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A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2, 4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of Mycobacterium austroafricanum was found. Mycobacterium sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including n-alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl t-butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.

The ice-nucleation protein (Inp) is a glycosyl phosphatidylinositol-anchored outer membrane protein found in some Gram-negative bacteria. Using Pseudomonas syringae inp as an anchoring motif, we investigated the functional display of a foreign protein, Zymomonas mobilis levansucrase (LevU), on the surface of Escherichia coli. The cells expressing Inp-LevU were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities, indicating the functional expression of Inp-LevU hybrid protein on the cell surface. The surface localization was further verified by immunofluorescence microscopy, fluorescence-activated cell sorting flow cytometry and immunogold electron microscopical examination. No growth inhibition or changes in the outer membrane integrity were observed upon the induction of fusion protein synthesis. Viability of the cells was also maintained over 48 hours in the stationary phase. Surface-displayed levansucrases were found to be resistant to the externally added proteases unless the cells were treated with EDTA. When the levansucrase-displayed cells were used as the enzyme source, levan (44 g/L) was efficiently synthesized from sucrose (130 g/L) with 34% (wt/wt) conversion yield, generating glucose (65 g/L) as a by-product.

Ice-nucleation protein (INP), an outer membrane protein from Pseudomonas syringae, is able to catalyze the ice crystal formation of supercooled water. It was exploited for anchoring of Bacillus subtilis carboxymethylcellulase (CMCase) on the surface of Escherichia coli. A surface anchoring vector, pGINP21M, was created that contains the multicloning sites including BamHI, SmaI and EcoRI at the end of the 3' flanking region encoding the C-terminus of INP instead of the stop codon for subcloning the foreign genes. The CMCase gene was in-frame subcloned for making INP-CMCase fusion proteins. The ability of this vector for directing the actual synthesis of INP-CMCase fusion proteins was confirmed by Western blotting analysis. CMCase targeted on the surface of cells was verified by measuring whole cell CMCase activity and ice-nucleation activity. CMCase activity was mainly detected on the cell surface whereas no enzyme activity was detected in the culture supernatant. Ice-nucleation activity was also maintained even if an INP-CMCase hybrid was made. This means that the fusion protein is functionally expressed and has its biological conformation on the surface. INP-CMCase fusion proteins were stable in the stationary phase. INP deleted of the repeating domain, thus producing no ice-nucleation activity, could also direct CMCase on the cell surface. This suggests that it has the secretion and targeting signal to the outer membrane.

The maintenance of a steady-state biofilm in a continuous-flow fixed-bed reactor, as a consequence of the reproduction-detachment of cells (an interfacial cell physiology phenomenon of steady-state biofilm) during the biodegradation of 2,4,6-trichlorophenol by Pseudomonas cells, was determined. After cell adhesion on an open-pore glass support, the biofilm was formed in a packed-bed recirculated reactor. After the steady-state biofilm was reached, the mechanisms of the interfacial cell detachment (at the biofilm-liquid interface) were determined. It was established that (i) the hydrophobicity of immobilized sessile cells (parent cells) increased (from 50 to 80%) as the dilution rate increased, while the hydrophobicity of detached suspended cells (daughter cells) remained constant (about 45%); and (ii) the immediately detached suspended cells showed a synchronized growth in about three generations. These results indicate that (i) the immobilized sessile and suspended detached cells grew synchronically at the end and at the beginning of the cell cycle, respectively; and (ii) the hydrophobicity difference of immobilized sessile and suspended detached cells permitted the cells detachment. Therefore, it is probable that independent of shear stress (due to recirculated flow), the synchronized growth and hydrophobicity of cells (which vary during the cell cycle) are the main factors permitting the maintenance of a steady-state xenobiotic-degrading biofilm reactor (in which the overall accumulation of biofilm is determined by the average growth rate of the biofilm cells minus the rate of detachment of cells from the biofilm).

