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Amino acid biosynthesis in mixed rumen cultures.

ABSTRACT: Mixed rumen micro-organisms, maintained in continuous culture readily incorporated labelled HCO3- and acetate into amino acids. Labelled propionate, in contrast, was utilized only for isoleucine biosynthesis, but failed to label other amino acids to any significant extent. Evidence was obtained showing that in these mixed, i.e. symbiotic, cultures foward tricarboxylic acid-cycle reactions only proceed to 2-oxoglutarate. 14C distribution in amino acids clearly shows that 2-oxoglutarate is not oxidized further by tricarboxylic acid-cycle enzymes. Instead, acetate is carboxylated to pyruvate which is then carboxylated to oxaloacetate. Oxaloacetate equilibrates with fumarate and thereby carbon atoms 1 and 4 as well as carbon atoms 2 and 3 are randomized. Evidence was also obtained for the carboxylation of propionate to 2-oxobutyrate, isovalerate to 4-methyl-2-oxopentanoate, phenylacetate and hydroxyphentlacetate to the corresponding phenyl- and hydroxyphenyl-pyruvic acids and succinate to 2-oxoglutarate. Of the amino acid precursors investigated, only 3-hydroxypyruvate, the precursor of serine, appeared to be synthesized via an oxidative step, i.e. 3-phosphoglyceric acid to 3-phosphohydroxypyruvic acid. Most 2-oxo precursors of amino acids in these organisms appear to be formed via reductive carboxylation of the precursor acid.

SUBMITTER: Sauer FD

PROVIDER: S-EPMC1165750 | biostudies-other | 1975 Sep

REPOSITORIES: biostudies-other

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Project description:Forages and concentrates have consistently distinct patterns of fermentation in the rumen, with forages producing more methane (CH4) per unit of digested organic matter (OM) and higher acetate to propionate ratio than concentrates. A mechanism based on the Monod function of microbial growth has been proposed to explain the distinct fermentation pattern of forages and concentrates, where greater dilution rates and lower pH associated with concentrate feeding increase dihydrogen (H2) concentration through increasing methanogens growth rate and decreasing methanogens theoretically maximal growth rate, respectively. Increased H2 concentration would in turn inhibit H2 production, decreasing methanogenesis, inhibit H2-producing pathways such as acetate production via pyruvate oxidative decarboxylation, and stimulate H2-incorporating pathways such as propionate production. We examined the hypothesis that equalizing dilution rates in serial rumen cultures would result in a similar fermentation profile of a high forage and a high concentrate substrate. Under a 2 × 3 factorial arrangement, a high forage and a high concentrate substrate were incubated at dilution rates of 0.14, 0.28, or 0.56 h-1 in eight transfers of serial rumen cultures. Each treatment was replicated thrice, and the experiment repeated in two different months. The high concentrate substrate accumulated considerably more H2 and formate and produced less CH4 than the high forage substrate. Methanogens were nearly washed-out with high concentrate and increased their initial numbers with high forage. The effect of dilution rate was minor in comparison to the effect of the type of substrate. Accumulation of H2 and formate with high concentrate inhibited acetate and probably H2 and formate production, and stimulated butyrate, rather than propionate, as an electron sink alternative to CH4. All three dilution rates are considered high and selected for rapidly growing bacteria. The archaeal community composition varied widely and inconsistently. Lactate accumulated with both substrates, likely favored by microbial growth kinetics rather than by H2 accumulation thermodynamically stimulating electron disposal from NADH into pyruvate reduction. In this study, the type of substrate had a major effect on rumen fermentation largely independent of dilution rate and pH.

Project description:GapMind is a Web-based tool for annotating amino acid biosynthesis in bacteria and archaea (http://papers.genomics.lbl.gov/gaps). GapMind incorporates many variant pathways and 130 different reactions, and it analyzes a genome in just 15 s. To avoid error-prone transitive annotations, GapMind relies primarily on a database of experimentally characterized proteins. GapMind correctly handles fusion proteins and split proteins, which often cause errors for best-hit approaches. To improve GapMind's coverage, we examined genetic data from 35 bacteria that grow in defined media without amino acids, and we filled many gaps in amino acid biosynthesis pathways. For example, we identified additional genes for arginine synthesis with succinylated intermediates in Bacteroides thetaiotaomicron, and we propose that Dyella japonica synthesizes tyrosine from phenylalanine. Nevertheless, for many bacteria and archaea that grow in minimal media, genes for some steps still cannot be identified. To help interpret potential gaps, GapMind checks if they match known gaps in related microbes that can grow in minimal media. GapMind should aid the identification of microbial growth requirements.IMPORTANCE Many microbes can make all of the amino acids (the building blocks of proteins). In principle, we should be able to predict which amino acids a microbe can make, and which it requires as nutrients, by checking its genome sequence for all of the necessary genes. However, in practice, it is difficult to check for all of the alternative pathways. Furthermore, new pathways and enzymes are still being discovered. We built an automated tool, GapMind, to annotate amino acid biosynthesis in bacterial and archaeal genomes. We used GapMind to list gaps: cases where a microbe makes an amino acid but a complete pathway cannot be identified in its genome. We used these gaps, together with data from mutants, to identify new pathways and enzymes. However, for most bacteria and archaea, we still do not know how they can make all of the amino acids.

