Acetate Accumulation Research Articles

Methanogenic community linked to organic acids fermentation from root exudates are affected by rice intensification in rotational soil systems

Rice paddy soils are an important biogenic source of methane, a relevant greenhouse gas. Rice rotation with upland crops and pastures affects hydrolytic and fermentative bacteria and methanogenic archaea by sequential exposition to oxic and anoxic conditions. There are several ways to reduce methane emissions from rice paddy soils. Here we examine the effect of intensification in rice rotations with upland crops upon the microbial community structure and methanogenesis when dicarboxylic acids exudated by rice root were amended in microcosms assays. Soils under rotations rice-pasture, rice-soybean and continuous rice, with the last two more intensive systems recently installed in the field experiment, were sampled. Amplicon sequencing of 16S rRNA gene analysis showed that the rotation system was a main driver of the microbial community structure, explaining 33%, 38% and 55% of the differences in the structure of Bacteria, Archaea and methanogens, respectively. The density of mcrA gene copies was significantly higher in continuous rice soil than in other soils, and the relative abundance of methanogenic archaea was 33% for rice monoculture, whereas represented 15% and 14% for the rice-pasture and rice-soybean soils, respectively. The incubation of soils with tartrate and succinate confirmed functional differences among soils since rice monoculture showed similar or higher methane production and significantly less acetate and propionate accumulation than other soils. The T-RFLP analysis of mcrA gene in soils and incubated soils revealed that Rice Cluster I increased after incubation in all rotational soils, whereas Methanosarcinaceae and Methanobacteriaceae remained relevant after incubation. Furthermore, a more stable community of methanogenic archaea was established in soil under continuous rice, since T-RF profiles of soils and incubated soils with dicarboxylic acids grouped together with 62% similarity, while communities from other rotational soils experimented a great shift after incubation with these substrates. Altogether, these results showed that the conversion of rice-pasture rotational fields to a more intensive rice crop production strongly impacts the methanogenic community structure and their potential for methane emission.

Chlamydomonas-Methylobacterium oryzae cooperation leads to increased biomass, nitrogen removal and hydrogen production

In the context of algal wastewater bioremediation, this study has identified a novel consortium formed by the bacterium Methylobacterium oryzae and the microalga Chlamydomonas reinhardtii that greatly increase biomass generation (1.22 g L−1·d−1), inorganic nitrogen removal (>99%), and hydrogen production (33 mL·L−1) when incubated in media containing ethanol and methanol. The key metabolic aspect of this relationship relied on the bacterial oxidation of ethanol to acetate, which supported heterotrophic algal growth. However, in the bacterial monocultures the acetate accumulation inhibited bacterial growth. Moreover, in the absence of methanol, ethanol was an unsuitable carbon source and its incomplete oxidation to acetaldehyde had a toxic effect on both the alga and the bacterium. In cocultures, both alcohols were used as carbon sources by the bacteria, the inhibitory effects were overcome and both microorganisms mutually benefited. Potential biotechnological applications in wastewater treatment, biomass generation and hydrogen production are discussed.

A rechargeable microbial electrochemical sensor for water biotoxicity monitoring

Microbial electrochemical sensor has been regarded as a promising technology for water biotoxicity monitoring, while its practical application is partially restricted by the requirement of organic substrate or power supply in real time. In the present study, rechargeable microbial electrochemical sensors were constructed, with the ex-situ preparation of bioanode and the in-situ preparation of biocathode. Acetate, formate, and hydrogen were identified as the charge carriers, and a Coulombic efficiency of 38% ± 18% and an energy efficiency of 2.2% ± 0.7% were obtained. The increase of charge current could decrease the energy efficiency as well as maximum acetate accumulation, while increase the H2 component. The increase of discharge resistor could enhance the energy efficiency, while had little impact on the Coulombic efficiency. Good reusability of rechargeable biosensors was observed with an injection of formaldehyde from 0% to 0.05%, but the sensors could not be fully recovered after the injection of 0.1% formaldehyde. The exposure of formaldehyde could affect the relative abundance of Acetobacterium, Acetoanaerobium, and Geobacter. When an abiotic cathode was used, the formate and hydrogen produced during the charging process were converted into acetate as an intermediate product during the discharging process. This study demonstrates the potential of the rechargeable microbial electrochemical system as a novel sensor for water biotoxicity monitoring.

