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Keywords:

  • cell membrane;
  • lipase;
  • Pseudomonas aeruginosa PseA;
  • solvent hydrophobicity;
  • solvent tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Organic solvents are generally considered to be microbicidal agents, owing to their toxic effects on the cell membrane. They tend to accumulate in bacterial membranes and severely disturb the membrane structure (Kieboom et al. 1998). In recent years, few micro-organisms able to grow in high solvent concentrations have been isolated and defined as solvent-tolerant microbes. These have drawn considerable attention as attractive biosystems for solvent bioremediation and biotransformation in nonaqueous media (Sardessai and Bhosle 2004; Tang et al. 2008; Gupta and Khare 2009). These microbes are also perceived to be excellent sources of enzymes functional in solvents (Isken and de Bont 1998). Properties of solvent tolerance have been encountered mainly in the genera Pseudomonas, Bacillus, Flavobacterium, Rhodococcus and Staphylococcus (Ogino and Ishikawa 2001; Fang et al. 2006).

The toxic effect of solvents is circumvented in these microbes through several adaptation strategies viz. (i) change in fatty acid conformation of the lipid bilayer from cis to trans and saturation of the fatty acid acyl chains, (ii) modification of the lipopolysaccharides of the outer membrane, (iii) modification of the cell surface hydrophobicity and (iv) active excretion of solvents through energy-dependent efflux pumps (Sikkema et al. 1995; Kieboom et al. 1998). Toluene efflux pumps are involved in solvent tolerance in several Gram-negative strains. The number of efflux pumps has been correlated with the degree of solvent tolerance in different Pseudomonas putida strains (Ramos et al. 2002).

In the present study, a solvent-tolerant strain of Pseudomonas aeruginosa PseA isolated by cyclohexane enrichment has been used (Gupta and Khare 2006). We have attempted to look into the possible adaptation mechanisms followed by this strain to survive under solvent-rich conditions. The effect of solvents on cell membrane, its surface hydrophobicity, cell morphology, cell size and permeability has been investigated by a combination of microscopic and biochemical techniques.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Micro-organism

Pseudomonas aeruginosa PseA, an organic solvent-tolerant micro-organism was maintained and subcultured as described previously (Gupta and Khare 2006). Inoculum was prepared by transferring loopful of this stock culture to the growth medium (nutrient broth) and cultivating at 30°C with shaking at 120 rev min−1 until the absorbance at 660 nm (A660) reached to 1·0.

Cultivation of PseA in the presence of solvents

Pseudomonas aeruginosa PseA was cultivated in 100 ml growth medium overlaid with 50 ml of solvents medium, without solvent served as control. Then, 1% (v/v) inoculum was used for seeding the medium. Incubation was carried out at 30°C and 140 rev min−1. Samples were withdrawn after 24 h, and growth was recorded as A660. These samples were centrifuged at 10 000 g for 10 min at 4°C. Lipase activity was determined in the cell-free supernatants using p-nitrophenyl palmitate as substrate (Kilcawley et al. 2002). One unit of enzyme activity is defined as the amount of enzyme releasing 1 nmol of p-nitrophenol.

Transmission electron microscopy (TEM)

Pseudomonas aeruginosa PseA was grown in the presence of 33% (v/v) tetradecane at 30°C. Cells of log phase (14 h) were centrifuged at 10 000 g for 10 min and processed for TEM as described by David et al. (1973). The images were recorded in TEM CM-10 model (Philips, Tokyo, Japan).

Cell membrane permeability

Permeability was followed as a function of the release of nucleic acid during the growth of PseA cells in the presence of solvents. The cells were grown on 100 ml medium supplemented with or without 50 ml solvents at 30°C and 140 rev min−1. Samples were withdrawn periodically and centrifuged at 10 000 g. Absorbance of cell-free supernatant was recorded at 260 nm on Specord 200 (Analytikjena, Jena, Germany).

Atomic force microscopy (AFM)

Pseudomonas aeruginosa was cultivated in the absence and presence of 50 ml tetradecane/cyclohexane at 30°C. Cells of log phase were harvested, washed and processed for AFM as described by Sullivan et al. (2005). The AFM images were recorded on AFM system (Nanoscope IIIa; Vecco Metrology Group, Santa Barbara, CA, USA) in contact mode with a scan rate of about 2·0 Hz.

