A gram-negative, rod-shaped, aerobe, capable of converting 2-propanol (isopropanol, IPA) to acetone was isolated from an oil/sump, and identified by 16 S rDNA analysis as Alcaligenes faecalis . Investigations showed this strain to be extremely solvent-tolerant and it was subsequently named ST1. In this study, A. faecalis ST1 cells were immobilized by entrapment in Ca-alginate beads (3 mm in diameter), and used in the bioconversion of high concentration IPA. The biodegradation rates and the corresponding microbial growth inside the beads were measured at four different IPA concentration ranges from 2 to 15 g l −1 . The maximum cell concentration obtained was 9.59 g dry cell weight (DCW) l −1 medium which equated to 66 g DCW l −1 gel, at an initial IPA concentration of 15 g l −1 after 216 h of incubation. A maximum biodegradation rate of 0.067 g IPA g cells −1 h −1 was achieved for 5 g l −1 IPA where an increase in IPA concentration to 38 g l −1 caused reduction in bead integrity. A modified growth medium was developed which allowed repeated use of the beads for more than 42 days without any loss of integrity and continued bioconversion activity.

The permeability barrier of the microbial cell envelope for substrates and products often causes very low reaction rates of whole cells. Therefore, it is of interest to develop an effective method to reduce this permeability barrier in order to increase product yields. Utilisation of pulse electric fields may improve amino acid release from Corynebacterium glutamicum by up to several orders of magnitude. In particular pulsed electric fields may change the cell/membrane's dielectric properties and induce the release of intracellular metabolites. In this study the parameters for successful electropermeabilization were determined and the viabilities of treated cells were examined. We also found that pulse treated cells not only maintained their viabilities but also their ability to reproduce, post-pulse treatment. Since electropermeabilized cells could maintain both their viabilities and ability to reproduce, we believe that this preliminary data may contribute to the optimization of fermentative production of amino acids and bioprocess enhancement through electropermeabilization and may be beneficial to industrial bioprocesses.

Selective and reversible permeabilization of the cell wall permeability barrier is the focus for many biotechnological applications. In this article, the basic principles for reversible membrane permeabilization, based on biological, chemical, and physical methods are reviewed. Emphasis is given to electroporation (electropermeabilization) which tends to be the most popular method for membrane permeabilization and for introduction of foreign molecules into the cells. The applications of this method in industrial processes as well as the critical factors and parameters which affect the success of this approach are discussed. The different strategies developed throughout the years for increased transformation efficiencies of the industrially important amino acid-overproducing bacterium Corynebacterium glutamicum, are also summarized.

The bioconversion of high concentration isopropanol (2-propanol, IPA) was investigated by a solvent tolerant strain of bacteria, which was identified as Sphingobacterium mizutae ST2 by partial 16S rDNA gene sequencing. This strain of bacteria exhibited the ability to utilise high concentration isopropanol as the sole carbon source, with mineralization occurring via an acetone intermediate into central metabolism. The biodegradative performance of this strain for IPA was examined over a 2–38 g l −1 concentration range, using specific growth rate (μ) and conversion rate analysis. Maximum specific growth rates (μ max ) of 0.0045 h −1 were routinely obtainable on IPA. In addition, the highest specific IPA degradation rate was obtained at a concentration of 7.5 g l −1 with a corresponding value of 0.045 g IPA g cells −1 h −1 . While the highest acetone yield reached its maximum value of 0.940 g acetone g IPA −1 at 7.5 g IPA l −1 . This is the first report on bioconversion of isopropanol at such high concentration by this solvent tolerant strain of S. mizutae and may allow its application in novel biocatalytic processes for effective biological conversion in two-phase solvent systems.

