Reduced Acetate Production Research Articles

Increasing degree of substitution inhibits acetate while promotes butyrate production during in vitro fermentation of citric acid-modified rice starch

Citric acid-modified starch functions as a resistant starch, while the combined effects of its fine molecular structure and degree of substitution on gut microbiota are not well understood. To this end, citric acid-modified starches with varying degrees of substitution were synthesized from rice starches with distinct molecular structures and their impact on gut microbiota composition and short-chain fatty acid (SCFA) production was analyzed. Notably, rice starch with a higher degree of substitution significantly reduced acetate production, while promoted butyrate production. Correlation analysis further suggested that amylopectin chains with 12 < DP ≤ 36 and amylose chains with 100 < DP ≤ 500 alter the growth of Faecalibacterium_prausnitzii and Bacteroides_vulgatus, consequentially determining the production of SCFAs. Collectively, these findings indicate that citric acid-modified rice starch with different degrees of substitution can target specific gut bacteria and SCFA production, thus conferring beneficial impact on human health.

Adaptive laboratory evolution and metabolic regulation of genetic Escherichia coli W3110 toward low-carbon footprint production of 5-aminolevulinic acid

BackgroundThe 5-aminolevulinic acid (5-ALA) is a prodrug that has been approved by FDA for photodynamic therapy of cancer. 5-ALA is intracellularly metabolized to protoporphyrin IX, which upon illumination with red light results in photooxidative damage to cells. However, microbial 5-ALA production is challenged by strain tolerance and limited metabolic engineering, while carbon capture is rarely conducted. In this study, E. coli W3110 was equipped with recombinant ALAS production and concomitant CO2 assimilation capacity for low-carbon footprint 5-ALA production. MethodsA stable E. coli strain expressing recombinant ALAS was further improved by adaptive laboratory evolution (ALE). Glycine distribution between biomass and 5-ALA was deciphered using C13 isotope analysis based on C13–2-glycine. Metabolic flux was enforced by overexpressing phosphoenolpyruvate carboxylase (ppc) and knockout of phosphate acetyltransferase (pta). The ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) were added to confer CO2 assimilation ability in RSSL strain. Significant findingsThe adaptive RL8 strain demonstrated 129% and 205% increase in biomass and 5-ALA, respectively. C13 isotope analysis revealed that RSSL assimilated CO2 into biomass and generated glycine from glucose for maximal 5-ALA production. Overexpression of ppc and knockout of pta led to energy conservation, reduced acetate production, thus increasing 5-ALA accumulation (9.53 g/L). Carbon uptake via RuBisCO/PRK significantly reduced CO2 emission to -2.32 g-CO2/g-DCW.

Plant extracts as natural modulators of gut microbiota community structure and functionality

The main objective of this work was to evaluate the effect that several plant extracts (currently sold as functional ingredients) have on gut microbiota community structure and functionality. Plant extracts were submitted to an in vitro digestion and fecal fermentation. Overall, plant extracts showed a marked inhibitory activity when compared to basal conditions. However, they also favored the growth of some bacteria such as Coprococcus and Butyricimonas, two butyrate producers. Especially interesting was tea extract which inhibited the growth of the genus Escherichia/Shigella, known to involve species related with gastrointestinal disorders. Additionally, tea extract increased the growth of Faecalibacterium, a known butyrate producer. Regarding short chain fatty acids production, while plant extracts reduced acetate production, butyrate was increased for most samples, especially tea extract. Propionate production was less affected in comparison with basal conditions. Fermentation by gut microbiota also modified the antioxidant capacity (assessed via DPPH, FRAP and Folin-Ciocalteu methods).

Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi)

Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2, H2) to a variety of fuels and chemicals through the Wood-Ljungdahl pathway. However, metabolic engineering and pathway elucidation in this microbe is limited by the lack of genetic tools to downregulate target genes. To overcome this obstacle, here we developed an inducible CRISPR interference (CRISPRi) system for C. ljungdahlii that enables efficient (> 94%) transcriptional repression of several target genes, both individually and in tandem. We then applied CRISPRi in a strain engineered for 3-hydroxybutyrate (3HB) production to examine targets for increasing carbon flux toward the desired product. Downregulating phosphotransacetylase (pta) with a single sgRNA led to a 97% decrease in enzyme activity and a 2.3-fold increase in titer during heterotrophic growth. However, acetate production still accounted for 40% of the carbon flux. Repression of aldehyde:ferredoxin oxidoreductase (aor2), another potential route for acetate production, led to a 5% reduction in acetate flux, whereas using an additional sgRNA targeted to pta reduced the enzyme activity to 0.7% of the wild-type level, and further reduced acetate production to 25% of the carbon flux with an accompanying increase in 3HB titer and yield. These results demonstrate the utility of CRISPRi for elucidating and controlling carbon flow in C. ljungdahlii.

Examining the Role of Gut Dysbiosis in Neuroinflammation and Hypertension in a Model of Obstructive Sleep Apnea

Obstructive sleep apnea (OSA), characterized by repeated closure of the upper airway during sleep, is a significant clinical problem and an independent risk factor for systemic hypertension. The importance of a healthy gut microbiota, and detriment of a dysbiotic microbiota, on host physiology is becoming increasingly evident. We have previously demonstrated that gut dysbiosis is associated with the development of hypertension in a rat model of OSA. Transplanting the dysbiotic microbiota from a hypertensive OSA rat into a normotensive rat induces hypertension, demonstrating a causal relationship between gut dysbiosis and hypertension. The mechanisms linking gut dysbiosis to hypertension are unknown. We tested the hypothesis that OSA‐induced gut dysbiosis impairs gut barrier function, causing systemic inflammation and neuroinflammation, which is linked to hypertension. Using an in vivo model of OSA, we exposed high fat fed rats to 2 weeks of sham or OSA (60 apneas/hr). Relative to sham rats, OSA led to a significant increase in TNFα mRNA expression in the cecum wall as well as decreased goblet cells/crypt in the cecum and colon (n=6, p&lt;0.05). OSA resulted in a 3‐fold increase in gut permeability (n=6, p&lt;0.05). Consistent with impaired gut barrier function and bacterial translocation, we observed increased bacterial 16S rRNA signatures in the mesenteric lymph node and visceral adipose tissue following OSA (n=4–7, p&lt;0.05). Flow cytometric analysis using whole brain revealed a significant increase in the number of activated microglia following OSA (n=4–6, p&lt;0.05). Short chain fatty acids play a key role in maintaining gut barrier integrity and regulating immune responses. We found that OSA significantly reduced the acetate concentraction in the cecum (n=5, p&lt;0.05). To increase short chain fatty acid production in the gut, rats were treated with a prebiotic (diet enriched with 20% resistant starch), or probiotic (Clostridium butyricum; 109 CFU by gavage every three days) during the 2 weeks of sham or OSA. Pre‐ and probiotics each prevented OSA‐induced decrease in cecal acetate concentration, prevented OSA‐induced loss of goblet cells, and prevented increased TNFα expression in the cecum of OSA rats (n=5–6, p&lt;0.05 for each). Additionally, pre‐ and probiotics prevented OSA‐induced activation of microglia in brain (n=6–8, p&lt;0.05). Importantly, both the pre‐ and probiotic successfully prevented the development of hypertension in OSA rats. To test the direct effects of acetate, we chronically infused 20μmol/(kg‐min) acetate into the cecum during the 2 weeks of sham or OSA. Cecal acetate infusion prevented OSA‐induced hypertension. These data demonstrate a causal role for gut dysbiosis in the development of hypertension, which involves reduced acetate production, gut barrier disruption, bacterial translocation, and neuroinflammation. Our data suggest that manipulation of the gut microbiota may serve as a novel therapy in the prevention of hypertension.Support or Funding InformationThis project was funded by NINDS R01NS080531 (RMB), AHA 16SDG29970000 (DJD), and by Public Health Service grant DK56338, which funds the Texas Medical Center Digestive Diseases Center (DJD).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic engineering of Klebsiella pneumoniae J2B for co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Reduction of acetate and other by-products

