Differential uptake and translocation of perfluoroalkyl substances by vegetable roots and leaves: Insight into critical influencing factors.

Crop contamination of perfluoroalkyl substances (PFASs) may threaten human health, with root and leaves representing the primary uptake pathways of PFASs in crops. Therefore, it is imperative to elucidate the uptake characteristics of PFASs by crop roots and leaves as well as the critical influencing factors. In this study, the uptake and translocation of PFASs by roots and leaves of pak choi and radish were systematically explored based on perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), and perfluorooctane sulfonate (PFOS). Additionally, the roles of root Casparian strips, leaf stomata, and PFAS structures in the aforementioned processes were elucidated. Compared with pak choi, PFASs are more easily transferred to leaves after root uptake in radish, resulting from the lack of root Casparian strips. In pak choi root, the bioaccumulation of C4-C8 perfluoroalkyl carboxylic acids (PFCAs) showed a U-shaped trend with the increase of their carbon chain lengths, and the translocation potentials of individual PFASs from root to leaves negatively correlated with their chain lengths. The leaf uptake of PFOA in pak choi and radish mainly depended on cuticle sorption, with the evidence of a slight decrease in the concentrations of PFOA in exposed leaves after stomatal closure induced by abscisic acid. The leaf bioaccumulation of C4-C8 PFCAs in pak choi exhibited an inverted U-shaped trend as their carbon chain lengths increased. PFASs in exposed leaves can be translocated to the root and then re-transferred to unexposed leaves in vegetables. The longer-chain PFASs showed higher translocation potentials from exposed leaves to root. PFOS demonstrated a higher bioaccumulation than PFOA in crop roots and leaves, mainly due to the greater hydrophobicity of PFOS. Planting root vegetables lacking Casparian strips is inadvisable in PFAS-contaminated environments, in view of their higher PFAS bioaccumulation and considerable human intake.

Biomethanization of rigid packaging made entirely of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate): Mono- and co-digestion tests and microbial insights.

This study evaluates the anaerobic mesophilic mono- and co-digestion of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) plastic bottles as a proxy for rigid packaging materials. Initial tests showed a 97.3 ± 0.2 % reduction in weight and an observable alteration in the surface (thinning, color fading and pitting) of the PHBH bottles after eight weeks. Subsequent tests showed that PHBH squares (3 × 3 cm) produced 400 NmL-CH4/g-VSfed, at a slower rate compared to powdered PHBH but with similar methane yield. Co-digestion experiments with food waste, swine manure, or sewage sludge showed successful digestion of PHBH alongside organic waste (even at a high bioplastic loading of 20 % volatile solids basis), with methane production comparable to or slightly higher than that observed in mono-digestion. Molecular analyses suggested that the type of co-substrate influenced microbial activity and that methane production was mainly driven by hydrogenotrophic methanogenesis. These results suggest the potential for integrating rigid PHBH packaging into anaerobic digesters.

Enzyme Catalyzed Formation of CoA Adducts of Fluorinated Hexanoic Acid Analogues using a Long-Chain acyl-CoA Synthetase from Gordonia sp. Strain NB4-1Y.

Per and polyfluoroalkyl substances (PFAS) are a large family of anthropogenic fluorinated chemicals of increasing environmental concern. Over recent years, numerous microbial communities have been found to be capable of metabolizing some polyfluoroalkyl substances, generating a range of low-molecular-weight PFAS metabolites. One proposed pathway for the microbial breakdown of fluorinated carboxylates includes ß-oxidation, this pathway is initiated by the formation of a CoA adduct. However, until recently no PFAS-CoA adducts had been reported. In a previous study, we were able to use a bacterial medium-chain acyl-CoA synthetase (mACS) to form CoA adducts of fluorinated adducts of propanoic acid and pentanoic acid but were not able to detect any products of fluorinated hexanoic acid analogues. Herein, we expressed and purified a long-chain acyl-CoA synthetase (lACS) and a A461K variant of mACS from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that lACS can catalyze the formation of CoA adducts of 1:5 fluorotelomer carboxylic acid (FTCA), 2:4 FTCA and 3:3 FTCA, albeit with generally low turnover rates (<0.02 s-1) compared with the nonfluorinated hexanoic acid (5.39 s-1). In addition, the A461K variant was found to have an 8-fold increase in selectivity toward hexanoic acid compared with wild-type mACS, suggesting that Ala-461 has a mechanistic role in selectivity toward substrate chain length. This provides further evidence to validate the proposed activation step involving the formation of CoA adducts in the enzymatic breakdown of PFAS.

