Microbial interaction between the succinate-utilizing bacterium Phascolarctobacterium faecium and the gut commensal Bacteroides thetaiotaomicron.

A large variety of microbes are present in the human gut, some of which are considered to interact with each other. Most of these interactions involve bacterial metabolites. Phascolarctobacterium faecium hardly uses carbohydrates for growth and instead uses succinate as a substrate. This study investigated the growth behavior of the co-culture of the succinate-specific utilizer P. faecium and the succinogenic gut commensal Bacteroides thetaiotaomicron. Succinate production by B. thetaiotaomicron supported the growth of P. faecium and concomitant propionate production via the succinate pathway. The succinate produced was completely converted to propionate. This result was comparable with the monoculture of P. faecium in the medium supplemented with 1% (w/v) succinate. We analyzed the transcriptional response (RNA-Seq) between the mono- and co-culture of P. faecium and B. thetaiotaomicron. Comparison of the expression levels of genes of P. faecium between the mono- and co-cultured conditions highlighted that the genes putatively involved in the transportation of succinate were notably expressed under the co-cultured conditions. Differential expression analysis showed that the presence of P. faecium induced changes in the B. thetaiotaomicron transcriptional pattern, for example, expression changes in the genes for vitamin B12 transporters and reduced expression of glutamate-dependent acid resistance system-related genes. Also, transcriptome analysis of P. faecium suggested that glutamate and succinate might be used as sources of succinyl-CoA, an intermediate in the succinate pathway. This study revealed some survival strategies of asaccharolytic bacteria, such as Phascolarctobacterium spp., in the human gut.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

Increased carbon dioxide reduction to acetate in a microbial electrosynthesis reactor with a reduced graphene oxide-coated copper foam composite cathode.

Microbial electrosynthesis is a bioprocess where microbes reduce CO2 into multicarbon chemicals with electrons derived from the cathode of a bioelectrochemical reactor. Developing a highly productive microbial electrosynthesis reactor requires excellent electrical connection between the electrochemical setup, the cathode, and the microbes. Copper is a highly conductive cathode material widely employed in electrochemical apparatuses. However, the antimicrobial properties of copper limit its usage for bioelectrochemistry. Here, biocompatible reduced graphene oxide coated on copper foam is synthesized as a cathode material for the microbial electrosynthesis of acetate from CO2. Dense and electroactive Sporomusa ovata biofilms form on the surface of reduced graphene oxide-coated copper foam electrodes while only scattered and damaged cells cover uncoated copper electrodes. Besides the formation of metabolically-active biofilms, acetate production rate from CO2 is 21.3 and 43.5-fold higher with this novel composite cathode compared with an uncoated copper foam cathode and a reversed cathode made of reduced graphene oxide foam coated with copper, respectively. The results demonstrate that reduced graphene oxide can be employed as a biocompatible and conductive buffer between microbes and bactericidal electrode materials with excellent electrochemical property to enable highly performant microbial electrosynthesis.

Almond Consumption and Processing Affects the Composition of the Gastrointestinal Microbiota of Healthy Adult Men and Women: A Randomized Controlled Trial.

BACKGROUND: Almond processing has been shown to differentially impact metabolizable energy; however, the effect of food form on the gastrointestinal microbiota is under-investigated. OBJECTIVE: We aimed to assess the interrelationship of almond consumption and processing on the gastrointestinal microbiota. DESIGN: A controlled-feeding, randomized, five-period, crossover study with washouts between diet periods was conducted in healthy adults (n = 18). Treatments included: (1) zero servings/day of almonds (control); (2) 1.5 servings (42 g)/day of whole almonds; (3) 1.5 servings/day of whole, roasted almonds; (4) 1.5 servings/day of roasted, chopped almonds; and (5) 1.5 servings/day of almond butter. Fecal samples were collected at the end of each three-week diet period. RESULTS: Almond consumption increased the relative abundances of Lachnospira, Roseburia, and Dialister (p ≤ 0.05). Comparisons between control and the four almond treatments revealed that chopped almonds increased Lachnospira, Roseburia, and Oscillospira compared to control (p < 0.05), while whole almonds increased Dialister compared to control (p = 0.007). There were no differences between almond butter and control. CONCLUSIONS: These results reveal that almond consumption induced changes in the microbial community composition of the human gastrointestinal microbiota. Furthermore, the degree of almond processing (e.g., roasting, chopping, and grinding into butter) differentially impacted the relative abundances of bacterial genera.

