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Keywords:

  • desulfurization;
  • dibenzothiophene;
  • fatty acid methyl esters;
  • light gas oil;
  • Mycobacterium phlei

Abstract

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

Aim:  To compare few phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10.

Methods and Results:  In the present study, dibenzothiophene (DBT) desulfurizing activity, composition of fatty acids of cell membranes, DBT sulfone monoxygenase gene (bdsA) and the selection pressure applied during the growth and enrichment of the bacterial strains M. phlei SM120-1 and M. phlei GTIS10 were compared in our laboratory. The DBT desulfurization activity of M. phlei SM120-1 was found to be 0·17 ± 0·02 μmol 2-HBP min−1 (gram dry cell weight)−1 and that of the bacterial strain M. phlei GTIS10 was 1·09 ± 0·05 μmol 2-HBP min−1 (gram dry cell weight)−1. Fatty acid methyl ester analysis of cell membranes of these two bacterial strains in the presence of light gas oil showed that both the strains had different fatty acid profiles in their cell membranes. Comparison of the full gene sequences of the desulfurization gene bdsA in the two bacterial strains showed significant difference in the bdsA gene sequences. There was a significant difference observed in the selection pressure applied during the growth and enrichment of the two bacterial strains.

Conclusions:  The results of the comparative study of the bacterial strains, M. phlei SM120-1 and M. phlei GTIS10 showed that there were considerable differences in the phenotypic and genotypic characteristics of these two strains.

Significance and Impact of Study:  The present study would broaden the understanding of biodesulfurization trait at intra-species level.


Introduction

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

Combustion of petroleum fractions containing sulfurous compounds is one of the major causes of acid rain, resulting in serious environmental distress. As much as 70% of the sulfur in these fuels may be in the form of heterocyclic organic compounds, such as benzothiophene (BT), dibenzothiophene (DBT) and more complex thiophenes (Finnerty and Robinson 1986). DBT is generally accepted as the model sulfur compound present in fossil fuels. The conventional Hydrodesulfurization process could not remove recalcitrant organosulfur compounds such as alkylated dibenzothiophene (DBT) derivatives (Ohshiro and Izumi 2002). A variety of bacterial cultures possess the ability to selectively remove sulfur from dibenzothiophene and diesel (Kilbane and Le Borgne 2004). These bacterial strains desulfurise DBT through a sulfur specific degradation pathway (Oldfield et al. 1997) with selective cleavage of carbon–sulfur bonds.

Kayser et al. (2002) isolated a moderately thermophilic bacterial strain Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization, from soils contaminated with coal or petroleum hydrocarbons. We also had isolated a moderately thermophilic bacterial strain M. phlei SM120-1 from a dibenzothiophene amended soil microcosm, capable of desulfurization of dibenzothiophene and middle-distillate range fuels. Both of these thermophilic desulfurizing bacterial strains followed 4S pathway for DBT desulfurization as evident from the metabolites described in this study for M. phlei SM120-1 and as reported earlier for M. phlei GTIS10 by Kayser et al. (2002). We also compared the desulfurization abilities of the bacterial strains M. phlei GTIS10 and M. phlei SM120-1 with respect to DBT and light gas oil. In addition, we also attempted to compare the desulfurization gene, bdsA, membrane fatty acid profiles, the selection pressure applied during the growth and enrichment of the strains, in order to broaden the understanding of biodesulfurization trait at intra-species level.

Materials and methods

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

Source of bacteria and growth conditions

The desulfurizing bacterial strain M. phlei SM120-1 was isolated from a dibenzothiophene amended soil microcosm based on its ability to desulfurise dibenzothiophene and used in the present study. The bacterial strain was taken from the TERI's culture collection for hydrocarbon degrading bacteria, sponsored by Department of Biotechnology, Government of India. The moderately thermophilic bacterial strain M. phlei GTIS10 was kindly provided by Dr John J. Kilbane II, Gas Technology Institute, IL, (USA). The M. phlei strains SM120-1 and GTIS10 were cultivated in the sulfur free basal salts medium (BSM; Kilbane and Jackowski 1992) with 0·54 mmol DBT as the sole source of sulfur.

Biodesulfurization of light gas oil (LGO) by growing cells and resting cells of M. phlei

For the desulfurization experiments of the light gas oil using growing cells, 1% (v/v) of the cells of M. phlei SM120-1 and GTIS10 (OD660 ∼ 1·0) grown with DBT as sole source of sulfur were inoculated into 500-ml Erlenmeyer flasks containing 100 ml of AF medium (Furuya et al. 2001) with 2% (v/v) of LGO in separate experiments and the reaction was allowed to proceed for 7 days at 45°C with agitation at 200 rpm. Biodesulfurization of LGO by resting cells of the both the M. phlei strains was performed using the protocol described by Kirimura et al. (2001).