Production of 3-hydroxypropionic acid (3-HP) or 1,3-propanediol (1,3-PDO) production from glycerol is challenging due to the problems associated with cofactor regeneration, coenzyme B12 synthesis, and the instability of pathway enzymes. To address these complications, simultaneous production of 3-HP and 1,3-PDO, instead of individual production of each compound, was attempted. With over-expression of an aldehyde dehydrogenase, recombinant Klebsiella pneumoniae could co-produce 3-HP and 1,3-PDO successfully. However, the production level was unsatisfactory due to excessive accumulation of many by-products, especially acetate. To reduce acetate production, we attempted; (i) reduction of glycerol assimilation through the glycolytic pathway, (ii) increase of glycerol flow towards co-production, and (iii) variation of aeration rate. These efforts were partially beneficial in reducing acetate and improving co-production: 21g/L of 1,3-PDO and 43g/L of 3-HP were obtained. Excessive acetate (>150mM) was still produced at the end of bioreactor runs, and limited co-production efficiency.

Acetate Dissimilation and Assimilation in Mycobacterium tuberculosis Depend on Carbon Availability.

Mycobacterium tuberculosis persists inside granulomas in the human lung. Analysis of the metabolic composition of granulomas from guinea pigs revealed that one of the organic acids accumulating in the course of infection is acetate (B. S. Somashekar, A. G. Amin, C. D. Rithner, J. Troudt, R. Basaraba, A. Izzo, D. C. Crick, and D. Chatterjee, J Proteome Res 10:4186-4195, 2011, doi:http://dx.doi.org/10.1021/pr2003352), which might result either from metabolism of the pathogen or might be provided by the host itself. Our studies characterize a metabolic pathway by which M. tuberculosis generates acetate in the cause of fatty acid catabolism. The acetate formation depends on the enzymatic activities of Pta and AckA. Using actyl coenzyme A (acetyl-CoA) as a substrate, acetyl-phosphate is generated and finally dephosphorylated to acetate, which is secreted into the medium. Knockout mutants lacking either the pta or ackA gene showed significantly reduced acetate production when grown on fatty acids. This effect is even more pronounced when the glyoxylate shunt is blocked, resulting in higher acetate levels released to the medium. The secretion of acetate was followed by an assimilation of the metabolite when other carbon substrates became limiting. Our data indicate that during acetate assimilation, the Pta-AckA pathway acts in concert with another enzymatic reaction, namely, the acetyl-CoA synthetase (Acs) reaction. Thus, acetate metabolism might possess a dual function, mediating an overflow reaction to release excess carbon units and resumption of acetate as a carbon substrate. During infection, host-derived lipid components present the major carbon source at the infection site. β-Oxidation of fatty acids results in the formation of acetyl-CoA. In this study, we demonstrate that consumption of fatty acids by Mycobacterium tuberculosis activates an overflow mechanism, causing the pathogen to release excess carbon intermediates as acetate. The Pta-AckA pathway mediating acetate formation proved to be reversible, enabling M. tuberculosis to reutilize the previously secreted acetate as a carbon substrate for metabolism.

Engineering Escherichia coli for fumaric acid production from glycerol

The evolved mutant Escherichia coli E2 previously developed for succinate production from glycerol was engineered in this study for fumaric acid production under aerobic conditions. Through deletion of three fumarases, 3.65g/L fumaric acid was produced with the yield of 0.25mol/mol glycerol and a large amount of acetate was accumulated as the main byproduct. In order to reduce acetate production several strategies were attempted, among which increasing the flux of the anaplerotic pathways through overexpression of phosphoenolpyruvate carboxylase gene ppc or the glyoxylate shunt operon aceBA effectively reduced acetate and improved fumaric acid production. In fed-batch culture, the resulting strain EF02(pSCppc) produced 41.5g/L fumaric acid from glycerol with 70% of the maximum theoretical yield and an overall productivity of 0.51g/L/h.