Host-microbiome interactions in human type 2 diabetes following prebiotic fibre (galacto-oligosaccharide) intake.

Aberrant microbiota composition and function have been linked to several pathologies, including type 2 diabetes. In animal models, prebiotics induce favourable changes in the intestinal microbiota, intestinal permeability (IP) and endotoxaemia, which are linked to concurrent improvement in glucose tolerance. This is the first study to investigate the link between IP, glucose tolerance and intestinal bacteria in human type 2 diabetes. In all, twenty-nine men with well-controlled type 2 diabetes were randomised to a prebiotic (galacto-oligosaccharide mixture) or placebo (maltodextrin) supplement (5·5 g/d for 12 weeks). Intestinal microbial community structure, IP, endotoxaemia, inflammatory markers and glucose tolerance were assessed at baseline and post intervention. IP was estimated by the urinary recovery of oral 51Cr-EDTA and glucose tolerance by insulin-modified intravenous glucose tolerance test. Intestinal microbial community analysis was performed by high-throughput next-generation sequencing of 16S rRNA amplicons and quantitative PCR. Prebiotic fibre supplementation had no significant effects on clinical outcomes or bacterial abundances compared with placebo; however, changes in the bacterial family Veillonellaceae correlated inversely with changes in glucose response and IL-6 levels (r -0·90, P=0·042 for both) following prebiotic intake. The absence of significant changes to the microbial community structure at a prebiotic dosage/length of supplementation shown to be effective in healthy individuals is an important finding. We propose that concurrent metformin treatment and the high heterogeneity of human type 2 diabetes may have played a significant role. The current study does not provide evidence for the role of prebiotics in the treatment of type 2 diabetes.

Sustainable growth of Dehalococcoides mccartyi 195 by corrinoid salvaging and remodeling in defined lactate-fermenting consortia.

Corrinoids are essential cofactors of reductive dehalogenases in Dehalococcoides mccartyi, an important bacterium in bioremediation, yet sequenced D. mccartyi strains do not possess the complete pathway for de novo corrinoid biosynthesis. Pelosinus sp. and Desulfovibrio sp. have been detected in dechlorinating communities enriched from contaminated groundwater without exogenous cobalamin corrinoid. To investigate the corrinoid-related interactions among key members of these communities, we constructed consortia by growing D. mccartyi strain 195 (Dhc195) in cobalamin-free, trichloroethene (TCE)- and lactate-amended medium in cocultures with Desulfovibrio vulgaris Hildenborough (DvH) or Pelosinus fermentans R7 (PfR7) and with both in tricultures. Only the triculture exhibited sustainable dechlorination and cell growth when a physiological level of 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, was provided. In the triculture, DvH provided hydrogen while PfR7 provided corrinoids to Dhc195, and the initiation of dechlorination and Dhc195 cell growth was highly dependent on the growth of PfR7. Corrinoid analysis indicated that Dhc195 imported and remodeled the phenolic corrinoids produced by PfR7 into cobalamin in the presence of DMB. Transcriptomic analyses of Dhc195 showed the induction of the CbiZ-dependent corrinoid-remodeling pathway and BtuFCD corrinoid ABC transporter genes during corrinoid salvaging and remodeling. In contrast, another operon annotated to encode a putative iron/cobalamin ABC transporter (DET1174-DET1176) was induced when cobalamin was exogenously provided. Interestingly, a global upregulation of phage-related genes was observed when PfR7 was present. These findings provide insights into both the gene regulation of corrinoid salvaging and remodeling in Dhc195 when it is grown without exogenous cobalamin and microbe-to-microbe interactions in dechlorinating microbial communities.

