Fermentation of Xylan,Corn Fiber,or Sugars to Acetoin and Butanediol by Bacillus polymyxa Strains

Bacillus polymyxa can produce levo-butanediol, a potential biogradable anti-freeze, and ethanol, a fuel additive, using starch-based fermentations. To explore use of less expensive biomass fermentation substrates, we screened B. polymyxa strains for good growth on xylans. During aerobic growth on glucose, six selected xylanolytic strains produced mainly acetoin and butanediol plus lesser amounts of acetaldehyde and ethanol. Undesirable acetoin formation was eliminated by anaerobic growth on glucose, but substrate usage, butanediol, and other fermentation products were greatly reduced. High xylanase activity occurred with growth on xylans or corn fiber, and about 50–65% of oatspelt xylan and 25–35% of the corn fiber were used during aerobic growth, but unexpectedly no butanediol and only small levels of acetoin were produced. Aerobic growth on arabinose, arabinose plus glucose, or xylose plus glucose resulted in both acetoin and butanediol formation. Little or no butanediol was made from xylose alone. Growth on an acid hydrolysate of corn fiber that contained a mixture of these sugars resulted in the formation of acetoin, acetaldehyde, and ethanol, but very little butanediol. The data suggest B. polymyxa is limited in conversion of xylan-rich biomass sources or their hydrolysates to butanediol. This limitation might be overcome by using better cultivation conditions and/or genetically engineered strains.     

Natural-abundance isotope ratio mass spectrometry as a means of evaluating carbon redistribution during glucose-citrate cofermentation by Lactococcus lactis.

The cometabolism of citrate and glucose by growing Lactococcus lactis ssp. lactis bv. diacetylactis was studied using a natural-abundance stable isotope technique. By a judicious choice of substrates differing slightly in their 13C/12C ratios, the simultaneous metabolism of citrate and glucose to a range of compounds was analysed. These end-products include lactate, acetate, formate, diacetyl and acetoin. All these products have pyruvate as a common intermediate. With the objective of estimating the degree to which glucose and citrate metabolism through pyruvate may be differentially regulated, the delta13C values of the products accumulated over a wide range of concentrations of citrate and glucose were compared. It was found that, whereas the relative accumulation of different products responds to both the substrate concentration and the ratio between the substrates, the delta13C values of the products primarily reflect the availability of the two substrates over the entire range examined. It can be concluded that in actively growing L. lactis the maintenance of pyruvate homeostasis takes precedence over the redox status of the cells as a regulatory factor.     

Elementary mode analysis to study the preculturing effect on the metabolic state of Lactobacillus rhamnosus during growth on mixed substrates

Quantification of metabolism through elementary modes offers insights into the working of a metabolic network. We have determined the fluxes of elementary modes through linear optimization using the stoichiometry of the elementary modes as a constraint. We apply this methodology to obtain insights into the effect of preculturing on growth of Lactobacillus rhamnosus on medium containing mixed substrates. L. rhamnosus, a microaerophilic organism, produces flavor compounds such as diacetyl and acetoin during growth on glucose and citrate. The uptake of citrate has been shown to be sensitive to preculturing states of the cells. Elementary modes demonstrated that citrate was utilized by the organism as a sole carbon source. Further, both glucose and citrate was catabolized by this organism through aerobic and anaerobic routes. The flux analysis indicated that only 21 elementary modes were operational during growth of L. rhamnosus on glucose and citrate. Glucose specifically accounted for 6 elementary modes, while the remaining 15 involved citrate as substrate. The modes associated with glucose were mainly operational when cells were precultured on glucose. It was observed that all the 21 modes contributed to the fluxes when the cells were precultured on citrate. The NADH recycling through lactate formation and oxygen uptake were dependent on the preculturing state. The analysis also demonstrated that preculturing on citrate yielded better productivity of diacetyl and acetoin.     

Conversion of Pyruvate to Acetoin Helps To Maintain pH Homeostasis in Lactobacillus plantarum

Pyruvate is the substrate for diacetyl and acetoin synthesis by lactobacilli. Exogenous pyruvate stimulates acetoin production when glucose is present as an energy source. In Lactobacillus plantarum ATCC 8014, the energy derived from glucose via glycolysis generated a constant proton motive force of about -120 mV. At a low external pH, energized cells rapidly transported and accumulated pyruvate but did not do so when they were deenergized by nigericin. When large amounts of pyruvate were transported and subsequently accumulated internally, the cotransported protons rapidly lowered the internal pH. The conversion of pyruvate to acetoin instead of acidic end products contributed to the maintenance of pH homeostasis. This is the first report showing that the conversion of pyruvate to acetoin serves as a mechanism of pH homeostasis.     

