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Bioresource Technology 128 (2013) 215221

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Bioresource Technology
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Coal induced production of a rhamnolipid biosurfactant by Pseudomonas stutzeri,


isolated from the formation water of Jharia coalbed
Durgesh Narain Singh, Anil Kumar Tripathi
School of Biotechnology, Banaras Hindu University, Varanasi 221005, India

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

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D.N. Singh, A.K. Tripathi / Bioresource Technology 128 (2013) 215221

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.

toluene, xylene, carbon tertrachloride, and chloroform with cell


free culture supernatant. The samples were vortexed for 13 min
and E24 value was determined as described above. All the measurements were repeated in twice for two times and an average value
was obtained.

2. Methods

Plate assay for lipase activity was determined using tributyrin


agar plates (Lakshmipathy et al., 2010). The cultures were streaked
on the tributyrin agar plates (Himedia) and incubated at 37 C for
48 h. The plates were then examined for clear zone formation
around the colonies.

2.1. Isolation and identication of perchlorate reducing bacteria


An enrichment culture was set up for isolation of perchlorate
reducing bacteria with formation water collected from Jharia coalbed (Singh et al., 2012). The enrichment medium consisting of
(g l1) KH2PO4, 1.2 g; Na2HPO412H2O, 10.8 g; MgSO4 7H2O,
0.04 g; FeSO47H2O, 0.02 g; MnCl24H2O, 0.02 g; NH4C1, 3.0 g was
prepared anaerobically and 10 mM sodium acetate and 10 mM sodium perchlorate was added from sterile anoxic stock as electron
donor and acceptor, respectively. Finally, cysteine hydrochloride
was added to a nal concentration of 0.005% as reducing agent. Anoxic medium was inoculated with formation water and incubated
at 37 C for one week after which bacteria were isolated by standard serial dilution method on nutrient agar plates at 37 C. Identication of the isolate based on 16S rRNA gene sequence
(GenBank accession number JX429927) was carried out according
to Singh and Tripathi (2011).

2.4. Lipase production

2.5. Structural characterization of biosurfactant

Ability of the isolate to utilize and solubilize coal was detected


in mineral salt medium as described previously by Singh and
Tripathi (2011). Further, coal solubilization ability of the isolate was
detected in Yeast extract mannitol broth (YMB; Himedia). Flasks
containing YMB with and without coal (1% w/v) were inoculated
with equal number of overnight-grown culture and incubated at
37 C with shaking at 200 rpm for 7 days. Culture from each ask
was centrifuged at 10,000 rpm for 5 min to pellet the cells and
the cell free culture supernatant was used for spectrophotometric
monitoring of coal solubilization at 450 nm as described elsewhere
(Willmann and Fakoussa, 1997).

The carbohydrate group of the biosurfactant was assayed by


rhamnose test (Dubois et al., 1956). Briey, 500 ll of culture supernatant was mixed with 500 ll of 5% phenol solution and 2.5 ml of
concentrated sulfuric acid, and incubated for 15 min before
measuring absorbance at 490 nm using the UVVis spectrophotometer GeneSys (Thermo Scientic, USA). The amount of rhamnose was determined with a reference standard constructed with
L-rhamnose.
The chemical nature of the biosurfactant was determined by
haemolytic assay (Hazra et al., 2010) and growth inhibition of
Bacillus subtilis (Ito et al., 1971). Sterile disk soaked in cell free culture supernatant of YMB with and without coal was placed on the
agar plate containing 5% blood, and on the nutrient agar containing
a lawn of B. subtilis cells. Blood agar plates were incubated at room
temperature for 2 days and nutrient agar plates of B. subtilis at
37 C overnight to observe zone for haemolysis and growth inhibition, respectively.
For FTIR analysis the pH of the cell free supernatant was adjusted to 2.0 and incubated at 4 C overnight for precipitation of
the biosurfactant, which was then extracted with equal volume
of CHCl3:CH3OH (2:1) and evaporated on a rotary evaporator to
yield a viscous honey colored pellet (Hazra et al., 2010). The peaks
were recorded in the 4000 to 400 cm1 spectral region at a resolution 2 cm1, using 0.23 mm KBr liquid cells with Perkin Elmer
Spectrum Version 10.03.05.

