Higher Acetate Production Research Articles

Effect of Mutant and Engineered High-Acetate-Producing Saccharomyces cerevisiae var. boulardii Strains in Dextran Sodium Sulphate-Induced Colitis.

Acetate-producing Saccharomyces cerevisiae var. boulardii strains could exert improved effects on ulcerative colitis, which here, was preclinically evaluated in an acute dextran sodium sulphate induced model of colitis. Nine-week-old female mice were divided into 12 groups, receiving either drinking water or 2.75% dextran sodium sulphate for 7 days, combined with a daily gavage of various treatments with different levels of acetate accumulation: sham control (phosphate buffered saline, no acetate), non-probiotic control (Baker's yeast, no acetate), probiotic control (Enterol®, transient acetate), and additionally several Saccharomyces cerevisiae var. boulardii strains with respectively no, high, and extra-high acetate accumulation. Disease activity was monitored daily, and feces samples were collected at different timepoints. On day 14, the mice were sacrificed, upon which blood and colonic tissue were collected for analysis. Disease activity in inflamed mice was lower when treated with the high-acetate-producing strain compared to sham and non-probiotic controls. The non-acetate-producing strain showed higher disease activity compared to the acetate-producing strains. Accordingly, higher histologic inflammation was observed in non- or transient-acetate-producing strains compared to the sham control, whereas this increase was not observed for high- and extra-high-acetate-producing strains upon induction of inflammation. These anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes. Moreover, only the strain with the highest acetate production was superior in maintaining a stable gut microbial alpha-diversity upon inflammation. These findings support new possibilities for acetate-mediated management of inflammation in inflammatory bowel disease by administrating high-acetate-producing Saccharomyces cerevisae var. boulardii strains.

H2 mediated mixed culture microbial electrosynthesis for high titer acetate production from CO2

Microbial electrosynthesis (MES) converts CO2 into value-added products such as volatile fatty acids (VFAs) with minimal energy use, but low production titer has limited scale-up and commercialization. Mediated electron transfer via H2 on the MES cathode has shown a higher conversion rate than the direct biofilm-based approach, as it is tunable via cathode potential control and accelerates electrosynthesis from CO2. Here we report high acetate titers can be achieved via improved in situ H2 supply by nickel foam decorated carbon felt cathode in mixed community MES systems. Acetate concentration of 12.5 g L−1 was observed in 14 days with nickel-carbon cathode at a poised potential of −0.89 V (vs. standard hydrogen electrode, SHE), which was much higher than cathodes using stainless steel (5.2 g L−1) or carbon felt alone (1.7 g L−1) with the same projected surface area. A higher acetate concentration of 16.0 g L−1 in the cathode was achieved over long-term operation for 32 days, but crossover was observed in batch operation, as additional acetate (5.8 g L−1) was also found in the abiotic anode chamber. We observed the low Faradaic efficiencies in acetate production, attributed to partial H2 utilization for electrosynthesis. The selective acetate production with high titer demonstrated in this study shows the H2-mediated electron transfer with common cathode materials carries good promise in MES development.

Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus.

A genome-scale metabolic model, encompassing a total of 623 genes, 727 reactions, and 865 metabolites, was developed for Pyrococcus furiosus, an archaeon that grows optimally at 100°C by carbohydrate and peptide fermentation. The model uses subsystem-based genome annotation, along with extensive manual curation of 237 gene-reaction associations including those involved in central carbon metabolism, amino acid metabolism, and energy metabolism. The redox and energy balance of P. furiosus was investigated through random sampling of flux distributions in the model during growth on disaccharides. The core energy balance of the model was shown to depend on high acetate production and the coupling of a sodium-dependent ATP synthase and membrane-bound hydrogenase, which generates a sodium gradient in a ferredoxin-dependent manner, aligning with existing understanding of P. furiosus metabolism. The model was utilized to inform genetic engineering designs that favor the production of ethanol over acetate by implementing an NADPH and CO-dependent energy economy. The P. furiosus model is a powerful tool for understanding the relationship between generation of end products and redox/energy balance at a systems-level that will aid in the design of optimal engineering strategies for production of bio-based chemicals and fuels. IMPORTANCE The bio-based production of organic chemicals provides a sustainable alternative to fossil-based production in the face of today's climate challenges. In this work, we present a genome-scale metabolic reconstruction of Pyrococcus furiosus, a well-established platform organism that has been engineered to produce a variety of chemicals and fuels. The metabolic model was used to design optimal engineering strategies to produce ethanol. The redox and energy balance of P. furiosus was examined in detail, which provided useful insights that will guide future engineering designs.