Co-metabolism of citrate and glucose by Leuconostoc spp.: effects on growth, substrates and products

Growth, substrate utilization and product formation from glucose, citrate and a mixture of both substrates were studied in four strains of Leuconostoc spp. Citrate was not used as an energy source but was rapidly metabolized when glucose was present. The predictable amounts of D-lactate and ethanol were produced from glucose, although strains X2 and 7–1 gave lower yields of ethanol. In strains NCW1, S3 and X2, co-metabolism of both glucose and citrate resulted in stimulation of growth, decreased uptake of glucose, increased acetate and D-lactate production and lack of ethanol production compared with that obtained with glucose alone. Strain 7–1 showed only growth stimulation and increased acetate production. Diacetyl, acetoin or 2, 3-butylene glycol were not detected. In strain NCW1 citrate had a slightly inhibitory effect on the enzymes of the 'ethanol' leg of glucose metabolism. Except for strain 7–1, these observations are consistent with a switch in glucose metabolism from ethanol to acetate production.     

Glucose degradation, molar growth yields, and evidence for oxidative phosphorylation in Streptococcus agalactiae

In a complex medium with the energy source as the limiting nutrient factor and under anaerobic growth conditions, Streptococcus agalactiae fermented 75% of the glucose to lactic acid and the remainder to acetic and formic acids and ethanol. By using the adenosine triphosphate (ATP) yield constant of 10.5, the molar growth yield suggested 2 moles of ATP per mole of glucose from substrate level phosphorylation. Under similar growth conditions, pyruvate was fermented 25% to lactic acid, and the remainder was fermented to acetic and formic acids. The molar growth yield suggested 0.75 mole of ATP per mole of pyruvate from substrate level phosphorylation. Under aerobic growth conditions about 1 mole of oxygen was consumed per mole of glucose; about one-third of the glucose was converted to lactic acid and the remainder to acetic acid, acetoin, and carbon dioxide. Molar growth yields indicated 5 moles of ATP per mole of glucose. Estimates based on products of glucose degradation suggested that about one-half of the ATP was derived from substrate level phosphorylation and one-half from oxidative phosphorylation. Addition of 0.5 m 2,4-dinitrophenol reduced the growth yield to that occurring in the absence of oxygen. Aerobic pyruvate degradation resulted in 30% of the substrate becoming reduced to lactic acid and the remainder being converted to acetic acid and carbon dioxide, with small amounts of formic acid and acetoin. The molar growth yields and products found suggested that 0.70 mole of ATP per mole of pyruvate resulted from substrate level phosphorylation and 0.4 mole per mole of pyruvate resulted from oxidative phosphorylation.     

Impact of aeration on the metabolic end-products formed from glucose and galactose by Streptococcus lactis

Compared with cultures grown aerobically in batch culture with glucose, aerated cultures of lactic streptococci had a less homolactic type of metabolism when galactose was the carbohydrate source in batch cultures, or when glucose was limiting in chemostat cultures. Differences in end-products of sugar metabolism between aerated and unaerated cultures were observed. In addition to lactate, formate, acetate and ethanol were produced in anaerobic cultures, whereas acetate and acetoin were formed in aerated cultures. Acetate production in aerated cultures depended on lipoic acid, an essential cofactor of the pyruvate dehydrogenase complex. In a chemically defined medium with glucose as the energy substrate, lipoic acid (or acetate) was an essential growth factor. Formation of acetoin was inversely related to lipoic acid concentration in the growth medium. Although not observed in unaerated cultures, acetoin (and 2,3-butanediol) was produced in unaerated buffered suspensions metabolizing pyruvate. Aeration caused a modest increase in the activities of aP-acetolactate synthetase and phosphate acetyl trans-ferase, but it is unlikely that the increases were sufficient to account for the changes in end-products of sugar metabolism observed.     

Clostridium magnum sp. nov., a non-autotrophic homoacetogenic bacterium

A new mesophilic, sporeforming, strictly anaerboic bacterium was isolated form enrichments with 2,3-butanediol as sole substrate and pasteurized freshwater sediment as inoculum. Cells were large, motile rods, and elliptical spores were formed subterminally or centrally. They stained Gram-negative, but no typical outer membrane layer could be observed by electron microscopy of ultrathin sections. 2,3-Butanediol, acetoin, fructose, glucose, sucrose, xylose, malate and citrate served as substrates and were completely converted to acetate with concomitant reduction of carbon dioxide. Growth on glucose (t _dmin=1.4 h) was faster than on butanediol (t _dmin=3.6 h). No growth occurred on hydrogen/carbon dioxide, on formate or on methanol. The guanine plus cytosine content of the DNA was 29.1%. The new isolate is described as a new species, Clostridium magnum sp. nov.     

