Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Purification, characterization and some properties of diacetyl(acetoin) reductase from Enterobacter aerogenes

A new method, faster, milder and more efficient than the one previously described [Bryn, K., Hetland, O. & Stormer, F. C. (1971) Eur. J. Biochem, 18, 116-119], for purification of diacetyl(acetoin) reductase from Enterobacter aerogenes is proposed. The experiments carried out with the electrophoretically pure preparations obtained by this procedure show that the enzyme (a) produces L-glycols from the corresponding L-alpha-hydroxycarbonyls by reversible reduction of their oxo groups and also reduces the oxo group of uncharged alpha-dicarbonyls converting them into L-alpha-hydroxycarbonyls, and (b) is specific for NAD. This is a new enzyme for which we suggest the systematic name of L-glycol: NAD+ oxidoreductase and the recommended name of L-glycol dehydrogenase(NAD). The molecular mass, pI, affinity for substrates and pH profiles of this enzyme are also described.     

Effect of substrates on acetoin production by Torulopsis colliculosa and Enterobacter species.

Under optimal conditions, Torulopsis colliculosa NRRL 172 and Enterobacter B-87 (ATCC 27613) produced 50 to 500 mg of acetoin per g of substrate. Whereas cane molasses, gur, glucose, and sucrose were suitable substrates for acetoin production, lactose and mannitol supported very good growth but yielded little or no acetoin. Production of acetoin increased with increases in the concentration of glucose, yeast extract, and peptone. Combination of substrates and intermittent feeding of substrate failed to increase the yields.     

Formation of diacetyl and acetoin by Lactococcus lactis via aspartate catabolism

Aims:  To verify whether diacetyl can be produced by Lactococcus lactis via amino acid catabolism, and to investigate the impact of the pH on the conversion.
Methods and Results:  Resting cells of L. lactis were incubated in reaction media at different pH values, containing l -aspartic acid or l -alanine as a substrate. After incubation, the amino acid and metabolites were analysed by HPLC and GC/MS. At pH 5 about 75% of aspartic acid and only 40% of alanine was degraded to pyruvate via a transamination step that requires the presence of α-ketoglutarate in the medium, but diacetyl was only produced from aspartic acid. Three per cent of pyruvate was transformed to acetolactate of which 50% was converted into diacetyl. At pH 5·5 and above the pyruvate conversion into acetolactate was less efficient than at pH 5, and acetolactate was mainly decarboxylated to acetoin.
Conclusions:  Acetoin and diacetyl can be formed as a result of aspartate or alanine catabolism by L. lactis in the presence of α-ketoglutarate in the medium.
Significance and Impact of the Study:  Lactic acid bacteria exhibiting both glutamate dehydrogenase activity and high aspartate aminotransferase activity are expected to be good diacetyl producers during cheese ripening at pH close to 5.     

Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae

Enterobactercloacae was isolated from the gut of the wood feeding termite, Heterotermesindicola, and a 2.25-kb fragment conferring cellulase activity was cloned in Escherichiacoli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonasmobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was threefold higher than the amount obtained with the original E.cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.     

Effect of some environmental parameters on fermentative hydrogen production by Enterobacter cloacae DM11

Fermentative hydrogen production was carried out by Enterobacter cloacae DM11, using glucose as the substrate. The effects of initial substrate concentration, initial medium pH, and temperature were investigated. Results showed that at an initial glucose concentration of 1.0% (m/v), the molar yield of hydrogen was 3.31 mol (mol glucose)(-1). However, at higher initial glucose concentration, both the rate and cumulative volume of hydrogen production decreased. The pH of 6.5 +/- 0.2 at a temperature of 37 degrees C was found most suitable with respect to maximum rate of production of hydrogen in batch fermentation. Activation enthalpies of fermentation and that of thermal deactivation of the present process were estimated following a modified Arrhenius equation. The values were 47.34 and 118.67 kJ mol(-1) K(-1), respectively. The effect of the addition of Fe(2+) on hydrogen production was also studied. It revealed that the presence of iron (Fe(2+)) in the media up to a concentration of 20 mg L(-1) had a marginal enhancing effect on total hydrogen production. A simple model developed from the modified Gompertz equation was applied to estimate the hydrogen production potential, production rate, and lag-phase time in a batch process, based on the cumulative hydrogen production curves, using the software program Curve Expert 1.3.     

