Organic solvent adaptation of Gram positive bacteria: applications and biotechnological potentials

Organic-solvent-tolerant bacteria are considered extremophiles with different tolerance levels that change among species and strains, but also depend on the inherent toxicity of the solvent. Extensive studies to understand the mechanisms of organic solvent tolerance have been done in Gram-negative bacteria. On the contrary, the information on the solvent tolerance mechanisms in Gram-positive bacteria remains scarce. Possible shared mechanisms among Gram-(−) and Gram-(+) microorganisms include: energy-dependent active efflux pumps that export toxic organic solvents to the external medium; cis-to-trans isomerization of unsaturated membrane fatty acids and modifications in the membrane phospholipid headgroups; formation of vesicles loaded with toxic compounds; and changes in the biosynthesis rate of phospholipids to accelerate repair processes. However, additional physiological responses of Gram-(+) bacteria to organic solvents seem to be specific. The aim of the present work is to review the state of the art of responsible mechanisms for organic solvent tolerance in Gram-positive bacteria, and their industrial and environmental biotechnology potential.     

Proteomic analysis reveals the participation of energy- and stress-related proteins in the response of Pseudomonas putida DOT-T1E to toluene

Pseudomonas putida DOT-T1E is tolerant to toluene and other toxic hydrocarbons through extrusion of the toxic compounds from the cell by means of three efflux pumps, TtgABC, TtgDEF, and TtgGHI. To identify other cellular factors that allow the growth of P. putida DOT-T1E in the presence of high concentrations of toluene, we performed two-dimensional gel analyses of proteins extracted from cultures grown on glucose in the presence and in the absence of the organic solvent. From a total of 531 spots, 134 proteins were observed to be toluene specific. In the absence of toluene, 525 spots were clearly separated and 117 proteins were only present in this condition. Moreover, 35 proteins were induced by at least twofold in the presence of toluene whereas 26 were repressed by at least twofold under these conditions. We reasoned that proteins that were highly induced could play a role in toluene tolerance. These proteins, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, were classified into four categories: 1, proteins involved in the catabolism of toluene; 2, proteins involved in the channeling of metabolic intermediates to the Krebs cycle and activation of purine biosynthesis; 3, proteins involved in sugar transport; 4, stress-related proteins. The set of proteins in groups 2 and 3 suggests that the high energy demand required for solvent tolerance is achieved via activation of cell metabolism. The role of chaperones that facilitate the proper folding of newly synthesized proteins under toluene stress conditions was analyzed in further detail. Knockout mutants revealed that CspA, XenA, and Tuf-1 play a role in solvent tolerance in Pseudomonas, although this role is probably not specific to toluene, as indicated by the fact that all mutants grew more slowly than the wild type without toluene.     

Isolation of new toluene-tolerant marine strains of bacteria and characterization of their solvent-tolerance properties

Aims:  To isolate and characterize new marine bacteria capable of tolerating high concentrations of organic solvents, and to understand the toxic effects of these chemicals on marine bacteria. Methods and Results:  Five marine bacteria able to tolerate 0·1% (v/v) toluene were isolated and characterized on the basis of their growth and survival rates in the presence of different organic solvents. The toluene-tolerant marine bacteria identified in this study could not grow in the presence of 0·1% (v/v) of several organic solvents with a log P_ow higher than that of the toluene (which in theory should be less toxic than toluene). The mechanisms underlying solvent tolerance were explored. Conclusions:  Isolates of four different genera were identified as toluene-tolerant. Toxicity of a second phase of an organic solvent toward these isolates could not be predicted on the basis of the solvents’ log P_ow. Significance and Impact of the Study:  To improve the biodegradation rate of some water-insoluble compounds, double-phase bioreactors can be used. This type of bioreactor will require strains able to grow in a salt-containing environment and able to tolerate a second phase of an organic solvent.     

Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120DeltaC

Utilization of solvent tolerant bacteria as biocatalysts has been suggested to enable or improve bioprocesses for the production of toxic compounds. Here, we studied the relevance of solvent (product) tolerance and inhibition, carbon metabolism, and the stability of biocatalytic activity in such a bioprocess. Styrene degrading Pseudomonas sp. strain VLB120 is shown to be solvent tolerant and was engineered to produce enantiopure (S)-styrene oxide from styrene. Whereas glucose as sole source for carbon and energy allowed efficient styrene epoxidation at rates up to 97 micromol/min/(g cell dry weight), citrate was found to repress epoxidation by the engineered Pseudomonas sp. strain VLB120DeltaC emphasizing that carbon source selection and control is critical. In comparison to recombinant Escherichia coli, the VLB120DeltaC-strain tolerated higher toxic product levels but showed less stable activities during fed-batch cultivation in a two-liquid phase system. Epoxidation activities of the VLB120DeltaC-strain decreased at product concentrations above 130 mM in the organic phase. During continuous two-liquid phase cultivations at organic-phase product concentrations of up to 85 mM, the VLB120DeltaC-strain showed stable activities and, as compared to recombinant E. coli, a more efficient glucose metabolism resulting in a 22% higher volumetric productivity. Kinetic analyses indicated that activities were limited by the styrene concentration and not by other factors such as NADH availability or catabolite repression. In conclusion, the stability of activity of the solvent tolerant VLB120DeltaC-strain can be considered critical at elevated toxic product levels, whereas the efficient carbon and energy metabolism of this Pseudomonas strain augurs well for productive continuous processing.     

