Lactate cross-feeding between Bifidobacterium species and Megasphaera indica contributes to butyrate formation in the human colonic environment.

Butyrate, a physiologically active molecule, can be synthesized through metabolic interactions among colonic microorganisms. Previously, in a fermenting trial of human fecal microbiota, we observed that the butyrogenic effect positively correlated with the increasing Bifidobacterium population and an unidentified Megasphaera species. Therefore, we hypothesized that a cross-feeding phenomenon exists between Bifidobacterium and Megasphaera, where Megasphaera is the butyrate producer, and its growth relies on the metabolites generated by Bifidobacterium. To validate this hypothesis, three bacterial species (B. longum, B. pseudocatenulatum, and M. indica) were isolated from fecal cultures fermenting hydrolyzed xylan; pairwise cocultures were conducted between the Bifidobacterium and M. indica isolates; the microbial interactions were determined based on bacterial genome information, cell growth, substrate consumption, metabolite quantification, and metatranscriptomics. The results indicated that two Bifidobacterium isolates contained distinct gene clusters for xylan utilization and expressed varying substrate preferences. In contrast, M. indica alone scarcely grew on the xylose-based substrates. The growth of M. indica was significantly elevated by coculturing it with bifidobacteria, while the two Bifidobacterium species responded differently in the kinetics of cell growth and substrate consumption. Coculturing led to the depletion of lactate and increased the formation of butyrate. An RNA-seq analysis further revealed the upregulation of M. indica genes involved in the lactate utilization and butyrate formation pathways. We concluded that lactate generated by Bifidobacterium through catabolizing xylose fueled the growth of M. indica and triggered the synthesis of butyrate. Our findings demonstrated a novel cross-feeding mechanism to generate butyrate in the human colon.IMPORTANCEButyrate is an important short-chain fatty acid that is produced in the human colon through microbial fermentation. Although many butyrate-producing bacteria exhibit a limited capacity to degrade nondigestible food materials, butyrate can be formed through cross-feeding microbial metabolites, such as acetate or lactate. Previously, the literature has explicated the butyrate-forming links between Bifidobacterium and Faecalibacterium prausnitzii and between Bifidobacterium and Eubacterium rectale. In this study, we provided an alternative butyrate synthetic pathway through the interaction between Bifidobacterium and Megasphaera indica. M. indica is a species named in 2014 and is indigenous to the human intestinal tract. Scientific studies explaining the function of M. indica in the human colon are still limited. Our results show that M. indica proliferated based on the lactate generated by bifidobacteria and produced butyrate as its end metabolic product. The pathways identified here may contribute to understanding butyrate formation in the gut microbiota.

Unique roles of vaginal Megasphaera phylotypes in reproductive health.

The composition of the human vaginal microbiome has been extensively studied and is known to influence reproductive health. However, the functional roles of individual taxa and their contributions to negative health outcomes have yet to be well characterized. Here, we examine two vaginal bacterial taxa grouped within the genus Megasphaera that have been previously associated with bacterial vaginosis (BV) and pregnancy complications. Phylogenetic analyses support the classification of these taxa as two distinct species. These two phylotypes, Megasphaera phylotype 1 (MP1) and Megasphaera phylotype 2 (MP2), differ in genomic structure and metabolic potential, suggestive of differential roles within the vaginal environment. Further, these vaginal taxa show evidence of genome reduction and changes in DNA base composition, which may be common features of host dependence and/or adaptation to the vaginal environment. In a cohort of 3870 women, we observed that MP1 has a stronger positive association with bacterial vaginosis whereas MP2 was positively associated with trichomoniasis. MP1, in contrast to MP2 and other common BV-associated organisms, was not significantly excluded in pregnancy. In a cohort of 52 pregnant women, MP1 was both present and transcriptionally active in 75.4 % of vaginal samples. Conversely, MP2 was largely absent in the pregnant cohort. This study provides insight into the evolutionary history, genomic potential and predicted functional role of two clinically relevant vaginal microbial taxa.

Calcium salts of long-chain fatty acids from linseed oil decrease methane production by altering the rumen microbiome in vitro.

