪酸厌氧链延长生物合成的相互作用网络与调控策略

本综述系统阐述了中链脂肪酸（MCFAs）厌氧生物合成过程中微生物相互作用网络的核心机制与调控策略。文章聚焦电子供体-受体生成及链延长（CE）过程，揭示了功能类群间底物竞争与互营共生如何直接调控代谢通量方向性与效率。通过代谢模块化分区、电子传递强化、群体感应（QS）等多维互作机制，为定向微生物工程策略（如电发酵、碳材料添加）提供理论支撑，以提升产物选择性及系统稳定性，推动生物质资源高效增值。

　　

Abstract

The anaerobic biosynthesis of medium-chain fatty acids (MCFAs) as valorized bio-based chemicals relies on intricate and dynamic interaction networks within microbial communities. This review systematically summarizes the key mechanisms and regulatory strategies driving MCFA biosynthesis in terms of microbial interactions, with a focus on electron donor-acceptor generation and chain elongation (CE) processes. The functional stability and resilience of anaerobic fermentation systems are collectively sustained by microbial diversity via modular functional partitioning, metabolic complementarity, resilience against perturbations, and environmental adaptation. Notably, substrate competition and syntrophic symbiosis between functional taxa directly govern the directionality and efficiency of the metabolic flux. Carbon source preferences and environmental factors synergistically steer pathway selection, while exogenous interventions such as enhanced electron transfer or niche occupation optimize microbial cooperation. In addition, quorum sensing and electrochemical synergy further balance inter-species competition to achieve a dynamic equilibrium between metabolic branch inhibition and enrichment of CE consortia. These multidimensional interaction mechanisms provide high-purity electron donors and stable metabolic foundations for MCFA synthesis to guide directional microbial engineering strategies to enhance product yields. This study systematically summarized how microbial interaction networks drive efficient MCFA biosynthesis via a multi-scale coordination between various mechanisms, including metabolic flux partitioning control, environmental response feedback, and functional modularization design, providing a theoretical foundation for resolving critical challenges during anaerobic MCFA fermentation.

Introduction

Given the imperatives of economic and environmental sustainability, the development of renewable energy sources and green chemicals has emerged as a critical pathway toward achieving energy transition and fostering sustainable development. In this context, biomass is a renewable carbon-containing resource that has garnered considerable interest. Anaerobic fermentation is a green and efficient biomass resource utilization technology, wherein microbially mediated processes convert biomass into valuable liquid chemicals such as alcohols and carboxylic acids. Medium-chain fatty acids (MCFAs, C₆–C₁₂ carboxylic acids) such as caproic acid (C₆) and caprylic acid (C₈) are valued for their high energy density, low water solubility, and diverse bioactivities. As a result, they have found broad applications in biofuels, food additives, and fine chemicals. Conventional MCFA production via chemical synthesis or vegetable oil extraction often faces restricted feedstock availability, complex purification processes, and high costs (e.g., caproic acid is priced at 20,000–27,000 CNY·ton^?1). In contrast, chain elongation (CE) methods, which convert organic waste into MCFAs via anaerobic microbial fermentation, offer environmental sustainability and resource recovery, as well as industrial potential.

The anaerobic biosynthesis of MCFAs involves synergistic metabolic cascades within anaerobic microbial consortia, including electron donor generation (e.g., lactate, ethanol), electron acceptor formation (short-chain fatty acids, SCFA), and CE. This complex metabolic network involves coordinated interactions between lactic acid bacteria (LAB), ethanol-producing bacteria/fungi, and CE bacteria, significantly complicating the regulation of MCFA synthesis. Critically, competitive consumption of intermediate metabolites severely constrains MCFA synthesis efficiency and product selectivity. Studies indicate that 30–50?% of carbon flux in typical CE systems is diverted to non-target products via electron donor competition among functional microbiota, resulting in MCFA yields below 40?% of theoretical maxima. Furthermore, dynamic imbalances between metabolic intermediates may trigger the functional deterioration of microbial consortia or even system collapse. This is primarily driven by undissociated MCFAs, such as caproic acid (C₆). When its concentration exceeds 0.87?g·L^?1, it inhibits key bacteria by compromising their cell membrane integrity and forming a detrimental feedback loop. Thus, deciphering the metabolic networks and regulatory mechanisms governing carbon flux allocation during CE is a fundamental scientific hurdle for scaling this technology from lab-scale research to industrial viability.