Macrocyclic trichothecene toxins produced by a strain of Stachybotrys atra from Hungary.

A strain of Stachybotrys atra isolated from a field case of stachybotryotoxicosis in Hungary was cultured in Hungary. All of the compounds toxic to brine shrimp were separated from the culture extract by solvent partition, column chromatography, and preparative thin-layer chromatography. Two of the toxic compounds were identified as verrucarin J and satratoxin H by comparison with pure standards resolved by high-pressure liquid chromatography and characterized by mass spectrometry. Two other toxic components were identified as roriden E and satratoxin G on the basis of their mass spectra. The fifth toxic compound was identified as a macrocyclic trichothecene based on the following findings: a positive 4-(p-nitrobenzyl)pyridine color reaction, hydrolysis resulting in verrucarol verified by combined gas chromatography-mass spectrometry, and a characteristic trichothecene proton-nuclear magnetic resonance spectrum. This macrocyclic trichothecene has a molecular ion (528) identical to satratoxin H, and its mass spectrum is similar; however, its Rf value on Silica Gel G differs.     

Growth and physiological response of Arthrobacter protophormiae RKJ100 toward higher concentrations of o-nitrobenzoate and p-hydroxybenzoate

Bioremediation of sites that are heavily contaminated with pollutant chemicals is a challenge as most of the microorganisms cannot tolerate higher concentrations of toxic compounds. Only a few strains of the genus Pseudomonas have been studied for their tolerance toward the higher concentrations of aromatic pollutant compounds, a phenomenon that is accompanied by various physiological changes. In the present study we have characterized the growth response and physiological changes (adaptations) of a Gram-positive bacterium, Arthrobacter protophormiae RKJ100, toward the higher concentrations of two aromatic compounds, viz. o-nitrobenzoate (ONB) and p-hydroxybenzoate (PHB). Arthrobacter protophormiae RKJ100 could utilize 30 mM ONB and 50 mM PHB as sole sources of carbon and energy. It was capable of growth on higher concentrations of ONB (up to 200 mM) and PHB (up to 150 mM) when the cells were pre-exposed to lower concentrations of these compounds. The adaptive responses shown by the organism during growth on higher concentrations of these compounds were evident from significant changes in cellular fatty acid profiles. In addition, Bacterial Adhesion To Hydrocarbon (BATH) assay and scanning electron microscopy showed substantial increase in cell surface hydrophobicity and decrease in cell size of A. protophormiae RKJ100 when grown on ONB and PHB as compared to succinate-grown cells.     

Solvent-tolerant bacteria in biocatalysis

The toxicity of fine chemicals to the producer organism is a problem in several biotechnological production processes. In several instances, an organic phase can be used to extract the toxic product from the aqueous phase during a fermentation. With the discovery of solvent-tolerant bacteria, more solvents can now be used in such two-liquid water–solvent systems. We are gaining new insights into the mechanisms of bacterial solvent tolerance, such as the active efflux of solvents from the cytoplasmic membrane and solvent-impermeable outer membranes.     

Evolution of ammonia and urea tolerance in Drosophila melanogaster: resistance and cross-tolerance

We examined whether populations of Drosophila melanogaster could evolve a genetically based tolerance to high levels of toxic compounds (urea or ammonia) added to their larval food medium. We also examined whether tolerance to one compound may impart cross-tolerance to other compounds. Five populations selected for ammonia tolerance (AX), five populations selected for urea tolerance (UX), and five unselected controls (AUC) were assayed for developmental time, viability, and female fertility. These characteristics were measured on each of the 15 populations reared on one of three larval food conditions (plain banana-molasses, 0.35 M NH(4)Cl, or 0.266 M urea). On urea-supplemented media, the urea-selected populations developed fastest and expressed the highest viability; the ammonia-selected populations developed significantly faster and had a higher viability than the controls. Similarly, on ammonia-supplemented media, the ammonia-selected populations developed fastest and expressed the highest viability; the urea-selected populations developed significantly faster and had a higher viability than the controls. This suggests that a cross-tolerance exists for resisting different toxic compounds. Urea-selected females reared on urea-containing food media displayed superior fecundity, without any observable cross-tolerance effect. When all populations were reared on food containing 0.266 M urea, the urea-selected populations had the lowest levels of urea in their tissues. All populations reared on food containing 0.37 M ammonia or 0.266 M urea, contained more ammonia in their tissues than did populations reared on plain food.     