Antimicrobial Activity of Zymomonas mobilis Is Related to Its Aerobic Catabolism and Acid Resistance

Zymomonas mobilis is an ethanologenic, facultatively anaerobic alpha-proteobacterium, known for its inhibitory effect on the growth of a wide variety of microorganisms. This property might be interesting for the design of novel antimicrobials, yet it has negative implications for biotechnology, as it hinders the use of Z. mobilis as a producer microorganism in cocultivation. So far, the chemical nature of its inhibitory compound(s) has not been established. In the present study, we demonstrate that the putative inhibitor is a low-molecular-weight (below 3 kDa), thermostable compound, resistant to protease treatment, which is synthesized under aerobic conditions in Z. mobilis strains via the active respiratory chain. It is also synthesized by aerated nongrowing, glucose-consuming cells in the presence of chloramphenicol, thus ruling out its bacteriocin-like peptide nature. The inhibitory activity is pH-dependent and strongly correlated with the accumulation of propionate and acetate in the culture medium. Although, in Z. mobilis, the synthesis pathways of these acids still need to be identified, the acid production depends on respiration, and is much less pronounced in the non-respiring mutant strain, which shows low inhibitory activity. We conclude that propionate and acetate play a central role in the antimicrobial effects of Z. mobilis, which itself is known to bear high resistance to organic acids.

Integrated multi-omics analyses reveal the key microbial phylotypes affecting anaerobic digestion performance under ammonia stress

Ammonia inhibition is one of the most common causes of instability during the operation of commercial biogas plants. Here, the sensitivity of different functional bacteria to ammonia stress, the ability of functional bacteria to adapt to ammonia stress, and the key phylotypes affecting anaerobic digestion (AD) performance were studied by evaluating the process performance, active microbiome, and protein expression patterns during endogenous ammonia accumulation using integrated metagenomics and metaproteomics analyses. Acetate metabolism was most sensitive to ammonia stress, and the expression activity of methyl-CoM reductase of Methanothrix was inhibited at relatively low ammonia concentrations, which resulted in the accumulation of acetate and other short-chain volatile fatty acids (VFAs) through feedback effects. As the AD process progressed, the abundance of active Methanosarcina with high ammonia tolerance increased, and the activity of their enzymes related to acetoclastic methanation was significantly up-regulated, which resulted in the complete restoration of acetate metabolism and AD performance. Thus, microbial communities can cope with acetate metabolic repression through self-optimization. In contrast, when the total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) increased to 4846.95 mg N/L and 337.46 mg N/L, respectively, propionate (and no other VFAs) accumulated in the digester, which was accompanied by a decrease in methane yield by more than 65%. At this time, the abundance of active syntrophic propionate-oxidizing bacteria (SPOB) decreased by 52%, and the expression of key enzymes related to propionate degradation was significantly down-regulated. The proportion of down-regulated differentially expressed proteins in the dominant Pelotomaculum was as high as 94%, indicating the severe suppression of the growth of these functional bacteria as well as their inability to easily acclimate to ammonia stress. Thus, SPOB appeared to be the key microbial phylotypes affecting AD performance under ammonia stress. Ammonia inhibited the methylmalonyl-CoA pathway of Pelotomaculum by inhibiting the expression of succinyl-CoA synthase, which resulted in the suppression of syntrophic propionate oxidation. The results of this study provide new insights into the microbial mechanism of ammonia inhibition and identify the key phylotypes affecting AD performance under ammonia stress. Our findings also shed light on the microbial regulatory targets of nitrogen-rich waste anaerobic digesters.

