Hermann J. Heipieper

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

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 cis–trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism

Isomerization of cis to trans unsaturated fatty acids is a mechanism enabling Gram-negative bacteria belonging to the genera Pseudomonas and Vibrio to adapt to several forms of environmental stress. The extent of the isomerization apparently correlates with the fluidity effects caused, i.e. by an increase in temperature or the accumulation of membrane-toxic organic compounds. Trans fatty acids are generated by direct isomerization of the respective cis configuration of the double bond without a shift of its position. The conversion of cis unsaturated fatty acids to trans is apparently instrumental in the adaptation of membrane fluidity to changing chemical or physical parameters of the cellular environment. Such an adaptive mechanism appears to be an alternative way to regulate membrane fluidity when growth is inhibited, e.g. by high concentrations of toxic substances. The cis-trans isomerase (Cti) activity is constitutively present and is located in the periplasma, it requires neither ATP nor any other cofactor such as NAD(P)H or glutathione, and it operates in the absence of de novo synthesis of lipids. Its independence from ATP is in agreement with the negative free energy of the reaction. cti encodes a polypeptide with an N-terminal hydrophobic signal sequence, which is cleaved off during or shortly after the enzyme is transported across the cytoplasmic membrane to the periplasmic space. A functional heme-binding site of the cytochrome c-type was identified in the predicted Cti polypeptide and very recently, direct evidence was obtained that isomerization does not include a transient saturation of the double bond.

Biodegradation of diesel/biodiesel blends by a consortium of hydrocarbon degraders: Effect of the type of blend and the addition of biosurfactants

Biodegradation experiments for diesel/biodiesel blends in liquid cultures by-petroleum degrading microbial consortium showed that for low amendments of biodiesel (10%) the overall biodegradation efficiency of the mixture after seven days was lower than for petroleum diesel fuel. Preferential usage of methyl esters in the broad biodiesel concentration range and diminished biodegradation of petroleum hydrocarbons for 10% biodiesel blend was confirmed. Rhamnolipids improved biodegradation efficiency only for blends with low content of biodiesel. Emulsion formation experiments showed that biodiesel amendments significantly affected dispersion of fuel mixtures in water. The presence of rhamnolipids biosurfactant affected stability of such emulsions and altered cell surface properties of tested consortium.

Biotechnological processes for biodiesel production using alternative oils

As biodiesel (fatty acid methyl ester (FAME)) is mainly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. The use of waste lipids such as waste frying oils, waste fats, and soapstock has been proposed as low-cost alternative feedstocks. Non-edible oils such as jatropha, pongamia, and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast, and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole-cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered, and the purification of FAME is simplified using biological catalysts.

Effect of aliphatic alcohols on growth and degree of saturation of membrane lipids in Acinetobacter calcoaceticus

The adaptive responses of the bacterium Acinetobacter calcoaceticus to different aliphatic alcohols on the level of the membrane fatty acids were studied in detail. The toxicity of the aliphatic alcohols increased with an increasing hydrophobicity. As alcohols are known to increase the fluidity of the membrane they consequently should cause the same adaptive effect on membrane level. Yet, cells of A. calcoaceticus react completely different to the alcohols: in the presence of long-chained alcohols they increase their degree of saturation, while in the presence of short-chained alcohols they decrease the degree of saturation. So, there are no observable differences in the adaptive responses of bacteria with the so-called anaerobic pathway, like Escherichia coli and Pseudomonas putida, and the bacterium carrying the so-called aerobic pathway like A. calcoaceticus. These results strongly indicate a physico-chemical difference in the membrane effect of both the partitioning and localisation of the different alcohols into the membrane and the membrane adaptive responses of the bacteria to these effects.

Cells of Pseudomonas putida and Enterobacter sp. adapt to toxic organic compounds by increasing their size

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.

Cell wall adaptations of planktonic and biofilm Rhodococcus erythropolis cells to growth on C5 to C16 n-alkane hydrocarbons

Rhodococcus erythropolis was found to utilize C5 to C16 n-alkane hydrocarbons as sole source of carbon and energy when growing as planktonic or biofilm cells attached to polystyrene surfaces. Growth rates on even numbered were two- to threefold increased relatively to odd-numbered n-alkanes and depended on the chain length of the n-alkanes. C10-, C12-, C14-, and C16-n-alkanes gave rise to two- to fourfold increased maximal growth rates relative to C5- to C9-hydrocarbons. In reaction to the extremely poor water solubility of the n-alkanes, both planktonic and biofilm cells exhibited distinct adaptive changes. These included the production of surface active compounds and substrate-specific modifications of the physicochemical cell surface properties as expressed by the microbial adhesion to hydrocarbon- and contact angle-based hydrophobicity, as well as the zeta potential of the cells. By contrast, n-alkane-specific alterations of the cellular membrane composition were less distinct. The specificity of the observed autecological changes suggest that R. erythropolis cells may be used in the development and application of sound biocatalytic processes using n-alkanes as substrates or substrate reservoirs or for target-specific bioremediation of oils and combustibles, respectively.

Simultaneous Degradation of Atrazine and Phenol by Pseudomonas sp. Strain ADP: Effects of Toxicity and Adaptation

ABSTRACT 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, Na2-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 ∼35% 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.

Membrane vesicle formation as a multiple-stress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation.

ABSTRACT Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.

Bacterial metabolism of environmental arsenic--mechanisms and biotechnological applications.

Arsenic causes threats for environmental and human health in numerous places around the world mainly due to its carcinogenic potential at low doses. Removing arsenic from contaminated sites is hampered by the occurrence of several oxidation states with different physicochemical properties. The actual state of arsenic strongly depends on its environment whereby microorganisms play important roles in its geochemical cycle. Due to its toxicity, nearly all organisms possess metabolic mechanisms to resist its hazardous effects, mainly by active extrusion, but also by extracellular precipitation, chelation, and intracellular sequestration. Some microbes are even able to actively use various arsenic compounds in their metabolism, either as an electron donor or as a terminal electron acceptor for anaerobic respiration. Some microorganisms can also methylate inorganic arsenic, probably as a resistance mechanism, or demethylate organic arsenicals. Bioavailability of arsenic in water and sediments is strongly influenced by such microbial activities. Therefore, understanding microbial reactions to arsenic is of importance for the development of technologies for improved bioremediation of arsenic-contaminated waters and environments. This review gives an overview of the current knowledge on bacterial interactions with arsenic and on biotechnologies for its detoxification and removal.