Bacteria tolerant to organic solvents

The toxic effects that organic solvents have on whole cells is an important drawback in the application of these solvents in environmental biotechnology and in the production of fine chemicals by whole-cell biotransformations. Hydrophobic organic solvents, such as toluene, are toxic for living organisms because they accumulate in and disrupt cell membranes. The toxicity of a compound correlates with the logarithm of its partition coefficient with octanol and water (log P _ow). Substances with a log P _ow value between 1 and 5 are, in general, toxic for whole cells. However, in recent years different bacterial strains have been isolated and characterized that can adapt to the presence of organic solvents. These strains grow in the presence of a second phase of solvents previously believed to be lethal. Different mechanisms contributing to the solvent tolerance of these strains have been found. Alterations in the composition of the cytoplasmic and outer membrane have been described. These adaptations suppress the effects of the solvents on the membrane stability or limit the rate of diffusion into the membrane. Furthermore, changes in the rate of the biosynthesis of the phospholipids were reported to accelerate repair processes. In addition to these adaptation mechanisms compensating the toxic effect of the organic solvents, mechanisms do exist that actively decrease the amount of the toxic solvent in the cells. An efflux system actively decreasing the amount of solvents in the cell has been described recently. We review here the current knowledge about exceptional strains that can grow in the presence of toxic solvents and the mechanisms responsible for their survival. Received: January 22, 1998 / Accepted: February 16, 1998     

Real-time solvent tolerance analysis of pseudomonas sp. strain VLB120{Delta}C catalytic biofilms

Biofilms are ubiquitous surface-associated microbial communities embedded in an extracellular polymeric (EPS) matrix, which gives the biofilm structural integrity and strength. It is often reported that biofilm-grown cells exhibit enhanced tolerance toward adverse environmental stress conditions, and thus there has been a growing interest in recent years to use biofilms for biotechnological applications. We present a time- and locus-resolved, noninvasive, quantitative approach to study biofilm development and its response to the toxic solvent styrene. Pseudomonas sp. strain VLB120ΔC-BT-gfp1 was grown in modified flow-cell reactors and exposed to the solvent styrene. Biofilm-grown cells displayed stable catalytic activity, producing (S)-styrene oxide continuously during the experimental period. The pillar-like structure and growth rate of the biofilm was not influenced by the presence of the solvent. However, the cells experience severe membrane damage during styrene treatment, although they obviously are able to adapt to the solvent, as the amount of permeabilized cells decreased from 75 to 80% down to 40% in 48 h. Concomitantly, the fraction of concanavalin A (ConA)-stainable EPS increased, substantiating the assumption that those polysaccharides play a major role in structural integrity and enhanced biofilm tolerance toward toxic environments. Compared to control experiments with planktonic grown cells, the Pseudomonas biofilm adapted much better to toxic concentrations of styrene, as nearly 65% of biofilm cells were not permeabilized (viable), compared to only 7% in analogous planktonic cultures. These findings underline the robustness of biofilms under stress conditions and its potential for fine chemical syntheses.     

Characterization of the adaptive response to trichloroethylene-mediated stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Environmentally Friendly Approach to the Synthesis of 3-[Benzylideneamino]-2-methylquinazolin-4(3H)-one Derivatives and Calculation of Their Toxicity

Application of deep eutectic solvents in synthesis of different heterocyclic compounds was proven very efficient. These solvents are a new generation of green solvents showing excellent potential for different purposes, where they are used as environmentally acceptable substitute for toxic and volatile organic solvents. This research describes their application in the synthesis of series of quinazolinone Schiff bases in combination with microwave, ultrasound-assisted and mechanochemical methods. First, a model reaction was performed in 20 different deep eutectic solvents to find the best solvent and then reaction conditions (solvent, temperature and reaction time) were optimized for each method. Afterwards, 40 different quinazolinone derivatives were synthesized in choline chloride/malonic acid (1 : 1) DES by each method and compared by their yields. Here we show that deep eutectic solvents can be very efficient in the synthesis of quinazolinone derivatives as an excellent substitution for volatile organic solvents. With green chemistry approach in mind, we have also performed a calculation on compounds’ toxicity and solubility, showing that most of them possess toxic and mutagenic properties with low water solubility.     