Polyphosphate storage during sporulation in the gram-negative bacterium Acetonema longum.

Using electron cryotomography, we show that the Gram-negative sporulating bacterium Acetonema longum synthesizes high-density storage granules at the leading edges of engulfing membranes. The granules appear in the prespore and increase in size and number as engulfment proceeds. Typically, a cluster of 8 to 12 storage granules closely associates with the inner spore membrane and ultimately accounts for ∼7% of the total volume in mature spores. Energy-dispersive X-ray spectroscopy (EDX) analyses show that the granules contain high levels of phosphorus, oxygen, and magnesium and therefore are likely composed of polyphosphate (poly-P). Unlike the Gram-positive Bacilli and Clostridia, A. longum spores retain their outer spore membrane upon germination. To explore the possibility that the granules in A. longum may be involved in this unique process, we imaged purified Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Clostridium sporogenes spores. Even though B. cereus and B. thuringiensis contain the ppk and ppx genes, none of the spores from Gram-positive bacteria had granules. We speculate that poly-P in A. longum may provide either the energy or phosphate metabolites needed for outgrowth while retaining an outer membrane.

Substrate-limited co-culture for efficient production of propionic acid from flour hydrolysate.

Propionic acid is presently mainly produced by chemical synthesis. For many applications, especially in feed and food industries, a fermentative production of propionic acid from cheap and renewable resources is of large interest. In this work, we investigated the use of a co-culture to convert household flour to propionic acid. Batch and fed-batch fermentations of hydrolyzed flour and a process of simultaneous saccharification and fermentation were examined and compared. Fed-batch culture with substrate limitation was found to be the most efficient process, reaching a propionic acid concentration of 30 g/L and a productivity of 0.33 g/L*h. This is the highest productivity so far achieved with free cells on media containing flour hydrolysate or glucose as carbon source. Batch culture and culture with controlled saccharification and fermentation delivered significantly lower propionic acid production (17-20 g/L) due to inhibition by the intermediate product lactate. It is concluded that co-culture fermentation of flour hydrolysate can be considered as an appealing bioprocess for the production of propionic acid.

Interactions between oil substrates and glucose on pure cultures of ruminal lipase-producing bacteria.

The hydrolysis of free fatty acids from lipids is a prerequisite for biohydrogenation, a process that effectively saturates free fatty acids. Anaerovibrio lipolyticus 5s and Butyrivibrio fibrisolvens have long been thought to be the major contributors to ruminal lipolysis; however, Propionibacterium avidum and acnes recently have been identified as contributing lipase activity in the rumen. In order to further characterize the lipase activity of these bacterial populations, each was grown with three different lipid substrates, olive oil, corn oil, and flaxseed oil (3 %). Because different finishing rations contain varying levels of glycogen (a source of free glucose) this study also documented the effects of glucose on lipolysis. P. avidum and A. lipolyticus 5s demonstrated the most rapid rates (P < 0.05) of lipolysis for cultures grown with olive oil and flaxseed oil, respectively. A. lipolyticus, B. fibrisolvens, and P. avidum more effectively hydrolyzed flaxseed oil than olive oil or corn oil, especially in the presence of 0.02 % glucose. Conversely, P. acnes hydrolyzed corn oil more readily than olive oil or flaxseed oil and glucose had no effect on lipolytic rate. Thus, these bacterial species demonstrated different specificities for oil substrates and different sensitivities to glucose.

Growth inhibition of Sporomusa ovata by incorporation of benzimidazole bases into cobamides.

