Mixed Acid Fermentation Research Articles

Bio-hydrogen and methane production from two-phase anaerobic digestion of food waste under the scheme of acidogenic off-gas reuse.

Anaerobic hydrolysis of food wastes sourced from bakery (T1), Chinese restaurant (T2), western-style restaurant (T3), and wet market (T4) were performed in leach bed reactors under the scheme of acidogenic off-gas reuse in methanogenic reactor. Results showed that food waste in T3 achieved the highest hydrogen production of 61.0 L/kg·VSadded. Highest activity of hydrogenase in both leachate and digestate samples confirmed the superior performance of H2 production in T3. Mixed acid fermentation with domination of acetate and butyrate was observed in all four treatments, whereas variations in quantification and speciation of the acidogenic products were closely related to the composition of substrates. High volatile solids (VS) removal (76.7%) was observed in T3 while VS reduction rates of the other treatments ranged from 37 to 55%. High COD production of 0.65 gCOD /g·VSadded together with the reuse of elevated acidogenic off-gas ensured the highest specific CH4 production of 0.42 L/g·VSadded in T3.

Reversible and Selective Interconversion of Hydrogen and Carbon Dioxide into Formate by a Semiartificial Formate Hydrogenlyase Mimic.

The biological formate hydrogenlyase (FHL) complex links a formate dehydrogenase (FDH) to a hydrogenase (H2ase) and produces H2 and CO2 from formate via mixed-acid fermentation in Escherichia coli. Here, we describe an electrochemical and a colloidal semiartificial FHL system that consists of an FDH and a H2ase immobilized on conductive indium tin oxide (ITO) as an electron relay. These in vitro systems benefit from the efficient wiring of a highly active enzyme pair and allow for the reversible conversion of formate to H2 and CO2 under ambient temperature and pressure. The hybrid systems provide a template for the design of synthetic catalysts and surpass the FHL complex in vivo by storing and releasing H2 on demand by interconverting CO2/H2 and formate with minimal bias in either direction.

Comparison of metabolic response between the planktonic and air-dried Escherichia coli to electrolysed water combined with ultrasound by 1H NMR spectroscopy.

The antimicrobial effects of electrolysed water and ultrasound have been well reported; however, little attention was paid to their effects on the metabolite changes of bacteria in different states. In this study, the metabolomic variations of Escherichia coli ATCC 25922 in planktonic and adherent state (air-dried on stainless steel coupons) after the combination treatment of low-concentration acidic electrolysed water (AEW, free available chlorine (FAC): 4 mg/L) and ultrasound were characterised, by conducting multivariate data analysis based on nuclear magnetic resonance (NMR) spectroscopy. Overall, 43 metabolites were identified in two states of E. coli, including a wide range of amino acids, organic acids, nucleotides and their derivatives. The quantification of whole-cell metabolism in planktonic and air-dried cultures was quite different: air-dried E. coli exhibited more resistance to ultrasound and AEW treatments due to initiating a protective response against oxidative and acid stresses, which was not observed in planktonic E. coli, whose levels of all identified metabolites were decreased significantly after the combined treatment. Further pathway analysis revealed that alanine, aspartate and glutamate metabolism, glycolysis, pyruvate metabolism and tricarboxylic acid (TCA) cycle were changed significantly in planktonic culture, but to a less extent in air-dried culture, in which some shifts in glutamate decarboxylase (GAD) system and some shunts like mixed acid fermentation and pentose phosphate pathway were observed for maintaining metabolic balance. These findings suggest that NMR-based metabolomics strategy is promising in identifying different metabolic shifts in different states of bacteria. They also provide some guidance for food equipment sanitisation, especially for organic food processing.

Effect of pre-fermentation types on the potential of methane production and energy recovery from food waste

Two-phase anaerobic digestion (TPAD) is a commonly used method for recovering energy from food waste, even though the relationship between fermentation type and methane production has not to be thoroughly investigated. In this study, homolactic acid fermentation (HOLA), heterolactic acid fermentation (HELA), butyric acid fermentation (BUA), and mixed acid fermentation (MA) were used in the first phase, and the corresponding methane production levels were compared. HELA and MA resulted in the maximum methane yields of 290 and 287 ml per gram chemical oxygen demand (COD), respectively, but they were not significantly higher than the yield of 279 ml/g COD from single-phase anaerobic digestion (SPAD). During methanogenesis, BUA led to the fastest hydrolysis and methane production rates, followed by MA and HELA. In spite of the similar potential for methane production and energy recovery, TPAD using either BUA, MA, or HELA as the fermentation phase exhibited at least 50% greater methane production efficiency than SPAD. Overall, HELA and MA were found to be the best choices in terms of treatment efficiency and energy recovery.