The aerobic biological oxidation of 2-propanol (isopropyl alcohol, IPA) at extremely high concentrations in air by an enriched solvent-tolerant microbial consortium operating at ambient temperature was evaluated for six months. Solvent-tolerant microbial cells were immobilised onto porous glass pall rings and fed with either IPA or its metabolic product acetone as sole carbon source. Successful biofiltration of solvent vapour at a concentration of 24 g m(-3) was achieved with oxidation of up to 100% total inlet carbon. The maximum IPA mass loading and IPA elimination capacity (EC) was 1700 g m(-3) h(-1). This performance exceeds all previous values published in the literature for similar processes. A slip feed experiment, using acetone, was also performed in order to assess the substrate specificity performance. The biofilter responded successfully to a switch from acetone to IPA as sole carbon source, displaying little reduction in overall organic carbon removal.

The aerobic biodegradation of high concentrations of gaseous acetone streams by a previously enriched solvent-tolerant bacterial consortium within a 1.9 litre vapour-phase fixed bed biofilter was investigated. Acetone biodegradation rate was measured and the characteristics of the bacterial community were investigated. 16S rRNA sequencing was used subsequently to identify these strains. Successful gas-phase biofiltration of solvent vapour at loadings of up to 360 g m(-3)h(-1) was implemented; this is higher than any other reported elimination capacity for acetone in the recognised literature. The mixed solvent-tolerant bacterial consortium was immobilized on sintered glass rings as a bioprocess intensification strategy. Acetone was tracked as the sole carbon source within a minimal salts medium. Removal efficiencies of up to 100% acetone were successfully demonstrated by the biofiltration system. The long-term effect on biofilter performance was also investigated. It showed that the consortium was able to adapt to high concentrations of acetone vapour (17 g m(-3)) over an extended period with removal efficiency above 80%. This result demonstrates that the biofilter could deal with high concentrations of acetone efficiently. It also shows that the consortium had a significant ability to adapt to high-concentration feed streams, which may occur under industrial conditions.

Since the 1950s when Micrococcus glutamicus later renamed Corynebacterium glutamicum was discovered, the production of amino acids by fermentative methods has become an important aspect of industrial microbiology. Numerous studies to understand and improve the metabolic conditions leading to amino acid overproduction have been carried out. Most amino acids are currently produced by use of mutants that contain combinations of auxotrophic and regulatory mutations. L ‐Glutamic acid is the amino acid produced in the greatest quantities (10 6 tonnes per year) and Corynebacteria are central to its industrial production. However, further improvements to strain performance are difficult to obtain by empirical optimization and a more rational approach is required. The use of metabolic flux analysis provides valuable information regarding bottlenecks in the formation of desired metabolites. Such techniques have found application in elucidating flux control, provided insight into metabolic network function and developed methods to amplify or redirect fluxes in engineered bioprocesses. Hence, branch points in biosynthesis, precursor supply in fuelling reactions and export of metabolites can be manipulated, resulting in high glutamic acid overproduction by Corynebacterium glutamicum fermentations. In this review, in addition to reviewing the state of play in metabolic flux analysis for glutamate overproduction, the metabolic pathways involved in the production of L ‐glutamic acid, the mechanisms mediating its efflux and secretion as well as their manipulation to achieve higher glutamate production, are discussed. The link between metabolic flux and transmembrane transport of glutamic acid are also considered. Copyright © 2004 Society of Chemical Industry

Since the 1950s when Micrococcus glutamicus later renamed Corynebacterium glutamicum was discovered, the production of amino acids by fermentative methods has become an important aspect of industrial microbiology. Numerous studies to understand and improve the metabolic conditions leading to amino acid overproduction have been carried out. Most amino acids are currently produced by use of mutants that contain combinations of auxotrophic and regulatory mutations. L-Glutamic acid is the amino acid produced in the greatest quantities (10(6) tonnes per year) and Corynebacteria are central to its industrial production. However, further improvements to strain,performance are difficult to obtain by empirical optimization and a more rational approach is required. The use of metabolic flux analysis provides valuable information regarding bottlenecks in the formation of desired metabolites. Such techniques have found application in elucidating flux control, provided insight into metabolic network function and developed methods to amplify or redirect fluxes in engineered bioprocesses. Hence, branch points in biosynthesis, precursor supply in fuelling reactions and export of metabolites can be manipulated, resulting in high glutamic acid overproduction by Corynebacterium glutamicum fermentations. In this review, in addition to reviewing the state of play in metabolic flux analysis for glutamate overproduction, the metabolic pathways involved in the production of L-glutamic acid, the mechanisms mediating its efflux and secretion as well as their manipulation to achieve higher glutamate production, are discussed. The link between metabolic flux and transmembrane transport of glutamic acid are also considered. (C) 2004 Society of Chemical Industry.