Phenolyl cobamides are unique members of a class of cobalt-containing cofactors that includes vitamin B12 (cobalamin). Cobamide cofactors facilitate diverse reactions in prokaryotes and eukaryotes. Phenolyl cobamides are structurally and chemically distinct from the more commonly used benzimidazolyl cobamides such as cobalamin, as the lower axial ligand is a phenolic group rather than a benzimidazole. The functional significance of this difference is not well understood. Here we show that in the bacterium Sporomusa ovata, the only organism known to synthesize phenolyl cobamides, several cobamide-dependent acetogenic metabolisms have a requirement or preference for phenolyl cobamides. The addition of benzimidazoles to S. ovata cultures results in a decrease in growth rate when grown on methanol, 3,4-dimethoxybenzoate, H2 plus CO2, or betaine. Suppression of native p-cresolyl cobamide synthesis and production of benzimidazolyl cobamides occur upon the addition of benzimidazoles, indicating that benzimidazolyl cobamides are not functionally equivalent to the phenolyl cobamide cofactors produced by S. ovata. We further show that S. ovata is capable of incorporating other phenolic compounds into cobamides that function in methanol metabolism. These results demonstrate that S. ovata can incorporate a wide range of compounds as cobamide lower ligands, despite its preference for phenolyl cobamides in the metabolism of certain energy substrates. To our knowledge, S. ovata is unique among cobamide-dependent organisms in its preferential utilization of phenolyl cobamides.

Microbial community succession during lactate amendment and electron acceptor limitation reveals a predominance of metal-reducing Pelosinus spp.

The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N(2) gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.

Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation.

Two hallmarks of the Firmicute phylum, which includes the Bacilli and Clostridia classes, are their ability to form endospores and their "Gram-positive" single-membraned, thick-cell-wall envelope structure. Acetonema longum is part of a lesser-known family (the Veillonellaceae) of Clostridia that form endospores but that are surprisingly "Gram negative," possessing both an inner and outer membrane and a thin cell wall. Here, we present macromolecular resolution, 3D electron cryotomographic images of vegetative, sporulating, and germinating A. longum cells showing that during the sporulation process, the inner membrane of the mother cell is inverted and transformed to become the outer membrane of the germinating cell. Peptidoglycan persists throughout, leading to a revised, "continuous" model of its role in the process. Coupled with genomic analyses, these results point to sporulation as a mechanism by which the bacterial outer membrane may have arisen and A. longum as a potential "missing link" between single- and double-membraned bacteria.

Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture.

Milk fat depression in cows fed high-grain diets has been related to an increase in the concentration of trans-10 C(18:1) and trans-10,cis-12 conjugated linoleic acid (CLA) in milk. These fatty acids (FA) are produced as a result of the alteration in rumen biohydrogenation of dietary unsaturated FA. Because a reduction in ruminal pH is usually observed when high-concentrate diets are fed, the main cause that determines the alteration in the biohydrogenation pathways is not clear. The effect of pH (6.4 vs. 5.6) and dietary forage to concentrate ratios (F:C; 70:30 F:C vs. 30:70 F:C) on rumen microbial fermentation, effluent FA profile, and DNA concentration of bacteria involved in lipolysis and biohydrogenation processes were investigated in a continuous culture trial. The dual-flow continuous culture consisted of 2 periods of 8 d (5 d for adaptation and 3 d for sampling), with a 2 x 2 factorial arrangement of treatments. Samples from solid and liquid mixed effluents were taken for determination of total N, ammonia-N, and volatile fatty acid concentrations, and the remainder of the sample was lyophilized. Dry samples were analyzed for dry matter, ash, neutral and acid detergent fiber, FA, and purine contents. The pH 5.6 reduced organic matter and fiber digestibility, ammonia-N concentration and flow, and crude protein degradation, and increased nonammonia and dietary N flows. The pH 5.6 decreased the flow of C(18:0), trans-11 C(18:1) and cis-9, trans-11 CLA, and increased the flow of trans-10 C(18:1), C(18:2n-6), C(18:3n-3), trans-11,cis-15 C(18:2) and trans-10,cis-12 CLA in the 1 h after feeding effluent. The pH 5.6 reduced Anaerovibrio lipolytica (32.7 vs. 72.1 pg/10 ng of total DNA) and Butyrivibrio fibrisolvens vaccenic acid subgroup (588 vs. 1,394 pg/10 ng of total DNA) DNA concentrations. The high-concentrate diet increased organic matter and fiber digestibility, nonammonia and bacterial N flows, and reduced ammonia-N concentration and flow. The high-concentrate diet reduced trans-11 C(18:1) and trans-10 C(18:1), and increased C(18:2n-6), C(18:3n-3) and trans-10,cis-12 CLA proportions in the 1 h after feeding effluent. The increase observed in trans-10,cis-12 CLA proportion in the 1 h after feeding effluent due to the high-concentrate diet was smaller that that observed at pH 5.6. Results indicate that the pH is the main cause of the accumulation of trans-10 C(18:1) and trans-10, cis-12 CLA in the effluent, but the trans-10,cis-12 CLA proportion can be also affected by high levels of concentrate in the diet.