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis.

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

Solid neutral red/Nafion conductive layer on carbon felt electrode enhances acetate production from CO2 and energy efficiency in microbial electrosynthesis system

Renewable electricity-based microbial electrosynthesis can upgrade CO2 into value-added chemicals and simultaneously increase the number of biocatalysts by cell growth, helping to achieve sustainable carbon-negative processes. In most studies, the main strategy for improving the MES performance was to enhance H2-based electron uptake by decreasing the overpotential and electrical conductivity of the electrode. Less is known about the electrode-based direct electron uptake for CO2 conversion in MES. In this study, a solid neutral red/Nafion conductive layer was developed on the carbon electrode surface using a feasible dip and dry method. The modified electrode showed higher HER overpotential and lower capacitance but enhanced redox capability and hydrophobicity, which increased direct electron transport to the bacteria rather than hydrogen-based indirect electron delivery. The Neutral red/Nafion-implemented MES showed faster start-up, higher acetate production, and energy efficiency than the non-modified electrode.

Miniphocaeibacter halophilus sp. nov., an ammonium-tolerant acetate-producing bacterium isolated from a biogas system.

An anaerobic bacterial strain, designated AMB_01T, recovered from mesophilic propionate enrichment of a high-ammonia biogas digester, was characterised using phenotypic and molecular taxonomic methods. Cells of AMB_01T are coccus-shaped and often occur arranged as diplococci or sarcina. Growth occurred at 20–45 °C, initial pH 5.5–8.5 and with up to 0.7 M NH4Cl, with optimum growth at 37–42 °C and pH 8.0. AMB_01T achieved high cell density and highest acetate production when grown on carbohydrates, including monomers, disaccharides and polysaccharides, such as glucose, maltose, cellobiose and starch. The strain was also able to use amino acids and some organic acids and alcoholic compounds for growth. Acetate was formed as the main product and yeast was not required for growth. The major cellular fatty acids were summed feature 4 (iso-C17 : 1I and/or anteiso-C17 : 1B), C18 : 1ω7, C14 : 0, C16 : 0 and summed feature 3 (C16 : 1ω7 and/or iso-C15 : 0 2OH). The highest 16S rRNA gene sequence similarity found was with Miniphocaeibacter massiliensis (96.6 %), within the family Peptoniphilaceae , phylum Bacillota (Firmicutes). The genomic DNA G+C content was 29.0 mol%. An almost complete set of genes for the acetyl-CoA pathway was found. Genome comparisons between AMB_01T and close relatives showed highest digital DNA–DNA hybridisation to Finegoldia magna (23 %), highest average nucleotide identity with genome nucleotide and amino acid sequences to M. massiliensis (72 and 73 %, respectively) and highest average nucleotide identity (87 %) with Schnuerera ultunensis , indicating that AMB_01T represents a novel species. Analysis of genomic, chemotaxonomic, biochemical and physiological data confirmed that strain AMB_01T represents a novel species, for which the name Miniphocaeibacter halophilus sp. nov. is proposed. The type strain is AMB_01T (=DSM 110247T=JCM 39107 T).

Autotrophic Acetate Production under Hydrogenophilic and Bioelectrochemical Conditions with a Thermally Treated Mixed Culture.

Bioelectrochemical systems are emerging technologies for the reduction in CO2 in fuels and chemicals, in which anaerobic chemoautotrophic microorganisms such as methanogens and acetogens are typically used as biocatalysts. The anaerobic digestion digestate represents an abundant source of methanogens and acetogens microorganisms. In a mixed culture environment, methanogen’s inhibition is necessary to avoid acetate consumption by the presence of acetoclastic methanogens. In this study, a methanogenesis inhibition approach based on the thermal treatment of mixed cultures was adopted and evaluated in terms of acetate production under different tests consisting of hydrogenophilic and bioelectrochemical experiments. Batch experiments were carried out under hydrogenophilic and bioelectrochemical conditions, demonstrating the effectiveness of the thermal treatment and showing a 30 times higher acetate production with respect to the raw anaerobic digestate. Moreover, a continuous flow bioelectrochemical reactor equipped with an anion exchange membrane (AEM) successfully overcomes the methanogens reactivation, allowing for a continuous acetate production. The AEM membrane guaranteed the migration of the acetate from the biological compartment and its concentration in the abiotic chamber avoiding its consumption by acetoclastic methanogenesis. The system allowed an acetate concentration of 1745 ± 30 mg/L in the abiotic chamber, nearly five times the concentration measured in the cathodic chamber.

