Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.

The phenol-degrading solvent-tolerant bacterium Pseudomonas putida P8 changed its cell shape when grown in the presence of aromatic compounds such as phenol and 4-chlorophenol. The sizes of cells that had been growing after addition of different concentrations of the toxic compounds were measured using a coulter counter that calculates the sizes of the rod-shaped bacteria to diameters of virtual spheres. The cells showed an increase in the diameter depending on the toxic effects of the applied concentrations of both solvents. The same effect was measured for an alkanol degrading bacterium, Enterobacter sp. VKGH12, in the presence of n-butanol. The reaction of the cells to different concentrations of n-butanol was examined by scanning electron microscopy. With this technique it could be shown that the size of the bacteria increased with increasing concentrations of n-butanol. These changes in cell size were dependent on the cellular activity and occurred only after addition of non-lethal concentrations. In the presence of lethal concentrations that completely inhibited cell growth, the cell sizes were similar to those of cells without intoxication. Taking into account the mathematical formula for spherical and cylindrical diameter and surface, respectively, the cells reacted to the presence of organic solvents by decreasing the ratio between surface and volume of the cells and therefore reducing their relative surfaces. As the cell surface and especially the cytoplasmic membrane are the major targets for the toxic effects of membrane-active compounds, this reduction of the relative surface represents an adaptive response to the presence of such compounds.

The solvent-tolerant strain Pseudomonas putida DOT-T1E was grown in batch fermentations in a 5-liter bioreactor in the presence and absence of 10% (vol/vol) of the organic solvent 1-decanol. The growth behavior and cellular energetics, such as the cellular ATP content and the energy charge, as well as the cell surface hydrophobicity and charge, were measured in cells growing in the presence and absence of 1-decanol. Although the cells growing in the presence of 1-decanol showed an about 10% reduced growth rate and a 48% reduced growth yield, no significant differences were measured either in the ATP and potassium contents or in the energy charge, indicating that the cells adapted completely at the levels of membrane permeability and energetics. Although the bacteria needed additional energy for adaptation to the presence of the solvent, they were able to maintain or activate electron transport phosphorylation, allowing homeostasis of the ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities and more negative cell surface charges were observed in cells grown in the presence of 1-decanol. Both reactions occurred within about 10 min after the addition of the solvent and were significantly different after killing of the cells with toxic concentrations of HgCl 2 . This adaptation of the surface properties of the bacterium to the presence of solvents seems to be very similar to previously observed reactions on the level of lipopolysaccharides, with which bacteria adapt to environmental stresses, such as heat shock, antibiotics, or low oxygen content. The results give clear physiological indications that the process with P. putida DOT-T1E as the biocatalyst and 1-decanol as the solvent is a stable system for two-phase biotransformations that will allow the production of fine chemicals in economically sound amounts.

The strain Pseudomonas putida DOT-T1E was tested for its ability to tolerate second phases of different alkanols for their use as solvents in two-liquid-phase biotransformations. Although 1-decanol showed an about 10-fold higher toxicity to the cells than 1-octanol, the cells were able to adapt completely to 1-decanol only and could not be adapted in order to grow stably in the presence of a second phase of 1-octanol. The main explanation for this observation can be seen in the higher water and membrane solubility of 1-octanol. The hydrophobicity (log P) of a substance correlates with a certain partitioning of that compound into the membrane. Combining the log P value with the water solubility, the maximum membrane concentration of a compound can be calculated. With this simple calculation, it is possible to predict the property of an organic chemical for its potential applicability as a solvent for two-liquid-phase biotransformations with solventtolerant P. putida strains. Only compounds that show a maximum membrane concentration of less than 400 mM, such as 1-decanol, seem to be tolerated by these bacterial strains when applied in supersaturating concentrations to the medium. Taking into consideration that a solvent for a two-liquid-phase system should possess partitioning properties for potential substrates and products of a fine chemical synthesis, it can be seen that 1-decanol is a suitable solvent for such biotransformation processes. This was also demonstrated in shake cultures, where increasing amounts of a second phase of 1-decanol led to bacteria tolerating higher concentrations of the model substrate 3-nitrotoluene. Transferring this example to a 5-liter-scale bioreactor with 10% (vol/vol) 1-decanol, the amount of 3-nitrotoluene tolerated by the cells is up to 200-fold higher than in pure aqueous medium. The system demonstrates the usefulness of two-phase biotransformations utilizing solventtolerant bacteria.Biocatalysis using whole cells promised to play an important role in the industrial synthesis of fine chemicals, pharmaceuticals, and precursors for chemical syntheses. However, the number of successful processes using whole-cell biotransformations is very small so far because several factors limit the number of applications (29,30). One important limitation of a successful application of whole-cell biotechnological processes toward classical chemical synthesis is that many reactions of interest involve substrates or products that are extremely toxic for the bacteria (35). This problem can be solved by the application of an organic solvent phase that functions as a source and a sink for toxic organic substrates and products, respectively (36). For biotransformations with whole cells, the major advantage of an addition of a second organic phase lies within its ability to act as a sink for the substrates as well as in the continuous removal of products (19,21,36). In both cases, the presence of a solvent phase keeps the concentrations of substrates and toxins at a level that does not lead to toxic e...