The discovery of a highly solvent-tolerant strain of microalga, identified as Chlorella vulgaris by 16S rRNA gene sequencing and termed SDC1 is described here for the first time. Ability to grow in the presence of isopropanol (IPA) solvent was assessed through specific growth rate (μ) determination at IPA feed concentrations of up to 16gl−1 at 20°C. Specific growth rates between 0.0017 and 0.0038h−1 were obtained in the presence of 2–16gl−1 IPA, and a value of 0.0047h−1 evident under IPA-free conditions. Axenic cultures of C. vulgaris SDC1 also demonstrated heterotrophic bioconversion of IPA at these elevated concentrations, where acetone, the suggested metabolite, was monitored as an indicator of aerobic degradation of IPA. Comparison of C. vulgaris SDC1 growth characteristics was carried out against that of the type culture strain, C. vulgaris Beijerinck (CCAP211/11B), where the solvent-tolerant strain SDC1 displayed lower sensitivity to high IPA concentrations. An LD50 value was found to occur at initial IPA concentrations of 3.8gl−1 for the type strain in comparison with 11.25gl−1 for the solvent-tolerant SDC1 strain. Despite several studies discussing the existence of solvent-tolerant bacteria, this is the first time solvent-tolerance at such high IPA concentrations (to 16gl−1) has been successfully demonstrated by an isolated C. vulgaris strain in a mineral salts medium.

Since the 1950s the production of amino acids by fermentative methods has become a very important aspect of industrial microbiology, leading to numerous studies to understand and improve the metabolic conditions driving to amino acid overproduction. In this review, in addition to a brief historic background of Coryneform bacteria, the various strategies used for strain improvement, such as and the use of auxotrophic strains and regulatory mutants, are discussed. Metabolic pathways involved in the production of L-lysine by Corynebacterium glutamicum and the mechanisms mediating its efflux and secretion are discussed. Metabolic flux analysis, which is considered to be a very powerful tool providing valuable information regarding bottlenecks in the production of desired metabolites, is also covered in relation to lysine secretion in conjunction with the significance of transport mechanisms. (C) 2004 Elsevier Ltd. All rights reserved.

The aerobic biodegradation of high-concentration, to 24 g l –1 , 2-propanol (IPA) by a thermophilic isolate ST3, identified as Bacillus pallidus , was successfully carried out for the first time. This solvent-tolerant B. pallidus utilized IPA as the sole carbon source within a minimal salts medium. Cultivation was carried out in 100-ml shake flasks at 60°C and compared with cultivation within a 1-l stirred tank reactor (STR). Specific growth rate ( µ ) was about 0.2 h –1 for both systems, with a maximum cell density of 2.4×10 8 cells ml –1 obtained with STR cultivation. During exponential growth and stationary phase, IPA biodegradation rates were found to be 0.14 and 0.02 g l –1 h –1 , respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l –1 h –1 were achievable in the STR. Generation of acetone, the major intermediate in aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Acetone levels reached a maximum of 2.2–2.3 g l –1 after 72 and 58 h for 100-ml and 1-l systems, respectively. Both IPA and acetone were completely removed from the medium following 160 and 175 h, respectively, during STR growth, although this was not demonstrated within shake-flask reactions. Growth of B. pallidus on acetone or IPA alone demonstrated that the maximum growth rate ( µ ) obtainable was 0.247 h –1 at 4 g l –1 acetone and 0.202 h –1 at 8 g l –1 IPA within shake-flask cultivation. These results indicate the potential of the solvent-tolerant thermophile B. pallidus ST3 in the bioremediation of hot solvent-containing industrial waste streams.