Effects of Disrupting Acetate Formation Pathways in Corynebacterium acetoacidophilum on Succinate Production Under Oxygen Deprivation

In order to reduce acetate production and improve succinate production and explore the main acetate formation pathways of C.acetoacidophilum,gene of pta,ackA,ctfA and aceE,which encoded phosphotransacetylase,acetate kinase,CoA-transfease and pyruvate dehydrogenase complex in C.acetoacidophilum were disrupted respectively by the means of homologous recombination.In C.acetoacidophilum△ldhA△pta-ackA,the titer of acetate,gluocse consumption rate,and both yields of acetate and succinate were found to be similar to that of C.acetoacidophilum AldhA.Furthermore,the titer and yield of acetate decreased by81.4%and 77.2%,respectively,the glucose consumption rate decreased by 28.3%and the yield of succinate increased by 25.3%in C.acetoacidophilum △ldhA△ctfA△pta-ackA.In addition,when gene aceE was deleted,C.acetoacidophilum △ldhA△aceE almost produced no acetate,the glucose consumption rate decrease by 35.6%and the yield of succinate increased by 34.7%.Under oxygen deprivation,almost all acetate formed through acetyl-CoA in C.acetoacidophilum.Gene pta,ackA and ctfA were the main genes of acetate formation pathways from acetyl-CoA.Disrupting gene aceE could cut off acetate synthesis in C.acetoacidophilum,and effectively increase succinate production.

Modification of glycolysis and its effect on the production ofl-threonine inEscherichia coli

High concentrations of acetate, the main by-product of Escherichia coli (E. coli) high cell density culture, inhibit bacterial growth and L-threonine production. Since metabolic overflux causes acetate accumulation, we attempted to reduce acetate production by redirecting glycolysis flux to the pentose phosphate pathway by deleting the genes encoding phosphofructokinase (pfk) and/or pyruvate kinase (pyk) in an L-threonine-producing strain of E. coli, THRD. pykF, pykA, pfkA, and pfkB deletion mutants produced less acetate (9.44 ± 0.83, 3.86 ± 0.88, 0.30 ± 0.25, and 6.99 ± 0.85 g/l, respectively) than wild-type THRD cultures (19.75 ± 0.93 g/l). THRDΔpykF and THRDΔpykA produced 11.05 and 5.35 % more L-threonine, and achieved a 10.91 and 5.60 % higher yield on glucose, respectively. While THRDΔpfkA grew more slowly and produced less L-threonine than THRD, THRDΔpfkB produced levels of L-threonine (102.28 ± 2.80 g/l) and a yield on glucose (0.34 g/g) similar to that of THRD. The dual deletion mutant THRDΔpfkBΔpykF also achieved low acetate (7.42 ± 0.81 g/l) and high L-threonine yields (111.37 ± 2.71 g/l). The level of NADPH in THRDΔpfkA cultures was depressed, whereas all other mutants produced more NADPH than THRD did. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of L-threonine.

Effects of different tannin-rich extracts and rapeseed tannin monomers on methane formation and microbial protein synthesis in vitro

