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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Soil salinization is a serious environmental hazard, limiting plant growth and production in different agro-ecological zones worldwide. Diethyl aminoethyl hexanoate (DA-6) as an essential plant growth regulator (PGR) exhibits a beneficial role in improving crop growth and stress tolerance. However, the DA-6-regulated effect and mechanism of salt tolerance in plants are still not fully understood. The objective of current study was to disclose salt tolerance induced by DA-6 in relation to changes in water and redox balance, photosynthetic function, ionic homeostasis, and organic metabolites reprogramming in white clover (Trifolium repens)., Results: A prolonged duration of salt stress caused water loss, impaired photosynthetic function, and oxidative injury to plants. However, foliar application of DA-6 significantly improved osmotic adjustment (OA), photochemical efficiency, and cell membrane stability under salt stress. In addition, high salinity induced massive accumulation of sodium (Na), but decreased accumulation of potassium (K) in leaves and roots of all plants. DA-6-treated plants demonstrated significantly higher transcript levels of genes involved in uptake and transport of Na and K such as VP1, HKT8, SOS1, NHX2, NHX6, and SKOR in leaves as well as VP1, HKT1, HKT8, H + -ATPase, TPK5, SOS1, NHX2, and SKOR in roots. Metabolomics analysis further illustrated that DA-6 primarily induced the accumulation of glucuronic acid, hexanoic acid, linolenic acid, arachidonic acid, inosose, erythrulose, galactopyranose, talopyranose, urea, 1-monopalmitin, glycerol monostearate, campesterol, stigmasterol, and alanine., Conclusions: The DA-6 significantly up-regulated transcript levels of multiple genes associated with increased Na + compartmentalization in vacuoles and Na + sequestration in roots to reduce Na + transport to photosynthetic organs, thereby maintaining Na + homeostasis under salt stress. The accumulation of many organic metabolites induced by the DA-6 could be attributed to enhanced cell wall and membrane structural stability and functionality, OA, antioxidant defense, and downstream signal transduction in leaves under salt stress. The present study provides a deep insight about the synergistic role of DA-6 in salt tolerance of white clover., (© 2024. The Author(s).)

Aim: To investigate the effect of pyruvate and glucose on leucine transamination and 3-methylbutanal production by Lactococcus lactis, including the comparison with cells possessing glutamate dehydrogenase (GDH) activity., Methods and Results: Lactococcus lactis cells were incubated in chemically defined medium (CDM) with the pH controlled at 5.2 to mimic cheese conditions. Pyruvate supplementation stimulated the production of the key flavour compound 3-methylbutanal by 3-4 times after 72 h of incubation. Concurrently, alanine production increased, demonstrating the involvement of pyruvate in transamination reactions. Glucose-metabolizing cells excreted α-ketoisocaproic acid and produced even 3 times more 3-methylbutanal after 24 h than pyruvate-supplemented cells. Conjugal transfer technique was used to transfer the plasmid pGdh442 carrying the gdh gene encoding for GDH to L. lactis. Introducing GDH did not stimulate the excretion of α-ketoisocaproic acid and the production of 3-methylbutanal., Conclusions: These results demonstrate that Lactococcus uses pyruvate to transaminate leucine into α-ketoisocaproic acid which supports 3-methylbutanal production. Surprisingly, GDH activity did not stimulate leucine transamination and 3-methylbutanal production., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

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 β-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.

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-CH 4 /g-VS fed , 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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R.A.B. and T.B. report a relationship with Société des Produits Nestlé S.A. that includes: employment. The authors declare the following financial interests relationships which may be considered as potential competing interests: O.G.-D., L.J.M.-M., and R.M. reports financial support was provided by Société des Produits Nestlé S.A., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Strong-flavor Baijiu, a type of Chinese liquor, is produced through anaerobic solid-state fermentation in a sealed mud pit. Ethyl caproate, the characteristic flavor compound of strong-flavor Baijiu, is influenced by caproic acid-producing bacteria in the pit mud. To better understand the formation of caproic acid, this study investigated the microbial composition and physicochemical parameters of pit mud from different layers (top, middle, and bottom) in Hubei and Sichuan provinces, China. The results revealed that Caproiciproducens plays a key role in caproic acid production by using lactic acid as a substrate, with its abundance increasing with the depth of the pit mud. A strain Caproiciproducens sp. R1, isolated from the pit mud, was shown to produce caproic acid from lactic acid within an initial pH range of 5.5-9.0 and lactic acid concentrations of 1 %-5 % (m/v). In addition, inoculation of strain R1 into Huangshui (a lactic acid-rich liquid from Baijiu production) resulted in 40.72 mM caproic acid production. This study demonstrates that Caproiciproducens plays a crucial role in caproic acid production from lactic acid during the fermentation process of strong-flavor Baijiu., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acetobacterium woodii and Megasphaera hexanoica were co-cultured for caproic acid (CA) production from lactic acid (LA) and CO 2 . Also, various concentrations (1 g/L, 3 g/L, 5 g/L, and 10 g/L) of Zero Valent Iron (ZVI) were supplied to study its impact on the co-culture system. In flask experiments, 10 g/L LA and 1.0 bar CO 2 produced 0.6 g/L CA with some biomass growth. ZVI increased LA consumption and CA production. Indeed, 3 g/L ZVI boosted CA production by 186 % and biomass accumulation by 103 %, suggesting that ZVI controls the carbon flux. Subsequent automated bioreactor studies showed that 3 g/L ZVI produced 1.842 g/L CA at stable pH, compared to 0.969 g/L without ZVI (control). Further, metabolic activity showed that both bacteria could directly use H 2 , generated by ZVI (3 g/L), as electron donor. Higher ZVI concentrations (10 g/L) resulted in Fe 2+ causing excessive oxidation pressure on M. hexanoica, with its carbon flux flowing preferentially towards biomass. Enzyme assays confirmed that A. woodii preferred 10 g/L ZVI while M. hexanoica preferred 3 g/L for optimal bioconversion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 H 2 and O 2 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., Competing Interests: L.T.A. has ownership in Capro-X, Inc., which is a start-up company that is commercializing a chain-elongating biotechnology production platform.