The ability of a previously enriched microbial population to utilize isopropanol (IPA) as the sole carbon source within a minimal salts medium is studied. The advantage of prior enrichment procedures for the improvement of IPA biodegradation performance is demonstrated for an IPA concentration of up to 24 g L(-1). Results showing the interrelationship between temperature and substrate utilization and inhibition levels at temperatures of between 2 degrees C and 45 degrees C are examined. Models of inhibition based on enzyme kinetics are assessed via nonlinear analysis, in order to accurately represent the growth kinetics of this solvent-tolerant mixed culture. The model that best describes the data is the Levenspiel substrate inhibition model, which can predict the maximum substrate level above which growth is completely limited. This is the first report of IPA treatment of up to 24 g L(-1) by an aerobic solvent-tolerant population.

The aerobic biodegradation of high liquid phase concentrations of 2-propanol (IPA) by a previously enriched solvent-tolerant bacterial consortium within a 1.9 l fed-batch three phase fixed bed bioreactor was investigated. Solvent concentrations of up to 7.9 g l(-1) were investigated. Previously enriched solvent-tolerant bacterial cells were immobilised onto porous glass cylinders as a means obioprocess intensification. Bioreactor start-up and acclimation was studied anacetone concentration tracked as an indicator of IPA utilization, as the sole carbon source within a minimal salts medium (MSM). The initial batch treatment of IPA exhibited a biodegradation rate of 0.11 g l(-1) h(-1) prior to biofilm formation Biofilm growth during the second batch treatment was consistent with an increase in metabolic activity and an IPA biodegradation rate of 0.34 g l(-1), followed by a reduction of biodegradation rate to a constant value of 0.078 g l(-1) h(-1) after 650 h. A maximum acetone generation rate of 1.3 g l(-1) h(-1) was obtained during the fourth IPA addition although the maximum acetone biodegradation rate of 0.38 g l(-1) h(-1) was observed during the initial IPA addition. It is proposed that the metabolic lag resulting from switching from alcohol dehydrogenase to acetone carboxylase is a major rate-limiting step in the deep oxidation of IPA to acetone. The results demonstrate the potential of a previously enriched solvent-tolerant bacterial consortium in fixed bed bioreactor systems, for the aerobic treatment of concentrated solvent-containing wastestreams.

The aerobic biodegradation and catalytic oxidation of vapor-phase 2-propanol (IPA) were investigated. The catalytic oxidation of IPA was carried out over zinc, copper, and chromium oxide catalysts prepared via a sol-gel technique in a fixed-bed reactor operated at atmospheric pressure and in the temperature range of 25-165 degreesC. The activity of the catalysts was measured by means of the light-off temperature (defined as 50% conversion of IPA). The light-off temperatures of zinc oxide, copper oxide, and chromium oxide are 90, 100, and 110 degreesC, respectively. The results indicate that, at relatively low temperature (40-100 degreesC), IPA was partially oxidized, which resulted in acetone formation. The maximum acetone selectivity varied between 30 and 97% at ca. 100 degreesC, depending on the types of catalyst. For the biodegradation study, enriched solvent-tolerant bacterial cells were immobilized onto porous glass cylinders within a biofilter. Successful biofiltration of high solvent vapor concentrations of up to 34 g m(-3) was achieved. An average IPA elimination capacity of up to 280 g m(-3) h(-1) was demonstrated by this biofiltration system. A slip feed experiment, using acetone, was investigated in order to assess the substrate specificity performance. The results show that the biofilter can deal with an alteration in feed composition and display no major reduction in the elimination performance. This paper shows that the concentration and compound distribution from the exit of a catalytic partial oxidation process are consistent with the inlet conditions of a gas-phase biofilter containing a solvent-tolerant microbial consortium. This points the way toward a potential integrated biofiltration-catalytic combustion system for the overall enhanced pollution abatement performance.