Carriage of Shiga toxin phage profoundly affects Escherichia coli gene expression and carbon source utilization

BackgroundEnterohemorrhagic Escherichia coli (E. coli) are intestinal pathogenic bacteria that cause life-threatening disease in humans. Their cardinal virulence factor is Shiga toxin (Stx), which is encoded on lambdoid phages integrated in the chromosome. Stx phages can infect and lysogenize susceptible bacteria, thus either increasing the virulence of already pathogenic bacterial hosts or transforming commensal strains into potential pathogens. There is increasing evidence that Stx phage-encoded factors adaptively regulate bacterial host gene expression. Here, we investigated the effects of Stx phage carriage in E. coli K-12 strain MG1655. We compared the transcriptome and phenotype of naive MG1655 and two lysogens carrying closely related Stx2a phages: ϕO104 from the exceptionally pathogenic 2011 E. coli O104:H4 outbreak strain and ϕPA8 from an E. coli O157:H7 isolate.ResultsAnalysis of quantitative RNA sequencing results showed that, in comparison to naive MG1655, genes involved in mixed acid fermentation were upregulated, while genes encoding NADH dehydrogenase I, TCA cycle enzymes and proteins involved in the transport and assimilation of carbon sources were downregulated in MG1655::ϕO104 and MG1655::ϕPA8. The majority of the changes in gene expression were found associated with the corresponding phenotypes. Notably, the Stx2a phage lysogens displayed moderate to severe growth defects in minimal medium supplemented with single carbon sources, e.g. galactose, ribose, L-lactate. In addition, in phenotype microarray assays, the Stx2a phage lysogens were characterized by a significant decrease in the cell respiration with gluconeogenic substrates such as amino acids, nucleosides, carboxylic and dicarboxylic acids. In contrast, MG1655::ϕO104 and MG1655::ϕPA8 displayed enhanced respiration with several sugar components of the intestinal mucus, e.g. arabinose, fucose, N-acetyl-D-glucosamine. We also found that prophage-encoded factors distinct from CI and Cro were responsible for the carbon utilization phenotypes of the Stx2a phage lysogens.ConclusionsOur study reveals a profound impact of the Stx phage carriage on E. coli carbon source utilization. The Stx2a prophage appears to reprogram the carbon metabolism of its bacterial host by turning down aerobic metabolism in favour of mixed acid fermentation.

Wetland Sediments Host Diverse Microbial Taxa Capable of Cycling Alcohols.

Alcohols are commonly derived from the degradation of organic matter and yet are rarely measured in environmental samples. Wetlands in the Prairie Pothole Region (PPR) support extremely high methane emissions and the highest sulfate reduction rates reported to date, likely contributing to a significant proportion of organic matter mineralization in this system. While ethanol and isopropanol concentrations up to 4 to 5 mM in PPR wetland pore fluids have been implicated in sustaining these high rates of microbial activity, the mechanisms that support alcohol cycling in this ecosystem are poorly understood. We leveraged metagenomic and transcriptomic tools to identify genes, pathways, and microorganisms potentially accounting for alcohol cycling in PPR wetlands. Phylogenetic analyses revealed diverse alcohol dehydrogenases and putative substrates. Alcohol dehydrogenase and aldehyde dehydrogenase genes were included in 62 metagenome-assembled genomes (MAGs) affiliated with 16 phyla. The most frequently encoded pathway (in 30 MAGs) potentially accounting for alcohol production was a Pyrococcus furiosus-like fermentation which can involve pyruvate:ferredoxin oxidoreductase (PFOR). Transcripts for 93 of 137 PFOR genes in these MAGs were detected, as well as for 158 of 243 alcohol dehydrogenase genes retrieved from these same MAGs. Mixed acid fermentation and heterofermentative lactate fermentation were also frequently encoded. Finally, we identified 19 novel putative isopropanol dehydrogenases in 15 MAGs affiliated with Proteobacteria, Acidobacteria, Chloroflexi, Planctomycetes, Ignavibacteriae, Thaumarchaeota, and the candidate divisions KSB1 and Rokubacteria We conclude that diverse microorganisms may use uncommon and potentially novel pathways to produce ethanol and isopropanol in PPR wetland sediments.IMPORTANCE Understanding patterns of organic matter degradation in wetlands is essential for identifying the substrates and mechanisms supporting greenhouse gas production and emissions from wetlands, the main natural source of methane in the atmosphere. Alcohols are common fermentation products but are poorly studied as key intermediates in organic matter degradation in wetlands. By investigating genes, pathways, and microorganisms potentially accounting for the high concentrations of ethanol and isopropanol measured in Prairie Pothole wetland sediments, this work advanced our understanding of alcohol fermentations in wetlands linked to extremely high greenhouse gas emissions. Moreover, the novel alcohol dehydrogenases and microbial taxa potentially involved in alcohol metabolism may serve biotechnological efforts in bioengineering commercially valuable alcohol production and in the discovery of novel isopropanol producers or isopropanol fermentation pathways.

