Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

Strategies for enzymatic esterification in organic solvents: comparison of microaqueous, biphasic, and micellar systems.

The kinetics of butyl butyrate synthesis by a lipase from Mucor miehei in different types of organic media were investigated. The three systems studied were a microaqueous medium containing enzyme in suspension in hexane, a water-hexane two-phase system, and reverse micelles. The synthesis of butyl butyrate was possible in all cases because of a favorable partition of the ester into the organic solvent. A sufficient stirring rate was necessary to achieve good reaction rates in the case of the liquid-liquid biphasic medium. The effect of water content was different according to the type of system used. The dependence of reaction rate and of conversion yield on enzyme and substrate concentrations was also investigated. From an applied point of view, the best performances were obtained with either microaqueous or liquid-liquid two-phase systems. The use of reverse micelles can be advocated only in particular conditions, such as low enzyme concentration, compatible with the specific constraints it involves.

Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR.

AIMS: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. METHODS AND RESULTS: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (

Biodegradation of tert-butyl alcohol and related xenobiotics by a methylotrophic bacterial isolate.

A new aerobic bacterial strain, CIP 1-2052, isolated from an activated sludge sample, was able to use tert-butyl alcohol (TBA), a product of methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) degradation, as its sole carbon and energy source. Cobalt ions stimulated TBA mineralization. The maximum growth and TBA degradation rates were 0.032 +/- 0.004 h(-1) and 35.8 +/- 8.5 mg TBA x g(-1) (cell dry mass) per h, respectively. The growth yield on TBA was 0.54 +/- 0.02 g x g(-1). Strain CIP 1-2052 exhibited a particular substrate specificity towards alcohols. It degraded tertiary alcohols, TBA and tert-amyl alcohol (TAA), but neither their primary and secondary alcohol homologues, nor ethanol. However, one-carbon compounds, namely methanol and formate, were degraded by strain CIP 1-2052, showing the methylotrophic nature of this isolate. The properties of this new strain suggest that it could be used for bioremediation of contaminated aquifers.

Microbial degradation and fate in the environment of methyl tert-butyl ether and related fuel oxygenates.

Oxygenates, mainly methyl tert-butyl ether (MTBE), are commonly added to gasoline to enhance octane index and improve combustion efficiency. Other oxygenates used as gasoline additives are ethers such as ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), and alcohols such as tert-butyl alcohol (TBA). As a result of its wide use, MTBE has been detected, mainly in the USA, in groundwater and surface waters, and is a cause of concern because of its possible health effects and other undesirable consequences. MTBE is a water-soluble and mobile compound that generates long pollution plumes in aquifers impacted by gasoline releases from leaking tanks. Field observations concur in estimating that, because of recalcitrance to biodegradation, natural attenuation is slow (half-life of at least 2 years). However, quite significant advances have been made in recent years concerning the microbiology of the degradation of MTBE and other oxygenated gasoline additives. The recalcitrance of these compounds results from the presence in their structure of an ether bond and of a tertiary carbon structure. For the most part, only aerobic microbial degradation systems have been reported so far. Consortia capable of mineralizing MTBE have been selected. Multiple instances of the cometabolism of MTBE with pure strains or with microflorae, growing on n-alkanes, isoalkanes, cyclohexane or ethers (diethyl ether, ETBE), have been described. MTBE was converted into TBA in all cases and was sometimes further degraded, but it was not used as a carbon source by the pure strains. However, mineralization of MTBE and TBA by several pure bacterial strains using these compounds as sole carbon and energy source has recently been reported. The pathways of metabolism of MTBE involve the initial attack by a monooxygenase. In several cases, the enzyme was characterized as a cytochrome P-450. After oxygenation, the release of a C -unit as formaldehyde or formate leads to the production of TBA, which can be converted to 2-hydroxyisobutyric acid and further metabolized. Developments in microbiology make biological treatment of water contaminated with MTBE and other oxygenates an attractive possibility. Work concerning ex situ treatment in biofilters by consortia and by pure strains, and involving or not cometabolism, is under way. Furthermore, the development of in situ treatment processes is a promisinggoal.

Acetone and Butanol Production by Clostridium acetobutylicum in a Synthetic Medium.

The effect of the component concentrations of a synthetic medium on acetone and butanol fermentation by Clostridium acetobutylicum ATCC 824 was investigated. Cell growth was dependent on the presence of Mg, Fe, and K in the medium. Mg and Mn had deleterious effects when in excess. Ammonium acetate in excess caused acid fermentation. The metabolism was composed of two phases: an acid phase and a solvent one. Low concentrations of glucose allowed the first phase only. The theoretical ratio of the conversion of glucose to solvents, which was 28 to 33%, was obtained with the following medium: MgSO(4), 50 to 200 mg/liter; MnSO(4), 0 to 20 mg/liter; KCl, 0.015 to 8 g/liter (an equivalent concentration of K was supplied in the form of KH(2)PO(4) and K(2)HPO(4)); FeSO(4), 1 to 50 mg/liter; ammonium acetate, 1.1 to 2.2 g/liter; para-aminobenzoic acid, 1 mg/liter; biotin, 0.01 mg/liter; glucose, 20 to 60 g/liter.

Roles of tert-butyl formate, tert-butyl alcohol and acetone in the regulation of methyl tert-butyl ether degradation by Mycobacterium austroafricanum IFP 2012.

Mycobacterium austroafricanum IFP 2012 is a Gram-positive strain able to grow on methyl tert-butyl ether (MTBE) as a sole carbon and energy source. The effect of two downstream metabolites of MTBE, tert-butyl formate (TBF) and tert-butyl alcohol (TBA) on MTBE degradation was investigated using resting cells. The addition of low concentrations of TBF decreased the MTBE degradation rate by about 30%. In contrast, the addition of TBA did not have a significant effect on MTBE degradation rate, even at high concentrations; and it was also shown that TBA degradation occurred only once MTBE was exhausted. At neutral pH, TBF hydrolysis involved mainly an esterase-type activity regulated by the presence of TBA. The TBF degradation rate was about four times lower than the MTBE degradation rate. Furthermore, acetone was identified as an intermediate during TBA degradation. An acetone mono-oxygenase activity, inhibited by methimazole but not by acetylene, was suggested. It was different from the MTBE/TBA mono-oxygenase and, thus, acetone did not appear to compete with MTBE and TBA for the same enzyme. These new results show that the metabolic regulation of the early steps of MTBE degradation by M. austroafricanum IFP 2012 is complex, involving inhibition and competition phenomena.