This study used 16S rRNA gene sequencing and metatranscriptomic analysis to comprehensively illustrate how ammonia stress influenced medium-chain fatty acids (MCFA) biosynthesis. MCFA synthesis was inhibited at total ammonia nitrogen (TAN) concentrations above 1000 mg N/L. TAN stress hindered organic hydrolysis, acidification, and volatile fatty acids elongation. Chain-elongating bacteria (e.g., Clostridium_sensu_stricto_12, Clostridium_sensu_stricto_1, Caproiciproducens) abundance remained unchanged, but their activity decreased, partially due to the increased reactive oxygen species. Metatranscriptomic analysis revealed reduced activity of enzymes critical for MCFA production under TAN stress. Fatty acid biosynthesis pathway rather than reverse β-oxidation pathway primarily contributed to MCFA production, and was inhibited under TAN stress. Functional populations likely survived TAN stress through osmoprotectant generation and potassium uptake regulation to maintain osmotic pressure, with NADH-ubiquinone oxidoreductase potentially compensating for ATP loss. This study enhances understanding of MCFA biosynthesis under TAN stress, aiding MCFA production system stability and efficiency improvement.

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Producing medium chain fatty acids (MCFAs) from waste activated sludge (WAS) is crucial for sustainable chemical industries. This study addressed the electron donor requirement for MCFAs production by inoculating Lactobacillus at varying concentrations (7.94 × 1010, 3.18 × 1011, and 6.35 × 1011 cell/L) to supply lactate internally. Interestingly, the highest MCFAs yield (∼2000 mg COD/L) occurred at the lowest Lactobacillus inoculation. Higher inoculation concentrations redirected more carbon from WAS towards alcohols production rather than MCFAs generation, with up to 2852 mg COD/L alcohols obtained under 6.35 × 1011 cell/L inoculation. Clostridium dominance and increased genes abundance for substrate hydrolysis, lactate conversion, and MCFAs/alcohol production collectively enhanced WAS-derived MCFAs and alcohols synthesis after Lactobacillus inoculation. Overall, the strategy of Lactobacillus inoculation regulated fermentation outcomes and subsequent carbon recovery in WAS, presenting a sustainable technology to achieve liquid bio-energy production from underutilized wet wastes.

Chain elongating bacteria are a unique guild of strictly anaerobic bacteria that have garnered interest for sustainable chemical manufacturing from carbon-rich wet and gaseous waste streams. They produce C6-C8 medium-chain fatty acids which are valuable platform chemicals that can be used directly, or derivatized to service a wide range of chemical industries. However, the application of chain elongating bacteria for synthesizing products beyond C6-C8 medium-chain fatty acids has not been evaluated. In this study, we assess the feasibility of expanding the product spectrum of chain elongating bacteria to C9-C12 fatty acids, along with the synthesis of C6 fatty alcohols, dicarboxylic acids, diols, and methyl ketones. We propose several metabolic engineering strategies to accomplish these conversions in chain elongating bacteria and utilize constraint-based metabolic modelling to predict pathway stoichiometries, assess thermodynamic feasibility, and estimate ATP and product yields. We also evaluate how producing alternative products impacts the growth rate of chain elongating bacteria via resource allocation modelling, revealing a trade-off between product carbon length and class versus cell growth rate. Together, these results highlight the potential for using chain elongating bacteria as a platform for diverse oleochemical biomanufacturing and offer a starting point for guiding future metabolic engineering efforts aimed at expanding their product range.

Recently, there has been notable interest in researching and industrially producing medium-chain carboxylic acids (MCCAs) like n-caproate and n-caprylate via chain elongation process. This study presents a comprehensive assessment of the behavior and MCCA production profiles of Clostridium kluyveri in batch and continuous modes, at different ethanol:acetate molar ratios (1.5:1, 3.5:1 and 5.5:1). The highest n-caproate concentration, 12.9 ± 0.67 g/L (92.9 ± 1.39 % MCCA selectivity), was achieved in batch mode at a 3.5:1 ratio. Interestingly, higher ratios favored batch mode selectivity over continuous mode when this was equal or higher to 3.5:1. Steady state operation yielded the highest n-caproate (9.5 ± 0.13 g/L) and n-caprylate (0.35 ± 0.020 g/L) concentrations at the 3.5:1 ratio. Increased ethanol:acetate ratios led to a higher excessive ethanol oxidation (EEO) in both operational modes, potentially limiting n-caproate production and selectivity, especially at the 5.5:1 ratio. Overall, this study reports the efficient MCCA production of both batch and continuous modes by C. kluyveri.

The depletion of fossil fuels has prompted an urgent search for alternative chemicals from renewable sources. Current technology in medium chain fatty acids (MCFAs) production though chain elongation (CE) is becoming increasingly sustainable, hence the motivation for this review, which provides the detailed description, insights and analysis of the metabolic pathways, substrates type, inoculum and fermentation process. The main rate-limiting steps of microbial MCFAs production were comprehensively revealed and the corresponding innovative solutions were also critically evaluated. Innovative strategies such as substrate pretreatment, electrochemical regulation, product separation, fermentation parameter optimization, and electroactive additives have shown significant advantages in overcoming the rate-limiting steps. Furthermore, novel regulatory strategies such as quorum sensing and electronic bifurcation are expected to further increase the MCFAs yield. Finally, the techno-economic analysis was carried out, and the future research focuses were also put forward.