Antimicrobial susceptibilities and clinical sources of Dialister species.

Seventy-four strains representing the four species of the genus Dialister were isolated from various clinical samples. Dialister pneumosintes and Dialister micraerophilus were the two mainly encountered species. Fifty-five isolates were tested against 14 antimicrobial agents. Decreased susceptibilities to piperacillin, metronidazole, macrolides, fluoroquinolones, and rifampin were demonstrated. The clinical impact of these decreased susceptibilities remains to be investigated but should prompt microbiologists to perform antimicrobial susceptibility testing for clinically important Dialister spp.

Lipopolysaccharides of anaerobic beer spoilage bacteria of the genus Pectinatus--lipopolysaccharides of a Gram-positive genus.

Bacteria of the genus Pectinatus emerged during the seventies as contaminants and spoilage organisms in packaged beer. This genus comprises two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis; both are strict anaerobes. On the basis of genomic properties the genus is placed among low GC Gram-positive bacteria (phylum Firmicutes, class Clostridia, order Clostridiales, family Acidaminococcaceae). Despite this assignment, Pectinatus bacteria possess an outer membrane and lipopolysaccharide (LPS) typical of Gram-negative bacteria. The present review compiles the structural and compositional studies performed on Pectinatus LPS. These lipopolysaccharides exhibit extensive heterogeneity, i.e. several macromolecularly and structurally distinct LPS molecules are produced by each strain. Whereas heterogeneity is a common property in lipopolysaccharides, Pectinatus LPS have been shown to contain exceptional carbohydrate structures, consisting of a fairly conserved core region that carries a large non-repetitive saccharide that probably replaces the O-specific chain. Such structures represent a novel architectural principle of the LPS molecule.

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets.

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.

Effects of a strain of Saccharomyces cerevisiae (Levucell SC1), a microbial additive for ruminants, on lactate metabolism in vitro.

The effect of Levucell SC, a strain of Saccharomyces cerevisiae marked as a feed additive for ruminants, was investigated in vitro on lactate metabolism by the ruminal bacteria Streptococcus bovis and Megasphaera elsdenii. The coculture between 10(7) live cells x mL(-1) of SC and a Streptococcus bovis strain in the presence of glucose reduced lactate production by the bacterial strain. Live yeast cells were able to compete with Streptococcus bovis for glucose utilization in strictly anaerobic conditions, so less glucose was available for the bacterium. SC also stimulated L-lactate utilization by a strain of M. elsdenii. The effect depended on the concentration of yeast cells added. Bacterial growth and fermentation end-product concentrations were also increased in the presence of SC. Some amino acids and vitamins, but not dicarboxylic acids, stimulated the bacterial specific activity of L-lactate uptake. SC was able to provide amino acids to M. elsdenii. In a coculture of Streptococcus bovis and M. elsdenii on glucose, the reduction of lactate concentration was improved by SC, the same trend being observed when maltose or soluble starch were used as carbon and energy source. These results indicate that SC can be a very useful tool to reduce lactate accumulation in vitro during fermentation of soluble sugars.