Evaluation of bio-hydrogen production using rice straw hydrolysate extracted by acid and alkali hydrolysis

This study was conducted to investigate the properties of hydrolysates obtained from acid and alkali hydrolysis and to evaluate the feasibility of employing them for bio-hydrogen production. High sugar concentrations of 16.8 g/L and 13.3 g/L were present in 0.5% and 1.0% H2SO4 hydrolysates, respectively. However, H2SO4 hydrolysis resulted in large amounts of short-chain fatty acids (SCFAs) and furan derivatives, which were removed by detoxification. In bio-hydrogen production, 1.0% H2SO4 hydrolysate showed a 55.6 mL of highest hydrogen production and 1.14 mol-H2/mol-hexose equivalentadded of hydrogen yield. In control and 1.0% NaOH hydrolysate, 29.7 mL and 36.9 mL of hydrogen were produced, respectively. Interestingly, relatively high acetate and butyrate production resulted in lactate reduction. Also, NH4OH hydrolysate produced less than 10 mL of hydrogen. Thus, these results indicate that hydrogen production and metabolite distribution can vary depending on the sugars and by-product composition in the hydrolysate.

Feed restriction and supplementing with propylene glycol, monensin sodium and rumen-protected choline chloride in periparturient Ghezel ewes: Implications on production and performance of ewes and their offspring

This study was conducted to evaluate the effects of supplementation of propylene glycol, monensin sodium and rumen-protected choline chloride to the late gestational restricted diet on milk production and composition, rumen fermentation and dry matter intake of ewes and their offspring performance. During the final trimester of pregnancy, 48 Ghezel ewes were randomly assigned to one of the 6 treatments: ad libitum feeding (CG, control); feed restriction (FR, 60% of ad libitum intake); feed restriction + propylene glycol (67 g/d) (FR-PG); feed restriction + propylene glycol + monensin sodium (30 mg/d) (FR-MS); feed restriction + propylene glycol + rumen-protected choline chloride (6 g/d) (FR-RPC); feed restriction + propylene glycol + monensin sodium + rumen-protected choline chloride (FR-PMC). Milk samples were collected during the first 4 weeks of lactation and analyzed for fat, protein and solids non-fat contents. Ruminal fluid was sampled on day 125 of pregnancy for the determination of volatile fatty acids and ammonia-nitrogen concentration. Dry matter intake of ewes was calculated daily according to the differences between feed provided and refused. The average daily gain of lambs was recorded until 30 days postnatal and weight on day 87 was considered as weaning weight. The data were analyzed in a completely randomized design using SAS v.9.3. The results showed that FR-RPC and FR-PMC ewes presented approximately a higher milk production (P < 0.05), milk protein and solids non-fat percentages (P < 0.01) than other ewes in almost all recording points. During the first and second weeks of lactation, the milk produced by FR ewes had the lowest fat percentage among all experimental groups (P < 0.0001). The lowest ruminal pH and ammonia-nitrogen concentration was found in FR and FR-MS ewes (P < 0.0001). Ewes in the CG and FR groups produced lower levels of total volatile fatty acids than other experimental groups (P < 0.0001). Besides, compared with other treatments, choline chloride supplementation caused a higher molar proportion of propionate (P < 0.001) while FR treatment resulted in a higher acetate production (P < 0.001) and acetate to propionate ratio (P < 0.01) compared to FR-RPC and FR-PMC treatments. Lambs born from FR-RPC ewes presented a greater average daily gain than those born from other ewes (P < 0.0001). Lambs reared by FR-PMC and FR ewes presented the highest and lowest weaning weight, respectively (P < 0.0001). It can be concluded that although feed restriction diminished animal production and offspring performance, simultaneous administration of propylene glycol, monensin sodium and rumen-protected choline chloride in restricted diets alleviated these negative effects and improved ewes milk production and offspring growth performance.