Application of IWA Anaerobic Digestion Model No. 1 to simulate butyric acid, propionic acid, mixed acid, and ethanol type fermentative systems using a variable acidogenic stoichiometric approach

IWA Anaerobic Digestion Model No. 1 (ADM1) is the most widely recognised and popular mathematical model for anaerobic digestion processes. However, the application of ADM1 to acidogenic fermentation is limited by its use of constant stoichiometry to describe the formation of products via carbohydrate fermentation. This study presents a modification of ADM1 using a variable acidogenic stoichiometric approach in which the hydrogen partial pressure (pH2) and pH are used to predict and regulate the acidogenic process. The fermentation of ethanol and its kinetics were introduced into the model structure. Experimental data from mixed acid-type fermentation in a 28.4 L anaerobic baffled reactor (ABR) fed with a sucrose solution with a chemical oxygen demand of 4000 mg L−1 were used to calibrate the model parameters. Two case studies involving continuous ethanol-type fermentation in an ABR and a continuous stirring tank reactor (CSTR) were used to validate the approach. The modified model achieved good predictions of the experimental data collected from butyric acid, propionic acid, mixed acid, and ethanol-type fermentation in the ABR and CSTR using the standard ADM1 parameter values without any parameter fitting beyond implementation of the variable acidogenic stoichiometry. The pH2 and pH thresholds in butyric acid, propionic acid, mixed acid, and ethanol-type fermentation could be predicted using this model, which was shown to be a valid mathematical tool for the regulation of fermentation type.

Experimental design and syntrophic microbial pathways for biofuel production from sugarcane bagasse under thermophilic condition

The molecular mechanisms behind the bioconversion of sugarcane bagasse into biofuel by promising metabolic pathways were studied, suggesting that proteolytic, cellulolytic and methanogenic microorganisms such as Coprothermobacter, Clostridium, and Methanothermobacter, respectively, took an important syntrophic role in lignocellulosic-derived fuel production. The mixed acid fermentation was the main route to the acetic, formic, butyric, and propionic acid production by acid-forming bacteria. Some aspects of biotechnological application of such metabolic pathways were evaluated from a central composite design, in which the effect of incubation temperature (from 45.8 to 74.2 °C) and yeast extract concentration (from 0.58 to 3.42 g/L) on hydrogen production were assessed. The interaction between these factors significantly affected the hydrogen production, which reached the highest value (17.3 mmol/L) using 3.42 g/L of yeast extract at 60 °C, and favored a plastic and physiological diverse microbial community related to bioconversion of SCB.