H2 competition between homoacetogenic bacteria and methanogenic archaea during biomethanation from a combined experimental-modelling approach

Competition for H2 between homoacetogenic bacteria and methanogenic archaea is commonly faced in biological biogas upgrading process reducing the potential for high methane production capacities. In the present work, different feeding regimes were examined using anaerobic inocula that were adapted and non-adapted to the gaseous feedstock. Adapted inoculum could compensate the increased pressure at all examined levels and especially, produced above 95% of the theoretical methane without accumulating acetate at 0.2 atm. On the contrary, acetate accumulation was detected when a non-adapted inoculum was used. Microbial adaptation to elevated pressures can be applied as a means to improve production capacities. Thermodynamic analysis showed that hydrogenotrophic methanogenesis had always lower permissive H2 partial pressures (0.43–0.85 Pa) compared to homoacetogenesis (3.20–7.46 Pa) at the examined pH (neutral vs alkaline) and pressure levels (i.e., 0.2–2.0 atm). An unstructured kinetic model was developed to study the influence of the examined variables in the hydrogenotrophic, homoacetogenesis and aceticlastic pathways. The kinetic parameters of the proposed model were estimated from experimental results and the goodness of the fitting was assessed by the coefficient of determination which indicated that the model describes efficiently the dynamics of the different compounds involved in the investigated pathways.

Exogenous N-hexanoyl-L-homoserine lactone assists in upflow anaerobic sludge blanket recovery from acetate accumulation via aceticlastic methanogens enrichment

Volatile fatty acids (VFAs) accumulation caused by shock loading severely hampers the performance and stability of anaerobic digestion. An upflow anaerobic sludge blanket (UASB) was operated to examine its performance, sludge properties, and microbial community behavior during shock loading and recovery with exogenous N-hexanoyl-L-homoserine lactone (C6-HSL). After shock loading, chemical oxygen demand (COD) removal was significantly reduced from 79.09% to 65.63%. The abundance of Methanosarcinales also significantly decreased, which resulted in acetate accumulation (1,163.55 mg/L). Sludge granules disintegrated along with the decrease in extracellular polymeric substances (EPS). After supplying 1 μg/L C6-HSL, COD removal resumed to 75.10%. Furthermore, C6-HSL enhanced the abundance and metabolic activity of aceticlastic methanogens, decreased acetate concentration to 146.87 mg/L, improved EPS secretion, and caused the re-assembly of disintegrated sludges to form large granules. These results advanced our understanding of microbial community behavior and provided an attractive strategy for restoration of UASB recovered from shock loading.

Flux balance analysis for overproduction of organic acids by Synechocystis sp. PCC 6803 under dark anoxic condition

Cyanobacteria have recently been considered as potential organisms for metabolic engineering with an objective to improve its ability to synthesize biofuel precursors and other value-added products. A modified version of the genome-scale metabolic model of Synechocystis sp. PCC 6803 model organism was used to theoretically demonstrate the flux distribution in the central carbon metabolism towards optimal accumulation of organic acids, namely succinate, acetate, lactate and malate. We further validate the model using experimental data reported by Hasunuma et al. Met. Engg. Comm., 2016, 3, 130–141 for organic acid synthesis under dark anoxic conditions from glycogen accumulated during photoautotrophic growth conditions by sequestering atmospheric CO2. We show that key limiting factors are redox balance causing gluconeogenesis, availability of CO2 and channeling of carbon flux to TCA cycle, in addition to the rate of breakdown of glycogen. Sodium bicarbonate supplementation enhances succinate production flux by eliminating the CO2 limitations for the phosphoenolpyruvate (PEP) to oxaloacetate biochemical reaction catalyzed by PEP carboxylase. A combined strategy of supplementation with NaHCO3 and increased temperature can only offer ~25% of the theoretical maximum succinate production. We suggest possible metabolic pathway interventional approaches to improve the succinate productivity.

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling.

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (1H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.