Estimation of desulfurization efficiency of the bacterial strains towards dibenzothiophene

Cell growth was measured spectrophotometrically (Shimadzu, Kyoto, Japan) at 660 nm. Desulfurization efficiency of both the M. phlei strains was estimated using high-performance liquid chromatography (type LC-10A; Shimadzu) as described by Furuya et al. (2001) and also by gas chromatography (GC, type GC-2010; Shimadzu) using the protocol described by Kirimura et al. (2004).

Estimation of desulfurization efficiency of the bacterial strains towards LGO

For estimation of extent of desulfurization of LGO after the biodesulfurization reaction, the culture broth containing the biodesulfurized petroleum fraction (LGO or diesel) was centrifuged at 24 000 g for 1 h at 20°C, and the oil phase was collected as the upper fraction. The total sulfur contents of the LGO after biodesulfurization was determined by total sulfur analyser (ANTEK, 7000 V; Antek, Houston, TX, USA).

Profiling of major membrane fatty acids of the M. phlei strains

The fatty acid profiling of the two M. phlei strains SM120-1 and GTIS10 was performed using the protocol described by Kaushik (2002). The chromatography was accomplished using Claurus 500 gas chromatograph (Perkin Elmer, Fremont, CA, USA) coupled with Claurus 500 mass spectrometer (Perkin Elmer). A PE-Elite-5 capillary column (ID 0·32 mm, length 15 m and 0·25 μm film thickness) was used with helium as the carrier gas. The oven temperature was initially kept at 80°C and was increased to 300°C at a rate of 5°C min−1, with a final holding time of 20 min at 300°C. The mass spectrometer was operated at 70 eV of electron ionization energy. The injector and detector temperatures were set at 200 and 300°C respectively. The fatty acid profiling was carried out by comparison of the chromatograms with various mass spectral libraries as well as with chromatograms of authentic standards.

PCR amplification, cloning, sequencing and phylogenetic analysis of bdsA gene

Genomic DNA from M. phlei SM120-1 was used as the template for the amplification of bdsA gene using PCR, following the protocol described by Kayser et al. (2002). The 1·2 kb PCR amplified bdsA gene was purified with GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Piscataway, NJ, USA) and later ligated with pGEM T-easy vector (Promega, Madison, WI, USA) and the ligation mixture was used to transform Escherichia coli JM109 using electroporation (Pulse 2·5 kV/25 mpd/200 ohm). Plasmids from the positive clones were isolated using Kurabo Automated DNA extraction system (Kurabo; Shimadzu) and the sequence was determined for an 1139-bp region of the amplified 1·2 kb bdsA gene. Sequence assembly and analysis were performed using GENETYX-MAC ver 10·1 (SDC, Tokyo, Japan). Sequence comparisons were performed with the FASTA program of the DNA Data Bank of Japan (DDBJ) and the blast program of the National Center for Biotechnology Information (NCBI; Bethesda, MD, USA). The multiple sequence alignment of deduced amino acid residues was performed using clustalw (Thompson et al. 1994) program of Genome Net facility at the Kyoto University Bioinformatics Center, Japan.

Nucleotide sequence accession numbers

The nucleotide sequences of bdsA gene of the bacterial strain M. phlei SM120-1 was submitted to GenBank (NCBI) under accession number, DQ223653.

Chemicals

DBT, 2-HBP were purchased from Tokyo Kasei (Tokyo, Japan). Tri reagent was purchased from Sigma-Aldrich Japan K.K, Tokyo, Japan. All other reagents were of analytical grade and commercially available. The hydrodesulfurized LGO was kindly supplied by the Japan Cooperation Center, Petroleum (Shizuoka).

Results

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

Estimation of desulfurization efficiency of the M. phlei strains SM120-1 and M. phlei GTIS10

The bacterial strain M. phlei SM120-1 desulfurized DBT to 2-HBP, following the 4 S pathway of desulfurization. The end product 2-HBP present in the culture filtrate had been identified by GC-MS (Fig. 1). Based on the HPLC analysis results, DBT desulfurization efficiency of the bacterial strain M. phlei SM120-1 was estimated to be 0·17 ± 0·02 μmol 2-HBP min−1 (gram dry cell weight)−1. Whereas, DBT desulfurization activity of the strain M. phlei GTIS10 was estimated to be 1·09 ± 0·05 μmol 2-HBP min−1 (gram dry cell weight)−1. The M. phlei strain SM120-1 could grow well in AF medium (Furuya et al. 2003) containing LGO as sole source of sulfur and reach an OD value of 1·2 (OD660 ∼ 1·2) with in 96 h. The growing cells of the M. phlei SM120-1 could also desulfurise light gas oil from initial sulfur content of 508–151 ppm with in 7 days at 45°C accounting for 70% of total sulfur removal from the LGO. The bacterial strain GTIS10 was unable to grow in AF medium containing LGO as sole source of sulfur as evidenced by the poor growth of the strain (OD660 ∼ 0·4) under similar conditions. The resting cells of the bacterial strain M. phlei SM120-1 desulfurized LGO from 224 to 76 ppm within six consecutive reactions of 24 h, whereas, the resting cells of the bacterial strain M. phlei GTIS10 could reduce sulfur to 210 ppm from an initial concentration of 224 ppm and no further reduction in sulfur was evidenced even after extended reaction times.