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., (© 2024 Institute of Food Technologists.)

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&#95;sensu&#95;stricto&#95;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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Carbon dioxide (CO 2 ) plays a crucial role in carbon chain elongation with ethanol serving as an electron donor. In this study, the impacts of various carbonates on CO 2 concentration, hexanoic acid production, and microbial communities during ethanol-butyric acid fermentation were explored. The results showed that the addition of MgCO 3 provided sustained inorganic carbon and facilitated interspecific electron transfer, thereby increasing hexanoic acid yield by 58%. MgCO 3 and NH 4 HCO 3 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 CaCO 3 group increased by 19% and 15%, respectively. The NaHCO 3 group exhibited high headspace CO 2 concentration, promoting acetogenic bacteria enrichment while reducing the abundance of Clostridium&#95;sensu&#95;stricto&#95;12. The batch addition of NaHCO 3 accelerated the synthesis of hexanoic acid and increased its production by 26%. The relative abundance of Clostridium&#95;sensus&#95;stricto&#95;12 was positively correlated with hexanoic acid production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

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.

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., Competing Interests: Declaration of Competing Interest All authors have read the submitted version of the manuscript. The authors have no conflict of interest in relation to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaC AR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaC AR 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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 β-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

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&#95;f&#95;Dysgonomonadaceae, Massilibacterium, and Seramator were the predominant bacteria. Functional enzyme analysis showed that high butyrate content strengthened the fatty acid biosynthesis pathway and reverse β-oxidization pathway. The findings showed the importance of butyrate in CCE for caproate production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 β 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 (PhaC BP-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 PhaC BP-M-CPF4 through N-terminal truncation., Results: The N-terminal truncated mutants of PhaC BP-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 PhaC BP-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 PhaC BP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaC BP-M-CPF4 G8. PhaC BP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (M w ) of PHA produced due to lower expression of the truncated PhaC BP-M-CPF4 . Transformants harbouring PhaC BP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaC BP-M-CPF4 G8 produced higher M w PHA in mostly single larger PHA granules with comparable production as the full-length PhaC BP-M-CPF4 ., Conclusion: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, M w , 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 M w in mostly single larger granules., (© 2024. The Author(s).)

CO 2 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 CO 2 loading rates (Low = 0.5 L CO2 ·L -1 ·d -1 , Medium = 1.0 L CO2 ·L -1 ·d -1 , High = 2.0 L CO2 ·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 CO 2 has a positive effect on chain elongation. The maximum production of caproate and CH 4 were observed at a low CO 2 loading rate. Caproate production reached 1.88 g COD·L -1 ·d -1 with a selectivity of 62.55 %, while CH 4 production reached 129.7 ml/d, representing 47.4 % of the total. Metagenomic analysis showed that low loading rate of CO 2 favored the enrichment of Clostridium kluyveri, with its abundance being 3.8 times higher than at of high CO 2 loading rate. Metatranscriptomic analysis revealed that high CO 2 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 β-oxidation pathway, CO 2 uptake pathway and hydrogenotrophic methanogenesis pathway were reduced at high CO 2 loading rate. These findings provide insight into the underlying mechanisms of how CO 2 affects chain elongation, and it could be a crucial reason for the poor performance of chain elongation systems with high endogenous CO 2 production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cannabinoids are important therapeutical molecules for human ailments, cancer treatment, and SARS-CoV-2. The central cannabinoid, cannabigerolic acid (CBGA), is generated from geranyl pyrophosphate and olivetolic acid by Cannabis sativa prenyltransferase (CsPT4). Despite efforts to engineer microorganisms such as Saccharomyces cerevisiae (S. cerevisiae) for CBGA production, their titers remain suboptimal because of the low conversion of hexanoate into olivetolic acid and the limited activity and stability of the CsPT4. To address the low hexanoate conversion, we eliminated hexanoate consumption by the beta-oxidation pathway and reduced its incorporation into fatty acids. To address CsPT4 limitations, we expanded the endoplasmic reticulum and fused an auxiliary protein to CsPT4. Consequently, the engineered S. cerevisiae chassis showed a marked improvement of 78.64-fold in CBGA production, reaching a titer of 510.32 ± 10.70 mg l -1 from glucose and hexanoate., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Hexanoate is a valuable chemical that can be produced by microorganisms that convert short-chain- to medium-chain carboxylic acids through a process called chain elongation. These microorganisms usually produce mixtures of butyrate and hexanoate from ethanol and acetate, but direct conversion of ethanol to hexanoate is theoretically possible. Steering microbial communities to ethanol-only elongation to hexanoate circumvents the need for acetate addition and simplifies product separation. The biological feasibility of ethanol elongation to hexanoate was validated in batch bioreactor experiments with a Clostridium kluyveri-dominated enrichment culture incubated with ethanol, acetate and butyrate in different ratios. Frequent liquid sampling combined with high-resolution off-gas measurements allowed to monitor metabolic behavior. In experiments with an initial ethanol-to-acetate ratio of 6:1, acetate depletion occurred after ± 35 h of fermentation, which triggered a metabolic shift to direct conversion of ethanol to hexanoate despite the availability of butyrate (± 40 mCmol L -1 ). When only ethanol and no external electron acceptor was supplied, stable ethanol to hexanoate conversion could be maintained until 60-90 mCmol L -1 of hexanoate was produced. After this, transient production of either acetate and butyrate or butyrate and hexanoate was observed, requiring a putative reversal of the Rnf complex. This was not observed before acetate depletion or in presence of low concentrations (40-60 mCmol L -1 ) of butyrate, suggesting a stabilizing or regulatory role of butyrate or butyrate-related catabolic intermediates. This study sheds light on previously unknown versatility of chain elongating microbes and provides new avenues for optimizing (waste) bioconversion for hexanoate production., (© 2023. Springer Nature Limited.)