Alcoholic ketoacidosis: an underdiagnosed entity

Alcoholic ketoacidosis (AKA) is a common and underdiagnosed cause of metabolic acidosis in patients with an alcohol-related disorder. It is a cause of non-hypoxic hyperlactatemia and its identification is particularly interesting in order to not multiply medical exams. Indeed, AKA will be cured by a simple management. Clinically, patients have digestive signs such as nausea, vomiting and abdominal pain after a bout of heavy drinking following by few days of fasting. On clinical examination, they have no impairment of consciousness and most often have a relatively well-preserved general condition. Biologically, there is a metabolic acidosis with an elevated anion gap associated with electrolyte disturbances and moderate hyperlactatemia. The pathophysiology of this acidosis is complex and involves an accumulation of ketone bodies, lactate and acetate. This diagnosis is unknown and often delayed or missed while its identification is essential in order to not confuse it with diabetic ketoacidosis. In fact, insulin treatment is at major risk of hypoglycemia, sometimes fatal, in these malnourished patients. The treatment consists of vitamin therapy and the correction of hydro-electrolyte disorders. Insulin and bicarbonate infusion should be avoided. The evolution is favorable and the acidosis is corrected in a few hours after this treatment. The risk is the recurrence of episodes. The purpose of this article is to describe the clinical, biological and pathophysiology of AKA so that it will be better recognized and its management more quickly implemented.

Volatile Profile and Biosynthesis of Post-harvest Apples are Affected by the Mechanical Damage.

Mechanical damage to fruit causes flavor changes during post-harvest supply chains. It is important to identify the main volatiles and explore their biosynthesis mechanism. In this study, the volatile changes in apples caused by mechanical damage were analyzed by gas chromatography-ion mobility spectrometry. Hexanal and ethyl acetate were accumulated and identified as potential volatile biomarkers to detect damaged apples. The study on the lipoxygenase (LOX) pathway and transcription factors (TFs) shows that mechanical damage up-regulated the expression of MdLOX-like, MdLOX3b, MdLOX7b, MdLOX7c, MdLOX2a, and MdAAT in the LOX pathway and that of one MYB TF (MdMYB-like), five ERF TFs (MdERF073, MdERF003, MdERF114, MdERF15, and MdERF2), and five WRKY TFs (MdWRKY23, MdWRKY17, MdWRKY46, MdWRKY48, and MdWRKY71). Notably, MdAAT was significantly correlated to MdMYB-like, MdWRKY23, MdWRKY71, MdERF15, and MdERF2. Thus, TFs may attribute to the accumulation of hexanal and ethyl acetate by regulating the expression of LOX pathway-related genes.

Synthetic biosensor accelerates evolution by rewiring carbon metabolism toward a specific metabolite.

Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolve cells under the selective condition toward the acquisition of genotypes that optimally reallocate cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determine that mutations in the conserved regions of proteins involved in global transcriptional regulation alter the expression of several genes associated with central carbon metabolism. These changes rewire central carbon flux toward the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.

Temperature Phased Anaerobic Digestion at the Intermediate Zone of 45 °C: Performances, Stability and Pathogen Deactivation

Temperature phased anaerobic digestion (TPAD) systems with conventional sequences (first stage of 55 ℃ and second stage of 35 ℃) have been widely studied. However, very limited studies were available on TPAD system with the first stage operated at the intermediate zone of 45 °C, mainly due to the notion that limited microbial activity occurs within this zone. The objective of this research was to evaluate the performance, stability and the capability of 45 °C TPAD in producing class A biosolids, in comparison to a conventional TPAD. Four combinations of TPAD systems were studied, 45 ℃ TPAD 2.5/10 (1st stage solids retention time (SRT) 2.5 days/2nd stage SRT 10 days), 45 ℃ TPAD 7.5/10, 55 ℃ TPAD 2.5/10 and 55 ℃ TPAD 7.5/10. Among all, 45 ℃ TPAD 7.5/10 was found to have the best performances, attributed to its high volatile solids (VS) destruction (58%), minimal acetate accumulation (127 mg/L), high methane yield (0.58 m3 CH4/kg VS removed), high COD destruction solid COD (sCOD; 74% and total COD (tCOD) 54%) and minimal free NH3 content (67.5 mg/L). As for stability, stable pH distribution, high alkalinity content and low VFA to alkalinity ratio, indicated a well-buffered system. Additionally, the system had also able to produce class A biosolids. Therefore, proved that TPAD system operated at the intermediate zone of 45 ℃ can perform better than the conventional TPAD, hence, highlighting its economic advantage.

