Principal component analysis applied to bacterial cell behaviour in the presence of organic solvents

The behaviour of cells of Rhodococcus erythropolis DCL14, Xanthobacter Py2, Arthrobacter simplex and Mycobacterium sp. NRRL B-3805, in biphasic systems containing different organic solvents was evaluated and compared. The data, obtained mainly by fluorescence microscopy and image analysis, was interpreted using principal components analysis (PCA). With this technique, the variability of the data could be summarised in 7 components, representing 75.8% of the variance of the data. Over a third of the variance could be explained by the first two principal components which represent solvent toxicity. Apparently this is the major factor influencing cell behaviour in an organic:aqueous system. However, factors such as substrate concentration, cell adaptation ability (resulting in morphological changes and aggregation or separation of cells) and membrane composition (specific to each strain) also play an important role in cell resistance to solvent toxicity. The results regarding cell shape indicate that loss of viability occurs, in the tested bacterial strains, after incorporation of molecules of solvent in the cellular membrane. This should result in an increase in membrane fluidity, and thus, in an alteration of cell shape. The ability to form “self-defence” clusters was observed to be different amongst the four strains. X. Py2 showed, in general, a low tendency to form aggregates under the tested conditions; A. simplex and R. erythropolis aggregated mainly in the presence of low log P solvents; and Mycobacterium. sp. cells showed a high ability to aggregate.     

Principal Components Analysis as a Tool to Summarise Biotransformation Data: Influence on Cells of Solvent Type and Phase Ratio

The effect of some solvents, present in different amounts, upon whole cells of Rhodococcus erythropolis DCL14 carrying out the biotransformation of (-)-carveol to (-)-carvone was studied. The solvents tested were ethyl butyrate, n-hexane, cyclohexane, iso-octane, n-dodecane, dimethyl sulfoxide, bis(2-ethylhexyl) phthalate and FC-70. The volumes of each solvent corresponded to organic:aqueous phase ratios of 0.0005, 0.0025, 0.005, 0.025 and 0.2. To assess any potential solvent protection towards substrate toxicity, assays were carried out at two initial carveol concentrations (15 and 50 mM). Carvone accumulation was followed by gas chromatography. Cell viability, several aspects of cell morphology and the ability to form clusters were monitored by fluorescence microscopy. Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions. PCA using the different volumes of each solvent as variable suggests that the variability of the observations can be summarised in six components which represent 79.4% of the variance of the data. Conversely, using cell and solvent data to perform the PCA, 97.1% of the variance of the data can be summarised in three components, the first two capturing 91.0% of the information. These components seem to represent solvent toxicity and a protective effect of the solvent from carveol toxicity.     

Solvent toxicity in organic-aqueous systems analysed by multivariate analysis

The effect of several solvents present in a biphasic reaction system on cells of Rhodococcus erythropolis DCL14, Xanthobacter Py2, Arthrobacter simplex and Mycobacterium sp. NRRL B-3805 was evaluated. These four strains have been widely exploited, from bioremediation to the production of fine chemicals, in two-phase reaction media. The solvents tested were ethyl butyrate, n-hexane, cyclohexane, iso-octane, n-dodecane, DMSO, bis(2-ethylhexyl) phthalate and fluorinert FC-70. The cell population was monitored by fluorescence microscopy and analysis of the images captured provided single-cell-level information on cell viability, morphological factors of both viable and non-viable cells, and on the number of viable cells in clusters. These data, and those concerning the initial carveol concentration, the carbon source used during growth, the adaptation time to the solvent prior to substrate addition, and the properties of the organic solvent, were interpreted using principal components analysis (PCA). For R. erythropolis, X. Py2, and A. simplex, between 70.1 and 80.4% of the variability of the data could be explained by six principal components, while 86.7% of the variance in the results obtained with Mycobacterium sp. could be represented by seven principal components. In all cases, solvent toxicity could explain over a third of the variability in the data. R. erythropolis cells were able to maintain their viability under harsh conditions. A period of contact between cells and solvent, prior to the addition of substrate, was prejudicial for R. erythropolis and A. simplex cells, at least for the most toxic solvents, but was beneficial for X. Py2 cells. The number of Mycobacterium sp. cells in clusters was lower after an adaptation period compared to the number of cells in aggregates when the substrate was added at time zero. The substrates transformed by R. erythropolis and A. simplex cells increased the toxicity of the system by decreasing the log P of the reaction mixture. Hydrocortisone was responsible for a reduction in the ability of A. simplex to respond to stress conditions. Mycobacterium sp. cells were apparently unaffected by -sitosterol. The results obtained are useful for the design of more efficient two-phase reaction systems where solvent toxicity may be overcome in order to increase cell productivity.Paper presented at the International Conference on Trends in Monitoring and Control of Life Science Applications, 7–8 October 2002, Lyngby, Denmark.     

