BACKGROUND: The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.
RESULTS: Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW-1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW- 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.
CONCLUSIONS: Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

With the growth of the aquaculture industry, antibiotic residues in treated wastewater have become a serious ecological threat. The effects of supplementation with diethyl aminoethyl hexanoate (DA-6) on the removal of tetracycline (TC), ciprofloxacin (CPFX), and sulfamonomethoxine (SMM) from aquaculture wastewater by different microalgae-based systems were examined and systematically analyzed. The results demonstrated that C. vulgaris -S395-2-C. rosea symbiont performed best under 0.2 mg L-1 antibiotic treatment for antibiotic removal. At 10-7 M, DA-6 significantly enhanced C. vulgaris-S395-2-C. rosea symbiont removal of CPFX and SMM at 0.20 mg L-1. The removal of TC, CPFX and SMM by this strain under optimal conditions was 99.2 ± 0.4 %, 86.3 ± 6.3 %, and 91.3 ± 5.7 %, respectively. These results suggest that DA-6 may act on microalgae-bacteria-fungi three-phase symbionts for the removal of multiple antibiotics from aquaculture wastewater.

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.

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.

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.

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.

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.

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.

A novel thermotolerant caproic acid-producing bacterial strain, Clostridium M1NH, was successfully isolated from sewage sludge. Ethanol and acetic acid at a molar ratio of 4:1 proved to be the optimal substrates, yielding a maximum caproic acid production of 3.5 g/L. Clostridium M1NH exhibited remarkable tolerance to high concentrations of ethanol (up to 5% v/v), acetic acid (up to 5% w/v), and caproic acid (up to 2% w/v). The strain also demonstrated a wide pH tolerance range (pH 5.5-7.5) and an elevated temperature optimum between 35 and 40 °C. Phylogenetic analysis based on 16S rRNA gene sequences revealed that Clostridium M1NH shares a 98% similarity with Clostridium luticellarii DSM 29923 T. The robustness of strain M1NH and its efficient caproic acid production from low-cost substrates highlight its potential for sustainable bio-based chemical production. The maximum caproic acid yield achieved by Clostridium M1NH was 1.6-fold higher than that reported for C. kluyveri under similar fermentation conditions. This study opens new avenues for valorizing waste streams and advancing a circular economy model in the chemical industry.

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.