Variability in n-caprylate and n-caproate producing microbiomes in reactors with in-line product extraction.

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 ß-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.

Comprehensive comparative study on n-caproate production by Clostridium kluyveri: batch vs. continuous operation modes.

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.

Inoculation of Zaopei with biofortification: Inducing variation in community structure and improving flavor compounds in Nongxiangxing baijiu.

The deterioration of the quality of raw liquor caused by the low content of ethyl hexanoate in Nongxiangxing baijiu has become a pervasive problem in the baijiu industry. Therefore, this study attempted to increase the synthesis of ethyl hexanoate by microorganisms with high esterase activity to increase Zaopei fermentation. The results showed that biofortification was a feasible and important way to improve the quality of the raw liquor and increase the ethyl hexanoate content. Adding Bacillus subtilis, Staphylococcus epidermidis, and Millerozyma farinosa for biofortified fermentation disturbed the microbial community structure of Zaopei and increased the abundance of Wickerhamomyces, Saccharomyces, and Thermoascus. The contents of ethyl hexanoate, ethyl valerate, ethyl caprylate, and ethyl heptanoate also increased noticeably in baijiu. The results of E-nose and sensory analysis tested and verified that the baijiu in the fortified group had better flavor characteristics.

High-cell-density fed-batch strategy to manufacture tailor-made P(HB-co-HHx) by engineered Ralstonia eutropha at laboratory scale and pilot scale.

The transition towards a sustainable bioeconomy requires the development of highly efficient bioprocesses that enable the production of bulk materials at a competitive price. This is particularly crucial for driving the commercialization of polyhydroxyalkanoates (PHAs) as biobased and biodegradable plastic substitutes. Among these, the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) shows excellent material properties that can be tuned by regulating its monomer composition. In this study, we developed a high-cell-density fed-batch strategy using mixtures of fructose and canola oil to modulate the molar composition of P(HB-co-HHx) produced by Ralstonia eutropha Re2058/pCB113 at 1-L laboratory scale up to 150-L pilot scale. With cell densities >100 g L-1 containing 70-80 wt% of PHA with tunable HHx contents in the range of 9.0-14.6 mol% and productivities of up to 1.5 g L-1 h-1, we demonstrate the tailor-made production of P(HB-co-HHx) at an industrially relevant scale. Ultimately, this strategy enables the production of PHA bioplastics with defined material properties on the kilogram scale, which is often required for testing and adapting manufacturing processes to target diverse applications.

Bacterial Growth Modulatory Effects of Two Branched-Chain Hydroxy Acids and Their Production Level by Gut Microbiota.

Branched-chain hydroxy acids (BCHAs), produced by lactic acid bacteria, have recently been suggested as bioactive compounds contributing to the systemic metabolism and modulation of the gut microbiome. However, the relationship between BCHAs and gut microbiome remains unclear. In this study, we investigated the effects of BCHAs on the growth of seven different families in the gut microbiota. Based on in vitro screening, both 2-hydroxyisovaleric acid (HIVA) and 2-hydroxyisocaproic acid (HICA) stimulated the growth of Lactobacillaceae and Bifidobacteriaceae, with HIVA showing a significant growth promotion. Additionally, we observed not only the growth promotion of probiotic Lactobacillaceae strains but also growth inhibition of pathogenic B. fragilis in a dosedependent manner. The production of HIVA and HICA varied depending on the family of the gut microbiota and was relatively high in case of Lactobacillaceae and Lachnosporaceae. Furthermore, HIVA and HICA production by each strain positively correlated with their growth variation. These results demonstrated gut microbiota-derived BCHAs as active metabolites that have bacterial growth modulatory effects. We suggest that BCHAs can be utilized as active metabolites, potentially contributing to the treatment of diseases associated with gut dysbiosis.