Nano zero-valent iron (NZVI) has been shown to effectively enhance the chain elongation (CE) process, addressing the issue of limited yield of medium-chain carboxylic acids (MCCA) from organic wastewater. However, the specific impact of NZVI on the metabolism of CE bacteria (CEB) is not well understood. In this study, it was aimed to investigate the mechanism by which an optimal concentration of NZVI influences CE metabolism, particularly in relation to ethanol oxidation, electron transfer, and MCCA synthesis. This was achieved through single-factor influence experiments and metagenomic analysis. The results showed that the addition of 1 g/gVSS NZVI achieved the highest MCCA yield (n-caproic acid + n-octanoic acid) at 2.02 g COD/L, which was 4.9 times higher than the control. This improvement in MCCA production induced by NZVI was attributed to several factors. Firstly, NZVI facilitated the oxidation of acetaldehyde, leading to its reduced accumulation in the system (from 18.4 % to 5.8 %), due to the optimized chemical environment created by NZVI corrosion, including near-neutral pH and a more reductive oxidation-reduction potential (ORP). Additionally, the inherent conductivity property of NZVI and the additional Fe ions released during corrosion improved the electron transfer efficiency between CEB. Lastly, both the composition of microbial communities and the abundance of unique enzyme genes confirmed the selective stimulation of NZVI on the reverse β-oxidation (RBO) pathway. These findings provide valuable insights into the role of NZVI in CEB metabolism and its potential application for enhancing MCCA production in CE bioreactors.

In this study, wine lees and waste activated sludge (WAS) were co-fermented for the first time in a 4:1 ratio (COD basis) at 20, 40, 70 and 100 gCOD/L, in batch at 37 °C and pH 7.0. The substrates were successfully converted to caproate (C6) and heptanoate (C7) with a high selectivity (40.2 % at 40 gCOD/L). The rapidly-growing chain-elongating microbiome was enriched inClostridiaceaeandOscillospiraceae, representing together 3.4-8.8 % of the community. Substrate concentrations higher than 40 gCOD/L negatively affected C6 and C7 selectivities and yields, probably due to microbial inhibition by high ethanol concentrations (15.82-22.93 g/L). At 70 and 100 gCOD/L, chain elongation shifted from ethanol-based to lactate-based, with a microbiome enriched in the lactic acid bacteriaRoseburia intestinalis(27.3 %) andEnterococcus hirae(13.8 %). The partial pressure of H2(pH2) was identified by thermodynamic analysis as a fundamental parameter for controlling ethanol oxidation and improving C6 and C7 selectivities.

Medium-chain carboxylates (MCCs) are used in various industrial applications. These chemicals are typically extracted from palm oil, which is deemed not sustainable. Recent research has focused on microbial chain elongation using reactors to produce MCCs, such as n-caproate (C6) and n-caprylate (C8), from organic substrates such as wastes. Even though the production of n-caproate is relatively well-characterized, bacteria and metabolic pathways that are responsible for n-caprylate production are not. Here, three 5 L reactors with continuous membrane-based liquid-liquid extraction (i.e., pertraction) were fed ethanol and acetate and operated for an operating period of 234 days with different operating conditions. Metagenomic and metaproteomic analyses were employed. n-Caprylate production rates and reactor microbiomes differed between reactors even when operated similarly due to differences in H2 and O2 between the reactors. The complete reverse β-oxidation (RBOX) pathway was present and expressed by several bacterial species in the Clostridia class. Several Oscillibacter spp., including Oscillibacter valericigenes, were positively correlated with n-caprylate production rates, while Clostridium kluyveri was positively correlated with n-caproate production. Pseudoclavibacter caeni, which is a strictly aerobic bacterium, was abundant across all the operating periods, regardless of n-caprylate production rates. This study provides insight into microbiota that are associated with n-caprylate production in open-culture reactors and provides ideas for further work.IMPORTANCEMicrobial chain elongation pathways in open-culture biotechnology systems can be utilized to convert organic waste and industrial side streams into valuable industrial chemicals. Here, we investigated the microbiota and metabolic pathways that produce medium-chain carboxylates (MCCs), including n-caproate (C6) and n-caprylate (C8), in reactors with in-line product extraction. Although the reactors in this study were operated similarly, different microbial communities dominated and were responsible for chain elongation. We found that different microbiota were responsible for n-caproate or n-caprylate production, and this can inform engineers on how to operate the systems better. We also observed which changes in operating conditions steered the production toward and away from n-caprylate, but more work is necessary to ascertain a mechanistic understanding that could be predictive. This study provides pertinent research questions for future work.

Despite the increased research efforts aimed at understanding iron-based conductive materials (CMs) for facilitating chain elongation (CE) to produce medium chain fatty acids (MCFAs), the impact of these materials on microbial community functions and the adaptation mechanisms to their biotoxicity remain unclear. This study found that the supply of zero-valent iron (ZVI) and magnetite enhanced the MCFAs carbon-flow distribution by 26 % and 52 %, respectively. Metagenomic analysis revealed the upregulation of fatty acid metabolism, pyruvate metabolism and ABC transporters with ZVI and magnetite. The predominant functional microorganisms were Massilibacterium and Tidjanibacter with ZVI, and were Petrimonas and Candidatus_Microthrix with magnetite. Furthermore, it was demonstrated that CE microorganisms respond and adapt to the biotoxicity of iron-based CMs by adjusting Two-component system and Quorum sensing for the first time. In summary, this study provided a new deep-insight on the feedback mechanisms of CE microorganisms on iron-based CMs.