image

Figure 1. Mass spectrograph depicting 2-HBP (m/z 170) in the culture filtrate of the bacterial strain Mycobacterium phlei SM120-1. The molecular mass of 2-HBP (m/z 170) and DBT (m/z 184) are indicated in the figure along with the structures.

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Comparison of major membrane fatty acid profiles of M. phlei SM120-1 and M. phlei GTIS10

Fatty acid methyl ester analysis (FAME) using GCMS showed that major fatty acid groups present in the cell membranes of both the strains were belonging to hexadecanoic and octadecanoic fatty acids, when DBT was the sole sulfur source for bacterial growth (Fig. 2a). A distinct change in the major fatty acid profiles was observed when light gas oil was used as the sole source of sulfur for growth of the bacterial strains used in the present study. FAME analysis showed that the major membrane fatty acid profile of the bacterial strain GTIS10 was found to be octadecanoic group, when LGO was provided as sole sulfur source. The major membrane fatty acid profile of the bacterial strain SM120-1 showed the presence of four straight chain fatty acid groups, viz. hexadecanoic, heptadecanoic, octadecanoic and nonadecanoic fatty acids (Fig. 2b), when LGO was provided as the sole source of sulfur for the bacterial growth.

image

Figure 2. (a) Parts of capillary gas chromatogram depicting major membrane fatty acid profiles of the bacterial strains GTI and SM120 -1 grown using DBT as sole sulfur source. (b) Major membrane fatty acid profiles of the bacterial strains GTIS10 and SM120 -1 grown using diesel as sole sulfur source.

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Comparative analysis of desulfurization gene bdsA of the bacterial strains M. phlei SM120-1 and M. phlei GTIS10

PCR-based screening for the presence of bdsA of the bacterial strain SM120-1, resulted in the amplification of 1·2 kb bdsA amplicon. Further, cloning and sequencing of an 1139 bp region of 1·2 kb bdsA amplicon was performed for elucidation of the homology of bdsA gene with the desulfurization gene dszA of M. phlei GTIS10. The deduced amino acid residues of bdsA gene of the bacterial strain M. phlei SM120-1 revealed a homology level of 11·96% with that of dszA of Rhodococcus rhodochrous IGTS8 (Fig. 3).

image

Figure 3. Multiple sequence alignments of the deduced amino acid sequences of the genes bdsA (a) and dszA (b) of Mycobacterium phlei SM120-1 and Rhodococcus rhodochrous IGTS8. The amino acid residues, which are identical, are indicated by white letters in black boxes. The amino acid residues, which are partially identical, are indicated by shadow boxes.

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Discussion

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

The DBT desulfurization efficiency of the canonical strain M. phlei GTIS10 was estimated to be 1·09 ± 0·05 μmol 2-HBP min−1 (gram dry cell weight)−1, which was approximately seven times higher than the DBT desulfurization efficiency of the bacterial strain SM120-1. But, experiments on desulfurization of LGO reveal that the strain SM120-1 could grow well using LGO as sole sulfur source and remove 70% of total sulfur from LGO with in 7 days at 45°C. Whereas, the M. phlei strain GTIS10 was unable to utilise LGO as sole source of sulfur as evidenced by poor growth of the bacterial strain under similar conditions. The resting cells of the strain SM120-1 desulfurized LGO to an extent of 66% of total sulfur removal. The resting cells of the bacterial strain GTIS10 removed total sulfur content of LGO only to a negligible amount (<10%). The earlier workers had proved that differences in desulfurization phenotypes do exist with in the same genus (Watanabe et al. 2002). In this study, we extend this observation of differences in phenotypic profiles of desulfurizing bacterial strains to the intra-species level. Further, based on the results of desulfurization experiments with DBT and LGO using the two Mycobacterium strains, we propose that DBT desulfurization efficiency alone could not be considered as an indicator for the ability of a bacterial strain to desulfurise petroleum fractions.