Plant roots are exposed to hypoxia in waterlogged soils, and they are further challenged by specific phytotoxins produced by microorganisms in such conditions. One such toxin is hexanoic acid (HxA), which, at toxic levels, causes a strong decline in root O 2 consumption. However, the mechanism underlying this process is still unknown. We treated pea (Pisum sativum L.) roots with 20 mM HxA at pH 5.0 and 6.0 for a short time (1 h) and measured leakage of key electrolytes such as metal cations, malate, citrate and nonstructural carbohydrates (NSC). After treatment, mitochondria were isolated to assess their functionality evaluated as electrical potential and O 2 consumption rate. HxA treatment resulted in root tissue extrusion of K + , malate, citrate and NSC, but only the leakage of the organic acids and NSC increased at pH 5.0, concomitantly with the inhibition of O 2 consumption. The activity of mitochondria isolated from treated roots was almost unaffected, showing just a slight decrease in oxygen consumption after treatment at pH 5.0. Similar results were obtained by treating the pea roots with another organic acid with a short carbon chain, that is, butyric acid. Based on these results, we propose a model in which HxA, in its undissociated form prevalent at acidic pH, stimulates the efflux of citrate, malate and NSC, which would, in turn, cause starvation of mitochondrial respiratory substrates of the Krebs cycle and a consequent decline in O 2 consumption. Cation extrusion would be a compensatory mechanism in order to restore plasma membrane potential., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Carboxylate chain elongation can create value-added bioproducts from the organic waste. The effects of Pt@C on chain elongation and associated mechanisms were investigated in simulated sequencing batch reactors. 5.0 g/L of Pt@C greatly increased the synthesis of caproate, with an average yield of 21.5 g COD/L, which was 207.4% higher than the trial without Pt@C. Integrated metagenomic and metaproteomic analyses were used to reveal the mechanism of Pt@C-enhanced chain elongation. Pt@C enriched chain elongators by increasing the relative abundance of dominant species by 115.5%. The expression of functional genes related to chain elongation was promoted in the Pt@C trial. This study also demonstrates that Pt@C may promote overall chain elongation metabolism by enhancing CO 2 uptake of Clostridium kluyveri. The study provides insights into the fundamental mechanisms of how chain elongation can perform CO 2 metabolism and how it can be enhanced by Pt@C to upgrade bioproducts from organic waste streams., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Caproate, an important medium-chain fatty acid, can only be synthesized by limited bacterial species by using ethanol, lactate, or certain saccharides. Caproicibacterium lactatifermentans is a promising caproate producer due to its glucose and lactate utilization capabilities. However, the global cellular responses of this bacterium to different carbon sources were not well understood. Here, C. lactatifermentans showed robust growth on glucose but more active caproate synthesis on lactate. Comparative transcriptome revealed that the genes involved in reverse β-oxidation for caproate synthesis and V-type ATPase-dependent ATP generation were upregulated under lactate condition, while several genes responsible for biomass synthesis were upregulated under glucose condition. Based on metabolic pathway reconstructions and bioenergetics analysis, the biomass accumulation on glucose condition may be supported by sufficient supplies of ATP and metabolite intermediates via glycolysis. In contrast, the ATP yield per glucose equivalent from lactate conversion into caproate was only 20% of that from glucose. Thus, the upregulation of the reverse β-oxidation genes may be essential for cell survival under lactate conditions. Furthermore, the remarkably decreased lactate utilization was observed after glucose acclimatization, indicating the negative modulation of lactate utilization by glucose metabolism. Based on the cotranscription of the lactate utilization repressor gene lldR with sugar-specific PTS genes and the opposite expression patterns of lldR and lactate utilization genes, a novel regulatory mechanism of glucose-repressed lactate utilization mediated via lldR was proposed. The results of this study suggested the molecular mechanism underlying differential physiologic and metabolic characteristics of C. lactatifermentans grown on glucose and lactate. IMPORTANCE Caproicibacterium lactatifermentans is a unique and robust caproate-producing bacterium in the family Oscillospiraceae due to its lactate utilization capability, whereas its close relatives such as Caproicibacterium amylolyticum , Caproiciproducens galactitolivorans, and Caproicibacter fermentans cannot utilize lactate but produce lactate as the main fermentation end product. Moreover, C. lactatifermentans can also utilize several saccharides such as glucose and maltose. Although the metabolic versatility of the bacterium makes it to be a promising industrial caproate producer, the cellular responses of C. lactatifermentans to different carbon sources were unknown. Here, the molecular mechanisms of biomass synthesis supported by glucose utilization and the cell survival supported by lactate utilization were revealed. A novel insight into the regulatory machinery in which glucose negatively regulates lactate utilization was proposed. This study provides a valuable basis to control and optimize caproate production, which will contribute to achieving a circular economy and environmental sustainability.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as a commercial bioplastic due to its biodegradability, thermoplastic and mechanical properties. The properties of this copolymer are greatly affected by the composition of 3HHx monomer. One of the most efficient ways to modulate the composition of 3HHx monomer in P(3HB-co-3HHx) is by manipulating the (R)-3HHx-CoA monomer supply. In this study, a new (R)-specific enoyl-CoA hydratase originating from a non-PHA producer, Streptomyces sp. strain CFMR 7 (PhaJ Ss ), was characterized and found to be effective in supplying 3HHx monomer during in vivo production of P(3HB-co-3HHx) copolymer. The P(3HB-co-3HHx) copolymer produced from the Cupriavidus necator transformant that harbors phaJ Ss , PHB - 4/pBBR1-C BP-M-CPF4 J Ss , showed enhanced 3HHx incorporation of up to 11 mol% without affecting the P(3HB-co-3HHx) production when palm oil was used as the carbon source. In addition, both k cat and k cat /K m of PhaJ Ss were higher toward the C6 than the shorter C4 substrates, underscoring the preference for 3-hydroxyhexanoyl-CoA. These results suggest that PhaJ Ss has a significant ability to supply 3HHx monomers for PHA biosynthesis via β-oxidation and can be applied for metabolic engineering of robust PHA-producing strains., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Soybean isoflavones (SIs) are widely found in food and herbal medicines. Although the pharmacological activities of SIs have been widely reported, their effects on the intestinal microecology of normal hosts have received little attention. Five-week-old Kunming (KM) mice were administered SIs (10 mg/kg/day) for 15 days. Food intake, body weight, and digestive enzyme activity were measured. Small intestine microbiota, including lumen-associated bacteria (LAB) and mucosa-associated bacteria (MAB), were analyzed using 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing. Short-chain fatty acids (SCFAs) were analyzed using gas chromatography-mass spectrometry (GC-MS). The results showed that the mice that consuming SIs showed a higher food intake but a lower body weight gain rate than that of normal mice. Sucrase, cellulase, and amylase activities reduced, while protease activity increased after SIs intervention. Moreover, SIs increased the intestinal bacterial diversity in both LAB and MAB of normal mice. The composition of LAB was more sensitive to SIs than those of MAB. Lactobacillus , Adlercreutzia , Coprococcus , Ruminococcus , Butyricicoccus , and Desulfovibrio were the differential bacteria among the LAB of mice treated with SIs. In addition, acetic acid, valeric acid, isobutyric acid, isovaleric acid, and caproic acid decreased, while butyric acid and propionic acid increased in the mice treated with SIs. Taken together, SIs are beneficial for weight control, even in short-term interventions. The specific mechanism is related to regulating the gut microbiota, changing digestive enzyme activities, and further affecting carbohydrate absorption and metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huang, Zheng, Hui and Xie.)

