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h i g h l i g h t s
" Pseudomonas stutzeri was isolated from the formation water of an Indian coalbed.
" The P. stutzeri isolate produced biosurfactant in response to coal supplementation.
" The coal induced biosurfactant produced by P. stutzeri was a rhamnolipid.
" P. stutzeri produced more biosurfactant with lignite than bituminous or anthracite.
a r t i c l e
i n f o
Article history:
Received 7 August 2012
Received in revised form 23 October 2012
Accepted 24 October 2012
Available online 3 November 2012
Keywords:
Pseudomonas
Biosurfactant
Rhamnolipid
Coal solubilization
Coalbed methane
a b s t r a c t
A strain of Pseudomonas stutzeri was isolated form an enrichment of perchlorate reducing bacteria from
the formation water collected from an Indian coalbed which solubilized coal and produced copious
amount of biosurfactant when coal was added to the medium. It produced maximum biosurfactant with
lignite coal followed by olive oil and soybean oil which was able to emulsify several aromatic hydrocarbons including kerosene oil, diesel oil, hexane, toluene etc. Haemolytic test, growth inhibition of Bacillus
subtilis and FTIR analysis showed rhamnolipid nature of the biosurfactant. The stability of the coal
induced biosurfactant in pH range of 48 and up to 25% NaCl concentration and 100 C temperature suggests that due to its ability to produce biosurfactant and solubilize coal P. stutzeri may be useful in the
coalbed for in situ biotransformation of coal into methane and in the bioremediation of PAHs from oil contaminated sites including marine environments.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
India is the 5th largest proven reservoir of coal in the world utilization of which as energy source results into atmospheric pollution including emission of green house gases (Hayward, 2010). In
situ microbial conversion of coal into methane gas is considered
an environment friendly alternative to produce the clean energy
source. Thus, microbial biotransformation of coal into simpler,
low molecular weight product is considered an economic and
effective way of improving the utility of coal. A variety of microorganisms including fungi (Hatakka, 1994), bacteria (Standberg and
Lewis, 1988) and actinomycetes (Quigley et al., 1989a) are capable
of coal solubilization/degradation. Studies suggested the mechanism involved in coal biotransformation mainly comprises of (1)
microbial production of alkaline substances (Quigley et al.,
1989b), (2) biocatalysts especially produced by fungi (Hatakka,
Corresponding author. Address: Laboratory of Bacterial Genetics, School of
Biotechnology, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India.
Tel.: +91 542 2368331, mobile: +91 9451525811; fax: +91 542 2368693.
E-mail address: tripathianil@rediffmail.com (A.K. Tripathi).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.10.127
1994), (3) metal ion chelators and surface active agents (Fakoussa,
1988; Polman et al., 1994). Surfactant decreases the coal surface
tension and increases the solubility of coal in aqueous solutions
(Fakoussa, 1988; Yuan et al., 2006). It is quite possible that both
enzymatic and non enzymatic mechanisms may be involved in coal
biotransformation process in the same microorganism. In a study it
was shown that chemically synthesized surfactants and surfactant
from Bacillus licheniformis and Candida bombicoal were able to solubilize different portion of coal (Breckenridge and Polman, 1994;
Polman et al., 1994).
Biosurfactants can be used in coal dust control, enhancement of
gas permeability of coal, removal of coal ash and bioremediation of
soil and groundwater contaminated with hydrocarbon. Microorganisms producing biosurfactant can participate in oil degradation.
Alternatively, they can function in a bacterial consortium, supplying the emulsier for other bacteria that carry out the degradation
of hydrocarbons (Ron and Rosenberg, 2002). In addition biosurfactant can be used in petroleum industry for transportation of crude
oil, enhanced oil recovery by increasing the apparent solubility of
petroleum components and effectively reducing the interfacial
216
tensions of oil and water in situ (Singh et al., 2006). In this study we
have described isolation and identication of a rhamnolipid producing Pseudomonas stutzeri strain from the formation water of
an Indian coalbed, which produced maximum biosurfactant in
presence of coal. We have shown here that the ability to produce
biosurfactant is responsible for the coal solubilization ability of this
bacterium.