Effect of static magnetic field on microbial community during anaerobic digestion

Dairy wastewater is characterized by high concentration of organic compounds and is commonly used for energy production. Methods for enhancement of biogas production include application of magnetizers on the digester to induce static magnetic field (SMF). The study aimed at investigation of Bacteria and Archaea communities during anaerobic digestion of model dairy wastewater exposed to SMF. Magnetic field caused a significant increase in methane production to 373.2 mL/g VS compared to 200.2 mL/g VS in a control reactor and methane content to 56.8% compared to 49.1% in a control reactor. In both reactors, the biomass was dominated by Trichococcus sp. The relative abundance of lactic acid bacteria was of about 10% higher in the reactor exposed to SMF. This higher number of Lactobacillales resulted from a higher acetate production, which additionally caused enhanced growth of Methanosarcinacaea in the reactor exposed to SMF. SMF also stimulated the growth of hydrogenotrophic methanogens.

Enrichment of salt-tolerant CO2–fixing communities in microbial electrosynthesis systems using porous ceramic hollow tube wrapped with carbon cloth as cathode and for CO2 supply

Microbial inocula from marine origins are less explored for CO2 reduction in microbial electrosynthesis (MES) system, although effective CO2–fixing communities in marine environments are well-documented. We explored natural saline habitats, mainly salt marsh (SM) and mangrove (M) sediments, as potential inoculum sources for enriching salt-tolerant CO2 reducing community using two enrichment strategies: H2:CO2 (80:20) enrichment in serum vials and enrichment in cathode chamber of MES reactors operated at −1.0 V vs. Ag/AgCl. Porous ceramic hollow tube wrapped with carbon cloth was used as cathode and for direct CO2 delivery to CO2 reducing communities growing on the cathode surface. Methanogenesis was dominant in both the M- and SM-seeded MES and the methanogenic Archaea Methanococcus was the most dominant genus. Methane production was slightly higher in the SM-seeded MES (4.9 ± 1.7 mmol) compared to the M-seeded MES (3.8 ± 1.1 mmol). In contrast, acetate production was almost two times higher in the M-seeded MES (3.1 ± 0.9 mmol) than SM-seeded MES (1.5 ± 1.3 mmol). The high relative abundance of the genus Acetobacterium in the M-seeded serum vials correlates with the high acetate production obtained. The different enrichment strategies affected the community composition, though the communities in MES reactors and serum vials were performing similar functions (methanogenesis and acetogenesis). Despite similar operating conditions, the microbial community composition of M-seeded serum vials and MES reactors differed from the SM-seeded serum vials and MES reactors, supporting the importance of inoculum source in the evolution of CO2-reducing microbial communities.

Current metabolic perspective on malnutrition in obesity: towards more subgroup-based nutritional approaches?

Lifestyle intervention may be effective in reducing type 2 diabetes mellitus incidence and cardiometabolic risk. A more personalised nutritional approach based on an individual or subgroup-based metabolic profile may optimise intervention outcome. Whole body insulin resistance (IR) reflects defective insulin action in tissues such as muscle, liver, adipose tissue, gut and brain, which may precede the development of cardiometabolic diseases. IR may develop in different organs but the severity may vary between organs. Individuals with more pronounced hepatic IR have a distinct plasma metabolome and lipidome profile as compared with individuals with more pronounced muscle IR. Additionally, genes related to extracellular modelling were upregulated in abdominal subcutaneous adipose tissue in individuals with more pronounced hepatic IR, whilst genes related to inflammation as well as systemic low-grade inflammation were upregulated in individuals with primarily muscle IR. There are indications that these distinct IR phenotypes may also respond differentially to dietary macronutrient composition. Besides metabolic phenotype, microbial phenotype may be of importance in personalising the response to diet. In particular fibres or fibre mixtures, leading to a high distal acetate and SCFA production may have more pronounced effects on metabolic health. Notably, individuals with prediabetes may have a reduced response to diet-induced microbiota modulation with respect to host insulin sensitivity and metabolic health outcomes. Overall, we need more research to relate metabolic subphenotypes to intervention outcomes to define more optimal diets for individuals with or predisposed to chronic metabolic diseases.