Growth of Gram-negative bacteria in the presence of organic solvents

The growth behavior of Gram-negative bacteria when exposed to high concentrations (50% v/v) of water-insoluble organic solvents was investigated. The solvents were chosen according to their polarity values as denoted by a logarithmically expressed parameter log P, where P is the partition coefficient of a given solvent in an equimolar mixture of octanol and water. The cell growth was measured by the number of colonies developed on a solid agar medium in direct contact with the solvents. All 31 strains tested showed characteristic growth patterns. The survival and subsequent growth of bacteria increased with the increase in the log P value and was found to be strain specific. For all the strains, 100% cell growth was reached from 0% within 0.1–0.4 log P units. Log P₅₀ values, defined as the log P values at which 50% of the cells form colonies, were determined for each bacterial strain. On the whole, Pseudomonas strains were found to be more resistant to apolar solvents than all other bacteria tested. This resistance was dependent not only on the polarities but also on the toxic nature of different organic solvents, the cell membrane components, and to a limited extent, the growth medium. A tenfold increase in the Mg²⁺ concentration in the growth medium enhanced the solvent resistance of E. coli but had no such effect on Pseudomonads. In general, different growth temperatures had no impact on the solvent resistance of the Gram-negative bacteria tested.     

Principal Components Analysis as a Tool to Summarise Biotransformation Data: Influence on Cells of Solvent Type and Phase Ratio

The effect of some solvents, present in different amounts, upon whole cells of Rhodococcus erythropolis DCL14 carrying out the biotransformation of (?)-carveol to (?)-carvone was studied. The solvents tested were ethyl butyrate, n-hexane, cyclohexane, iso-octane, n-dodecane, dimethyl sulfoxide, bis(2-ethylhexyl) phthalate and FC-70. The volumes of each solvent corresponded to organic:aqueous phase ratios of 0.0005, 0.0025, 0.005, 0.025 and 0.2. To assess any potential solvent protection towards substrate toxicity, assays were carried out at two initial carveol concentrations (15 and 50 mM). Carvone accumulation was followed by gas chromatography. Cell viability, several aspects of cell morphology and the ability to form clusters were monitored by fluorescence microscopy. Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions. PCA using the different volumes of each solvent as variable suggests that the variability of the observations can be summarised in six components which represent 79.4% of the variance of the data. Conversely, using cell and solvent data to perform the PCA, 97.1% of the variance of the data can be summarised in three components, the first two capturing 91.0% of the information. These components seem to represent solvent toxicity and a protective effect of the solvent from carveol toxicity.     

The bioreduction of a β-tetralone to its corresponding alcohol by the yeast Trichosporon capitatum MY1890 and bacterium Rhodococcus erythropolis MA7213 in a range of ionic liquids

The stereospecific reduction of 6-Br-β-tetralone to its corresponding alcohol (S)-6-Br-β-tetralol was carried out by the yeast Trichosporon capitatum MY1890 and by the bacterium Rhodococcus erythropolis MA7213, using a range of ionic liquids chosen for the diversity of their composition. The decrease in cell viability of both types of cell upon exposure to ionic liquids was found to be between that determined for cells residing purely in fermentation media, and cells residing in a two-phase mixture of media and organic solvent (toluene). For T. capitatum MY1890 bioconversions, the water miscible hydrophilic ionic liquid [Emim][TOS] gave a reaction profile comparable to that observed in the previously studied water-ethanol (10% v/v) system, in terms of overall rate of reaction (0.2 g (prod) L^-1 h^-1) and conversion (100%). Of the hydrophobic ionic liquids evaluated, [Oc₃MeN][BTA] gave the best conversion of 60%, but at a much reduced rate, suggesting solute mass transfer from the ionic liquid phase was rate limiting. For bioconversions carried out with R. erythropolis MA7213 employing 20% v/v [Emim][TOS] as a co-solvent, the conversion yield doubled, and a four-fold increase in initial rate was found compared to the standard ethanol co-solvent. This was attributed to improved cell viability and reduced aggregation of the R. erythropolis MA7213 compared to T. capitatum MY1890. Overall, this study demonstrates the feasibility of using ionic liquids for whole cell biocatalysis, however, no obvious link is apparent between the physico-chemical properties of ionic liquids, their influence on cell viability, and their efficacy as media for bioconversions.     

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.     

Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia Sp. and the gram-negative bacteria Acinetobacter and Pseudomonas Sp

The toxicity of homologous series of organic solvents has been investigated for the gram-positive bacteria, Arthrobacter sp. and Nocardia sp., and the gram-negative bacteria, Acinetobacter sp. and Pseudomonas sp. The hydrophobicity of the solvent, expressed by its logP(octanol), proves to be a good measure for the toxicity of solvents in a two-phase system. The transition from toxic to nontoxic solvents occurs between logP(octanol) 3 and 5 and depends on the homologous series. No correlation has been found between the hydrophobicity of the substituent on the alkyl backbone of the solvent and the location of the transition point in toxicity. The logP(octanol), above which all solvents are nontoxic, is used to express the solvent tolerance of the bacteria. In general, the solvent tolerance of gram-negative bacteria is found to be slightly higher than that of gram-positive bacteria, but this does not hold for all homologous series of organic solvents investigated.Because the toxicity effects of organic solvents in a two-phase system can be ascribed to molecular as well as phase toxicity effects, molecular toxicity effects were investigated separately in a one-phase system with subsaturating amounts of organic solvent. The solvent concentration in the aqueous phase, at which 50% of the metabolic activity of the bacteria is lost, is used to express solvent toxicity. This concentration is found to be similar for the gram-positive Arthrobacter and the gram-negative Acinetobacter. Assuming the critical membrane concentration theory (G. J. Osborne et al. Enzyme Microb. Technol. 1990, 12: 281-291) to be valid, it can be concluded that differences in solvent tolerance between these two bacteria, cannot be ascribed to differences in response to molecular toxicity. Prediction of the toxicity of any solvent, using the critical membrane theory, appears to be possible in the case of alkanols or alkyl acetates. However, prediction of the toxicity of ethers appears to be impossible. (c) 1993 John Wiley & Sons, Inc.     

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues—value precursors in the synthesis of modern glucocorticoids starting from 9-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and ¹H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.     

Dielectric Spectroscopy: a Rapid Method for the Determination of Solvent Biocompatibility During Biotransformations

Dielectric spectroscopy provides a convenient means of determining the degree of intactness of biological cells. 4-terminal dielectric measurements of suspensions of Saccharomyces cerevisiae at 0.4 MHz show that, as with all other biological cells, these organisms possess a substantial β-dispersion. The additional of octanol to such suspensions causes a rapid decrease in the electrical capacitance of the suspension, which parallels the cellular viability as determined by methylene blue staining. The kinetics of cell death are determined in part by the rate of dissolution of the organic solvent in the aqueous phase. The toxicity of several organic solvents to S. cerevisiae is studied using this technique, and is found to be dependent upon the polarity of the solvent. The present method provides a simple and rapid means for assessing the biocompatibility of solvents used in biotransformations.     

Electrophoretic studies on the Anisakis simplex complex (Ascaridida:Anisakidae) from the Mediterranean and North-East Atlantic

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and 1986. Electrophoretic studies on the Anisakis simplex complex (Ascaridida: Anisakidae) from the Mediterranean and North-East Atlantic. International Journal for Parasitology 16: 633–640. The genetic variation of the sibling species Anisakis simplex A and A. simplex B was investigated by electrophoretic analysis of 22 gene-enzyme systems. The two species are reproductively isolated and no gene flow takes place between them. Three loci, Sod, Adk-2 and Lap-1, show distinct alleles in A. simplex A and A. simplex B, allowing their reliable identification both at the larval and adult stages. A fourth locus, Got, appears to be diagnostic at the 95% level. The value of Nei's genetic distance found between A. simplex A and A. simplex B is 0.28. Parameters of genetic variability (He, P, A) are given for both species. The geographic distribution of A. simplex A and A. simplex B appears to be mainly Mediterranean for the former, and mainly North Atlantic for the latter. Several paratenic hosts (fish and squid) and one cetacean definitive host are identified for each of the two species. The names A. pegreffii and A. simplex are tentatively proposed for A. simplex A and A. simplex B respectively.     

Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms

Anaerobic bacteria such as the solventogenic clostridia can ferment a wide range of carbon sources (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to produce carboxylic acids (acetic and butyric) and solvents such as acetone, butanol, and ethanol (ABE). The fermentation process typically proceeds in two phases (acidogenic and solventogenic) in a batch mode. Poor solvent resistance by the solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability of ABE production by fermentation. The toxic effect of solvents, especially butanol, limits the concentration of these solvents in the fermentation broth, limiting solvent yields and adding to the cost of solvent recovery from dilute solutions. The accepted dogma is that toxicity in the ABE fermentation is due to chaotropic effects of butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnological whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents on inhibition of cell metabolism (nutrient transport, ion transport, and energy metabolism); (2) cell membrane fluidity, death, and solvent tolerance associated with the ability of cells to tolerate high concentrations of solvents without significant loss of cell function; and (3) strategies for overcoming poor solvent resistance in acetone and butanol-producing microorganisms.     