Changes in development and ultrastructure of Aspergillus niger mycelium, strain with increased tolerance to toxic substances from beet molasses.

Effects of a defoamer and toxic molasses compounds on development and ultrastructure of A. niger mycelium, strain Z, characterized by high tolerance to these substances and producing citric acid in surface fermentation on proper molasses media with 70% yield were presented. Spumol BJ in concentration of 5 microliters/100 cm3 as well as toxic molasses compounds stimulated the process of swelling and germinating of conidia. Moreover, giant conidia, unable to germinate, appeared. Developing mycelium with dispersed hyphae became mucilaginous after 17-20 h culture, which indicated the process of sinking but after 24 h some part of the mycelium developed normally. Electron microscopic observations of mycelium developing in the presence of the toxic substances showed along with electron-transparent cytoplasm in a consequence of decrease in ribosome number, changes in ultrastructure of mitochondria. It may be assumed that one of the reasons of the above described abnormalities in development and ultrastructure of mycelium was a disturbance of respiration processes. The appearance of deposits of electron-dense material in mitochondria suggested the existence of a defence mechanism, eliminating toxic substances.     

Characterization of the Adaptive Response to Trichloroethylene-Mediated Stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Tolerance to 1-pentene-3-ol and to 1-pentene-3-one in relation to alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities inDrosophila melanogaster

The detoxification of 1-pentene-3-ol (pentenol) and 1-pentene-3-one (pentenone) by Drosophila melanogaster adult flies has been studied in two homozygous lines for the AdhF and AdhS alleles (LRC lines), in their respective lines selected for tolerance to ethanol (LRSe lines) and in a homozygous strain for the Adhn4 null allele. For each line, the genotype and sex LDs50 of both compounds were estimated. Then, in order to explain the differences in LD50, both alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities were assayed. In addition, the effects of pentenone on AKR activity were also studied. Our results show that ADH-positive flies exhibit a much higher sensitivity to pentenol than ADH-null flies. However, both ADH-positive and ADH-null flies show a similar tolerance to pentenone. Our results show that flies selected for improving tolerance to ethanol also have increased tolerance to pentenol (FF and SS flies) and pentenone (SS flies). However, this improved ability to tolerate pentenol and/or pentenone cannot be explained by changes in ADH or AKR activities. On the other hand, we have observed a beneficial effect of pentenol, but not of pentenone, in n4 flies. We also show that AKR activity is not modified by the administration of pentenone. These results suggest that, in the absence of ADH activity, pentenol may be transformed into a compound that is less toxic than pentenone and that pentenone itself might also be transformed into a less toxic compound.     

Isolation and characterization of Deinococcus geothermalis T27, a slightly thermophilic and organic solvent-tolerant bacterium able to survive in the presence of high concentrations of ethyl acetate

A solvent-tolerant, slightly thermophilic bacterium was isolated at 45 degrees C in the presence of toluene vapor provided as the sole carbon source. Strain T27 was identified as Deinococcus geothermalis T27. It could tolerate high concentrations of solvent provided as a nonaqueous layer (5% and 20%, v/v) to a cell suspension and had a remarkable ability to tolerate a broad range of solvents having log P(ow) values ranging from 5.6 of n-decane to as low as 0.7 of ethyl acetate. It was also able to utilize some of the solvents tested as a growth substrate at 45 degrees C. The addition of Ca(2+) ion, glucose and fructose partially promoted solvent tolerance. Cells exposed to ethyl acetate appeared to have a smaller size; however, the cell structure was not altered and was apparently well defined even after solvent shock. The tolerance of D. geothermalis T27 in the presence of high levels of toxic solvent stress at a comparatively high temperature indicated its potential use in biotechnological applications as well as bioremediation of xenobiotics.     

An investigation of the toxicity of insecticides to birds' eggs using the egg-injection technique

Twenty-five insecticides have been tested for their toxicity to hen embryos at various concentrations, using an egg-injection technique. Of the two major groups, the organophosphorus compounds are much more toxic than the organochlorine. Most organophosphorus compounds lower the hatch rate and cause teratogenic effects at 100 ppm. Most organochlorines do not harm the embryo at high dosages (up to 500 ppm), with notable exceptions to this among the cyclodienes. However, starvation of the hatched chicks suggests that the majority of organochlorine compounds can kill at this stage, though fed chicks survive. The solvent used affected toxicity; in general, insecticides are more toxic to the embryo when dissolved in corn oil than when dissolved in acetone. If these findings can be applied to wild birds, it can be assumed that the vast majority of insecticides are harmless to birds' eggs. They are either not toxic in the concentrations so far found in this country, or else unlikely to pass through the mother bird to the egg.