Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes

It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges–Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l⁻¹) and propionic (4 mmol l⁻¹), whereas protonated lactic (4 mmol l⁻¹) and benzoic (0·16 mmol l⁻¹) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes . This system can be used to screen inhibitors for applications in food safety.     

Metabolic engineering of Bacillus subtilis for enhanced production of acetoin

Acetoin is widely used in food and other industries. A bdhA and acoA double-knockout strain of Bacillus subtilis produced acetoin at 0.72?mol/mol, a 16.4?% increased compared to the wild type. Subsequent overexpression of the alsSD operon enhanced the acetolactate synthase activity by 52 and 66?% in growth and stationary phases, respectively. However, deletion of pta gene caused little increase of acetoin production. For acetoin production by the final engineered strain, BSUW06, acetoin productivity was improved from 0.087?g/l?h, using M9 medium plus 30?g glucose/l under micro-aerobic conditions, to 0.273?g/h?l using LB medium plus 50?g glucose/l under aerobic conditions. In fermentor culture, BSUW06 produced acetoin up to 20?g/l.     

Aerobic and anaerobic metabolism of Listeria monocytogenes in defined glucose medium.

A defined medium with glucose as the carbon source was used to quantitatively determine the metabolic end products produced by Listeria monocytogenes under aerobic and anaerobic conditions. Of 10 strains tested, all produced acetoin under aerobic conditions but not anaerobic conditions. Percent carbon recoveries of end products, typified by strain F5069, were as follows: lactate, 28%; acetate, 23%; and acetoin, 26% for aerobic growth and lactate, 79%; acetate, 2%; formate, 5.4%; ethanol, 7.8%; and carbon dioxide, 2.3% for anaerobic growth. No attempt to determine carbon dioxide under aerobic growth conditions was made. The possibility of using acetoin production to assay for growth of L. monocytogenes under defined conditions should be considered.     

Aerobic Metabolism of Brochothrix thermosphacta Growing on Meat Surfaces and in Laboratory Media

Acetoin and acetic, isobutyric and isovaleric acids are major end-products, and important components of the spoilage odours, of Brochothrix thermosphacta growing aerobically on meat surfaces or in tryptone-based medium containing glucose, ribose or glycerol. Acetoin and acetic acid are probably derived entirely from the carbohydrates and isobutyric and isovaleric acids from valine and leucine respectively. Glucose and pH are both important factors in controlling the relative amounts of end-products, low glucose and near neutral pH favouring fatty acid formation, high glucose and lower pH values favouring acetoin formation.     

Metabolic engineering strategies for acetoin and 2,3-butanediol production: advances and prospects

Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and 2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology.     

The effect of propionic to acetic acid ratio on anaerobic-aerobic (low dissolved oxygen) biological phosphorus and nitrogen removal

In this paper, three lab-scale sequencing batch reactors (SBR-A, B, and C) operated with anaerobic/aerobic (low dissolved oxygen, 0.15-0.45 mg L(-1)) configuration were long-term cultured, respectively with single acetic acid and propionic/acetic acid of 1/1 and 2/1 (carbon molar ratio), and the comparisons of anaerobic and aerobic transformations of phosphorus and nitrogen among them were made. With the increase of propionic/acetic acid, lower anaerobic phosphorus release and higher phosphorus release to short-chain fatty acids uptake ratio were observed, and less anaerobic and aerobic transformations of glycogen and poly-3-hydroxybutyrate as well as total polyhydroxyalkanoates occurred, but the transformations of poly-3-hydroxyvalerate and poly-3-hydroxy-2-methyvalerate increased. The phosphorus removal efficiency was respectively 81, 94 and 97% in SBR-A, B and C. Almost all ammonium was removed and no significant nitrite was accumulated at different propionic/acetic acid ratios. However, the nitrate accumulation and total nitrogen removal were observed to be affected by propionic/acetic acid ratio. The total nitrogen removal efficiency was 61, 68 and 82%, and the aerobic end nitrate concentration was 8.05, 6.40 and 3.54 mg L(-1) in three SBRs, respectively. All the above studies indicated that the sole acetic acid caused more nitrate accumulation than propionic and acetic acids mixture, and a pertinent increase of wastewater propionic/acetic acid ratio was of benefit to both nitrogen and phosphorus removal in an anaerobic/aerobic (low dissolved oxygen) biological wastewater treatment process.     