Degradation of hydrocarbons and alcohols by Rhodococcus erythropolis DCL14: A comparison in scale performance

The degradation of alkanes and alcohols by Rhodococcus erythropolis DCL14 growing cells was tested at different reactor scales, ranging from a 2-L fermenter to 300 µL 96-well plates. The highest growth rates were attained in the fermenter, where pH, temperature and aeration rate, in particular, are monitored and controlled. However, the results acquired in reactors smaller than 100 mL indicate that these reactors may be successfully used in testing carbon sources, since the normalized results obtained with the different carbons sources were maintained in the 100-0.3 mL range. This study also shows the great potential of R. erythropolis DCL14 cells to degrade a wide range of alcohols and alkanes, which may be used in bioremediation of contaminated sites.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Optimal selection of organic solvents for biocompatible extraction of beta-carotene from Dunaliella salina

In the aim of beta-carotene biocompatible extraction, toxicity of various pure solvents belonging to different homologous series has been investigated for Dunaliella salina. The results showed that solvents having logP(oct) > 5 or having a molecular weight over 150 g/mol can be considered biocompatible for this microalga. The membrane critical solvent concentration for each series of solvents has been calculated applying Osborne's model, showing that the aliphatic chlorinated hydrocarbon is the most toxic family studied. Mixtures of a biocompatible solvent (decane) with a toxic solvent (CH(2)Cl(2), MEK, MTBE) have been studied. The beta-carotene extraction ability of CH(2)Cl(2)-decane mixture was found six times more efficient than with pure decane. It has been demonstrated that the extraction ability of solvent depends on its affinity with the product extracted and on its concentration incorporated in the cellular membrane.     

Asymmetric syntheses of nitroalkanols using Pseudomonas sp. lipase: a proposal for the selection of the solvent system of lipase-catalyzed transesterification

By lipase-catalyzed stereoselective transesterification using Amano AK from Pseudomonas sp., nitroalcohols such as 1-nitro-2-butanol and 1-nitro-3-methyl-2-butanol were synthesized enantioselectively with enantiomeric ratios (E values) of 20.9 and 12.5, respectively, in n-propyl ether. Various results were obtained during the lipase-catalyzed transesterification by changing the organic solvents that were used. The plots of the E values against the reciprocal of the dielectric constants () of the various organic solvents produced a bell-shaped curve which had a maximum E value for n-propyl ether (1/=0.3). The distance between the enzyme and the substrate might be changed in response to a change in the organic solvent.     

Antiviral activity of the volatile oils of Melissa officinalis L. against Herpes simplex virus type-2

Melissa officinalis L. (Lamiaceae) has been used in a variety of practical applications in medical science. Our objective in the current study was to determine the effects of the volatile oil components of M. officinalis on Herpes simplex virus type 2 (HSV-2) replication in HEp-2 cells. Four different concentrations (25, 50, 100, 150 and 200 μg/ml) of volatile oils were examined. Experiments were carried out using HEp-2 cells. M. officinalis volatile oil was found to be non-toxic to HEp-2 cells up to a concentration of 100 μg/ml. It was, however, found to be slightly toxic at a concentration over of 100 μg/ml. The antiviral activity of non-toxic concentrations against HSV-2 was tested. The replication of HSV-2 was inhibited, indicating that the M. officinalis L. extract contains an anti-HSV-2 substance.     

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     

Production of solvents (ABE fermentation) from whey permeate by continuous fermentation in a membrane bioreactor

A continuous bioreactor where cells were recycled using a cross-flow microfiltration (CFM) membrane plant was investigated for the production of solvents (ABE fermentation) from whey permeate using Clostridium acetobutylicum P262. A tubular CFM membrane plant capable of being backflushed was used.The continuous fermentations were characterized by cyclic solventogenic and acidogenic behaviour, and ultimately degenerated to an acidogenic state. Steady-state solvent production was obtained for only short periods. This degeneration is attributed to the complex morphological behaviour of this strain of organism on this substrate.It is postulated that to achieve steady-state solvent production over extended periods of time, it is necessary to maintain a balance among the various morphological cell forms, i.e. acid-producing vegetative cells, solvent-producing clostridial cells, and inert forms, e.g. spores.     

Cochliopodium barki n. sp. (Rhizopoda, Himatismenida) re-isolated from soil 30 years after its initial description

A strain of Cochliopodium isolated from grassland soil at Sourhope Research Station (Scotland, UK) was found to be identical to the strain “Cochliopodium sp.2” studied by Bark in 1973. We name it Cochliopodium barki. It belongs to a group of species (comprising also C. minus and Cochliopodium sp. “NYS strain”) with very similar scale pattern.