Microbial selection on mixtures of chlorinated and nonchlorinated compounds that are poorly soluble in water and/or toxic to growing microbial cells was examined in both biphasic aqueous-organic and monophasic aqueous systems. A biphasic system in which silicone oil was used as the organic phase permitted the acceleration of acclimation, leading to rapid selection and to an increase in xenobiotic compound degradation. In contrast, acclimation, selection, and degradation were very slow in the monophasic aqueous system. The variation in microbial growth rate with the degree of dispersion (i.e., dispersion at different silicone oil concentrations and agitation rates), and cell adhesion to the silicone oil indicate that the performance of the biphasic aqueous-organic system is dependent on the interfacial area between the two phases and that microbial activity is important at this interface. Therefore, the biphasic water-silicone oil system could be used for microbial selection in the presence of xenobiotic compounds that are toxic and have low water solubility.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Batch cultures of Pseudomonas mendocina, grown in rich medium with glucose excess, showed metabolic differences dependent upon whether the growth conditions were aerobic or anaerobic, with or without added electron acceptor. Under anaerobic conditions in the absence of nitrate, P. mendocina reached the stationary phase of growth after 2 or 3 days, followed by a stationary phase of 4 to 5 days. Under these conditions, a mixed-type fermentative metabolism (formic, lactic, and acetic acids) appeared. A fivefold-higher specific rate of glucose consumption and eightfold-higher production of organic acids, compared with aerobic cultures, were shown by this microorganism growing anaerobically in the absence of exogenous electron acceptors. The gradients of organic acid produced by P. mendocina under these conditions reached a maximum (lactate, 180 mV; formate, 150 mV; acetate, 215 mV) between days 2 and 3 of culture. The proton motive force (delta p) decreased during growth from -254 to -71 mV. The intracellular pH remained alkaline during the culture, reaching a steady-state value of 7.9. The gradients of organic acids apparently contributed to the generation of a delta p, which, according to the Energy Recycling Model (P. A. M. Michels, J. P. J. Michels, J. Boonstra, and W. N. Konings, FEMS Microbiol. Lett. 5:357-364, 1979), would produce an average energy gain of 1 or 1.5 mol of ATP equivalents per mol of glucose consumed with H+/ATP stoichiometry of 3 or 2, respectively. Low YATP and Yglucose values were observed, suggesting that an uncoupled metabolism exists; i.e., ATP produced by catabolic processes is not directly used for biomass synthesis. This metabolic uncoupling could be induced at least in part by organic acids and the ATP wastage could be induced by a membrane-bound ATPase involved in intracellular pH regulation.

Candida tropicalis grows on soluble starch, corn, and cassava powders without requiring that these substrates be previously hydrolyzed. C. tropicalis possesses the enzyme needed to hydrolyze starch, namely, an alpha-amylase. That property has been used to develop a fermentation process whereby C. tropicalis can be grown directly on corn or cassava powders so that the resultant mixture of biomass and residual corn or cassava contains about 20% protein, which represents a balanced diet for either animal fodder or human food. The fact that no extra enzymes are required to hydrolyze starch results in a particularly efficient way of improving the nutritional value of amylaceous products, through a single-step fermentation process.

Bacterial strains were selected for their capacity to assimilate and to transform ammonium-lignosulfonate. Modification of the methyl content and of low molecular weight alkyl functions were demonstrated by gas-chromatography and HLPC analysis. Most of these strains completely degraded simple phenolic compounds related to lignosulfonate without inhibition by the carbohydrates present in the liquor. Further investigation suggested that the enzymes involved in the fission of the aromatic nuclei were constitutive in the strains tested.

A method for the preparation of protoplasts from Candida tropicalis cultivated on n-tetradecane is described. This essentially consists of replacing the mannitol-sorbitol solution of the classical helicase technique by 1 m magnesium sulfate and lowering the pH to 4.1 during incubation in the presence of helicase. The protoplasts thus prepared behave like intact cells and are capable of consuming oxygen in the presence of n-tetradecane, n-decane, 1-decanol, and glucose.