Redirection of biochemical pathways for the enhancement of H2 production by Enterobacter cloacae

Improvement in H₂ production was achieved through redirection of metabolic pathways by blocking formation of alcohol and some organic acids in Enterobacter cloacae IIT-BT 08. The wild type strain was more susceptible to allyl alcohol (7 mM) and to the combined effect of NaBr and NaBrO₃ (40 mM each at pH 5.5) than were double mutants, with defects in both alcohol and organic acid formation pathways, which had higher H₂ yields (3.4 mol mol^–1 glucose) than the wild type strain (2.1 mol mol^–1 glucose).     

Production of 2,3-butanediol by newly isolated Enterobacter cloacae

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of l-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars). Received: 17 December 1998 / Revision received: 9 March 1999 / Accepted: 20 March 1999     

Exopolysaccharide production by a genetically engineered Enterobacter cloacae strain for microbial enhanced oil recovery

Microbial enhanced oil recovery (MEOR) is a petroleum biotechnology for manipulating function and/or structure of microbial environments existing in oil reservoirs for prolonged exploitation of the largest source of energy. In this study, an Enterobacter cloacae which is capable of producing water-insoluble biopolymers at 37 °C and a thermophilic Geobacillus strain were used to construct an engineered strain for exopolysaccharide production at higher temperature. The resultant transformants, GW3-3.0, could produce exopolysaccharide up to 8.83 g l⁻¹ in molasses medium at 54 °C. This elevated temperature was within the same temperature range as that for many oil reservoirs. The transformants had stable genetic phenotype which was genetically fingerprinted by RAPD analysis. Core flooding experiments were carried out to ensure effective controlled profile for the simulation of oil recovery. The results have demonstrated that this approach has a promising application potential in MEOR.     

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.     

Population genetics of the nomenspecies Enterobacter cloacae

The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known. Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E. cloacae complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E. cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71(R) were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C beta-lactamase. Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs. Sequence crowd xiii split into two groups after rpoB analysis. Only three strains formed a cluster with the type strain of E. cloacae, indicating that the minority of isolates identified as E. cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E. cloacae complex.     

Nonspecific induction of beta-lactamase in Enterobacter cloacae

Induction of beta-lactamase was monitored in a strain of Enterobacter cloacae exhibiting high resistance to most beta-lactam antibiotics. Large amounts of the enzyme were induced not only in the presence of beta-lactams, but also in the presence of other bicyclic molecules such as folic acid, thiamin, tryptophan or haemin. Moreover, complex media (such as Trypticase soy broth and Schaedler's broth) and various body fluids (serum, pleural fluid and cerebrospinal fluid) also possessed considerable induction potency. Neither 'specific' induction (by beta-lactams) nor 'non-specific' induction (by other bicyclic compounds) could be augmented by addition of exogenous cAMP. These findings indicate that inducible beta-lactamases deserve more attention, above all with respect to the development of resistance against third-generation cephalosporins.     

Characterization of an exopolysaccharide produced by a marine Enterobacter cloacae

An exopolysaccharide producing marine bacterium, Enterobacter cloacae, was isolated from marine sediment collected from Gujarat coast, India. Chemical investigation of exopolysaccharide (EPS 71 a) revealed that this exopolysaccharide was an acidic polysaccliaride containing high amount of uronic acid, fucose and sulfate which is rare for bacterial exopolysaccharides. EPS 71a was found to have fucose, galactose, glucose and glucuronic acid in a molar ratio of 2: 1: 1: 1.     

Enumeration of Enterobacter cloacae after chloramine exposure

Growth of Enterobacter cloacae on various media was compared after disinfection. This was done to examine the effects of monochloramine and chlorine on the enumeration of coliforms. The media used were TLY (nonselective; 5.5% tryptic soy broth, 0.3% yeast extract, 1.0% lactose, and 1.5% Bacto-Agar), m-T7 (selective; developed to recover injured coliforms), m-Endo (selective; contains sodium sulfite), TLYS (TLY with sodium sulfite), and m-T7S (m-T7 with sodium sulfite). Sodium sulfite in any medium improved the recovery of chloramine-treated E. cloacae. However, sodium sulfite in TLYS and m-T7S did not significantly improve the detection of chlorine-treated E. cloacae, and m-Endo was the least effective medium for recovering chlorinated bacteria. Differences in recovery of chlorine- and chloramine-treated E. cloacae are consistent with mechanistic differences between the disinfectants.