The reasons for the differences in desulfurization property of these two bacterial strains could be possibly because of the fact that the bacterial strain GTIS10 was isolated on dibenzothiophene and cultured on this substrate for >4 years (Kayser et al. 2002). This selection pressure may have rendered it incapable of growing in the presence of complex hydrocarbon fuels, whereas the bacterial strain M. phlei SM120-1 was continuously enriched on various diesel oils of Indian and Japanese origin for >3 years. Hence, it could be perceived that more oil tolerant strains can readily be obtained with the right selection pressure, such as continuous growth in the presence of complex hydrocarbon fuels.

The fatty acid methyl ester analysis of major membrane fatty acids of both bacterial strains showed that there was a clear difference in membrane fatty acid profiles, when LGO was used as the sole sulfur source. Apart from the octadecanoic group of fatty acids, which was found to be present in both strains, there were additional fatty acids, viz, hexadecanoic, heptadecanoic and nonadecanoic groups present in strain SM120-1, when LGO was supplied as a sole sulfur source. An increase in 10-methyl branched fatty acids was also observed as a response to increase in temperature in M. phlei (Suutari and Laakso 1993).

The strain M. phlei SM120-1 responded to the substrate change, from DBT to LGO, by changing its membrane fatty acid composition to the four straight chain saturated fatty acid groups mentioned in this study. Mycobacterial lipids had been studied intensively and there is evidence that 10-methyl octadecanoic acid, like tuberculostearic acid, are located in cell envelope lipids (Laneelle et al. 1990). It is known that lipoarabinomannan is one of the major components of cell envelope, and it traverses the cell wall of a mycobacterium. Moreover, tuberculostearic acid and palmitate are major acyl groups of the phophatidylinositol moiety, which anchors lipoarabinomannan to the cytoplasmic membrane (Hunter and Brennan 1990).

The strain GTIS10 was found to possess only the octadecanoic group of fatty acids as its major membrane fatty acid group. Based on this observation, we presume that the bacterial strain SM120-1 was able to change its fatty acid profiles when exposed to LGO, resulting in increased concentrations of lipoarabinomannan. This particular step could be responsible for the survival of the strain SM120-1 from the toxicity caused by LGO.

Understanding the molecular basis of bacterial desulfurization requires comparative analysis of nucleotide sequences of desulfurization genes and deduced amino acid sequences of enzymes from different species of bacteria (Kirimura et al. 2004). Further, Kayser et al. (2002), showed that the dszABC operon of M. phlei GTIS10 is 100% identical to the dszABC operon of R. rhodochrous IGTS8, also known as Rhodococcus erythropolis IGTS8. Hence, in this study, the dszA gene sequences of R. rhodochrous IGTS8 were used in place of M. phlei GTIS10 for the comparison of desulfurization genes of M. phlei SM120-1 and M. phlei GTIS10, because of lack of availability of the gene sequences of the later in the public database.

The homology of deduced amino acid residues of the bdsA gene was only 11·96% when compared with the dszA of R. rhodochrous IGTS8. According to the data available in the literature, the differences between bds and dsz operons (61% identity of bds and dsz operons based on DNA sequence and about 74% identity based on protein sequences; Kilbane and Le Borgne 2004) could be sufficient to account for the observed differences in specific activity, as observed in the present study.

For a commercial scale biodesulfurization process, biocatalyst longevity above 400 h is required (McFarland 1999), which is one or two orders of magnitude higher than the longevity of native cell biocatalysts. The low longevity is partly attributed to the low tolerance of the native hosts for the oil. It will be important to screen proper hosts possessing high tolerance for the oil (Galan et al. 2000). It is important to achieve both the rate and substrate range for achieving very low sulfur levels in diesel oil and other oil streams (Ishii et al. 2005).In the present study, though, the bacterial strain SM120-1 showed promising results in terms of desulfurization of LGO, the longevity of SM120-1 cells in the presence of LGO and the rate of desulfurization of organic sulfur from fuels was relatively low. Genetic engineering and directed evolution techniques might be helpful in enhancing the longevity as well as substrate range of the strain SM120-1, in developing a commercially viable process of LGO biodesulfurization.

Acknowledgements

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

This research work was supported by Department of Biotechnology, Ministry of Science and Technology, Government of India. We are thankful to R.K. Pachauri, Director General, TERI and Dr Vibha Dhawan, Director, TERI School of Advanced Studies for providing the infrastructure for carrying out this study. Thanks are also due to Jawaharlal Nehru Memorial Fund, New Delhi, India for providing Overseas Research fellowship to one of us. We thank Dr J. J. Kilbane Jr for providing us with the strains. We thank Prof K. Kirimura, Waseda University, Japan for his guidance and support to carry out a part of this work in his laboratory. We thank Nitu Sood for analysis of FAME data.

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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