To further clarify the enhancement mechanisms underlying anaerobic MCFA biosynthesis, recent reviews have focused on substrate type modulation, augmentation of CE microbial consortia, optimization of key environmental parameters, and product recovery technologies. These studies have highlighted the central role of reverse β-oxidation (RBO) and fatty acid biosynthesis (FAB). They have also noted the importance of genera such as Clostridiumand Caproiciproducens, as well as the influence of pH, electron donors, and hydrogen pressure. Strengthening strategies, such as the use of carbon-based materials, quorum sensing (QS), electron bifurcation, and electro-fermentation, have been combined with in situ product recovery methods. These investigations have provided initial insights into the core functional microorganisms and how their associated metabolic pathways drive CE. However, since the efficiency of MCFA biosynthesis depends on an intricate cross-stage microbial metabolic interaction network, further systematic investigations are warranted. It is essential to have a deeper understanding of the mechanisms governing cross-stage microbial interactions, dynamic regulation strategies for electron donors, and the plasticity of the metabolic network. Clarifying these integrated mechanisms is critical for improving MCFA product selectivity, system stability, and overall process sustainability.

Therefore, building upon current research advancements, this review systematically synthesizes the microbial interaction networks and regulatory strategies underpinning anaerobic MCFA biosynthesis. It delineates the microbial composition, functional partitioning, and cross-species interactions across the core stages of electron donor generation, electron acceptor generation, and CE. Furthermore, the principles governing the construction and regulatory characteristics of electron donor-dependent microbial interaction networks are analyzed, revealing that the dynamic profiling of microbial interactions forms the basis for directional regulation. These insights provide a theoretical foundation for the targeted enhancement of MCFA product selectivity and process sustainability, thereby offering critical advances for the efficient valorization of biomass resources. Furthermore, these approaches provide a scientific basis for the effective conversion of diverse organic waste streams, including agricultural residues, municipal sludge, livestock wastewater, and industrial organic effluents, into MCFA. This can promote resource recovery and environmental sustainability efforts within relevant industries.

Electron donor generation stage

In anaerobic fermentation systems, lactate and ethanol are critical electron donors during the microbial biosynthesis of MCFAs. The diversity of their metabolic pathways and cross-conversion of key intermediates profoundly influence the microbial interactions and CE efficiency.

Microbial interaction mechanisms during the donor-acceptor generation stage

The generation of electron donors and acceptors is essential for maintaining a functioning metabolic network. Microbial communities, including LAB, ethanol-producing bacteria/fungi and acidogenic bacteria, interact competitively and cooperatively to regulate the accumulation of key metabolites such as lactate, ethanol, and SCFA. These interactions, mediated by metabolic division of labor, electron transfer, and QS, determine the carbon flux distribution and overall system efficiency.

Microbial interaction mechanisms during the chain elongation stage

During the CE stage, microbial interactions determine the synthesis efficiency and product distribution of MCFA. In the presence of different electron donors, functional microorganisms establish multi-layered networks via metabolic division of labor, electron transfer, and thermodynamic regulation. Lactate-driven systems rely on metabolic coupling between producers and consumers. Ethanol-driven systems demonstrate a tripartite dynamic balance among ethanol-oxidizing bacteria, CE bacteria, and hydrogen-consuming partners. These interactions are further fine-tuned by environmental factors (e.g., pH, H₂ partial pressure) and exogenous interventions (e.g., carbon materials, applied voltage), which collectively optimize electron flow and suppress competing pathways.

Challenges and opportunities

Although the pivotal role of microbial interaction networks in anaerobic CE for MCFAs biosynthesis is being increasingly recognized, existing studies have predominantly relied on static multi-omics data or single-strain metabolic models. These approaches inadequately resolve the dynamic interaction patterns of functional consortia during electron donor generation and CE phases. Furthermore, the metabolic networks during MCFA biosynthesis involve complex cross-feeding and regulatory relationships that are highly sensitive to environmental perturbations. Future research should integrate real-time multi-omics, computational modeling, and advanced bioreactor design to elucidate the spatiotemporal dynamics of microbial interactions. This will enable the development of precision调控 strategies to enhance system resilience and product output.

Conclusions

This review systematically synthesized recent advancements in understanding microbial interactions during anaerobic fermentation for the biosynthesis of MCFAs. It elucidated how cooperative-competitive relationships within complex microbiota govern metabolic pathway selection and production efficiency. The analysis also revealed that microbial cross-talk operated through two-phase mechanisms (donor-acceptor generation and CE phases), where the functional division of labor, metabolic complementarity, and environmental adaptation collectively sustain system stability. By leveraging exogenous interventions (e.g., QS, electro-fermentation) and optimizing operational parameters, it is possible to steer carbon flux toward target MCFAs while minimizing metabolic branching. These insights provide a roadmap for designing robust microbial consortia and scaling up CE processes for sustainable bioproduction.