Calcium salts of long-chain fatty acids (CSFA) from linseed oil have the potential to reduce methane (CH4) production from ruminants; however, there is little information on the effect of supplementary CSFA on rumen microbiome as well as CH4 production. The aim of the present study was to evaluate the effects of supplementary CSFA on ruminal fermentation, digestibility, CH4 production, and rumen microbiome in vitro. We compared five treatments: three CSFA concentrations-0% (CON), 2.25% (FAL) and 4.50% (FAH) on a dry matter (DM) basis-15 mM of fumarate (FUM), and 20 mg/kg DM of monensin (MON). The results showed that the proportions of propionate in FAL, FAH, FUM, and MON were increased, compared with CON (P < 0.05). Although DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) digestibility decreased in FAL and FAH compared to those in CON (P < 0.05), DM digestibility-adjusted CH4 production in FAL and FAH was reduced by 38.2% and 63.0%, respectively, compared with that in CON (P < 0.05). The genera Ruminobacter, Succinivibrio, Succiniclasticum, Streptococcus, Selenomonas.1, and Megasphaera, which are related to propionate production, were increased (P < 0.05), while Methanobrevibacter and protozoa counts, which are associated with CH4 production, were decreased in FAH, compared with CON (P < 0.05). The results suggested that the inclusion of CSFA significantly changed the rumen microbiome, leading to the acceleration of propionate production and the reduction of CH4 production. In conclusion, although further in vivo study is needed to evaluate the reduction effect on rumen CH4 production, CSFA may be a promising candidate for reduction of CH4 emission from ruminants.

Exploring the Maternal and Infant Oral Microbiomes: A Pilot Study.

Setting the stage for good oral health early in life is critical to long-term oral and overall health. This exploratory study aimed to characterize and compare maternal and newborn oral microbiota among mother-infant pairs. Oral samples were collected from 34 pregnant African American women and their infants at 1 to 3 months of age. Extracted 16SrRNA genes were matched to the Human Oral Microbiome Database. Alpha and beta diversity differed significantly between overall maternal and infant microbiomes. Maternal or infant alpha diversity, however, was not differentiated by maternal gingival status. Several demographic and behavioral variables were associated with, but not predictive of, maternal oral microbiome alpha diversity. There was no association, however, among birth mode, feeding mode, and the infant oral microbiome. Megasphaera micronuciformis was the only periodontal pathogen detected among the infants. Notably, maternal gingival status was not associated with the presence/absence of most periodontal pathogens. This study provides an initial description of the maternal and infant oral microbiomes, laying the groundwork for future studies. The perinatal period presents an important opportunity where perinatal nurses and providers can provide oral assessment, education, and referral to quality dental care.

An Efficient New Process for the Selective Production of Odd-Chain Carboxylic Acids by Simple Carbon Elongation Using Megasphaera hexanoica.

The caproate-producing bacterium, Megasphaera hexanoica, metabolizes fructose to produce C2~C8 carbon-chain carboxylic acids using various electron acceptors. In particular, odd-chain carboxylic acids (OCCAs) such as valerate (C5) and heptanoate (C7), were produced at relatively high concentrations upon propionate supplementation. Using a statistical experimental design method, the optimal culture medium was established for the selective production of OCCAs among the total produced acids. In a medium containing 2.42 g L-1 sodium acetate and 18.91 g L-1 sodium propionate, M. hexanoica produced 9.48 g L-1 valerate, 2.48 g L-1 heptanoate, and 0.12 g L-1 caproate. To clarify the metabolism of the exogenous added propionate for OCCAs production, 13C tracer experiments were performed by supplementing the culture broth with [1,2,3-13C3] propionate. The metabolites analysis based on mass spectrometry showed that the propionate was only used to produce valerate and heptanoate without being participated in other metabolic pathways. Furthermore, the carbon elongation pathway in M. hexanoica was explained by the finding that the incorporation of propionate and acetate in the produced valerate occurred in only one orientation.

Human gut bacteria as potent class I histone deacetylase inhibitors in vitro through production of butyric acid and valeric acid.

Overexpression of histone deacetylase (HDAC) isoforms has been implicated in a variety of disease pathologies, from cancer and colitis to cardiovascular disease and neurodegeneration, thus HDAC inhibitors have a long history as therapeutic targets. The gut microbiota can influence HDAC activity via microbial-derived metabolites. While HDAC inhibition (HDI) by gut commensals has long been attributed to the short chain fatty acid (SCFA) butyrate, the potent metabolic reservoir provided by the gut microbiota and its role in host physiology warrants further investigation in a variety of diseases. Cell-free supernatants (CFS) of 79 phylogenetically diverse gut commensals isolated from healthy human donors were screened for their SCFA profile and their total HDAC inhibitory properties. The three most potent HDAC inhibiting strains were further evaluated and subjected to additional analysis of specific class I and class II HDAC inhibition. All three HDAC inhibitors are butyrate producing strains, and one of these also produced substantial levels of valeric acid and hexanoic acid. Valeric acid was identified as a potential contributor to the HDAC inhibitory effects. This bacterial strain, Megasphaera massiliensis MRx0029, was added to a model microbial consortium to assess its metabolic activity in interaction with a complex community. M. massiliensis MRx0029 successfully established in the consortium and enhanced the total and specific HDAC inhibitory function by increasing the capacity of the community to produce butyrate and valeric acid. We here show that single bacterial strains from the human gut microbiota have potential as novel HDI therapeutics for disease areas involving host epigenetic aberrations.