We studied the pattern of the cis-trans isomerization of unsaturated fatty acids in cells of Pseudomonas putida S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond. Direct evidence that isomerization does not include a transient saturation of the double bond was obtained. In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome c type.Notwithstanding the toxicity of aromatic solvents to most microorganisms, bacteria that are able to tolerate high concentrations of these compounds in their environment do exist, and most of them belong to the genus Pseudomonas (11). One of the solvent adaptation mechanisms enabling these Pseudomonas strains to grow in the presence of membrane-disrupting compounds is the isomerization of cis-to trans-unsaturated fatty acids (for reviews, see references 1 and 13). The extent of the isomerization apparently correlates with the toxicity and the concentration of such organic compounds in the membrane (7,8).cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12. The cis-trans isomerase activity is constitutively present in Pseudomonas, does not require ATP or other cofactors like NAD(P)H or glutathione, and works in the absence of de novo synthesis of lipids (2,5,7,15). Its independence from ATP is consistent with the negative free energy of the cis-to-trans isomerization (19). The enzyme has been purified from the periplasmic fraction of Pseudomonas oleovorans (17) and Pseudomonas sp. strain E3 (16) and has been isolated as a His-tagged P. putida P8 protein heterologously expressed in Escherichia coli (9). The cis-trans isomerase gene cloned and sequenced from P. putida P8 (10, 21) and P. putida DOT-T1E (12) made evident that the isomerase has an N-terminal hydrophobic signal sequence, which is cleaved off after the enzyme has been targeted to the periplasmic space. Holtwick et al. (9) provided evidence that the enzyme is a cytochrome c-type protein, as they found CXXCH, a heme-binding site (14), in the predicted Cti polypeptide. For an enzyme preparation from Pseudomonas sp. strain E3, which is presumably homologous to the cti gene product of P. putida P8, it was suggested that iron (probably Fe 3ϩ ) plays a crucial role in the catalytic reaction (16). Despite the wealth of available information, the biochemical mechanism of the solvent-induced regulation of isomerase activity, in relation to membrane homeostasis (18), still remains obscure.(Molecular biological investigations presented in this paper are part of the research conducted by Angelika von Wallbrunn in partial fulfillment of the requirements for a Ph.D. from the University of Münster, Münster, Germany.)Cultivation of the strain and supplementation with deuterated oleic acid. P. putida S12 (4) was cultivated in a mineral medium (3) with 15 mM glucose as the sole carbon source. Cells were grown in 100-ml shake cultures in a hor...

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na 2 -succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to ϳ35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.

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Rhodococcus erythropolis DCL14 has the ability to convert the terpene (-)-carveol to the valuable flavour compound (-)-carvone when growing on a wide range of carbon sources. To study the effect of carbon and energy sources such as alkanes, alkanols and terpenes on the biotechnological process, the cellular adaptation at the level of fatty acid composition of the membrane phospholipids and the (-)-carvone production were examined. All tested carbon sources caused a dose-dependent increase in the degree of saturation of the fatty acids. The exception was observed with short-chain alcohols such as methanol and ethanol, to which the cells adapted with a concentration-dependent decrease in the saturation degree of the membrane phospholipids. This influence of the different carbon sources on the rigidity of the cell membrane also had an impact on the (-)-carvone productivity of the strain.