The rumen simulation technique (RUSITEC) was used to investigate the effect of ergot alkaloids (EA) and a mycotoxin deactivating product (Biomin AA; MDP) on nutrient digestion, ruminal fermentation parameters, total gas, methane, and microbial nitrogen production. Ruminal fermentation vessels received a feedlot finishing diet of 90:10 concentrate:barley silage (DM basis). Using a randomized complete block design, treatments were assigned (n = 4 vessels/treatment) within two RUSITEC apparatuses in a 2 × 2 factorial arrangement. Treatments included: (1) control (CON) diet (no EA and no MDP); (2) CON diet + 1 g/d MDP; (3) CON diet + 20 mg/kg EA; and (4) CON diet + 20 mg/kg EA + 1 g/d MDP. The study was conducted over 14 d with 7 d of adaptation and 7 d of sample collection. Data were analyzed in SAS using PROC MIXED including fixed effects of EA, MDP, and the EA×MDP interaction. Random effects included RUSITEC apparatus and cow rumen inoculum (n = 4). Ergot alkaloids decreased dry matter (DMD) (P = 0.01; 87.9 vs. 87.2%) and organic matter disappearance (OMD) (P = 0.02; 88.8 vs. 88.4%). Inclusion of MDP increased OMD (P = 0.01; 88.3 vs. 88.9%). Neutral detergent fiber disappearance (NDFD) was improved with MDP; however, an EA×MDP interaction was observed with MDP increasing (P < 0.001) NDFD more with EA diet compared to CON. Acetate proportion decreased (P = 0.01) and isovalerate increased (P = 0.03) with EA. Consequently, acetate:propionate was reduced (P = 0.03) with EA. Inclusion of MDP increased total volatile fatty acid (VFA) production (P < 0.001), and proportions of acetate (P = 0.03) and propionate (P = 0.03), and decreased valerate (P < 0.001), isovalerate (P = 0.04), and caproate (P = 0.002). Treatments did not affect (P ≥ 0.17) ammonia, total gas, or methane production (mg/d or mg/g of organic matter fermented). The inclusion of MDP reduced (P < 0.001) microbial nitrogen (MN) production in the effluent and increased (P = 0.01) feed particle-bound MN. Consequently, total MN decreased (P = 0.001) with MDP. In all treatments, the dominant microbial phyla were Firmicutes, Bacteroidota, and Proteobacteria, and the major microbial genus was Prevotella. Inclusion of MDP further increased the abundance of Bacteroidota (P = 0.04) as it increased both Prevotella (P = 0.04) and Prevotellaceae&#95;UCG-003 (P = 0.001). In conclusion, EA reduced OMD and acetate production due to impaired rumen function, these responses were successfully reversed by the addition of MDP., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Feeding 100% forage rape to sheep consistently lowers methane emissions per unit of intake (CH4/DMI) compared to those fed 100% ryegrass pasture. However, forage rape is usually supplemented with other feeds, which might impact the mitigation potential provided by forage rape. The objective of this study was to determine the effect of substituting ryegrass with graded levels of forage rape in the diet of lambs on methane emissions and rumen fermentation characteristics. Seventy wether lambs (n = 14/treatment) were fed a ryegrass-based pasture substituted with 0%, 25%, 50%, 75%, and 100% of forage rape (Brassica napus; FR0, FR25, FR50, FR75, and FR100, respectively) on a dry matter basis. Methane emissions and dry matter intake were measured for 48 h in respiration chambers and a rumen fluid sample was collected. CH4/DMI decreased (P < 0.01) with increasing forage rape inclusion in the diet so that sheep fed FR100 and FR75 emitted 34% and 11% less, respectively, than those fed FR0. CH4/DMI differences for lambs fed FR25 and FR50 were much smaller (<6%) relative to FR0. The pH of rumen fluid decreased (P < 0.01) at higher levels of forage rape inclusion in the diet (FR75 and FR100) compared to low levels of inclusion (FR0, F25, and F50). The proportion of ruminal acetate was least in FR100 (30%) followed by FR75 (10%), FR50 (8%), and FR25 (4%) compared with FR0 (P < 0.001). The proportion of propionate plus succinate was greater for FR100 (+40%), FR75 (+28%), and FR50 (+29%) compared with FR0, with FR25 intermediate (P < 0.001). The methanol concentration, and ethanol and propanol proportions in rumen fluid were greater for FR100 compared with any other treatment (P < 0.001). In conclusion, CH4/DMI decreased at high levels of forage rape inclusion in the diet and especially feeding FR100 was associated with a pronounced shift in rumen fermentation profile, with a significant presence of succinate, ethanol, propanol, methanol, valerate, and caproate., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Pit mud microbial consortia play crucial roles in the formation of Chinese strong-flavor baijiu's key flavor-active compounds, especially butyric and caproic acids. Clostridia , one of the abundant bacterial groups in pit mud, were recognized as important butyric and caproic acid producers. Research on the interactions of the pit mud microbial community mainly depends on correlation analysis at present. Interaction between Clostridium and other microorganisms and its involvement in short/medium-chain fatty acid (S/MCFA) metabolism are still unclear. We previously found coculture of two clostridial strains isolated from pit mud, Clostridium fermenticellae JN500901 ( C. 901) and Novisyntrophococcus fermenticellae JN500902 ( N. 902), could enhance S/MCFA accumulation. Here, we investigated their underlying interaction mechanism through the combined analysis of phenotype, genome, and transcriptome. Compared to monocultures, coculture of C. 901 and N. 902 obviously promoted their growth, including shortening the growth lag phase and increasing biomass, and the accumulation of butyric acid and caproic acid. The slight effects of inoculation ratio and continuous passage on the growth and metabolism of coculture indicated the relative stability of their interaction. Transwell coculture and transcriptome analysis showed the interaction between C. 901 and N. 902 was accomplished by metabolite exchange, i.e., formic acid produced by C. 901 activated the Wood-Ljungdahl pathway of N. 902, thereby enhancing its production of acetic acid, which was further converted to butyric acid and caproic acid by C. 901 through reverse β-oxidation. This work demonstrates the potential roles of mutually beneficial interspecies interactions in the accumulation of key flavor compounds in pit mud. IMPORTANCE Microbial interactions played crucial roles in influencing the assembly, stability, and function of the microbial community. The metabolites of pit mud microbiota are the key to flavor formation of Chinese strong-flavor baijiu. So far, researches on the interactions of the pit mud microbial community have been mainly based on the correlation analysis of sequencing data, and more work needs to be performed to unveil the complicated interaction patterns. Here, we identified a material exchange-based mutualistic interaction system involving two fatty acid-producing clostridial strains ( Clostridium fermenticellae JN500901 and Novisyntrophococcus fermenticellae JN500902) isolated from pit mud and systematically elucidated their interaction mechanism for promoting the production of butyric acid and caproic acid, the key flavor-active compounds of baijiu. Our findings provide a new perspective for understanding the complicated interactions of pit mud microorganisms.

