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.     

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.     

A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.     

Industrial potential of organic solvent tolerant bacteria

Most bacteria and their enzymes are destroyed or inactivated in the presence of organic solvents. Organic solvent tolerant bacteria are a relatively novel group of extremophilic microorganisms that combat these destructive effects and thrive in the presence of high concentrations of organic solvents as a result of various adaptations. These bacteria are being explored for their potential in industrial and environmental biotechnology, since their enzymes retain activity in the presence of toxic solvents. This property could be exploited to carry out bioremediation and biocatalysis in the presence of an organic phase. Because a large number of substrates used in industrial chemistry, such as steroids, are water-insoluble, their bioconversion rates are affected by poor dissolution in water. This problem can be overcome by carrying out the process in a biphasic organic-aqueous fermentation system, wherein the substrate is dissolved in the organic phase and provided to cells present in the aqueous phase. In bioprocessing of fine chemicals such as cis-diols and epoxides using such cultures, organic solvents can be used to extract a toxic product from the aqueous phase, thereby improving the efficiency of the process. Bacterial strains reported to grow on and utilize saturated concentrations of organic solvents such as toluene can revolutionize the removal of such pollutants. It is now known that enzymes display striking new properties in the presence of organic solvents. The role of solvent-stable enzymes in nonaqueous biocatalysis needs to be explored and could result in novel applications.     

Cellular response mechanisms in Pseudomonas aeruginosa PseA during growth in organic solvents

Aims:  Solvent-tolerant bacteria have emerged as a new class of micro-organisms able to grow at high concentrations of toxic solvents. Such bacteria and their solvent-stable enzymes are perceived to be useful for biotransformations in nonaqueous media. In the present study, the solvent-responsive features of a lipase–producing, solvent-tolerant strain Pseudomonas aeruginosa PseA have been investigated to understand the cellular mechanisms followed under solvent-rich conditions.
Methods and Results:  The solvents, cyclohexane and tetradecane with differing log P -values (3·2 and 7·6 respectively), have been used as model systems. Effect of solvents on (i) the cell morphology and structure (ii) surface hydrophobicity and (iii) permeability of cell membrane have been examined using transmission electron microscopy, atomic force microscopy and other biochemical techniques. The results show that (i) less hydrophobic (low log P -value) solvent cyclohexane alters the cell membrane integrity and (ii) cells adapt to organic solvents by changing morphology, size, permeability and surface hydrophobicity. However, no such changes were observed in the cells grown in tetradecane.
Conclusions:  It may be concluded that P. aeruginosa PseA responds differently to solvents of different hydrophobicities. Bacterial cell membrane is more permeable to less hydrophobic solvents that eventually accumulate in the cytoplasm, while highly hydrophobic solvents have lesser tendency to access the membrane.
Significance and Impact of the Study:  To the best of our knowledge, these are first time observations that show that way of bacterial solvent adaptability depends on nature of solvent. Difference in cellular responses towards solvents of varying log P -values (hydrophobicity) might prove useful to search for a suitable solvent for carrying out whole-cell biocatalysis.     

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.     

Organic Solvent-Tolerant Bacterium Which Secretes an Organic Solvent-Stable Proteolytic Enzyme

A bacterial strain which can be grown in a medium containing organic solvents and can secrete a proteolytic enzyme was isolated and identified as Pseudomonas aeruginosa. The strain was derived by the following two-step procedures: high proteolytic enzyme producers were first isolated by the usual method, and then the organic solvent-tolerant microorganism was selected from these high-rate proteolytic enzyme producers. The proteolytic activity of the supernatant of the culture was stable in the presence of various organic solvents. The stability of the enzyme in the presence of organic solvents, of which the values of the logarithm of the partition coefficient (log P) were equal to or more than 3.2, was almost the same as that in the absence of organic solvents. It is expected that both the solvent-tolerant microorganism and the solvent-stable enzyme produced by this strain can be used as catalysts for reactions in the presence of organic solvents.     

细菌的有机溶剂耐受机制

有机溶剂有严重破坏微生物正常生理功能的毒害作用,但是研究工作者发现有些细菌能够在较高有机溶剂浓度下依赖独特的耐受机制得以生存,这种机制的发现大大鼓舞了工业菌尤其是溶剂生产菌和毒性有机物降解菌的工业适应性改造研究。以下概述了有机溶剂对细胞毒性作用机制,并在根据参数logP衡量不同溶剂对细胞的毒性程度的基础上,重点总结了溶剂耐受菌耐受有机溶剂的机制,即膜上顺反异构、增加饱和脂肪酸的比率、改变极性头部、外膜的生理变化、细胞的形态变化、胞内溶剂的降解和泵出等,结合本课题组在筛选溶剂耐受菌株和提高现有菌株溶剂耐受性研究方面的经验,希望对重要工业微生物溶剂耐受相关的生理功能进行更深入地研究,提高微生物的工业适应性。     

Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent’s toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.     

Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase

A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only approximately 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.     

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     

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.     

Isolation and characterization of BTEX tolerant and degrading Pseudomonas putida BCNU 106

Bacterial strains growing in river sediments were screened to identify an organic solvent-tolerant strain of Pseudomonas. Using this screen, Pseudomonas sp. BCNU 106 was isolated on the basis of its ability to grow on benzene, toluene, ethylbenzene, and three xylene isomers, o-, m- and p-xylene, as its sole carbon source. BCNU 106 was identified as a gram-negative, rod-shaped aerobic and mesophilic bacterium, which grew in liquid media containing high concentrations of organic solvents. 16S rDNA analysis classified BCNU 106 as a new member of the genus Pseudomonas. BCNU 106 was distinguishable from other Pseudomonas strains that are tolerant to organic solvents in that the isolate had the ability to utilize all three xylene isomers as well as benzene, toluene and ethylbenzene. The unique properties of the isolate such as solvent-tolerance and the ability to degrade xylene isomers may have important implications for the efficient treatment of solvent wastes.     

Solvent tolerance in Gram-negative bacteria

Bacteria have been found in all niches explored on Earth, their ubiquity derives from their enormous metabolic diversity and their capacity to adapt to changes in the environment. Some bacterial strains are able to thrive in the presence of high concentrations of toxic organic chemicals, such as aromatic compounds, aliphatic alcohols and solvents. The extrusion of these toxic compounds from the cell to the external medium represents the most relevant aspect in the solvent tolerance of bacteria, however, solvent tolerance is a multifactorial process that involves a wide range of genetic and physiological changes to overcome solvent damage. These additional elements include reduced membrane permeabilization, implementation of a stress response programme, and in some cases degradation of the toxic compound. We discuss the recent advances in our understanding of the mechanisms involved in solvent tolerance.     

Bioproduction of vanillin using an organic solvent-tolerant <Emphasis Type="Italic">Brevibacillus agri</Emphasis> 13

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).     

Whole cell biocatalysis in nonconventional media

Summary In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.     

Microbiology of the oil fly, Helaeomyia petrolei

Helaeomyia petrolei larvae isolated from the asphalt seeps of Rancho La Brea in Los Angeles, Calif., were examined for microbial gut contents. Standard counts on Luria-Bertani, MacConkey, and blood agar plates indicated ca. 2 x 10(5) heterotrophic bacteria per larva. The culturable bacteria represented 15 to 20% of the total population as determined by acridine orange staining. The gut itself contained large amounts of the oil, had no observable ceca, and maintained a slightly acidic pH of 6.3 to 6.5. Despite the ingestion of large amounts of potentially toxic asphalt by the larvae, their guts sustained the growth of 100 to 1,000 times more bacteria than did free oil. All of the bacteria isolated were nonsporeformers and gram negative. Fourteen isolates were chosen based on representative colony morphologies and were identified by using the Enterotube II and API 20E systems and fatty acid analysis. Of the 14 isolates, 9 were identified as Providencia rettgeri and 3 were likely Acinetobacter isolates. No evidence was found that the isolates grew on or derived nutrients from the asphalt itself or that they played an essential role in insect development. Regardless, any bacteria found in the oil fly larval gut are likely to exhibit pronounced solvent tolerance and may be a future source of industrially useful, solvent-tolerant enzymes.     

Biodesulfurization in biphasic systems containing organic solvents

Biphasic systems can overcome the problem of low productivity in conventional media and have been exploited for biocatalysis. Solvent-tolerant microorganisms are useful in biotransformation with whole cells in biphasic reactions. A solvent-tolerant desulfurizing bacterium, Pseudomonas putida A4, was constructed by introducing the biodesulfurizing gene cluster dszABCD, which was from Rhodococcus erythropolis XP, into the solvent-tolerant strain P. putida Idaho. Biphasic reactions were performed to investigate the desulfurization of various sulfur-containing heterocyclic compounds in the presence of various organic solvents. P. putida A4 had the same substrate range as R. erythropolis XP and could degrade dibenzothiophene at a specific rate of 1.29 mM g (dry weight) of cells(-1) h(-1) for the first 2 h in the presence of 10% (vol/vol) p-xylene. P. putida A4 was also able to degrade dibenzothiophene in the presence of many other organic solvents at a concentration of 10% (vol/vol). This study is a significant step in the exploration of the biotechnological potential of novel biocatalysts for developing an efficient biodesulfurization process in biphasic reaction mixtures containing toxic organic solvents.