Tannins, polyphenolic compounds found in plants, are known to complex with proteins of feed and rumen bacteria. This group of substances has the potential to reduce methane production either with or without negative effects on digestibility and microbial yield. In the first step of this study, 10 tannin-rich extracts from chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed, and four rapeseed tannin monomers (pelargonidin, catechin, cyanidin and sinapinic acid) were used in a series of in vitro trials using the Hohenheim gas test, with grass silage as substrate. The objective was to screen the potential of various tannin-rich extracts to reduce methane production without a significant effect on total gas production (GP). Supplementation with pelargonidin and cyanidin did not reduce methane production; however, catechin and sinapinic acid reduced methane production without altering GP. All tannin-rich extracts, except for tara extract, significantly reduced methane production by 8% to 28% without altering GP. On the basis of these results, five tannin-rich extracts were selected and further investigated in a second step using a Rusitec system. Each tannin-rich extract (1.5 g) was supplemented to grass silage (15 g). In this experiment, nutrient degradation, microbial protein synthesis and volatile fatty acid production were used as additional response criteria. Chestnut extract caused the greatest reduction in methane production followed by valonea, grape seed and sumach, whereas myrabolan extract did not reduce methane production. Whereas chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production. However, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry matter and organic matter was also reduced by tannin supplementation, and no differences were found between the tannin-rich extracts. CP degradation and ammonia-N accumulation in the Rusitec were reduced by tannin treatment. The amount and efficiency of microbial protein synthesis were not significantly affected by tannin supplementation. The results of this study indicated that some tannin-rich extracts are able to reduce methane production without altering microbial protein synthesis. We hypothesized that chestnut and valonea extract have the greatest potential to reduce methane production without negative side effects.

Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering

Background2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production.ResultsWe first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH) pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 g·l-1) and yield (0.113 g·g-1) were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition.ConclusionsWith the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.

Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

BackgroundPlasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production.ResultsSuch a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date.ConclusionThese results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that reduce acetate production would also be advantageous. The results further suggest that using some other means for plasmid selection than antibiotic resistance, or at least weakening the marker's expression, would be beneficial because it would allow more precursor metabolites, energy, and reducing power to be put toward plasmid production. Thus far, the impact of eliminating Pyk activity has been explored experimentally, with significantly higher plasmid yields resulting.

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37 degrees C. At 42 degrees C, its plasmid content was ninefold higher than JM101 at 37 degrees C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp(uv), under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.

Kinetics of butyric acid fermentation of glucose and xylose by Clostridium tyrobutyricum wild type and mutant

The kinetics of butyric acid fermentation by Clostridium tyrobutyricum at pH 6.0 and 37 °C were studied with the wild type ATCC 25755 and its mutant PPTA-Em, which was obtained from integrational mutagenesis to inactivate the chromosomal pta gene, encoding phosphotransacetylase (PTA). The potential of using this mutant to improve butyric acid production from glucose and xylose was evaluated in both free and immobilized cell fermentations. Compared to the wild type, in free cell fermentations PPTA-Em produced 15% more butyrate (0.38 g/g versus 0.33 g/g) from both glucose and xylose, at much higher concentrations (37.2 g/L versus 22.9 g/L from glucose and 33.5 g/L versus 19.4 g/L from xylose). The increased butyrate production in the mutant can be attributed to the reduced acetate production as well as reduced specific growth rate. The acetate yield in the mutant was reduced by 13.5% (0.058 g/g versus 0.067 g/g) and 32% (0.045 g/g versus 0.066 g/g) from glucose and xylose, respectively. The mutant's specific growth rate was reduced by 36% (0.137 h −1 versus 0.214 h −1) on glucose and 26% (0.086 h −1 versus 0.116 h −1) on xylose. A fibrous-bed bioreactor (FBB) was used to immobilize PPTA-Em mutant cells and further improve butyric acid production. The final butyric acid concentrations in fed-batch fermentations reached 49.9 g/L from glucose and 51.6 g/L from xylose, with the butyrate yield increased to 0.44 g/g glucose and 0.45 g/g xylose. As evidenced by the greatly increased butyrate/acetate ratio in the final product profile, it is concluded that the mutant's metabolic pathway has been shifted to favor butyrate production due to the knockout of pta gene even though acetate production remains at a significant level. The observed metabolic shift is corroborated by the changed protein expression patterns as seen in two-dimensional protein electrophoresis and SDS-PAGE.