Chimeric polyhydroxyalkanoate synthase PhaCAR is characterized by the capacity to incorporate unusual glycolate (GL) units and spontaneously synthesize block copolymers. The GL and 3-hydroxybutyrate (3HB) copolymer synthesized by PhaCAR is a random-homo block copolymer, poly(GL-ran-3HB)-b-poly(3HB). In the present study, medium-chain-length 3-hydroxyhexanoate (3HHx) units were incorporated into this copolymer using PhaCAR for the first time. The coenzyme A (CoA) ligase from Pseudomonas oleovorans (AlkK) serves as a simple 3HHx-CoA supplying route in Escherichia coli from exogenously supplemented 3HHx. NMR analyses of the obtained polymers revealed that 3HHx units were randomly connected to 3HB units, whereas GL units were heterogeneously distributed. Therefore, the polymer is composed of 2 segments: P(3HB-co-3HHx) and P(GL-co-3HB-co-3HHx). The thermal and mechanical properties of the terpolymer indicate no contiguous P(3HB) segments in the material, consistent with the NMR results. Therefore, PhaCAR synthesized the novel block copolymer P(3HB-co-3HHx)-b-P(GL-co-3HB-co-3HHx), which is the first block polyhydroxyalkanoate copolymer comprising 2 copolymer segments., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Drosophila sechellia is a dietary specialist endemic to the Seychelles islands that has evolved to consume the fruit of Morinda citrifolia. When ripe, the fruit of M. citrifolia contains octanoic acid and hexanoic acid, two medium-chain fatty acid volatiles that deter and are toxic to generalist insects. Drosophila sechellia has evolved resistance to these volatiles allowing it to feed almost exclusively on this host plant. The genetic basis of octanoic acid resistance has been the focus of multiple recent studies, but the mechanisms that govern hexanoic acid resistance in D. sechellia remain unknown. To understand how D. sechellia has evolved to specialize on M. citrifolia fruit and avoid the toxic effects of hexanoic acid, we exposed adult D. sechellia, D. melanogaster and D. simulans to hexanoic acid and performed RNA sequencing comparing their transcriptional responses to identify D. sechellia specific responses. Our analysis identified many more genes responding transcriptionally to hexanoic acid in the susceptible generalist species than in the specialist D. sechellia. Interrogation of the sets of differentially expressed genes showed that generalists regulated the expression of many genes involved in metabolism and detoxification whereas the specialist primarily downregulated genes involved in the innate immunity. Using these data, we have identified interesting candidate genes that may be critically important in aspects of adaptation to their food source that contains high concentrations of HA. Understanding how gene expression evolves during dietary specialization is crucial for our understanding of how ecological communities are built and how evolution shapes trophic interactions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Per- and polyfluorinated substances (PFAS), ubiquitously present in the environment and biota, are transferred to the fetus via the placenta. PFAS can be distinguished, among other things, by their different carbon chain lengths and functional groups. The aim of this study was to provide comprehensive evidence on PFAS transfer rates across the human placental barrier by means of a meta-analysis based upon a systematic review. The available literature up to April 2021 was reviewed and transplacental transfer efficiencies (TTEs) of PFAS assessed. A total of 39 studies reporting data on 20 PFAS were included in the systematic review. Of these, 20 studies with data on 19 compounds were included in the meta-analysis. Comprehensive Meta-Analysis (CMA v3.0) was used for quantitative, statistical analyses with random effects models. A curvilinear relationship was found with short and long chains of perfluorocarboxylic acids (PFCAs) exhibiting higher TTE than compounds with intermediate chain length. Among the less well studied PFAS, perfluorohexanoic acid (PFHxA), 6:2 fluorotelomersulfonic acid (6:2 FTS) and perfluorobutanoic acid (PFBA) stood out the most with a high TEEs. The dependence of TTEs on chain length and functional group is clearly shown in this first meta-analysis on PFAS transfer across the human placenta. More data on effects of less well studied PFAS in pregnant women and neonates are needed to assess the potential risk for fetal exposure.