2. Methods
For screening of the biosurfactant activity, the culture supernatant was taken from the asks containing YMB supplemented with
and without coal. Cells were separated by centrifugation at
12,000 rpm for 10 min and cell free culture supernatant was used
for tests. Reduction in the surface tension of medium was measured by du Nouy ring type tentiometer (Krauss, Germany) (Chandran and Das, 2010). Triton X-100 was used as positive control for
the measuring reduction in the surface tension of water. Emulsifying ability of the culture supernatant was measured by vortexing
equal volume of cell free culture supernatant and kerosene oil for
13 min and determining the percentage of volume occupied by
the emulsion. The mixture was allowed to settle for 24 h and the
height of the emulsion was measured to determine the emulsication index (E24 values) which is described as ratio of the height of
emulsied zone (EZ) to total height (TH); [E24 = (EZ/TH) 100]
(Bonilla et al., 2005; Sriram et al., 2011a). Chemical surfactants
such as Tween 80, Triton-X 100 (Himedia) and 10% sodium dodecyl
sulfate (SDS; Merck) were used as positive test control (Chandran
and Das, 2010). Further, the ability of cell free supernatant to emulsify different hydrophobic substrates was tested by mixing equal
volume of kerosene, diesel, olive oil, soyabean oil, hexane, benzene,
217
218
Fig. 1. Phylogenetic (neighbor-joining) tree based on 16S rRNA gene sequence showing relationship of the formation water isolate with other members of the genus
Pseudomonas. Accession numbers of selected sequences are given in parentheses. Scale bar represents 0.005 substitutions per nucleotide position.
biosurfactants when grown with different substrates such as glycerol, mannitol, fructose, glucose, n-parafns and different vegetable oils (Desai and Banat, 1997).
3.4. Effect of different hydrocarbons on the biosurfactant producing
ability of P. stutzeri-BHU
Fig. 2. (a) Comparison of the emulsifying ability of the coal induced biosurfactant
produced by Pseudomonas stutzeri using kerosene oil with the other known
surfactants. (b) The ability of coal induced biosurfactant to emulsify different
hydrocarbons. Error bar indicates SD.
219
variteties of coal like bituminous and anthracite coal. Low rank lignite coal is relatively more hydrophilic in nature and is more porous than the higher rank coals. Therefore, low rank coals are more
susceptible to biosolubilization than high ranking coals (Catcheside
and Ralph, 1999) due to their relative richness in hydroxyl and
dihydroxy aromatic structures and carboxyl groups which are
known to facilitate biodegradaiton. Since Pseudomonas species
are known to carry out oxidative ring cleavage of aromatic and
polyaromatic hydrocarbons, P. stutzeri-BHU isolated from the
Jharia coalbed might solubilize low rank coal by oxidative mechanisms as well as by producing biosurfactant. Extracellularly
produced oxidative enzymes (peroxidases) were earlier shown to
facilitate the solubilization of lignite coal as the addition of lignite
to the culture medium increased the activities of peroxidases capable of attacking lignite (Willmann and Fakoussa, 1997).
3.5. Effect of nitrogen sources on biosurfactant production
Since lignite coal induced maximum production of the rhamnolipid by P. stutzeri-BHU we examined the effect of different nitrogen sources on biosurfactant production by P. stutzeri-BHU with
lignite coal. Although P. stutzeri-BHU grew much better on NH4Cl,
NH4NO3 and NaNO3 in YMB amended with 1% coal it produced signicantly lower levels of biosurfactant than that produced in YMB
medium containing 1% coal (without nitrogen sources). Addition of
NaNO3, NH4Cl and NH4NO3 reduced the biosurfactant production
by <1/2, <1/5 and <1/12, respectively. Similarly, E24 values decreased to 1020% after the addition of different nitrogen sources
(Table 1). As observed by us in P. stutzeri-BHU, the production of
rhamnolipid biosurfactant in P. aeruginosa was also adversely affected by the availability of nitrogen sources in the medium
(Ramana and Karanth, 1989). Similarly, nitrogen limitation was
shown to increase biosurfactant production in C. tropicalis IIP-4
(Singh et al., 1990) and Nocardia strain SFC-D (Kosaric et al.,
1990). Among the nitrogen sources tested, NaNO3 showed the least
inhibition but the emulsifying ability of the biosurfactant produced
by NaNO3 supplemented cultures was highly reduced. It is likely
that NaNO3 supplemented cultures produced some secondary
metabolites which interfere in the emulsication (Bonilla et al.,
2005).