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters.

Absolute quantification of intracellular coenzyme A (CoA), coenzyme A disulfide, and short-chain acyl-coenzyme A thioesters was addressed by developing a tailored metabolite profiling method based on liquid chromatography in combination with tandem mass spectrometric detection (LC-MS/MS). A reversed phase chromatographic separation was established which is capable of separating a broad spectrum of CoA, its corresponding derivatives, and their isomers despite the fact that no ion-pairing reagent was used (which was considered as a key advantage of the method). Excellent analytical figures of merit such as high sensitivity (LODs in the nM to sub-nM range) and high repeatability (routinely 4 %; N = 15) were obtained. Method validation comprised a study on standard purity, stability, and recoveries during sample preparation. Uniformly labeled U(13)C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.

Rapid changes in key ruminal microbial populations during the induction of and recovery from diet-induced milk fat depression in dairy cows.

The ruminant provides a powerful model for understanding the temporal dynamics of gastrointestinal microbial communities. Diet-induced milk fat depression (MFD) in the dairy cow is caused by rumen-derived bioactive fatty acids, and is commonly attributed to the changes in the microbial population. The aim of the present study was to determine the changes occurring in nine ruminal bacterial taxa with well-characterised functions, and abundance of total fungi, ciliate protozoa and bacteria during the induction of and recovery from MFD. Interactions between treatment and time were observed for ten of the twelve populations. The total number of both fungi and ciliate protozoa decreased rapidly (days 4 and 8, respectively) by more than 90% during the induction period and increased during the recovery period. The abundance of Streptococcus bovis (amylolytic) peaked at 350% of control levels on day 4 of induction and rapidly decreased during the recovery period. The abundance of Prevotella bryantii (amylolytic) decreased by 66% from day 8 to 20 of the induction period and increased to the control levels on day 12 of the recovery period. The abundance of Megasphaera elsdenii and Selenomonas ruminantium (lactate-utilising bacteria) increased progressively until day 12 of induction (>170%) and decreased during the recovery period. The abundance of Fibrobacter succinogenes (fibrolytic) decreased by 97% on day 4 of induction and increased progressively to an equal extent during the recovery period, although smaller changes were observed for other fibrolytic bacteria. The abundance of the Butyrivibrio fibrisolvens/Pseudobutyrivibrio group decreased progressively during the induction period and increased during the recovery period, whereas the abundance of Butyrivibrio hungatei was not affected by treatment. Responsive taxa were modified rapidly, with the majority of changes occurring within 8 d and their time course was similar to the time course of the induction of MFD, demonstrating a strong correlation between changes in ruminal microbial populations and MFD.

Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii.

Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421-432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2 O2 , whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2 O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles' heel of anaerobes when exposed to air.

Lipidomics as an important key for the identification of beer-spoilage bacteria.

Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was used for characterizing intact plasmalogen phospholipid molecules in beer-spoilage bacteria. Identification of intact plasmalogens was carried out using collision-induced dissociation and the presence of suitable marker molecular species, both qualitative and quantitative, was determined in samples containing the anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method had a limit of detection at 1 pg for the standard, i.e. 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and be linear in the range of four orders of magnitude from 2 pg to 20 ng. This technique was applied to intact plasmalogen extracts from the samples of contaminated and uncontaminated beer without derivatization and resulted in the identification of contamination of beer by Megasphaera and Pectinatus bacteria. The limit of detection was about 830 cells of anaerobic bacteria, i.e. bacteria containing natural cyclopropane plasmalogenes (c-p-19:0/15:0), which is the majority plasmalogen located in both Megasphaera and Pectinatus. The SIM ESI-MS method has been shown to be useful for the analysis of low concentration of plasmalogens in all biological samples, which were contaminated with anaerobic bacteria, e.g. juice, not only in beer. Significance and impact of the study: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) using collision-induced dissociation was used to characterize intact plasmalogen phospholipid molecules in beer-spoilage anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method has a detection limit of 1 pg for the standard 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and is linear within four orders of magnitude (2 pg to 20 ng). The limit of detection was about 830 cells of bacteria containing natural cyclopropane plasmalogen (c-p-19:0/15:0). SIM ESI-MS method is useful for analyzing low concentrations of plasmalogens in biological samples contaminated with anaerobic bacteria, e.g. beer or juice.

Effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 on ruminal pH and the lactic acid concentration of simulated rumen acidosis in vitro.

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis.

Comparative genome analysis of Megasphaera sp. reveals niche specialization and its potential role in the human gut.

With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563) and BL7 (DSM25562), isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460) highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the "glycobiome" based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes) amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate), vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the human host.

An in vitro model of the horse gut microbiome enables identification of lactate-utilizing bacteria that differentially respond to starch induction.

Laminitis is a chronic, crippling disease triggered by the sudden influx of dietary starch. Starch reaches the hindgut resulting in enrichment of lactic acid bacteria, lactate accumulation, and acidification of the gut contents. Bacterial products enter the bloodstream and precipitate systemic inflammation. Hindgut lactate levels are normally low because specific bacterial groups convert lactate to short chain fatty acids. Why this mechanism fails when lactate levels rapidly rise, and why some hindgut communities can recover is unknown. Fecal samples from three adult horses eating identical diets provided bacterial communities for this in vitro study. Triplicate microcosms of fecal slurries were enriched with lactate and/or starch. Metabolic products (short chain fatty acids, headspace gases, and hydrogen sulfide) were measured and microbial community compositions determined using Illumina 16S rRNA sequencing over 12-hour intervals. We report that patterns of change in short chain fatty acid levels and pH in our in vitro system are similar to those seen in in vivo laminitis induction models. Community differences between microcosms with disparate abilities to clear excess lactate suggest profiles conferring resistance of starch-induction conditions. Where lactate levels recover following starch induction conditions, propionate and acetate levels rise correspondingly and taxa related to Megasphaeraelsdenii reach levels exceeding 70% relative abundance. In lactate and control cultures, taxa related to Veillonellamontpellierensis are enriched as lactate levels fall. Understanding these community differences and factors promoting the growth of specific lactate utilizing taxa may be useful to prevent acidosis under starch-induction conditions.

ANOVA-like differential expression (ALDEx) analysis for mixed population RNA-Seq.

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.

Fermentation of alfalfa wet-fractionation liquids to volatile fatty acids by Streptococcus bovis and Megasphaera elsdenii.

"Green juice", obtained by squeezing fresh alfalfa leaves inoculated with lactic acid bacteria, was fermented at room temperature for 7-21 d to obtain 12-47 g lactic acid L(-1). Inoculation of green juice with Streptococcus bovis and incubation at 39°C reduced fermentation time to 8-12h. The resulting "brown juice" from either fermentation had a pH of ∼4.5 and a protein precipitate. Upon adjustment to pH 5.2-6.8 and inoculation with Megasphaera elsdenii, brown juice was fermented within 48 h to up to 18 g of mixed volatile fatty acids (VFA) L(-1). Single-stage fermentation of green juice by both species in coculture typically resulted in overgrowth of S. bovis and acid inhibition of M. elsdenii, inhibiting VFA production. Because the juice fermentations are conducted without sterilization or supplemental nutrients, they can potentially contribute to an integrated process featuring protein recovery and fermentation of fractionated solids to VFA and other products.

In situ biphasic extractive fermentation for hexanoic acid production from sucrose by Megasphaera elsdenii NCIMB 702410.

Hexanoic acid production by a bacterium using sucrose as an economic carbon source was studied under conditions in which hexanoic acid was continuously extracted by liquid-liquid extraction. Megasphaera elsdenii NCIMB 702410, selected from five M. elsdenii strains, produced 4.69 g l⁻¹ hexanoic acid in a basal medium containing sucrose. Production increased to 8.19 g l⁻¹ when the medium was supplemented by 5 g l⁻¹ sodium butyrate. A biphasic liquid-liquid extraction system with 10 % (v/v) alamine 336 in oleyl alcohol as a solvent was evaluated in a continuous stirred-tank reactor held at pH 6. Over 90 % (w/w) of the hexanoic acid in a 0.5 M aqueous solution was transferred to the extraction solvent within 10 h. Cell growth was not significantly inhibited by direct contact of the fermentation broth with the extraction solvent. The system produced 28.42 g l⁻¹ of hexanoic acid from 54.85 g l⁻¹ of sucrose during 144 h of culture, and 26.52 and 1.90 g l⁻¹ of hexanoic acid was accumulated in the extraction solvent and the aqueous fermentation broth, respectively. The productivity and yield of hexanoic acid were 0.20 g l⁻¹ h⁻¹ and 0.50 g g⁻¹ sucrose, respectively.