n -Caproic acid (CA) is gaining increased attention due to its high value as a chemical feedstock. Ruminococcaceae bacterium strain CPB6 is an anaerobic mesophilic bacterium that is highly prolific in its ability to perform chain elongation of lactate to CA. However, little is known about the genome-wide transcriptional analysis of strain CPB6 for CA production triggered by the supplementation of exogenous lactate. In this study, cultivation of strain CPB6 was carried out in the absence and presence of lactate. Transcriptional profiles were analyzed using RNA-seq, and differentially expressed genes (DEGs) between the lactate-supplemented cells and control cells without lactate were analyzed. The results showed that lactate supplementation led to earlier CA p,roduction, and higher final CA titer and productivity. 295 genes were substrate and/or growth dependent, and these genes cover crucial functional categories. Specifically, 5 genes responsible for the reverse β-oxidation pathway, 11 genes encoding ATP-binding cassette (ABC) transporters, 6 genes encoding substrate-binding protein (SBP), and 4 genes encoding phosphotransferase system (PTS) transporters were strikingly upregulated in response to the addition of lactate. These genes would be candidates for future studies aiming at understanding the regulatory mechanism of lactate conversion into CA, as well as for the improvement of CA production in strain CPB6. The findings presented herein reveal unique insights into the biomolecular effect of lactate on CA production at the transcriptional level.

Metabolic chemical reports have fundamentally changed the way researchers study glycosylation. However, when administered as per- O -acetylated sugars, reporter molecules can participate in nonspecific chemical labeling of cysteine residues termed S -glycosylation. Without detailed proteomic analyses, these labeling events can be indistinguishable from bona fide enzymatic labeling convoluting experimental results. Here, we report a solution in the synthesis and characterization of two reporter molecules functionalized at the anomeric position with hexanoic acid: 1-Hex-GlcNAlk and 1-Hex-6AzGlcNAc. Both reporters exhibit robust labeling over background with negligible amounts of nonspecific chemical labeling in cell lysates. This strategy serves as a template for the design of future reporter molecules allowing for more reliable interpretation of results.

The transformation of diverse feedstocks into medium-chain fatty acids (MCFAs) by mixed cultures is a promising biorefinery route because of the high value of MCFAs. A particular concern is how to maintain the microbial consortia in mixed cultures to achieve stable MCFA production. The Chinese strong aroma-type liquor ( Baijiu ) fermentation system continually produces caproic acid for decades through a spontaneous inoculation of anaerobes from pit mud into fermented grains. Therefore, illuminating the dominant caproate-producing bacterium (CPB) in pit mud and how the CPB is sustained in the spontaneous fermentation system will help to reveal the microbiological mechanisms of stable caproate production. Here, we examined pit mud samples across four Chinese strong aroma-type Baijiu -producing areas and found that a caproate-producing Caproicibacterium sp. was widely distributed in these distilleries, with relative abundance ranging from 1.4 to 35.5% and an average abundance of 11.4%. Through controlling carbon source availability, we obtained different simplified caproate-producing consortia and found that the growth advantage of Caproicibacterium sp. was highly dependent on glucose. Then, two strains, named Caproicibacterium sp. strain LBM19010 and Caproicibacterium sp. strain JNU-WLY1368, were isolated from pit mud of two regions. The metabolic versatility of this species utilizing starch, maltose, glucose, and lactate reflected its adaptability to the fermentation environment where these carbon sources coexist. The simultaneous utilization of glucose and lactate contributed to the balance between cell growth and pH homeostasis. This study reveals that multiple adaptation strategies employed by the predominant CPB promotes its stability and dominance in a saccharide- and lactate-rich anaerobic habitat. IMPORTANCE The Chinese strong aroma-type liquor ( Baijiu ) fermentation environment is a typical medium-chain fatty acid-producing system with complex nutrients. Although several studies have revealed the correlation between microbial community composition and abiotic factors, the adaptation mechanisms of dominant species to abiotic environment are still unknown in this special anaerobic habitat. This study identified the predominant CPB in Chinese strong aroma-type Baijiu fermentation system. Metabolic versatility and flexibility of the dominant CPB with a small-size genome indicated that this bacterium can effectively exploit available carbon and nitrogen sources, which could be a key factor to promote its ecological success in a multispecies environment. The understanding of growth and metabolic features of the CPB responsible for its dominance in microbial community will not only contribute to the improvement of Chinese strong aroma-type Baijiu production but also expand its potential industrial applications in caproate production.

Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In the present study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA into butyrate and caproyl-CoA into caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8-times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse β-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp346 and Ala351 have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production., (© 2021 The Author(s).)