Targeted microbial collaboration to enhance key flavor metabolites by inoculating Clostridium tyrobutyricum and Saccharomyces cerevisiae in the strong-flavor Baijiu simulated fermentation system.

Ethyl hexanoate and ethyl butyrate are indispensable flavor metabolites in strong-flavor Baijiu (SFB), but batch production instability in fermenting grains can reduce the quality of distilled Baijiu. Biofortification of the fermentation process by designing a targeted microbial collaboration pattern is an effective method to stabilize the quality of Baijiu. In this study, we explored the metabolism under co-culture liquid fermentation with Clostridium tyrobutyricum DB041 and Saccharomyces cerevisiae YS219 and investigated the effects of inoculation with two functional microorganisms on physicochemical factors, flavor metabolites, and microbial communities in solid-state simulated fermentation of SFB for the first time. The headspace solid-phase microextraction-gas chromatography-mass spectrometry results showed that ethyl butyrate and ethyl hexanoate significantly increased in fermented grain. High-throughput sequencing analysis showed that Pediococcus, Lactobacillus, Weissella, Clostridium_sensu_stricto_12, and Saccharomyces emerged as the dominant microorganisms at the end of fermentation. Co-occurrence analysis showed that ethyl hexanoate and ethyl butyrate were significantly correlated (|r| > 0.5, P < 0.05) with a cluster of interactions dominated by lactic acid bacteria (Pediococcus, Lactobacillus, Weissella, and Lactococcus), which was driven by the functional C. tyrobutyricum and S. cerevisiae. Mantel test showed that moisture and reducing sugars were the main physicochemical factor affecting microbial collaboration (|r| > 0.7, P < 0.05). Taken together, the collaborative microbial pattern of inoculation with C. tyrobutyricum and S. cerevisiae showed positive results in enhancing typical flavor metabolites and the synergistic effects of microorganisms in SFB.

Hexanoic acid production and microbial community in anaerobic fermentation: Effects of inorganic carbon addition.

Carbon dioxide (CO2) plays a crucial role in carbon chain elongation with ethanol serving as an electron donor. In this study, the impacts of various carbonates on CO2 concentration, hexanoic acid production, and microbial communities during ethanol-butyric acid fermentation were explored. The results showed that the addition of MgCO3 provided sustained inorganic carbon and facilitated interspecific electron transfer, thereby increasing hexanoic acid yield by 58%. MgCO3 and NH4HCO3 inhibited the excessive ethanol oxidation and decreased the yield of acetic acid by 51% and 42%, respectively. The yields of hexanoic acid and acetic acid in the CaCO3 group increased by 19% and 15%, respectively. The NaHCO3 group exhibited high headspace CO2 concentration, promoting acetogenic bacteria enrichment while reducing the abundance of Clostridium_sensu_stricto_12. The batch addition of NaHCO3 accelerated the synthesis of hexanoic acid and increased its production by 26%. The relative abundance of Clostridium_sensus_stricto_12 was positively correlated with hexanoic acid production.

Analysis of ester-producing performance in high-yield ethyl hexanoate yeast and the effect on metabolites in bio- enhanced Daqu, the starter for Baijiu and other traditional fermented foods.

AIMS: Ethyl hexanoate, one of the key flavor compounds in strong-flavor Baijiu. To improve the content of ethyl hexanoate in strong-flavor Baijiu, a functional strain with high yield of ethyl hexanoate was screened and its ester-producing performance was studied. METHODS AND RESULTS: Upon identification, the strain was classified as Candida sp. and designated as ZY002. Under optimal fermentation conditions, the content of ethyl hexanoate synthesized by ZY002 can be as high as 170.56 mg L-1. A fermentation test was carried out using the ZY002 strain bioaugmented Daqu to verify the role of the strain applied to Baijiu brewing. It was found that strain ZY002 could not only improve the moisture and alcohol contents of fermented grains but also diminish the presence of reducing sugar and crude starch. Furthermore, it notably amplified the abundance of flavor compounds. CONCLUSION: In this study, Candida sp. ZY002 with a high yield of ethyl hexanoate provided high-quality strain resources for the actual industrial production of Baijiu.

Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Elevated caproic acid production from one-stage anaerobic fermentation of organic waste and its selective recovery by electro-membrane process.

A constructed microbial consortia-based strategy to enhance caproic acid production from one-stage mixed-fermentation of glucose was developed, which incubated with acidogens (Clostridium sensu stricto 1, 11 dominated) and chain elongators (including Clostridium sensu stricto 12, Sporanaerobacter, and Caproiciproducens) acclimated from anaerobic sludge. Significant product upgrading toward caproic acid (8.31 g‧L-1) and improved substrate degradation was achieved, which can be greatly attributed to the lactic acid platform. Whereas, a small amount of caproic acid was observed in the control incubating with acidogens, with an average concentration of 2.09 g‧L-1. The strategy accelerated the shape and cooperation of the specific microbial community dominated by Clostridium sensu stricto and Caproiciproducens, which thereby contributed to caproic acid production via the fatty acid biosynthesis pathway. Moreover, the tailored electrodialysis with bipolar membrane enabled progressive up-concentration and acidification, allowing selective separation of caproic acid as an immiscible product with a purity of 82.58 % from the mixture.

Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaCAR for the Biosynthesis of Poly[(R)-3-hydroxybutyrate-co-6-hydroxyhexanoate].

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCAR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCAR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB-co-6HHx) synthesis in Escherichia coli. As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB-co-6HHx) decreased as the 6HHx fraction increased.

Caproicibacter sp. BJN0012, a potential new species isolated from cellar mud for caproic acid production from glucose.

Acids play a crucial role in enhancing the flavor of strong-aroma Baijiu, and among them, caproic acid holds significant importance in determining the flavor of the final product. However, the metabolic synthesis of caproic acid during the production process of Baijiu has received limited attention, resulting in fluctuations in caproic acid content among fermentation batches and generating production instability. Acid-producing bacteria found in the cellar mud are the primary microorganisms responsible for caproic acid synthesis, but there is a lack of research on the related microbial resources and their metabolic properties. Therefore, it is essential to identify and investigate these acid-producing microorganisms from cellar mud to ensure stable caproic acid synthesis. In this study, a unique strain was isolated from the cellar mud, exhibiting a 98.12 % similarity in its 16 S rRNA sequence and an average nucleotide identity of 79.57 % with the reference specie, together with the DNA-DNA hybridization of 23.20 % similarity, confirming the distinct species boundaries. The strain was able to produce 1.22 ± 0.55 g/L caproic acid from glucose. Through genome sequencing, annotation, and bioinformatics analysis, the complete pathway of caproic acid synthesis from glucose was elucidated, and the catalytic mechanism of the key thiolase for caproic acid synthesis was investigated. These findings provide useful fundamental data for revealing the metabolic properties of caproic acid-producing bacteria found in cellar mud.

Efficient caproic acid production from lignocellulosic biomass by bio-augmented mixed microorganisms.

Producing caproic acid via carboxylate platform is an environmentally-friendly approach for treating lignocellulosic agricultural waste. However, its implementation is still challenged by low product yields and selectivity. A microbiome named cellulolytic acid-producing microbiome (DCB), proficient in producing cellulolytic acid, was successfully acquired and shows promise for producing high-level caproic acid. In this study, a bioaugmentation method utilizing Clostridium kluyveri is proposed to enhance caproic acid yield of DCB using rice straw. With exogenous ethanol, bioaugmentation with Clostridium kluyveri significantly improved the caproic acid concentration and selectivity by 7 times and 4.5 times, achieving 12.9 g/L and 55.1 %, respectively. The addition of Clostridium kluyveri introduced reverse ß-oxidation pathway, a more efficient caproic acid production pathway. Meanwhile, bioaugmentation enriched the bacteria proficient in degrading straw and producing short-chain fatty acids, providing more substrates for caproic acid production. This study provides potential bioaugmentation strategies for optimizing caproic acid yield from lignocellulosic biomass.