Ability of Acidaminococcus fermentans to oxidize trans-aconitate and decrease the accumulation of tricarballylate, a toxic end product of ruminal fermentation.

Mixed ruminal bacteria convert trans-aconitate to tricarballylate, a tricarboxylic acid which chelates blood divalent cations and decreases their availability (J. B. Russell and P. J. Van Soest, Appl. Environ. Microbiol. 47:155-159, 1984). Decreases in blood magnesium in turn cause a potentially fatal disease known as grass tetany. trans-Aconitate was stoichiometrically reduced to tricarballylate by Selenomonas ruminantium, a common ruminal bacterium in grass-fed ruminants (J. B. Russell, Appl. Environ. Microbiol. 49:120-126, 1985). When mixed ruminal bacteria were enriched with trans-aconitate, a trans-aconitate-oxidizing bacterium was also isolated (G. M. Cook, F. A. Rainey, G. Chen, E. Stackebrandt, and J. B. Russell, Int. J. Syst. Bacteriol. 44:576-578, 1994). The trans-aconitate-oxidizing bacterium was identified as Acidaminococcus fermentans, and it converted trans-aconitate to acetate, a nontoxic end product of ruminal fermentation. When S. ruminantium and A. fermentans were cocultured with trans-aconitate and glucose, tricarballylate never accumulated and all the trans-aconitate was converted to acetate. Continuous-culture studies (dilution rate, 0.1 h-1) likewise indicated that A. fermentans could outcompete S. ruminantium for trans-aconitate. When mixed ruminal bacteria were incubated in vitro with 10 mM trans-aconitate for 24 h, 45% of the trans-aconitate was converted to tricarballylate. Tricarballylate production decreased 50% if even small amounts of A. fermentans were added to the incubation mixes (0.01 mg of protein per mg of mixed bacterial protein). When A. fermentans (2 g of bacterial protein) was added directly to the rumen, the subsequent conversion of trans-aconitate to tricarballylate decreased 50%, but this effect did not persist for more than 18 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of an Aspergillus oryzae fermentation extract and other factors on lactate utilization by the ruminal bacterium Megasphaera elsdenii.

The objective of this study was to determine the effects of an Aspergillus oryzae fermentation extract (Amaferm) as well as other factors on lactate utilization by the ruminal bacterium Megasphaera elsdenii B159. Addition of Amaferm or a filter-sterilized Amaferm filtrate stimulated L-lactate uptake by both M. elsdenii and the ruminal selenomonad strain H18. Growth of M. elsdenii in medium that contained DL-lactate (2 g/L), Trypticase, and yeast extract was only slightly stimulated by the addition of 5% (vol/vol) Amaferm filtrate after 24 h. However, growth of M. elsdenii in a similar medium lacking Trypticase and yeast extract was increased over twofold by the addition of either 2 or 5% (vol/vol) Amaferm filtrate. These results suggest that Amaferm provides growth factors (i.e., amino acids, B vitamins) to support growth of M. elsdenii on lactate. There was no inhibition of L-lactate uptake when lactate-grown cells of M. elsdenii were incubated with excess (10 mM) glucose, sucrose, or maltose. In addition, when cells were grown on glucose, sucrose, or maltose rather than lactate there was little difference in L-lactate uptake, suggesting that L-lactate transport in M. elsdenii is not subject to catabolite repression by these soluble sugars. Both K+ and Na+ had little effect on L-lactate uptake. Uptake was unaffected at extracellular pH values between 6.0 and 8.0, whereas pH values of 5.0 and 4.0 increased uptake. In addition, L-lactate uptake was inhibited between 34 and 61% by protonophores. These results suggest that protons may be involved in the uptake of L-lactate by M. elsdenii B159.

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.