Microbial adhesion to hydrocarbon (MATH) test

Pseudomonas aeruginosa PseA cells of log phase cultivated in the absence and presence of solvents at 30°C were centrifuged at 8000 g and resuspended in saline (0·8% NaCl) to A660 = 0·6. The suspension was analysed by the MATH test as described by Aono and Kobayashi (1997).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We have previously described isolation and characterization of a solvent-tolerant strain P. aeruginosa PseA (Gupta and Khare 2006). It was observed to be an extracellular lipase producer in the absence of solvents (Gaur et al. 2008). In the present study, lipase production is checked in the solvent-supplemented medium. Figure 1 shows the level of lipase secretion when PseA was grown in the presence of solvents of varying log P-values. Appreciable lipase activity was observed only in the medium supplemented with tetradecane (Log P 7·6). Lipase production was negligible in other solvents-supplemented media, lowest being in cyclohexane (lowest log P).

image

Figure 1.  Lipase production by Pseudomonas aeruginosa PseA in the presence of solvents. Pseudomonas aeruginosa PseA was cultivated in nutrient medium with solvents of varying log P-values. Samples were withdrawn after 24 h, and growth was recorded as A660. Lipase activity was determined in the cell-free supernatants using p-nitrophenyl palmitate as substrate. Numbers in bracket represent the log P-value of that solvent. (empty bars) Growth and (-○-) lipase activity.

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To reveal changes occurring at the membrane level in the presence of cyclohexane and tetradecane, transmission electron micrographs of PseA cells were recorded. In case of the cells grown in the presence of tetradecane, the cell membrane was intact (Fig. 2b) as that in control (Fig. 2a, without solvent), and no accumulation of solvent was seen in the cytoplasm.

image

Figure 2.  Transmission electron micrographs of Pseudomonas aeruginosa PseA. Pseudomonas aeruginosa PseA was grown in the absence and presence of solvents (33%, v/v). Cells of log phase were processed for transmission electron microscopy as described. (a) Cells in the absence of tetradecane; (b) cells in the presence of tetradecane (exposure: 31 000).

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In general, solvents exert their toxic effect by altering the cell membrane permeability. The release of nucleic acid components into the medium during the growth of PseA in the presence of cyclohexane and tetradecane was recorded as function of time. Results in Fig. 3 demonstrate that in case of cyclohexane, the release of internal cellular components increased with time. However, no such release was observed in control and tetradecane-supplemented medium.

image

Figure 3.  Nucleic acid release by Pseudomonas aeruginosa PseA. Pseudomonas aeruginosa cells were grown in nutrient medium in the absence (control) and presence of 33% (v/v) solvents. Samples were withdrawn periodically, and absorbance of cell-free supernatant was recorded at 260 nm. (-•-) Control; (-○-) tetradecane and (-Δ-) cyclohexane.

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To further substantiate the difference in the PseA cells grown in cyclohexane and tetradecane, AFM was carried out. Figure 4(a–c) shows that morphology of P. aeruginosa PseA cells is affected in the presence of cyclohexane. The image statistics of PseA at a scan size of 2 μm showed reduction in cell size in case of cyclohexane from a root mean square (RMS) value of 26·09 (in control cells grown in the absence of solvents) to 20·52 nm (data not shown), whereas no significant changes were observed in case of tetradecane (RMS = 27·01 nm). Therefore, the cells grown in tetradecane were similar to the control cells.

image

Figure 4.  Atomic force microscopy of Pseudomonas aeruginosa PseA. Pseudomonas aeruginosa was cultivated in nutrient medium supplemented with tetradecane or cyclohexane. Cells of log phase were harvested, washed and processed for AFM as described. (a) Control (without solvent); (b) cells grown in tetradecane; (c) cells grown in cyclohexane.