Microbial Diversity and Organic Acid Production of Guinea Pig Faecal Samples

The guinea pig (Cavia porcellus) or cavy is a grass-eating rodent. Its main diet consists of grass or hay, which comprises cellulose, hemicellulose, lignin and their derivatives. Here, the microbial diversity of faecal samples of two guinea pigs and microbial enrichments made with substrates, including starch waste and dried grass, were investigated along with organic acid production profiles. The microbial communities of the faecal samples were dominated by the phyla Bacteroidetes (40%) and Firmicutes (36%). Bacteroidales S24-7 (11% in Cavy 1 and 21% in Cavy 2) was the most abundant order. At genus level, many microorganisms remained unclassified. Different carbon sources were used for organic acid production in faecal enrichments. The dominant bacterial groups in the secondary enrichments with dried grass, starch waste and xylose were closely related to Prevotella and Blautia. Acetate was the predominant organic acid from all enrichments. The organic acid production profiles corresponded to a mixed acid fermentation but differed depending on the substrate. Eight phylogenetically different isolates were obtained, including a novel Streptococcus species, strain Cavy grass 6. This strain had a low abundance (1%) in one of the faecal samples but was enriched in the dried grass enrichment (3%). Cavy grass 6, a fast-growing heterolactic bacterium, ferments cellobiose to lactate, acetate, formate and ethanol. Our results show that cavy faecal samples can be applied as microbial source for organic acid production from complex organic substrates. The cavy gut contains many as-yet-uncultivated bacteria which may be appropriate targets for future studies.

Effect of temperature on the fermentation of starch by two high efficient H2 producers

The batch fermentative hydrogen production using starch was conducted to investigate the influence of temperature (25 °C-40 °C) on the co-cultures system of Bacillus cereus ATCC 14579 (T) [Strain A] and Brevundimonas naejangsanensis BIO-TAS2-2(T) [Strain B]. The results showed that the optimal temperature for hydrogen production was 35 °C in both the sole and the mixed culture fermentation. At this temperature, the co-culturing system increased the substrate consumption rate by 34 % and 70 % compared with the Strain A and Strain B fermentation, respectively. The co-culturing system enhanced the hydrogen production yield by 52 % and 62 % compared with the Strain A and Strain B fermentation, respectively. The analysis of soluble metabolites during the fermentation process indicated that the mixed acid fermentation conducted in two strains and co-culturing system was dominated by butyric acid type fermentation at the optimal temperature. The Formate pathway probably exists in Strain A, and the PFOR pathway probably exists in Strain B. Both strains A and B play a synergistic role in hydrogen production at 35 °C using starch as substrate.

Активация альтернативного дыхания с внутренним акцептором электронов при анаэробной утилизации глюкозы у штаммов Escherichia coli с нарушенной способностью к брожению

The activation of alternative respiration with an internal electron acceptor upon anaerobic utilization of glucose in Escherichia coli strains with the impaired fermentation ability has been studied. It was found out that the respiration processes utilizing pyruvic acid as an endogenous electron acceptor can markedly contribute to the maintenance of the anaerobic redox balance in E. coli strains deficient in mixed acid fermentation pathways. The sequential inactivation of the pathways of anaerobic dissimilation of pyruvate and impairment of the functionality of the reductive branch of the tricarboxylic acid cycle led to the increase in the contribution of the respiratory formation of lactic acid and alanine to the biosynthesis of the reduced products of anaerobic glucose utilization by the strains from 11 to 54%. The analysis of the enantiomeric composition of the lactic acid and alanine secreted by the strains demonstrated that D-lactate dehydrogenase (Dld), L-lactate dehydrogenase (LctD), and D-alanine dehydrogenase (DadA) participated in the biosynthesis of the respective compounds. Escherichia coli, glucose, fermentation, respiration, metabolic engineering, pyruvate, lactate, alanine. The work was supported by a grant from the Russian Foundation for Basic Research (Project no. 18-04-01222)

Actinobaculum massiliense Proteome Profiled in Polymicrobial Urethral Catheter Biofilms.