ATP requirements for benzoic acid tolerance in Zygosaccharomyces bailii

AIMS: To calculate the energetic requirements for benzoic acid tolerance in Zygosaccharomyces bailii in chemostat experiments. METHODS AND RESULTS: A 5.6-l stirred-tank chemostat was used. The yield of ATP (Y(ATP)) was calculated under nitrogen atmosphere, assuming equimolar ATP and ethanol production. Under these conditions Y(ATP), equal to 20 g mol(-1) of ATP, was not affected by the acid, whereas the maintenance coefficient (m(ATP)) increased from 1.0 mmol of ATP g(-1) h(-1) in the absence of the acid to 4.8 in the presence of 0.67 mmol l(-1) undissociated benzoic acid. These ATP requirements were similar to those found in Saccharomyces cerevisiae with other weak acids. CONCLUSIONS: No significant differences have been found in the energy expended to cope with the acid between sensitive and tolerant species. Therefore, the main difference between tolerant and sensitive species could rely on cellular features that would not need extra energy in terms of ATP. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential mechanisms involved in the tolerance to weak acids in yeasts have been extensively studied but their actual relevance has not been assessed. Our results suggest that future efforts should concentrate on nonexpending energy features as membrane permeability and metabolic tolerance in the cytoplasm.     

The effect of pH and temperature on haemolysin production by Listeria species

Acetoin reductase (EC 1.1.1.4) from Kluyveromyces marxianus var. marxianus NRRL Y-1196 was found to possess the highest specific activity (3.64 units/mg protein) of the four cultures studied. The enzyme was NADH-dependent and catalysed the conversion of acetoin to 2,3-butanediol. It was stable at 40°C for 30 min, but lost 50% cf its activity after 15 min at 50°C. The optimum pH for the enzymatic reduction of acetoin was 7.0. The K _m values of the crude enzyme for acetoin and NADH were determined to be 0.57 mmol/l and 0.045 mmol/l, respectively.     

Production of PHA from starchy wastewater via organic acids

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60-80%), propionic (10-30%) and butyric (5-40%) acids. The total volatile fatty acids reached 4000 mg l(-1) at a chemical oxygen demand (COD) loading rate of 25-35 g l(-1) day(-1). A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l(-1) day(-1) gave a high propionic acid content (29% wt) and a high rate around 26 g l(-1) day(-1) led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80-100 mg NH(3) per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8-9 g l(-1)) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.     

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production byPropionibacterium acidipropionici

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor withPropionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 g/l propionic acid and 12.8 g/l acetic acid were obtained in about 146 h in laboratory medium with 79 g/l glucose added over five feeding periods. The highest concentration of propionic acid, 45 g/l, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 g/l propionic acid and 11 g/l acetic acid were produced in 108 h from 59.4 g/l total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30–37 g/l propionic acid and 13 g/l acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.Journal paper J-16303 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 3122.     

Precursors of the major end products of aerobic metabolism of Brochothrix thermosphacta

To further our understanding of off-odour production by Brochothrix thermosphacta , the nature and origins of its end products have been compared during aerobic growth in complex and in minimal, defined medium. In complex medium glucose is the major precursor of acetoin and acetic acid but alanine may be an additional minor source. Iso butyric, iso valeric (3-methylbutyric) and 2-methylbutyric acids are derived exclusively from valine, leucine and iso leucine, respectively. In minimal defined medium although the same end products are produced they are all derived from glucose.     

Inhibition of enterobacteria and Listeria growth by lactic, acetic and formic acids

Minimum inhibitory concentrations (MIC) of undissociated lactic, acetic and formic acids were evaluated for 23 strains of enterobacteria and two of Listeria monocytogenes. The evaluation was performed aerobically and anaerobically in a liquid test system at pH intervals of between 4.2 and 5.4. Growth of the enterobacteria was inhibited at 2–11 mmol 1⁻¹, 0.5–14 mmol 1⁻¹ and 0.1–1.5 mmol 1⁻¹ of undissociated lactic, acetic and formic acids, respectively. The MIC value was slightly lower with anaerobic conditions compared with aerobic conditions. The influence of protons on the inhibition was observed for acetic acid at the low pH values. Undissociated lactic acid was 2 to 5 times more efficient in inhibiting L. monocytogenes than enterobacteria. Acetic acid had a similar inhibitory action on L. monocytogenes compared with enterobacteria. Inorganic acid (HCl) inhibited most enterobacteria at pH 4.0; some strains, however, were able to initiate growth to pH 3.8. The results indicate that the values of undissociated acid which occur in a silage of pH 4.1–4.5 are about 10–100 times higher than required in order to protect the forage from the growth of enterobacteria and L. monocytogenes.     

Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 10⁹ cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h^–1 could be used, which gave higher volumetric productivities (0.96 g l^–1 h^–1 for propionic acid and 0.26 g l^–1 h^–1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.Journal paper no. J- 15614 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3122     

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Inhibition of Listeria monocytogenes by acetate, benzoate and sorbate: weak acid tolerance is not influenced by the glutamate decarboxylase system

Aims:  Weak acids are widely used by the food industry to prevent spoilage and to inhibit the growth of pathogenic micro-organisms. In this study the inhibitory effects of three commonly used weak acids, acetic acid, benzoic acid and sorbic acid, on the growth of Listeria monocytogenes were investigated.
Methods and Results:  In a chemically defined medium at pH 6·4 benzoic acid had the greatest inhibitory effect (50% inhibition of growth at 4 mmol l⁻¹), while acetate was the least inhibitory (50% inhibition of growth at 50 mmol l⁻¹). Mutants lacking either sigmaB (Δ sigB ) or two of the glutamate decarboxylase systems (Δ gadAB ) were used to investigate the contribution these systems make to weak acid tolerance in L. monocytogenes .
Conclusions:  The stress-inducible sigma factor sigmaB (σ^B) was not required for protection against acetate and played only a minor role in tolerating benzoate and sorbate. The glutamate decarboxylase system, which plays an important role in tolerating inorganic acids, played no significant role in the ability of L. monocytogenes to tolerate these weak acids, and neither did the presence of glutamate in the growth medium.
Significance and Impact of the Study:  These results suggest that the effectiveness of weak acid preservatives in food will not be compromised by the presence of glutamate, at least under mildly acidic conditions.     

Compartmentalizing metabolic pathway in Candida glabrata for acetoin production

Acetoin, a valuable compound, has high potential as a biochemical building block. In this study, subcellular metabolic engineering was applied to engineer the mitochondrion of Candida glabrata for acetoin production. With the aid of mitochondrial targeting sequences, a heterologous acetoin pathway was targeted into the mitochondria to increase the enzyme concentrations and level of intermediate, followed by coupling with the mitochondrial pyruvate carrier (MPC) to increase the availability of mitochondrial pyruvate. As a result, the strain comprising the combination of the mitochondrial pathway and MPC could yield approximately 3.26 g/L of acetoin, which was about 59.8% higher than that produced by the cytoplasmic pathway. These results provided a new insight into the metabolic engineering of C. glabrata for acetoin production, and offered a potential platform to improve the performance of engineered pathways.     

Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor

Cultures using nitrate as the terminal electron acceptor were conducted in Schaeffer's medium to evaluate the growth performance and metabolic profiles of Bacillus subtilis, and its potential to express the aprE (subtilisin) gene under anoxic conditions. Nitrate was converted to ammonia through nitrite reduction; and different product profiles were observed during the growth phase when nitrate was added at various concentrations (4-24 mM) to Schaeffer's medium containing glucose (4 g l(-1)). If nitrate was not limiting, then acetic acid and acetoin were accumulated, suggesting a limitation of reduced cofactors but, if nitrate became limiting, then lactic acid and butanediol were accumulated, suggesting an excess of reduced cofactors. Due to a strong lysis at the onset of the end of the growth phase, sporulation frequency and aprE expression were negligible in anaerobic batch cultures. Fed-batch fermentation allowed the development of a stationary phase through a continuous supply of glucose and nitrate. In this case, sporulation frequency was almost null, but interestingly aprE expression was similar to that found in aerobic cultures.     

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.     

Effect of short-chain acids on the carboxymethylcellulase activity of the ruminal bacterium<Emphasis Type="Italic">Ruminococcus albus</Emphasis>

The addition of 100-300 mmol/L of acetic, propionic, butyric or lactic acids (short-chain acids), or of acetic, propionic, and butyric acids (volatile fatty acids, VFA) mixtures increased the degradation of carboxymethyl cellulose (CMC) by R. albus (7.5 to 46 and 6 to 39 %, respectively). Differences among individual acids were observed at 300 mmol/L whereas VFA mixtures differed at 100 mmol/L. When assayed at the same concentration, CMCase activity was increased less by NaCl than by the short-chain acids, whereas ethylene glycol decreased the activity. Since osmolarity and/or ionic strength changes in the medium cannot completely account for the observed increases of carboxymethylcellulase (CMCase) activity, it is suggested that the anions of short-chain acids produce changes in the reaction media polarity that contribute to the effects observed. Alterations in the media could also bring about conformational changes in CMCase leading to increased rates of reaction and subsequent increases in CMC degradation. Finally, explanations for the observed phenomena based on the direct effect of the compounds tested on the cellulosome complex, its domains, and/or its component enzymes are proposed.