Volatile Fatty Acid Production from Baker’s Yeast Industry Effluent

Baker’s Yeast Industry (BYI) effluent was one of the strongest wastewaster type that contains high amount of organic matter, salinity, nitrogen and sulphate. These characteristics were unfavorable for the application of wastewater treatment. For this reason, a recovery-oriented approach was applied for the appropriate conditions to increase the Volatile Fatty Acid (VFA) production potential of BYI. In this concept, hydrolysis anaerobic Membrane Bioreactor was used to produce VFAs. The hydrolysis reactor was operated between 2.5 and 13.4 days in Hydraulic Retention Time, at a thermophilic temperature (55 °C) and at pH of 4.5–8 for 858 days. The highest biomass concentration was 26 g L−1 in the reactor, and mainly acetate, propionate and butrate production was observed. The highest acetate production was seen at pH 7 with an average concentration of 25,930 mg L−1. The highest total VFA production yield was 0.98 g VFA–COD g−1 COD. It was concluded that carrying out hydrolysis and acidification reactions at neutral pH was better as long as methane production could be avoided. In addition, a VFA-rich stream with free suspended solids could be obtained with the 0.05 µ pore diameter submerged membrane. Population dynamics remained quite stable and rich in biomass consortia throughout the study. It was suggested that a high commercial value could be obtained, especially by recovering acetate from the hydrolysis reactor’s outlet stream.

Co-culture-based biological carbon monoxide conversion by Citrobacter amalonaticus Y19 and Sporomusa ovata via a reducing-equivalent transfer mediator

The biological conversion of carbon monoxide (CO) has been highlighted for the development of a C1 gas biorefinery process. Despite this, the toxicity and low reducing equivalent of CO uptake make biological conversion difficult. The use of synthetic co-cultures is an alternative way of enhancing the performance of CO bioconversion. This study evaluated a synthetic co-culture consisting of Citrobacter amalonaticus Y19 and Sporomusa ovata for acetate production from CO. In this consortium, the CO2 and H2 produced by the water-gas shift reaction of C. amalonaticus Y19, were utilized further by S. ovata. Higher acetate production was achieved in the co-culture system compared to the monoculture counterparts. Furthermore, syntrophic cooperation via various reducing equivalent carriers provided new insights into the synergistic metabolic benefits with a toxic and refractory substrate, such as CO. This study also suggests an appropriate model for examining the syntrophic interaction between microbial species in a mixed community.

High efficiency microbial electrosynthesis of acetate from carbon dioxide by a self-assembled electroactive biofilm

Microbial electrosynthesis (MES) is a biocathode-driven process, producing high-value chemicals from CO2. Here, an in situ self-assembled graphene oxide (rGO)/biofilm was constructed, in MES, for high efficient acetate production. GO has been successfully reduced by electroautotrophic bacteria for the first time. An increase, of 1.5 times, in the volumetric acetate production rate, was obtained by self-assembling rGO/biofilm, as compared to the control group. In MES with rGO/biofilm, a volumetric acetate production rate of 0.17gl−1d−1 has been achieved, 77% of the electrons consumed, were recovered and the final acetate concentration reached 7.1gl−1, within 40days. A three-dimensional rGO/biofilm was constructed enabling highly efficient electron transfer rates within biofilms, and between biofilm and electrode, demonstrating that the development of 3D electroactive biofilms, with higher extracellular electron transfer rates, is an effective approach to improving MES efficiency.

Modulation of calcium carbonate precipitation by exopolysaccharide in Bacillus sp. JH7.

Extracellular polymeric substance (EPS) is proposed to facilitate calcium ion supersaturation through its nucleation effect during the microbially induced calcium carbonate precipitation (MICP) process. However, the supersaturation effect of Ca2+ via EPS in MICP has not been clearly demonstrated. Enhanced exopolysaccharide production of the alkali- and halotolerant MICP-capable bacteria, Bacillus sp. JH7, was achieved through glycerol addition. This was demonstrated by measuring cellular precipitation and Congo red binding. Interestingly, field emission scanning electron microscopy and energy-dispersive X-ray spectrometry analysis demonstrated that there was no MICP under glycerol-amended conditions. Although glycerol promoted exopolysaccharide capture of Ca2+ ions, Ca2+ embedded onto EPS did not participate in MICP formation. The pH was reduced in glycerol-added media, which led us to analyze high acetate production under our test conditions. Purified glycerol-induced exopolysaccharide showed a higher capacity of Ca2+ capture than the control. Quantitative RT-PCR analysis showed that three genes involved in exopolysaccharide production were highly upregulated by glycerol. The amounts of three detected monosaccharides (arabinose, glucose, and mannose) were altered by glycerol. Cell hydrophobicity measurements indicated that glycerol could confer more hydrophilic characteristics to cells, which might enhance Ca2+ binding onto EPS. Unexpectedly, our data demonstrated, for the first time, that glycerol could promote exopolysaccharide and acetate production under our test condition, which could inhibit MICP by reducing the availability of free Ca2+.