Lactate-driven chain elongation (LCE) has emerged as a new biotechnology to upgrade organic waste streams into a valuable biochemical and fuel precursor, medium - chain carboxylate, n -caproate. Considering that a low cost of downstream extraction is critical for biorefinery technology, a high concentration of n -caproate production is very important to improve the scale-up of the LCE process. We report here that in a nonsterile open environment, the n -caproate concentration was increased from the previous record of 25.7 g·liter -1 to a new high level of 33.7 g·liter -1 (76.8 g chemical oxygen demand [COD]·liter - 1 ), with the highest production rate being 11.5 g·liter -1 ·day -1 (26.2 g COD·liter - 1 ·day -1 ). In addition, the LCE process remained stable, with an average concentration of n -caproate production of 20.2 ± 5.62 g·liter -1 (46.1 ± 12.8 g COD·liter - 1 ) for 780 days. Dynamic changes in taxonomic composition integrated with metagenomic data reveal the microbial ecology for long-term production of high concentrations of n -caproate: (i) the core microbiome is related to efficient functional groups, such as Ruminococcaceae (with functional strain CPB6); (ii) the core bacteria can maintain stability for long-term operation; (iii) the microbial network has relatively low microbe-microbe interaction strength; and (iv) low relative abundance and variety of competitors. The network structure could be shaped by hydraulic retention time (HRT) over time, and long-term operation at an HRT of 8 days displayed higher efficacy. IMPORTANCE Our research revealed the microbial network of the LCE reactor microbiome for n- caproate production at high concentrations, which will provide a foundation for designing or engineering the LCE reactor microbiome to recover n -caproate from organic waste streams in the future. In addition, the hypothetical model of the reactor microbiome that we proposed may offer guidance for researchers to find the underlying microbial mechanism when they encounter low-efficiency n -caproate production from the LCE process. We anticipate that our research will rapidly advance LCE biotechnology with the goal of promoting the sustainable development of human society., (Copyright © 2021 American Society for Microbiology.)

Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) are still at higher risk for cardiovascular diseases (CVDs) that are mediated by chronic inflammation. Identification of novel inflammatory mediators with the inherent potential to be used as CVD biomarkers and also as therapeutic targets is critically needed for better risk stratification and disease management in PLWH. Here, we investigated the expression and potential role of the multi-isoform proinflammatory cytokine IL-32 in subclinical atherosclerosis in PLWH (n=49 with subclinical atherosclerosis and n=30 without) and HIV- controls (n=25 with subclinical atherosclerosis and n=24 without). While expression of all tested IL-32 isoforms (α, β, γ, D, ϵ, and θ) was significantly higher in peripheral blood from PLWH compared to HIV- controls, IL-32D and IL-32θ isoforms were further upregulated in HIV+ individuals with coronary artery atherosclerosis compared to their counterparts without. Upregulation of these two isoforms was associated with increased plasma levels of IL-18 and IL-1β and downregulation of the atheroprotective protein TRAIL, which together composed a unique atherosclerotic inflammatory signature specific for PLWH compared to HIV- controls. Logistic regression analysis demonstrated that modulation of these inflammatory variables was independent of age, smoking, and statin treatment. Furthermore, our in vitro functional data linked IL-32 to macrophage activation and production of IL-18 and downregulation of TRAIL, a mechanism previously shown to be associated with impaired cholesterol metabolism and atherosclerosis. Finally, increased expression of IL-32 isoforms in PLWH with subclinical atherosclerosis was associated with altered gut microbiome (increased pathogenic bacteria; Rothia and Eggerthella species) and lower abundance of the gut metabolite short-chain fatty acid (SCFA) caproic acid, measured in fecal samples from the study participants. Importantly, caproic acid diminished the production of IL-32, IL-18, and IL-1β in human PBMCs in response to bacterial LPS stimulation. In conclusion, our studies identified an HIV-specific atherosclerotic inflammatory signature including specific IL-32 isoforms, which is regulated by the SCFA caproic acid and that may lead to new potential therapies to prevent CVD in ART-treated PLWH., Competing Interests: JK has stock ownership in Bristol-Myers Squibb, Johnson & Johnson, Medtronic, Merck and Pfizer. J-PG was employed by the company Caprion Inc. (now CellCarta). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 El-Far, Durand, Turcotte, Larouche-Anctil, Sylla, Zaidan, Chartrand-Lefebvre, Bunet, Ramani, Sadouni, Boldeanu, Chamberland, Lesage, Baril, Trottier, Thomas, Gonzalez, Filali-Mouhim, Goulet, Martinson, Kassaye, Karim, Kizer, French, Gange, Ancuta, Routy, Hanna, Kaplan, Chomont, Landay and Tremblay.)

The retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt), which is a promising therapeutic target for immune diseases, is a major transcription factor of genes related to psoriasis pathogenesis, such as interleukin (IL)-17A, IL-22, and IL-23R. Inspired by the co-crystal structure of RORγt, a 6-oxo-4-phenyl-hexanoic acid derivative 6a was designed, synthesized, and identified as a ligand of RORγt. The structure-activity relationship (SAR) studies in 6a, which focus on the improvement of its membrane permeability profile by introducing chlorine atoms, led to finding 12a, which has a potent RORγt inhibitory activity and a favorable pharmacokinetic profile., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Short-chain fatty acid esters are important flavor chemicals in Chinese traditional fermented Baijiu. Monascus purpureus was recognized as an important microorganism contributing to ester synthesis. However, the molecular basis for ester synthesis was still lacking. The present work combined genome sequencing, transcriptome sequencing, gene library construction, and enzyme engineering to discover a novel catalyst from M. purpureus (isolated from Baijiu fermentation starter). Enzyme LIP05, belonging to the α/β hydrolase family, was identified to synthesize short-chain fatty acid esters under aqueous phase. After deleting the lid domain of LIP05, the synthesis of ethyl pentanoate, ethyl hexanoate, ethyl octanoate, or ethyl decanoate was achieved. Ethyl octanoate with the highest conversion ratio of 93.7% was obtained with the assistance of ultrasound. The study reveals the molecular basis for synthesizing short-chain fatty acid esters by M. purpureus and will promote the application of the species or the enzyme in food industry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Poly(ε-caprolactone) (PCL) is commonly used in devices for tissue reconstruction due to its biocompatibility and suitable mechanical properties. However, its high crystallinity and hydrophobicity do not favor cell adhesion and difficult polymer bioresorption. To improve these characteristics, the development of engineered scaffolds for tissue regeneration, based on poly(globalide- co -ε-caprolactone) (PGlCL) covalently bonded with N -acetylcysteine (PGlCL-NAC) was proposed. The scaffolds were obtained from polymer blends of PCL and PGlCL-NAC, using the electrospinning technique. The use of PGlCL-NAC allowed for the modification of the physical and chemical properties of PCL electrospun scaffolds, including an expressive reduction in the fiber's diameter, hydrophobicity, and crystallinity. All electrospun scaffolds showed no cytotoxicity against fibroblasts (McCoy cells). In vitro biocompatibility assays showed that all tested scaffolds provided high cell viability and proliferation in short-term (NRU, MTT, and nuclear morphology assays) and long-term (clonogenic assay) assays. Nevertheless, PGlCL-NAC based scaffolds have favored the survival and proliferation of the cells in comparison to PCL scaffolds. Cell adhesion on the scaffolds assessed by electronic microscopy images confirmed this behavior. These results suggest that the incorporation of PGlCL-NAC in scaffolds for tissue regeneration could be a promising strategy to improve cell-surface interactions and contribute to the development of more efficiently engineered biomedical devices.