A cell engineering approach to enzyme-based fed-batch fermentation

BackgroundA fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation.ResultsA putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent.ConclusionsThis study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation.

TCA Cycle and Its Relationship with Clavulanic Acid Production: A Further Interpretation by Using a Reduced Genome-Scale Metabolic Model of Streptomyces clavuligerus.

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of β-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.

Increasing salinity concentrations determine the long-term participation of methanogenesis and sulfidogenesis in the biodigestion of sulfate-rich wastewater

The competition between sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) depends on several factors, such as the COD/SO42− ratio, sensitivity to inhibitors and even the length of the operating period in reactors. Among the inhibitors, salinity, a characteristic common to diverse types of industrial effluents, can act as an important factor. This work aimed to evaluate the long-term participation of sulfidogenesis and methanogenesis in the sulfate-rich wastewater process (COD/SO42− = 1.6) in an anaerobic structured-bed reactor (AnSTBR) using sludge not adapted to salinity. The AnSTBR was operated for 580 d under mesophilic temperature (30 °C). Salinity levels were gradually increased from 1.7 to 50 g-NaCl L−1. Up to 35 g-NaCl L−1, MA and SRB equally participated in COD conversion, with a slight predominance of the latter (53 ± 11%). A decrease in COD removal efficiency associated with acetate accumulation was further observed when applying 50 g-NaCl L−1. The sulfidogenic pathway corresponded to 62 ± 17% in this case, indicating the inhibition of MA. Overall, sulfidogenic activity was less sensitive (25%-inhibition) to high salinity levels compared to methanogenesis (100%-inhibition considering the methane yield). The wide spectrum of SRB populations at different salinity levels, namely, the prevalence of Desulfovibrio sp. up to 35 g-NaCl L−1 and the additional participation of the genera Desulfobacca, Desulfatirhabdium, and Desulfotomaculum at 50 g-NaCl−1 explain such patterns. Conversely, the persistence of Methanosaeta genus was not sufficient to sustain methane production. Hence, exploiting SRB populations is imperative to anaerobically remediating saline wastewaters.

Characteristics of the process of biohydrogen production from simple and complex substrates with different biopolymer composition

The work investigated the characteristics of the dark fermentation (DF) process of a number of simple (starch, sunflower oil, peptone, both separately and mixed) and complex (dog food, pig feed, sewage sludge) substrates using a mixed culture of microorganisms, with a controlled pH (5.5), at 55 °C. Peptone and sunflower oil were characterized by the lowest production of H2, namely 5.0 and 2.3 ml H2/g COD, respectively. The specific hydrogen yield from starch was 1.55 mol H2/mol hexose. The addition of peptone and sunflower oil to starch reduced the specific yield of hydrogen from starch by 23%. A large difference in hydrogen production was observed during DF of complex substrates. The specific hydrogen yield from dog food was 46.5 ml H2/g COD or 143.4 ml H2/g carbohydrates; from pig feed – 32.1 ml H2/g COD or 91.6 ml H2/g carbohydrates; and from sewage sludge – 9.3 ml H2/g COD or 98.0 ml H2/g carbohydrates. Possible relationships between the biopolymer composition of substrates and characteristics of the DF process were analyzed using Spearman's rank correlation coefficients. The concentration of carbohydrates, as well as the ratio of carbohydrates/proteins and carbohydrates/fats, were the main factors influencing the high specific yield of H2, its content in biogas, as well as the ratio of H2/soluble metabolites. The concentration of proteins had a statistically significant positive effect on the accumulation of acetate and succinate, and carbohydrates - on the accumulation of caproate.