Ethyl acetate production by the elusive alcohol acetyltransferase from yeast

Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains.

Bioelectrochemical conversion of CO2 to chemicals: CO2 as a next generation feedstock for electricity-driven bioproduction in batch and continuous modes.

The recent concept of microbial electrosynthesis (MES) has evolved as an electricity-driven production technology for chemicals from low-value carbon dioxide (CO2) using micro-organisms as biocatalysts. MES from CO2 comprises bioelectrochemical reduction of CO2 to multi-carbon organic compounds using the reducing equivalents produced at the electrically-polarized cathode. The use of CO2 as a feedstock for chemicals is gaining much attention, since CO2 is abundantly available and its use is independent of the food supply chain. MES based on CO2 reduction produces acetate as a primary product. In order to elucidate the performance of the bioelectrochemical CO2 reduction process using different operation modes (batch vs. continuous), an investigation was carried out using a MES system with a flow-through biocathode supplied with 20 : 80 (v/v) or 80 : 20 (v/v) CO2 : N2 gas. The highest acetate production rate of 149 mg L-1 d-1 was observed with a 3.1 V applied cell-voltage under batch mode. While running in continuous mode, high acetate production was achieved with a maximum rate of 100 mg L-1 d-1. In the continuous mode, the acetate production was not sustained over long-term operation, likely due to insufficient microbial biocatalyst retention within the biocathode compartment (i.e. suspended micro-organisms were washed out of the system). Restarting batch mode operations resulted in a renewed production of acetate. This showed an apparent domination of suspended biocatalysts over the attached (biofilm forming) biocatalysts. Long term CO2 reduction at the biocathode resulted in the accumulation of acetate, and more reduced compounds like ethanol and butyrate were also formed. Improvements in the production rate and different biomass retention strategies (e.g. selecting for biofilm forming micro-organisms) should be investigated to enable continuous biochemical production from CO2 using MES. Certainly, other process optimizations will be required to establish MES as an innovative sustainable technology for manufacturing biochemicals from CO2 as a next generation feedstock.

High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources.

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.

Dynamic change of pH in acidogenic fermentation of cheese whey towards polyhydroxyalkanoates production: Impact on performance and microbial population

Polyhydroxyalkanoates (PHA) are a sustainable alternative to conventional plastics that can be obtained from industrial wastes/by-products using mixed microbial cultures (MMC). MMC PHA production is commonly carried out in a 3-stage process of acidogenesis, PHA culture selection and accumulation. This research focused on the possibility of tailoring PHA by controlling the acidogenic reactor operating conditions, namely pH, using cheese whey as model feedstock. The objective was to investigate the impact that dynamically varying the acidogenic pH, when targeting different PHA monomer profiles, had on the performance and microbial community profile of the anaerobic reactor. To accomplish this, an anaerobic reactor was continuously operated under dynamic pH changes, ranging from pH 4 to 7, turning to pH 6 after each change of pH. At pH 6, lactate and acetate were the dominant products (41–48% gCOD basis and 22–44% gCOD basis, respectively). At low pH, lactate production was higher while at high pH acetate production was favoured. Despite the dynamic change of pH, the fermentation product composition at pH 6 was always similar, showing the resilience of the process, i.e. when the same pH value was imposed, the culture produced the same metabolic products independently of the history of changes occurring in the system. The different fermentation product fractions led to PHAs of different compositions. The microbial community, analysed by high throughput sequencing of bacterial 16S rRNA gene fragments, was dominated by Lactobacillus, but varied markedly when subjected to the highest and lowest pH values of the tested range (4 and 7), with increase in the abundance of Lactococcus and a member of the Candidate Division TM7. Different bacterial profiles obtained at pH 6 during this dynamic operation were able to produce a consistent profile of fermentation products (and consequently a constant PHA composition), demonstrating the community’s functional redundancy.