Perfluoroalkyl substances (PFAS) represent one of the most recalcitrant class of compounds of emerging concern and their removal from water is a challenging goal. In this study, we investigated the removal efficiency of three selected PFAS from water, namely, perfluorooctanoic acid (PFOA), perfluorohexanoic acid (PFHxA) and pefluorooctanesulfonic acid (PFOS) using a custom-built non-thermal plasma generator. A modified full factorial design (with 2 levels, 3 variables and the central point in which both quadratic terms and interactions between couple of variables were considered) was used to investigate the effect of plasma discharge frequency, distance between the electrodes and water conductivity on treatment efficiency. Then, the plasma treatment running on optimized conditions was used to degrade PFAS at ppb level both individually and in mixture, in ultrapure and groundwater matrices. PFOS 1 ppb exhibited the best degradation reaching complete removal after 30 min of treatment in both water matrices (first order rate constant 0.107 min -1 in ultrapure water and 0.0633 min -1 in groundwater), while the degradation rate of PFOA and PFHxA was slower of around 65% and 83%, respectively. During plasma treatment, the production of reactive species in the liquid phase (hydroxyl radical, hydrogen peroxide) and in the gas phase (ozone, NO x ) was investigated. Particular attention was dedicated to the nitrogen balance in solution where, following to NO x hydrolysis, total nitrogen (TN) was accumulated at the rate of up to 40 mg N L -1 h -1 .

'Nanguo' pear is particularly renowned for its fragrance. Esters are the main components of its aroma, which are synthesized primarily by the LOX pathway. We identified the main volatile esters and critical gene family members involved in the LOX pathway by monitoring their variation accompanying post-harvest ripening and examining their roles through principal component analysis (PCA), partial least-square regression (PLSR), and correlation analysis. In pears ripening to the optimum taste period (OTP), components and contents of volatile esters reached a peak, of which ethyl butanoate, ethyl hexanoate, and hexyl acetate were most prominent. Linoleic acid and linolenic acid contents rose greatly until OTP and then declined; the activities of LOX, alcohol dehydrogenase (ADH), and alcohol acyltransferase (AAT) increased progressively until the OTP. Among the genes involved in LOX-pathway, the expressions of PuLOX3, PuADH3, and PuAAT contributed most to changes of total ester and main esters in 'Nanguo' pears., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aims: The objective of this study was to evaluate the potential of hexanoic acid (Hex) as a silage additive., Methods and Results: The effect of Hex, Lactobacillus plantarum (Lp) and their combination (Hex + Lp) on the aerobic stability of napier grass silage was investigated. Napier grass was ensiled without additives (C) or with Lp, Hex or Hex + Lp for 60 days followed by 7 days of aerobic exposure. After 60 days of ensiling, the Lp silage had the lowest pH and the highest lactic acid (LA) concentration among all silage, whereas the highest water-soluble carbohydrates (WSC) content was observed in Hex + Lp silage, followed by Hex silage. After 60 days of ensiling, the population of yeasts in C and Lp silages was > 3·0 log 10 CFU per g FW, while that of Hex and Hex + Lp was <2·0 log 10 CFU per g FW. During aerobic exposure, the pH in the Lp and C silages increased (P < 0·05) above 7·0 on day 5. The pH of Hex silage was the lowest among all silages on day 3 and 5, followed by a significant (P < 0·05) increase until 7 days of aerobic exposure. There were no significant changes in pH and AA of Hex + Lp silage over the duration of aerobic exposure. The concentrations of LA in C, Lp and Hex silages decreased while that of Hex + Lp silage remained stable after 3 days of aerobic exposure. The Hex delayed the decline of WSC contents and the increase in yeasts over the aerobic exposure period., Conclusion: Addition of Hex (97 h) and Hex + Lp (>168 h) improved aerobic stability (P < 0·05) as compared to the control (83 h)., Significance and Impact of the Study: Hex or in combination with L. plantarum inhibited the proliferation of yeasts during aerobic exposure of napier grass. Thus, Hex is an alternative antifungal additive to improve aerobic stability., (© 2020 The Society for Applied Microbiology.)

Children with inflammatory bowel diseases (IBD) are particularly vulnerable to infection with Clostridioides difficile (CDI). IBD and IBD + CDI have overlapping symptoms but respond to distinctive treatments, highlighting the need for diagnostic biomarkers. Here, we studied pediatric patients with IBD and IBD + CDI, comparing longitudinal data on the gut microbiome, metabolome, and other measures. The microbiome is dysbiotic and heterogeneous in both disease states, but the metabolome reveals disease-specific patterns. The IBD group shows increased concentrations of markers of inflammation and tissue damage compared with healthy controls, and metabolic changes associate with susceptibility to CDI. In IBD + CDI, we detect both metabolites associated with inflammation/tissue damage and fermentation products produced by C. difficile. The most discriminating metabolite found is isocaproyltaurine, a covalent conjugate of a distinctive C. difficile fermentation product (isocaproate) and an amino acid associated with tissue damage (taurine), which may be useful as a joint marker of the two disease processes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)