Actinobaculum massiliense, a Gram-positive anaerobic coccoid rod colonizing the human urinary tract, belongs to the taxonomic class of Actinobacteria. We identified A. massiliense as a cohabitant of urethral catheter biofilms (CB). The CBs also harbored more common uropathogens, such as Proteus mirabilis and Aerococcus urinae, supporting the notion that A. massiliense is adapted to a life style in polymicrobial biofilms. We isolated a clinical strain from a blood agar colony and used 16S rRNA gene sequencing and shotgun proteomics to confirm its identity as A. massiliense. We characterized this species by quantitatively comparing the bacterial proteome derived from in vitro growth with that of four clinical samples. The functional relevance of proteins with emphasis on nutrient import and the response to hostile host conditions, showing evidence of neutrophil infiltration, was analyzed. Two putative subtilisin-like proteases and a heme/oligopeptide transporter were abundant in vivo and are likely important for survival and fitness in the biofilm. Proteins facilitating uptake of xylose/glucuronate and oligopeptides, also highly expressed in vivo, may feed metabolites into mixed acid fermentation and peptidolysis pathways, respectively, to generate energy. A polyketide synthase predicted to generate a secondary metabolite that interacts with either the human host or co-colonizing microbes was also identified. The product of the PKS enzyme may contribute to A. massiliense fitness and persistence in the CBs.

Inactivation of Malic Enzymes Improves the Anaerobic Production of Four-Carbon Dicarboxylic Acids by Recombinant Escherichia coli Strains Expressing Pyruvate Carboxylase

The genes maeA and maeB, encoding NADH- and NADPH-dependent malic enzymes, have been deleted in a recombinant Escherichia coli strain with inactivated mixed-acid fermentation pathways and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. During anaerobic glucose utilization, the parental strain synthesized malic, fumaric, and succinic acids as the main fermentation end products, while pyruvic acid was accumulated as the main by-product resulting from the functioning of the pyruvate–oxaloacetate–malate–pyruvate futile cycle. Upon individual deletions of the maeA and maeB genes, the mutant strains converted glucose into four-carbon dicarboxylic acids with increased efficiency still secreting notable amounts of pyruvic acid. The combined inactivation of both malic enzymes in the constructed strain significantly elevated the portion of malic, fumaric, and succinic acids among the fermentation end products with a concomitant decrease in the secretion of pyruvic acid and other by-products due to the abolishment of the action of the futile cycle competing with the target biosynthetic processes.

Anaerobic Degradation of the Plant Sugar Sulfoquinovose Concomitant With H2S Production: Escherichia coli K-12 and Desulfovibrio sp. Strain DF1 as Co-culture Model.

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is produced by plants and other phototrophs and its biodegradation is a relevant component of the biogeochemical carbon and sulfur cycles. SQ is known to be degraded by aerobic bacterial consortia in two tiers via C3-organosulfonates as transient intermediates to CO2, water and sulfate. In this study, we present a first laboratory model for anaerobic degradation of SQ by bacterial consortia in two tiers to acetate and hydrogen sulfide (H2S). For the first tier, SQ-degrading Escherichia coli K-12 was used. It catalyzes the fermentation of SQ to 2,3-dihydroxypropane-1-sulfonate (DHPS), succinate, acetate and formate, thus, a novel type of mixed-acid fermentation. It employs the characterized SQ Embden-Meyerhof-Parnas pathway, as confirmed by mutational and proteomic analyses. For the second tier, a DHPS-degrading Desulfovibrio sp. isolate from anaerobic sewage sludge was used, strain DF1. It catalyzes another novel fermentation, of the DHPS to acetate and H2S. Its DHPS desulfonation pathway was identified by differential proteomics and demonstrated by heterologously produced enzymes: DHPS is oxidized via 3-sulfolactaldehyde to 3-sulfolactate (SL) by two NAD+-dependent dehydrogenases (DhpA, SlaB); the SL is cleaved by an SL sulfite-lyase known from aerobic bacteria (SuyAB) to pyruvate and sulfite. The pyruvate is oxidized to acetate, while the sulfite is used as electron acceptor in respiration and reduced to H2S. In conclusion, anaerobic sulfidogenic SQ degradation was demonstrated as a novel link in the biogeochemical sulfur cycle. SQ is also a constituent of the green-vegetable diet of herbivores and omnivores and H2S production in the intestinal microbiome has many recognized and potential contributions to human health and disease. Hence, it is important to examine bacterial SQ degradation also in the human intestinal microbiome, in relation to H2S production, dietary conditions and human health.