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Another important index to relate interaction of solvents with cell membrane is the hydrophobicity of cell surface. Microbial adhesion to hydrocarbon test was performed to see the changes in cell surface hydrophobicity as a function of solvent hydrophobicity. Results show that the cell surface hydrophobicity increased with increasing hydrophobicity of the solvent. The MATH value for control (without solvent) was found to be 15%. This value increased to 20% when P. aeruginosa was grown in cyclohexane (log P 3·2) and 30% in tetradecane (log P 7·6).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The production of lipase in tetradecane-supplemented medium and no production in cyclohexane-supplemented medium by P. aeruginosa PseA signified some differences in the bacterial response to the type of solvent it encountered. Cyclohexane and tetradecane were continued as model solvent systems to study the difference in PseA cellular changes/adaptations.

The solvents profoundly affect membrane integrity and structure, thereby altering the permeability and incurring toxicity. Transmission electron micrographs of PseA cells grown in the presence of tetradecane showed no apparent changes in cell membrane, in contrary to cyclohexane, where accumulation of solvent in cytoplasm with a disturbed cell membrane has been reported (Gupta and Khare 2006). This infers that the two solvents with different hydrophobicities: cyclohexane (log P 3·2) and tetradecane (log P 7·6) are tolerated by PseA through different adaptation mechanisms.

The effect of these solvents on cell membrane permeability was also studied. The release of nucleic acid components into the medium in the presence of cyclohexane and not in tetradecane supports the results of TEM, which showed changes in the PseA cell membrane in cyclohexane-supplemented medium but not the tetradecane supplemented. Similar effect of phenol on Escherichia coli membrane leading to release of internal cellular components has been reported by Heipieper et al. (1991). Gram-negative bacteria are known to have porin (proteins) embedded in the lipopolysaccharides matrix of outer membrane. These proteins act as a barrier for hydrophobic compounds and big molecules (Neumann et al. 2006). In case of P. aeruginosa, such proteins might be responsible for inaccessibility of highly hydrophobic and larger tetradecane molecules across the membrane whereas providing easy passage to less hydrophobic and smaller cyclohexane molecules.

AFM, a powerful technique for investigating nanometric physicochemical and mechanical properties of the cell surface (Sullivan et al. 2005), was used to study the PseA cells. Cyclohexane caused the cells to aggregate. This effect has been seen as one of the tolerance mechanisms reported in case of E. coli to survive in the presence of phenol (Heipieper et al. 1991). Aggregation of mutant Bradyrhizobium japonicum cells caused by the deficiency of lipopolysaccharides components on the cell surface has also been reported previously (Park and So 2000).

PseA cells showed a decrease in cell size when grown in cyclohexane. This may be attributed to more affinity of less hydrophobic molecules (cyclohexane) for the available water in the membrane. Similar reduction in P. aeruginosa cell size as a result of growth on crude oil medium has been observed earlier (Norman et al. 2002).

When bacteria are grown in an aqueous-organic biphasic system, the cell surface properties are modified to combat the toxic effects of solvents. Higher cell surface hydrophobicity in tetradecane medium as indicated by higher MATH values explains it to be a better way to survive under highly hydrophobic condition. Alteration in surface properties of P. aeruginosa under environmental stress viz. high temperature and oxygen stress conditions have been observed previously (Sabra et al. 2003). Increase in cell surface hydrophobicity of Pseudomonas sp. TIS1-127 by decreasing the cultivation temperature from 37 to 28°C has also been observed (Hori et al. 2009), confirming cell surface behavior to be one of the mechanisms for survival under altered culture conditions.

All the above results about solvent adaptability of P. aeruginosa PseA indicate that (i) the presence of less hydrophobic (low Log P) solvent causes changes in cell membrane leading to release in cell components (ii) cells adapt to organic solvents by changing morphology, size, permeability and surface hydrophobicity and (iii) solvent adaptability depends on the nature of solvent used, and solvents with higher hydrophobicity are better tolerated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The financial support by Department of Biotechnology (DBT) and Department of Science and Technology (DST), Ministry of Human Resource Development (MHRD), Government of India, is gratefully acknowledged. Author RG is thankful to IIT Delhi and Council for Scientific and Industrial Research (Government of India) for research fellowship.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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