Metagenomic analysis towards understanding the effects of ammonia on chain elongation process for medium chain fatty acids production.

The production of medium chain fatty acids (MCFAs) through chain elongation (CE) from organic wastes/wastewater has attracted much attention, while the effects of a common inhibitor-ammonia has not been elucidated. The mechanism of ammonia affecting CE was studied by metagenomic. The lag phase duration of caproate production was increased, and the maximum caproate production rate was decreased by 43.4 % at 4 g-N/L, as compared to 0 g-N/L. And hydrochar (HC) alleviated the inhibition of ammonia at 4 g-N/L. Metagenomic analysis indicated that ammonia induced UBA4085 sp.FDU78 as the dominant microorganism, and metabolic reconstruction revealed its potential CE ability. Furthermore, ammonia inhibited the reverse ß oxidation pathway and Acetyl-CoA production pathway. The tolerance of UBA4085 sp.FDU78 to ammonia was associated with the uptake of inorganic ions, energy conservation, and synthesis of osmoprotectants. The present study provided a deep-insight on the ammonia tolerance mechanism on the CE process.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Influence of acetate-to-butyrate ratio on carbon chain elongation in anaerobic fermentation.

This study investigated the effect of electron acceptor (EA) distribution (acetate to butyrate ratio) on the carbon chain elongation (CCE) process. The results showed that the higher content of butyrate in the initial material led to the higher production of caproate. The maximum production of caproate was 3.74 ± 0.30 g·L-1, which was obtained when only butyrate was added as EA. Little caproate but much butyrate was produced where only acetate was added as EA. This indicated that CCE bacteria preferentially selected acetate as the EA to produce butyrate, and butyrate could be selected as EA to produce caproate only when the acetate content was much lower than butyrate. Unclassified_f_Dysgonomonadaceae, Massilibacterium, and Seramator were the predominant bacteria. Functional enzyme analysis showed that high butyrate content strengthened the fatty acid biosynthesis pathway and reverse ß-oxidization pathway. The findings showed the importance of butyrate in CCE for caproate production.

CO2 uptake in ethanol-driven chain elongation system: Microbial metabolic mechanisms.

CO2 as a byproduct of organic waste/wastewater fermentation has an important impact on the carboxylate chain elongation. In this study, a semi-continuous flow reactor was used to investigate the effects of CO2 loading rates (Low = 0.5 LCO2·L-1·d-1, Medium = 1.0 LCO2·L-1·d-1, High = 2.0 LCO2·L-1·d-1) on chain elongation system Ethanol and acetate were utilized as the electron donor and electron acceptor, respectively. The results demonstrate that low loading rate of CO2 has a positive effect on chain elongation. The maximum production of caproate and CH4 were observed at a low CO2 loading rate. Caproate production reached 1.88 g COD·L-1·d-1 with a selectivity of 62.55 %, while CH4 production reached 129.7 ml/d, representing 47.4 % of the total. Metagenomic analysis showed that low loading rate of CO2 favored the enrichment of Clostridium kluyveri, with its abundance being 3.8 times higher than at of high CO2 loading rate. Metatranscriptomic analysis revealed that high CO2 loading rate induced oxidative stress in microorganisms, as evidenced by increased expression of heat shock proteins and superoxide dismutase genes. Further investigation suggested that genes associated with the reverse ß-oxidation pathway, CO2 uptake pathway and hydrogenotrophic methanogenesis pathway were reduced at high CO2 loading rate. These findings provide insight into the underlying mechanisms of how CO2 affects chain elongation, and it could be a crucial reason for the poor performance of chain elongation systems with high endogenous CO2 production.