Shifting product spectrum by pH adjustment during long-term continuous anaerobic fermentation of food waste

Anaerobic fermentation is widely used to recover different products from food waste, and in this study, the evolution of fermentation products and microbial community along with pH variation was investigated thoroughly using four long-term reactors. Lactic fermentation dominated the system at pH 3.2–4.5 with lactic acid concentration of 5.7–13.5 g/L, and Lactobacillus was the superior sort. Bifidobacteria increased significantly at pH 4.5, resulting in the increase of acetic acid. Butyric acid fermentation was observed at pH 4.7–5.0. Bifidobacterium, Lactobacillus, and Olsenella were still dominant, but the lactic acid produced by them was converted to volatile fatty acids (VFAs) rapidly by Megasphaera, Caproiciproducens, Solobacteria, etc. Mixed acid fermentation occurred at pH 6.0 with the highest concentration 14.2 g/L of VFAs, and the dominant Prevotella and Megasphaera converted substrates to VFAs directly. On the whole, pH 4.5 and 4.7 led to the highest hydrolysis rate of 50% and acidification rate of 45%.

Understanding the Role of Escherichia coli Hydrogenases and Formate Dehydrogenases in the FO F1 -ATPase Activity during the Mixed Acid Fermentation of Mixture of Carbon Sources.

During fermentation Escherichia coli produces di-hydrogen (H2 ) via reversible membrane-bound [Ni-Fe]-hydrogenases (Hyd). This study describes the total and N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity and H2 production at various pHs in E. coli wild type and mutants encoding Hyd enzymes and formate dehydrogenases (FDH) on fermentation of glucose, glycerol, and formate. The highest total ATPase activity was detected at pH 7.5 in hyaB hybC selC (lacking large subunits of Hyd-1 and Hyd-2 and FDH, respectively) triple mutant. This ATPase activity was mainly due to the proton-translocating ATPase but in FDH mutant the DCCD inhibition was less compared to wild type. Potassium ions stimulated total ATPase activity at pH 5.5 ~50% and ~35% in wild type and hypF (lacking all Hyd enzymes) mutant, respectively. Moreover, K+ also stimulated DCCD inhibited ATPase activity ~1.7-fold-2-fold in strains where FDH was absent only at pH 5.5. DCCD inhibited H2 production only at pH 5.5 in all assays. Taken together it is suggested that at low pH, FDH, and Hyd enzymes are linked with the FO F1 -ATPase for regulating and maintaining the cytoplasmatic pH and thus proton motive force generation. FDH and Hyd enzymes have impact on the FO F1 -ATPase activity depending on external pH and potassium ions. © 2018 IUBMB Life, 70(10):1040-1047, 2018.

A low-complexity metabolic network model for the respiratory and fermentative metabolism of Escherichia coli.

Over the last decades, predictive microbiology has made significant advances in the mathematical description of microbial spoiler and pathogen dynamics in or on food products. Recently, the focus of predictive microbiology has shifted from a (semi-)empirical population-level approach towards mechanistic models including information about the intracellular metabolism in order to increase model accuracy and genericness. However, incorporation of this subpopulation-level information increases model complexity and, consequently, the required run time to simulate microbial cell and population dynamics. In this paper, results of metabolic flux balance analyses (FBA) with a genome-scale model are used to calibrate a low-complexity linear model describing the microbial growth and metabolite secretion rates of Escherichia coli as a function of the nutrient and oxygen uptake rate. Hence, the required information about the cellular metabolism (i.e., biomass growth and secretion of cell products) is selected and included in the linear model without incorporating the complete intracellular reaction network. However, the applied FBAs are only representative for microbial dynamics under specific extracellular conditions, viz., a neutral medium without weak acids at a temperature of 37℃. Deviations from these reference conditions lead to metabolic shifts and adjustments of the cellular nutrient uptake or maintenance requirements. This metabolic dependency on extracellular conditions has been taken into account in our low-complex metabolic model. In this way, a novel approach is developed to take the synergistic effects of temperature, pH, and undissociated acids on the cell metabolism into account. Consequently, the developed model is deployable as a tool to describe, predict and control E. coli dynamics in and on food products under various combinations of environmental conditions. To emphasize this point,three specific scenarios are elaborated: (i) aerobic respiration without production of weak acid extracellular metabolites, (ii) anaerobic fermentation with secretion of mixed acid fermentation products into the food environment, and (iii) respiro-fermentative metabolic regimes in between the behaviors at aerobic and anaerobic conditions.