Flavonols enhanced production of anti-inflammatory substance(s) by Bifidobacterium adolescentis: prebiotic actions of galangin, quercetin, and fisetin.

The gut microbiota is capable of the bioconversion of flavonoids whereas influences of probiotic anaerobes on the bioactivities of flavonoids and vice versa are still unclear. Here, we investigated functional interactions with respect to the anti-inflammatory activity between flavonols and probiotic bacteria. Ten enteric (6 probiotic and 4 indigenous) bacteria were incubated with flavonols (galangin, kaempferol, quercetin, myricetin, and fisetin) under anaerobic conditions, and the supernatants were assessed for their effects on nitric oxide (NO) production in lipopolysaccaride-stimulated RAW264 cells. Although the conditioned medium from the flavonol mono-culture and almost all of the tested co-cultures failed to inhibit NO production, the medium from the Bifidobacterium adolescentis/flavonols (galangin, quercetin, and fisetin) co-culture highly suppressed NO production. This activity increased during the 1-6 H incubation in a time-dependent manner and was not observed in the co-culture using heat-inactivated B. adolescentis. Interestingly, when the B. adolescentis cell number was increased, the supernatant from the mono-culture of the bacteria showed NO suppression, suggesting that B. adolescentis may produce NO suppressant(s), and flavonols may have a promoting effect. These findings indicate that flavonols have a prebiotic-like effect on the anti-inflammatory activity of B. adolescentis.

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.

Quantitative analysis of growth and volatile fatty acid production by the anaerobic ruminal bacterium Megasphaera elsdenii T81.

Megasphaera elsdenii T81 grew on either DL-lactate or D-glucose at similar rates (0.85 h(-1)) but displayed major differences in the fermentation of these substrates. Lactate was fermented at up to 210-mM concentration to yield acetic, propionic, butyric, and valeric acids. The bacterium was able to grow at much higher concentrations of D-glucose (500 mM), but never removed more than 80 mM of glucose from the medium, and nearly 60 % the glucose removed was sequestered as intracellular glycogen, with low yields of even-carbon acids (acetate, butyrate, caproate). In the presence of both substrates, glucose was not used until lactate was nearly exhausted, even by cells pregrown on glucose. Glucose-grown cultures maintained only low extracellular concentrations of acetate, and addition of exogenous acetate increased yields of butyrate, but not caproate. By contrast, exogenous acetate had little effect on lactate fermentation. At pH 6.6, growth rate was halved by exogenous addition of 60 mM propionate, 69 mM butyrate, 44 mM valerate, or 33 mM caproate; at pH 5.9, these values were reduced to 49, 49, 18, and 22 mM, respectively. The results are consistent with this species' role as an effective ruminal lactate consumer and suggest that this organism may be useful for industrial production of volatile fatty acids from lactate if product tolerance could be improved. The poor fermentation of glucose and sensitivity to caproate suggests that this strain is not practical for industrial caproate production.

Lactate and acrylate metabolism by Megasphaera elsdenii under batch and steady-state conditions.

The growth of Megasphaera elsdenii on lactate with acrylate and acrylate analogues was studied under batch and steady-state conditions. Under batch conditions, lactate was converted to acetate and propionate, and acrylate was converted into propionate. Acrylate analogues 2-methyl propenoate and 3-butenoate containing a terminal double bond were similarly converted into their respective saturated acids (isobutyrate and butyrate), while crotonate and lactate analogues 3-hydroxybutyrate and (R)-2-hydroxybutyrate were not metabolized. Under carbon-limited steady-state conditions, lactate was converted to acetate and butyrate with no propionate formed. As the acrylate concentration in the feed was increased, butyrate and hydrogen formation decreased and propionate was increasingly generated, while the calculated ATP yield was unchanged. M. elsdenii metabolism differs substantially under batch and steady-state conditions. The results support the conclusion that propionate is not formed during lactate-limited steady-state growth because of the absence of this substrate to drive the formation of lactyl coenzyme A (CoA) via propionyl-CoA transferase. Acrylate and acrylate analogues are reduced under both batch and steady-state growth conditions after first being converted to thioesters via propionyl-CoA transferase. Our findings demonstrate the central role that CoA transferase activity plays in the utilization of acids by M. elsdenii and allows us to propose a modified acrylate pathway for M. elsdenii.