Acetoin production in growing Leuconostoc mesenteroides

Leuconostoc mesenteroides NCDO 518, provided with oxygen and pyruvate, preferentially used oxygen as accessory electron acceptor and converted pyruvate to acetoin. With glucose, 5.6 mM, as sole energy source only small amounts of acetoin were formed (0.08–0.21 mM). With glucose, 5.6 mM, and pyruvate, 20 mM, substantial amounts of acetoin were produced in growing, aerated cultures at pH 5 (2.8 mM, equivalent to 0.5 mol [mol glucose fermented]^–1). On exhaustion of glucose, growth ceased but metabolism of pyruvate continued with the formation of acetate and a little acetoin. In aerated cultures at pH 6 the general pattern was similar to that at pH 5 but less acetoin (0.6 mM) was formed during the growth phase and, after the exhaustion of glucose, pyruvate was converted very slowly to acetate only. Leuc. mesenteroides did not grow with pyruvate as sole energy source.     

Citrate uptake in exchange with intermediates in the citrate metabolic pathway in Lactococcus lactis IL1403

Carbohydrate/citrate cometabolism in Lactococcus lactis results in the formation of the flavor compound acetoin. Resting cells of strain IL1403(pFL3) rapidly consumed citrate while producing acetoin when substoichiometric concentrations of glucose or l-lactate were present. A proton motive force was generated by electrogenic exchange of citrate and lactate catalyzed by the citrate transporter CitP and proton consumption in decarboxylation reactions in the pathway. In the absence of glucose or l-lactate, citrate consumption was biphasic. During the first phase, hardly any citrate was consumed. In the second phase, citrate was converted rapidly, but without the formation of acetoin. Instead, significant amounts of the intermediates pyruvate and α-acetolactate, and the end product acetate, were excreted from the cells. It is shown that the intermediates and acetate are excreted in exchange with the uptake of citrate catalyzed by CitP. The availability of exchangeable substrates in the cytoplasm determines both the rate of citrate consumption and the end product profile. It follows that citrate metabolism in L. lactis IL1403(pFL3) splits up in two routes after the formation of pyruvate, one the well-characterized route yielding acetoin and the other a new route yielding acetate. The flux distribution between the two branches changes from 85:15 in the presence of l-lactate to 30:70 in the presence of pyruvate. The proton motive force generated was greatest in the presence of l-lactate and zero in the presence of pyruvate, suggesting that the pathway to acetate does not generate proton motive force.     

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis

C nuclear magnetic resonance (C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-C]citrate, [3-C]pyruvate, and [2-C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. alpha-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the alpha-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.     

Acetoin production in Leuconostoc mesenteroides NCDO 518

Abstract Cell suspensions of Leuconostoc mesenteroides NCDO 518 converted pyruvate to acetoin and a small amount of lactate and acetate. Acetoin was not produced from mixtures of pyruvate and glucose unless the ratio of pyruvate to glucose was greater than 2:1. In the presence of glucose, external pyruvate was first used as an electron acceptor, being reduced to lactate, and was converted to acetoin only after the exhaustion of glucose. Use of added pyruvate as an electron acceptor suppressed ethanol formation and the products of glucose fermentation were then lactate and acetate; 2 mol of pyruvate per mol of glucose were required to completely suppress ethanol formation. It is suggested that acetoin is produced by heterofermentative organisms when available pyruvate is in excess of that required for reoxidation of all NADH produced during glucose fermentation.     

Effect of preculturing conditions on growth of Lactobacillus rhamnosus on medium containing glucose and citrate

Lactobacillus rhamnosus can metabolize citrate through a citrate inducible transport system. The growth curves of L. rhamnosus on medium containing glucose and citrate was found to be highly dependent on preculturing conditions. It exhibited diauxic growth when precultured on glucose, but demonstrated simultaneous consumption when cultured on citrate. The maximum specific growth rate for cells growing on glucose + citrate was 0.38 h-1, which was higher than the growth rate on individual substrates (0.28 h-1). Simultaneous consumption also yielded higher net flavour compounds, diacetyl and acetoin. Flux analysis indicated that L. rhamnosus requires oxygen for balancing excess NADH through NADH oxidase. The flux analysis provided insights into the metabolic network of L. rhamnosus.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu

CitrLactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu. On the other hand, the Y(Glu) value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP(i). In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Engineering Bacillus subtilis for acetoin production from glucose and xylose mixtures

As a vital flavor compound, acetoin is extensively used in dairy products and drinks industry. In this study, Bacillus subtilis was engineered to metabolize glucose and xylose as substrates for acetoin production. Initially, gene araE from B. subtilis, encoding the xylose transport protein AraE, was placed under the control of the constitutive promoter P₄₃ for over-expression. Batch cultures showed that 10 g/L xylose was depleted completely in 32 h. Subsequently, genes xylA and xylB from Escherichia coli, encoding xylose isomerase and xylulokinase respectively, were introduced into B. subtilis, and the recombinant turned out to assimilate glucose and xylose without preference. In shake-flask fermentations, 5.5 g/L acetoin with a yield of 0.70 mol (mol sugar)⁻¹ was obtained by the optimum strain BSUL13 under microaerobic conditions, which offered a metabolic engineering strategy on engineering microbe as cell factory for the production of high-valued chemicals from renewable resource.     

Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation

Acetoin (3-hydroxy-2-butanone), a very popular food spice is now used in many industries (pharmaceuticals, chemicals, paint, etc.). In this study, an acetoin high producing strain, numbered as JNA-310, was newly isolated and identified as Bacillus subtilis which is safe on food industry, based on its physiological, biological tests and 16S rDNA sequence analysis. When glucose was used as carbon source in fermentation, the fermentation characterizations of this strain were analyzed, and a new phenomenon of reverse transforming 2,3-butanediol which was synthesized from glucose in the fermentation broth to acetoin was detected. Before 96 h, glucose which was mainly transformed to 2,3-butanediol and acetoin was totally consumed, and the yield of the two products were 41.7 and 21.0 g/l respectively. Acetoin was only a by product in the fermentation broth at prophase of fermentation. At the end of fermentation, the yield of acetoin was greatly improved and the yield of 2,3-butanediol was declined and the yield of them were about 42.2 and 15.8 g/l, respectively. The results indicated that 2,3-butanediol was reversely transformed to acetoin.     

Fermentation of polyethylene glycol via acetaldehyde in Pelobacter venetianus

Summary Pelobacter venetianus, a strictly anaerobic bacterium recently isolated with polyethylene glycol (PEG) as substrate, ferments PEG's with molecular masses of 106–40000, as well as acetoin, ethanolamine, choline, and ethoxyethanol, to acetate and ethanol. Ethylene glycol (EG) and acetaldehyde were fermented in the same manner at limiting concentrations in continuous culture. Growth with glycolaldehyde led to acetate as sole fermentation product. Acetaldehyde appeared as byproduct of PEG fermentation, and accumulated to high concentrations during degradation of PEG 4000 and PEG 6000. Utilization of PEG's was constitutive, whereas acetoin degradation was inducible. Acetaldehyde was shown to be the primary product of EG degradation, and inhibited utilization of other substrates. Enzymes involved in the fermentation of PEG, EG, acetoin, and glycolaldehyde were demonstrated in cell-free extracts, except for the PEG degrading enzyme and EG dehydrase. These results demonstrate that acetaldehyde plays a central role in the metabolism of Pelobacter venetianus. A scheme of intermediary metabolism and PEG degradation is discussed.Abbreviations EG ethylene glycol - Di-EG diethylene glycol - PEG (20 000) polyethylene glycol (molecular weight 20 000)     

The acetoin assimilation pathway of Pseudomonas putida KT2440 is regulated by overlapping global regulatory elements that respond to nutritional cues

Many microorganisms produce and excrete acetoin (3-hydroxy-2-butanone) when growing in environments that contain glucose or other fermentable carbon sources. This excreted compound can then be assimilated by other bacterial species such as pseudomonads. This work shows that acetoin is not a preferred carbon source of Pseudomonas putida, and that the induction of genes required for its assimilation is down-modulated by different, independent, global regulatory systems when succinate, glucose or components of the LB medium are also present. The expression of the acetoin degradation genes was found to rely on the RpoN alternative sigma factor and to be modulated by the Crc/Hfq, Cyo and PTS^Ntr regulatory elements, with the impact of the latter three varying according to the carbon source present in addition to acetoin. Pyruvate, a poor carbon source for P. putida, did not repress acetoin assimilation. Indeed, the presence of acetoin significantly improved growth on pyruvate, revealing these compounds to have a synergistic effect. This would provide a clear competitive advantage to P. putida when growing in environments in which all the preferred carbon sources have been depleted and pyruvate and acetoin remain as leftovers from the fermentation of sugars by other microorganisms.