Characterization of an organic-solvent-tolerant Brevibacillus agri strain 13 able to stabilize solvent/water emulsion

Brevibacillus agri strain 13 was isolated and characterized as a Gram-positive organic-solvent-tolerant bacterium able to grow at 45 °C. It can tolerate high concentrations (5% and 20%, v/v) of various organic solvents with a broad range of log P _ow when the organic solvent was provided as a nonaqueous layer. Although it can tolerate a number of aromatic solvents, it cannot utilize them as a sole carbon source. The surface characteristics of cells exposed to organic solvent were investigated using the bacterial adhesion to hydrocarbon test, a contact angle measurement, ζ potential determination, and fluorescence microscopy analysis and compared with that of nonexposed cells. The results showed that although it has a hydrophilic cell surface, it has a unique indigenous cell surface characteristic in which the cells can stabilize solvent-in-water emulsion by adhering to the solvent–water interface of the solvent droplets. The tolerance and predilection of B. agri strain 13 toward organic solvents may suggest its potential application as a whole-cell biocatalyst for the biotransformation process of water-immiscible substrate(s).     

Solvent selection for enhanced bioproduction of 3-methylcatechol in a two-phase partitioning bioreactor

The biotransformation of toluene to 3-methycatechol (3MC) via Pseudomonas putida MC2 was used as a model system for the development of a biphasic process offering enhanced overall volumetric productivity. Three factors were investigated for the identification of an appropriate organic solvent and they included solvent toxicity, bioavailability of the solvent as well as solvent affinity for 3MC. The critical log P (log P(crit)) of the biocatalyst was found to be 3.1 and log P values were used to predict a solvent's toxicity. The presence of various functional groups of candidate solvents were used to predict the absorption of 3MC and it was found that solvents possessing polarity showed an affinity towards 3MC. Bis (2-ethylhexyl) sebecate was selected for use in the biphasic system as it fulfilled all selection criteria. A two-phase biotransformation with BES and a 50% phase volume ratio, achieved an overall volumetric productivity of 440 mg 3MC/L-h, which was an improvement by a factor of approximately 4 over previously operated systems. Additional work focused on reducing the toluene feed in order to minimize possible toxicity and decrease loss of substrate (toluene), a result of volatilization. Toluene losses were reduced by a factor of 4, compared to previously operated systems, without suffering an appreciable loss in overall volumetric productivity.     

A protease stable in organic solvents from solvent tolerant strain of Pseudomonas aeruginosa

A solvent tolerant strain of Pseudomonas aeruginosa (PseA) was isolated from soil samples by cyclohexane enrichment in medium. The strain was able to sustain and grow in a wide range of organic solvents. The adaptation of P. aeruginosa cell towards solvents was seen at membrane level in transmission electron micrographs. It also secreted a novel protease, which exhibited remarkable solvent stability and retained most of the activity at least up to 10 days in the presence of hydrophobic organic solvents (log P > or = 2.0) at 25% (v/v) concentrations. The protease was able to withstand as high as 75% concentration of solvents at least up to 48 h. P. aeruginosa strain and its protease, both seem promising for solvent bioremediation, wastewater treatment and carrying out biotransformation in non-aqueous medium.     

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log P_octanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between ? 1.1 and 8.8 were examined. Organic solvents with log P_octanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL^?1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.     

Biphasic aqueous/organic biotransformation of acetaldehyde and benzaldehyde by Zymomonas mobilis pyruvate decarboxylase

Zymomonas mobilis pyruvate decarboxylase (PDC) transformed acetaldehyde and benzaldehyde into (R)-phenylacetylcarbinol (PAC), the precursor for the synthesis of ephedrine and pseudoephedrine. Organic solvents were screened for a biphasic biotransformation with the enzyme in an aqueous phase and the toxic substrates delivered through the organic phase. In the absence of substrates a second phase of 1-pentanol, hexadecane or MTBE (methyl tertiary-butyl ether) stabilized the PDC activity in comparison to a control without added solvent. Organic phase solvents for optimal PAC production had partitioning coefficient (log P) values between 0.8 and 2.8 (production of more than 8 mg PAC/ U PDC), however there was no correlation between enzyme stability and log P. Best PAC formation was observed with the eight tested alcohols, which in contrast to the other solvents allowed lower initial concentrations of toxic acetaldehyde (54-81 mM) in the aqueous phase. 1-pentanol, 1-hexanol, and isobutanol resulted in the highest specific PAC production of 11 mg PAC /U PDC. Without the addition of an organic phase, only 1.2 mg/U was formed.     

Modification Effects of Organic Solvents on Microenvironment of the Enzyme in Thermolysin-catalyzed Peptide Synthesis of N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine Methyl Ester

Thermolysin-catalyzed peptide synthesis using N-benzyloxycarbonyl)-l-phenylalanine (Z-Phe) and l-phenylalanine methyl ester (Phe-OMe) as substrates was done mainly in a water-organic one phase solvent system. The organic solvent content used was less than the saturation concentration in buffer. With organic solvents with high log P values, the enzymatic activity increased as the organic solvent content increased; but further increases in the organic solvent content decreased the enzymatic activity, showing an “organic activity” profile. On the other hand, with organic solvents of low log P values, the enzymatic reaction was inhibited even by the initial addition of organic solvents. When a correlation between maximum activities and logP values or Hildebrand solubility parameters was investigated, a linear correlation was obtained among the same category of organic solvents, but not between categories. This suggests that the direct effect of organic solvents on the microenvironment of the enzyme largely depends on the molecular structure of the solvents.     

Rules for optimization of biocatalysis in organic solvents

General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.     

Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump

Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log P(OW) of the solvent, where P(OW) is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of delta acrAB and/or delta tolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log P(OW) = 5.5). The delta tolC mutant was more sensitive to nonane than the delta acrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log P(OW) higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The delta tolC mutant accumulated n-nonane or decane (log P(OW) = 6. 0) more abundantly than the parent or the delta acrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log P(OW) in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane.     

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 hydrophilic and hydrophobic organic solvent mixture enhances enzyme stability in organic media

Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (~ >4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

Organic solvents for bioorganic synthesis

Summary The influence of solvents on enzymatic activity and stability was investigated. As a model reaction the -chymotrypsin-catalyzed esterification of N-acetyl-l-phenylalanine with ethanol was used. The enzyme was adsorbed on porous glass beads and used in various solvents. Small amounts of water were added to increase the enzymatic activity. These enzyme preparations obeyed. Michaelis-Menten kinetics. K _m,app decreased slightly with the log P value of the solvent while V _app increased markedly with the log P value. Log P values were also useful for generalizing the influence of solvents on enzyme stability. The enzyme preparations showed a markedly higher thermostability in dry solvents having log P values >0.7 than in less hydrophobic solvents.Also the operational stability was better in the more hydrophobic solvents. The amount of water added to the enzyme preparations greatly influenced the initial reaction rates. For some solvents optimal water contents were determined. The thermostability decreased with increasing water content.The observations are summarized in the conclusion that more hydrophobic solvents are preferable to less hydrophobic ones. The log P value gives a good guidance when selecting an organic solvent for enzymatic conversions.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

Use of non-conventional media inMorinda citrifolia cell cultures

Non-conventional media, containing organic solvents as supplement, were exploited to obtain the non-lethal product release of plant cell secondary metabolites, using suspension cultures ofMorinda citrifolia as model system. The results of our preliminary studies about solvent biocompatibility show that the discrimination between biocompatible and toxic solvents can be achieved by means of two parameters: log P and critical solvent concentration. The last one seems to be a better indicator of solvent toxicity for living cells.Abbreviations log P logarithm of P - P solvent partition coefficient in a standard system n-octanol/water     

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.     

Role of the Multidrug Efflux Systems of Pseudomonas aeruginosa in Organic Solvent Tolerance

Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in Pseudomonas aeruginosa. Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism. Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as n-hexane and p-xylene. Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of P. aeruginosa. Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent. These data suggest that the efflux of organic solvents is a factor in the tolerance of P. aeruginosa to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics.     

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.     

Relation Between the Dielectric Constant of Hydrophobic Cation Exchange Membrane and Membrane Permeability to Counterions

Filters made of cellulose acetate-nitrate when saturated with organic solvents and interposed between aqueous solutions form membranes which behave like cation exchangers. The diffusion coefficients of counterions in such membranes are strongly dependent upon the dielectric constant of the saturating solvent. The results obtained suggest that a linear relationship between the log of the cation's diffusion coefficient (or membrane conductance) and the reciprocal value of the dielectric constant of the saturating solvent exists. There is also a good correlation between the relative membrane permeability to organic cations and the solubility of the cations in the pure solvent phase. These studies indicate that there are two routes for cation movement through the membrane: (a) the bulk hydrophobic phase and (b) continuous narrow aqueous channels.     

Screening of organic solvents for bioprocesses using aqueous-organic two-phase systems

The application of conventional organic solvents has been essential in several steps of bioprocesses in order to achieve sufficient economic efficiency. The use of organic solvents is frequently used either to partly or fully replace water in the reaction medium or as a process aid for downstream separation.Nowadays, manufacturers are increasingly requested to avoid and substitute solvents with hazardous potential. Therefore, the solvent selection must account for potential environmental hazards, health and safety problems, in addition to fulfilling the ideal characteristics for application in a process.For the first time, criteria including Environment, Health and Safety (EHS), as well as the technical requirements for reaction and separation have been reviewed, collected and integrated in a single organic solvent screening strategy to be used as a guideline for narrowing down the list of solvents to test experimentally. Additionally, we have also included a solvent selection guide based on the methodology developed in the Innovative Medicines Initiative CHEM21 (IMI CHEM21) project and applied specifically to water-immiscible solvents commonly used in bioprocesses.     

Enhanced bioproduction of carvone in a two-liquid-phase partitioning bioreactor with a highly hydrophobic biocatalyst

The microbial biotransformation of (-)-trans-carveol to the flavor and fragrance compound (R)-(-)-carvone by Rhodococcus erythropolis DCL14 was carried out in a 3 L two phase partitioning bioreactor with an immiscible liquid second phase in an effort to improve upon the reactor performance achieved in a single aqueous phase system. The purpose of employing the liquid second phase is to minimize biotransformation rate inhibition due to the accumulation of the toxic substrate (cis-carveol) and product (carvone) in the aqueous phase. 1-Dodecene was chosen as the solvent for this application because it is biocompatible, non-biodegradable and has a superior affinity for the target product (carvone) relative to the other solvents tested. However, when 1-dodecene was used in the biotransformation, the extremely hydrophobic R. erythropolis DCL14 created an emulsion with the organic solvent with significant sequestering of the cells into the organic phase and negligible substrate conversion. To overcome these operational difficulties, silicone oil, which is considered a liquid polymer, was used with the aim of preventing emulsification and sequestration of cells in the non-aqueous phase. Although some emulsification of the water-silicone oil was again created by the cells, operability was improved and, in fed-batch mode, the system was able to convert approximately 2(1/2) times more carveol than a benchmark single aqueous phase system before substrate/product toxicity caused the biotransformation to stop. This study has demonstrated enhancement of a microbial biotransformation for the production of a high value nutraceutical compound via the use of a second partitioning phase, along with operational challenges arising from the use of a highly hydrophobic organism in such systems.