Project description:Synthesis gas (syngas) fermentation using acetogenic bacteria is an approach for production of bulk chemicals like acetate, ethanol, butanol, or 2,3-butandiol avoiding the fuel vs. food debate by using carbon monoxide, carbon dioxide, and hydrogen from gasification of biomass or industrial waste gases. Suffering from energetic limitations, yields of C4-molecules produced by syngas fermentation are quite low compared with ABE fermentation using sugars as a substrate. On the other hand, fungal production of malic acid has high yields of product per gram metabolized substrate but is currently limited to sugar containing substrates. In this study, it was possible to show that Aspergilus oryzae is able to produce malic acid using acetate as sole carbon source which is a main product of acetogenic syngas fermentation. Bioreactor cultivations were conducted in 2.5 L stirred tank reactors. During the syngas fermentation part of the sequential mixed culture, Clostridium ljungdahlii was grown in modified Tanner medium and sparged with 20 mL/min of artificial syngas mimicking a composition of clean syngas from entrained bed gasification of straw (32.5 vol-% CO, 32.5 vol-% H2, 16 vol-% CO2, and 19 vol-% N2) using a microsparger. Syngas consumption was monitored via automated gas chromatographic measurement of the off-gas. For the fungal fermentation part gas sparging was switched to 0.6 L/min of air and a standard sparger. Ammonia content of medium for syngas fermentation was reduced to 0.33 g/L NH4Cl to meet the requirements for fungal production of dicarboxylic acids. Malic acid production performance of A. oryzae in organic acid production medium and syngas medium with acetate as sole carbon source was verified and gave YP?S values of 0.28 g/g and 0.37 g/g respectively. Growth and acetate formation of C. ljungdahlii during syngas fermentation were not affected by the reduced ammonia content and 66 % of the consumed syngas was converted to acetate. The overall conversion of CO and H2 into malic acid was calculated to be 3.5 g malic acid per mol of consumed syngas or 0.22 g malic acid per gram of syngas.

Project description:Mitigation of enteric methane (CH4) emissions from ruminant livestock represents an opportunity to improve the sustainability, productivity, and profitability of beef and dairy production. Ruminal methanogenesis can be mitigated via two primary strategies: (1) alternative electron acceptors and (2) enzymatic inhibition of methanogenic pathways. The former utilizes the thermodynamic favorability of certain reactions such as nitrate/nitrite reduction to ammonia (NH3) while the latter targets specific enzymes using structural analogs of CH4 and methanogenic cofactors such as bromochloromethane (BCM). In this study, we investigated the effects of four additives and their combinations on CH4 production by rumen microbes in batch culture. Sodium nitrate (NaNO3), sodium sulfate (Na2SO4), and 3-nitro-1-propionate (3NPA) were included as thermodynamic inhibitors, whereas BCM was included as a enzymatic inhibitor. Individual additives were evaluated at three levels of inclusion in experiments 1 and 2. Highest level of each additive was used to determine the combined effect of NaNO3 + Na2SO4 (NS), NS + 3NPA (NSP), and NSP + BCM (NSPB) in experiments 3 and 4. Experimental diets were high, medium, and low forage diets (HF, MF, and LF, respectively) and consisted of alfalfa hay and a concentrate mix formulated to obtain the following forage to concentrate ratios: 70:30, 50:50, and 30:70, respectively. Diets with additives were placed in fermentation culture bottles and incubated in a water bath (39°C) for 6, 12, or 24h. Microbial DNA was extracted for 16S rRNA and ITS gene amplicon sequencing. In experiments 1 and 2, CH4 concentrations in control cultures decreased in the order of LF, MF, and HF diets, whereas in experiments 3 and 4, CH4 was highest in MF diet followed by HF and LF diets. Culture pH and NH3 in the control decreased in the order of HF, MF, to LF as expected. NaNO3 decreased (p < 0.001) CH4 and butyrate and increased acetate and propionate (p < 0.03 and 0.003, respectively). Cultures receiving NaNO3 had an enrichment of microorganisms capable of nitrate and nitrite reduction. 3NPA also decreased CH4 at 6h with no further decrease at 24 h (p < 0.001). BCM significantly inhibited methanogenesis regardless of inclusion levels as well as in the presence of the thermodynamic inhibitors (p < 0.001) while enriching succinate producers and assimilators as well as propionate producers (p adj < 0.05). However, individual inclusion of BCM decreased total short chain fatty acid (SCFA) concentrations (p < 0.002). Inhibition of methanogenesis with BCM individually and in combination with the other additives increased gaseous H2 concentrations (p < 0.001 individually and 0.028 in combination) while decreasing acetate to propionate ratio (p < 0.001). Only the cultures treated with BCM in combination with other additives significantly (padj < 0.05) decreased the abundance of Methanobrevibacter expressed as log fold change. Overall, the combination of thermodynamic and enzymatic inhibitors presented a promising effect on ruminal fermentation in-vitro, inhibiting methanogenesis while optimizing the other fermentation parameters such as pH, NH3, and SCFAs. Here, we provide a proof of concept that the combination of an electron acceptor and a methane analog may be exploited to improve microbial efficiency via methanogenesis inhibition.

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Amino acid biosynthesis in mixed rumen cultures.

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