Fish oils as potent rumen methane inhibitors and associated effects on rumen fermentation in vitro and in vivo

The effect of two fish oil (FO) types, FOa ( n-3-eicosapentanoic acid (EPA), 18.1%; n-3-docosahexanoic acid (DHA), 11.9%) and FOb (EPA, 5.4%; DHA, 7.5%) and quantity (0, 12.5, 25, 50, 75, 100 and 125 mg FO) on rumen fermentation patterns was evaluated with 24 and 48 h batch in vitro incubations and compared with fermentation shifts induced by soybean oil (SO). The 48 h incubation was essentially a batch culture as it was re-inoculated with rumen fluid, buffer and hay after 24 h. Shifts in rumen fermentation pattern were only observed during the second step of the 48 h incubation for the three oils. The inhibition of rumen methane (CH 4) production was influenced by both oil (FOa>SO>FOb) and concentration, although amounts higher than 75 or 25 mg for FOa and FOb, respectively, did not decrease further CH 4 production. A maximal CH 4 inhibition of 80% was observed. CH 4 inhibition seems proportional to the relative amount of polyunsaturated fatty acids (PUFA) and their rate of lipolysis. Lower CH 4 production was accompanied by increased propionate and reduced acetate production. SO supplementation was associated with lower net VFA productions (−17%), whereas none of the amounts of FO reduced VFA production. In vivo effects of rumen FOa injection (18 ml, twice daily at 1 h after feeding) on rumen fermentation and NDF digestibility were studied in a cross-over trial with four rumen canulated wethers, offered a hay/concentrate (65/35, w/w) diet, at their maintenance energy requirements. In vivo FOa injection did not alter faecal NDF digestibility (48.5±4.5% versus 46.5±8.2% for control and FOa, respectively, P>0.05), despite a lower 48 h rumen in sacco degradability (57.5±3.3% versus 51.6±5.8%, P<0.05). FOa-induced higher rumen propionate concentrations (211.8±18.0 versus 262.7±20.8 mmol/mol total VFA, P<0.05), suggesting a depression of rumen methanogenesis, confirmed during simultaneous in vitro incubations (344.2±43.4 versus 287.7±36.6 mmol/mol total VFA, P<0.05). Reduced biohydrogenation of FO PUFA, resulting in rumen accumulation of unesterified EPA and DHA might be nutritionally and ecologically relevant in terms of an increased post-ruminal supply of these PUFA for incorporation into animal products and through reduced rumen methanogenesis, the latter representing both an energy loss for the animal and an important greenhouse gas.

Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil.

Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U-(14)C]glucose and [2-(14)C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O). Addition of nitrate resulted in complete inhibition of CH(4) production from [U-(14)C]glucose and [2-(14)C]acetate. However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from [U-(14)C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of (14)CO(2) from [U-(14)C]glucose, but all increased the production of (14)CO(2) from [2-(14)C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced (14)CH(4) production from [2-(14)C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production.

Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil

Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U- 14C]glucose and [2- 14C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N 2O). Addition of nitrate resulted in complete inhibition of CH 4 production from [U- 14C]glucose and [2- 14C]acetate. However, addition of ferrihydrite or sulfate decreased the production of 14CH 4 from [U- 14C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of 14CO 2 from [U- 14C]glucose, but all increased the production of 14CO 2 from [2- 14C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced 14CH 4 production from [2- 14C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO 2 − and N 2O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH 4 towards CO 2 production.

Overproduction of acetyl-coenzyme A synthetase isoenzymes in respiring Saccharomyces cerevisiae cells does not reduce acetate production after exposure to glucose excess.

To investigate whether the production of acetate which occurs after exposure of respiring Saccharomyces cerevisiae cells to excess glucose can be reduced by overproduction of acetyl-CoA synthetase (ACS, EC 6.2.1.1), the ACS1 and ACS2 genes were introduced on multi-copy plasmids. For each isoenzyme, the level in glucose-limited chemostat cultures was increased by 3-6-fold, relative to an isogenic reference strain. However, ACS overproduction did not result in a reduced production of acetate after a glucose pulse (100 mmol l-1) to these cultures. This indicates that a limited capacity of ACS is not the sole cause of acetate accumulation in S. cerevisiae.