An experimental assessment of robust control and estimation of acetate concentration in Escherichia coli BL21(DE3) fed-batch cultures

Avoiding acetate accumulation is a major challenge in Escherichia coli fed-batch cultures, since it leads to an inhibition of the cell respiratory capacity. A closed-loop regulation ensuring a low acetate concentration offers a practical solution to maintain the cultures near the optimal operating conditions. In this work, a robust Generic Model Controller (GMC) is designed and implemented to regulate the acetate concentration in E. coli BL21 (DE3) fed-batch cultures. To compensate for model mismatch, disturbances, and measurement noise, a robust design using the LMI formalism is achieved, considering robustness and transient performance requirements. Since the acetate concentration is not available for on-line measurement, an Unscented Kalman Filter (UKF) is also designed and implemented to estimate the acetate concentration based on an on-line biomass measurement. The proposed GMC-UKF strategy is validated through simulation runs and experimental tests at lab-scale. The control strategy shows good performance in regulating the non-measured acetate concentration. The estimation of this latter signal from the biomass measurement is performed accurately by the UKF.

2,3-Butanediol synthesis from glucose supplies NADH for elimination of toxic acetate produced during overflow metabolism

Overflow metabolism-caused acetate accumulation is a major problem that restricts industrial applications of various bacteria. 2,3-Butanediol (2,3-BD) synthesis in microorganisms is an ancient metabolic process with unidentified functions. We demonstrate here that acetate increases and then decreases during the growth of a bacterium Enterobacter cloacae subsp. dissolvens SDM. Both bifunctional acetaldehyde/ethanol dehydrogenase AdhE-catalyzed ethanol production and acetate-induced 2,3-BD biosynthesis are indispensable for the elimination of acetate generated during overflow metabolism. 2,3-BD biosynthesis from glucose supplies NADH required for acetate elimination via AdhE-catalyzed ethanol production. The coupling strategy involving 2,3-BD biosynthesis and ethanol production is widely distributed in bacteria and is important for toxic acetate elimination. Finally, we realized the co-production of ethanol and acetoin from chitin, the second most abundant natural biopolymer whose catabolism involves inevitable acetate production through the coupling acetate elimination strategy. The synthesis of a non-toxic chemical such as 2,3-BD may be viewed as a unique overflow metabolism with desirable metabolic functions.

Study on relationship between bacterial diversity and quality of Huangjiu (Chinese Rice Wine) fermentation.

Huangjiu (Chinese rice wine) is brewed in an open environment, where bacteria play an important role during the fermentation process. In this study, bacterial community structure and composition changes in the fermented mash liquid of mechanized Huangjiu, well‐fermented manual Huangjiu (wines of good qualities), and poorly fermented manual Huangjiu (wines of poor qualities: spoilage, high acidity, low alcohol content) in different fermentation stages from Guyuelongshan Shaoxing Huangjiu company were analyzed via metagenomic sequencing. And bacterial metabolic difference was analyzed via gene prediction of metabolic pathway enzymes. The results showed that the bacterial diversity degree was abundant, and the number of bacterial species in every sample was approximately 200–400. Lactic acid bacteria (LAB) dominated the bacterial community of Huangjiu fermentation, and lactobacillus was predominant species in well‐fermented Huangjiu while Lactobacillus brevis had an absolute dominance in spoilage Huangjiu. Further, gene prediction revealed that transformation of malate to pyruvate and lactate anabolism was more active in mash liquid of well‐fermented manual Huangjiu, while acetate accumulation was stronger in mash liquid of poorly fermented manual Huangjiu, which explained acidity excess reason in poorly fermented Huangjiu at gene level.