3.6. Effect of temperature, pH and salinity on stability of the
biosurfactant
between hydrophilic enzymes and hydrophobic coal surface leading to an increased biosolubilization/biodegradation of the coal.
Alternatively, biosurfactants also bind and remove the metals
involved in ionic linkages in coal humates which increases the
number of free acidic groups and hence the increased solubility
of coal humates. Study of the mechanism of coal solubilization
by Coriolus versicolor has earlier shown that it produced oxalate
which sequesters polyvalent metal ions such as Ca2+, Fe3+, Al 3+,
Mg2+, which are involved in forming ionic linkages of coal humates
(Quigley et al., 1989b).
Low rank lignite coal caused maximum production of rhamnolipids and maximum coal solubilization when compared to the hard
Biosurfactant produced by P. stutzeri-BHU showed 100% emulsication in the temperature range of 10100 C, pH range of 4.08.0
and up to 25% NaCl. Emulsication was reduced to 50% after autoclaving or to 2550% when pH was decreased to 2.0 or increased to
10.0. Similarly, emulsication declined to 50% at >25% NaCl concentration (Fig. 4). Coal induced biosurfactant was stable up to
100 C, active in the pH range 410 and showed emulsication
even at 35% NaCl. These properties of the biosurfactant produced
by P. stutzeri-BHU suggest its potential utility in acid mine drainage
and enhanced microbial oil recovery. Stability of this biosurfactant
at high NaCl concentrations also suggests its utility in marine environment and or such environments where salt concentrations are
high (Chandran and Das, 2010).
3.7. Heavy metal tolerance of the strain
Heavy metal tolerance of P. stutzeri-BHU was evaluated by measuring the diameter of the zone of growth inhibition. The strain
was considered to be tolerant to the heavy metal if the diameter
of the zone of inhibition was 63 cm (Sriram et al., 2011a,b). Based
on this criteria P. stutzeri was tolerant to MnCl2, CuSO4, PbCl2,
FeSO4 and sodium arsenate, and sensitive to CdSO4, HgCl2, CoCl2,
220
Table 1
Effect of nitrogen sources on the production of biosurfactant by Pseudomonas stutzeri.
Sample
Rhamnose
(mg/ml)
Protein
(mg/ml)
Rhamnose mg
Protein mg
YMB
YMB + Coal
YMB + Coal + NH4Cl
YMB + Coal + NH4NO3
YMB + Coal + NaNO3
0.045
8.740
3.470
1.318
7.310
0.161
0.135
0.268
0.275
0.270
0.279
64.74
12.94
4.79
27.07
0
75
15
10
15
E24
E24/mg of rhamnose.
NiSO4 and ZnCl2 (Fig. 5). The ability of this bacterium to grow in the
presence of heavy metals such as copper, lead and arsenic and produce biosurfactant in response to different hydrocarbon makes it a
promising tool for bioremediation of hydrocarbons at heavy metal
polluted sites. Metal tolerant bacteria capable of producing rhamnolipid biosurfactants have been isolated and used to mobilize
arsenic from mine tailings (Wang and Mulligan, 2009) and bioremediation of heavy metals (Chandran and Das, 2010; Sriram
et al., 2011a).
4. Conclusion
Pseudomonas stutzeri-BHU isolated from Jharia coalbed produced copious amount of rhamnolipid biosurfactant in presence
of coal. Although this bacterium itself is not capable of utilizing
coal or its components, its ability to produce biosurfactant and solubilize coal might enable other microbes in degrading coal components and making it available to the microbial community for
various activities including methanogenesis. Biosurfactant had an
excellent emulsifying ability and showed stability over wide range
of temperature, pH and salinity suggesting its possible use in the
in situ biotransformation of coal into methane and bioremediation
of PAHs from the oil contaminated sites including marine
environments.
Acknowledgements
This work was supported by a grant from Department of Biotechnology, Government of India to AKT. DNS was supported by
a fellowship from University Grants Commission. We thank Institute of Reservoir Studies, ONGC, Ahmedabad for help in the collection of formation water and Prof. D.S. Pandey, Department of
Chemistry, Banaras Hindu University in FTIR analysis.
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