Role of hydrogenases 3 and 4 in Escherichia coli growth and H2 producing hydrogenase activity during anaerobic utilization of lactose

It is known that Escherichia coli produces hydrogen gas (H2) during mixed acid fermentation via hydrogenase (Hyd) enzymes utilizing sugars, alcohols or organic acids. However, there are many sugars including lactose which are not studied well. Therefore, H2 production and responsible Hyd enzymes activity and role have been investigated. During lactose fermentation (1–5 g L−1) by E. coli mainly Hyd-4 and partially Hyd-3 have been shown to affect cell growth and H2 production at different pHs. At 2 g L−1 lactose fermentation in hyfG (lacking large subunit of Hyd-4) mutant at pH 5.5 and 6.5 resulted in the decrease of specific growth rate ∼2.5-fold and ∼1.8-fold, respectively, compared to wild type. Hyd-3 contribution to cell growth was observed at pH 6.5 with 1 g L−1 and at pH 7.5 with 5 g L−1 lactose fermentation. In addition, external pH during 24 or 48 h was less acidified in hyfG mutant cells grown at pH 6.5 than in wild type. Moreover, the H2 yield in wild type cells was 6.00 ± 0.017 mmol L−1. Hyd-3 and Hyd-4 were responsible for H2 production at pH 7.5, 6.5 and 5.5, as shown by using hycE (lacking large subunit of Hyd-3) and hyfG single and hycE hyfB-R (lacking Hyd-3 large subunit and several subunits of Hyd-4) double mutants. These findings provide novel insights into the H2 producing activity of E. coli Hyd-3 and Hyd-4 during utilization of lactose and might be useful for improving H2 production from lactose or lactose containing various wastes.

Comparative Transcriptomic Analysis of Streptococcus thermophilus TH1436 and TH1477 Showing Different Capability in the Use of Galactose.

Streptococcus thermophilus is a species widely used in the dairy industry for its capability to rapidly ferment lactose and lower the pH. The capability to use galactose produced from lactose hydrolysis is strain dependent and most of commercial S. thermophilus strains are galactose-negative (Gal−), although galactose-positive (Gal+) would be more technologically advantageous because this feature could provide additional metabolic products and prevent galactose accumulation in foods. In this study, a next generation sequencing transcriptome approach was used to compare for the first time a Gal+ and a Gal− strain to characterize their whole metabolism and shed light on their different properties, metabolic performance and gene regulation. Transcriptome analysis revealed that all genes of the gal operon were expressed very differently in Gal+ and in the Gal− strains. The expression of several genes involved in mixed acid fermentation, PTS sugars transporter and stress response were found enhanced in Gal+. Conversely, genes related to amino acids, proteins metabolism and CRISPR associated proteins were under-expressed. In addition, the strains showed a diverse series of predicted genes controlled by the transcriptional factor catabolite control protein A (CcpA). Overall, transcriptomic analysis suggests that the Gal+ strain underwent a metabolic remodeling to cope with the changed environmental conditions.

Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen

Sharpea and Kandleria are associated with rumen samples from low-methane-emitting sheep. Four strains of each genus were studied in culture, and the genomes of nine strains were analysed, to understand the physiology of these bacteria. All eight cultures grew equally well with d-glucose, d-fructose, d-galactose, cellobiose, and sucrose supplementation. d-Lactate was the major end product, with small amounts of the mixed acid fermentation products formate, acetate and ethanol. Genes encoding the enzymes necessary for this fermentation pattern were found in the genomes of four strains of Sharpea and five of Kandleria. Strains of Sharpea produced traces of hydrogen gas in pure culture, but strains of Kandleria did not. This was consistent with finding that Sharpea, but not Kandleria, genomes contained genes coding for hydrogenases. It was speculated that, in co-culture with a methanogen, Sharpea and Kandleria might change their fermentation pattern from a predominately homolactic to a predominately mixed acid fermentation, which would result in a decrease in lactate production and an increase in formation of acetate and perhaps ethanol. However, Sharpea and Kandleria did not change their fermentation products when co-cultured with Methanobrevibacter olleyae, a methanogen that can use both hydrogen and formate, and lactate remained the major end product. The results of this study therefore support a hypothesis that explains the link between lower methane yields and larger populations of Sharpea and Kandleria in the rumens of sheep.