2.3. Screening of biosurfactant activity of culture supernatant

2.6. Production of biosurfactant with different hydrocarbons

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,

Production of biosurfactant with different hydrocarbon was


tested in YMB containing different hydrocarbons (1% w/v and v/
v) viz H2O2 treated coal (oxidized coal), raw coal, kerosene oil, diesel oil, olive oil, soyabean oil, naphthalene, anthracene and phenanthrene. Flasks were inoculated with equal number of cells
from an overnight grown culture. Inoculated asks were incubated
at 37 C at 200 rpm for 7 days. The concentration of rhamnolipid
was determined by measuring the amount of rhamonse (Dubois
et al. (1956) by the colorimetric phenol sulfuric acid method at
490 nm using the UVVis spectrophotometer GeneSys (Thermo
Scientic, USA). Protein content was measured as described by
Bradford method of protein estimation (Bradford, 1976). The E24
values of the cell free culture supernatant were measured after
equalization of the amount of rhamnolipid from each treatment.

2.2. Utilization and solubilization of coal

2.7. Production of biosurfactant with different types of coal


Production of biosurfactant with different types of coal was
evaluated as described above. YMB supplemented with different
types of coal viz Lignite, Bituminous and Anthracite (1% w/v) were
inoculated with equal numbers of cells from overnight grown

D.N. Singh, A.K. Tripathi / Bioresource Technology 128 (2013) 215221

culture. Inoculated asks were incubated at 37 C at 200 rpm for


7 days.
2.8. Effect of addition of nitrogen source on production of biosurfactant
Effect of nitrogen sources on biosurfactant production was evaluated in YMB supplemented with 1% lignite coal, to which different
nitrogen sources (1 g l1) viz NH4Cl, NH4NO3 and NaNO3 were
added. Flasks were inoculated with equal number of cells from
overnight grown culture and incubated at 37 C at 200 rpm for
7 days. Concentration of rhamnolipid and emulsifying ability of
the cell free supernatant was measured as described above.
2.9. Stability characterization of biosurfactant
To determine the thermal stability of the biosurfactant, the cell
free culture supernatant was incubated at different temperatures
(10100 C) for 15 min and cooled down to room temperature.
Similarly, pH of supernatant was adjusted to 210 to determine
the effect of pH on biosurfactant activity. The effect of addition
of different concentration of NaCl (535%, w/v) was also tested.
The E24 value of each treatment was determined as described
above (Chandran and Das, 2010).
2.10. Screening for heavy metal tolerance of isolate
Sensitivity of the isolate to the heavy metals was tested on LB
agar containing tetrazolium salt (50 lg/ml). The sterile disks saturated with 1 M metal salt solutions (70 ll) were placed in the center of the bacterial lawn and the plates were incubated overnight at
37 C. The diameter of the zone of inhibition was noted for corresponding metals (Sriram et al., 2011a).
3. Results and discussion
3.1. Isolation and identication of a P. stutzeri strain from a
perchlorate-reducing enrichment culture
In our previous study on cultivation independent analysis of the
microbial community present in the formation water of Jharia
coalbed we detected 16S rRNA gene sequences showing 95100%
identity with that of a perchlorate reducing bacterium, Dechloromonas, which is well known for its ability to degrade polyaromatic
hydrocarbons anaerobically (Singh et al., 2012). With a view to isolate Dechloromonas strains from the formation water of the Jharia
coalbed a perchlorate reducing enrichment culture was set up. A
16S rRNA gene sequence based analysis of the bacterial community
of this enrichment culture indicated predominance of bacteria
showing afnity to P. stutzeri, Pseudomonas chloritidismutans, Pseudomonas sp. me-13 and Chelatococcus sp. (data not shown). When
an inoculum from the enrichment culture was spread on the nutrient agar plates and incubated at 37 C for 3 days it showed dominance of yellow colored bacterial colonies. Identication of a ve
randomly selected yellow colored colonies based on nearly complete 16S rRNA gene sequence revealed 99% identity to P. stutzeri
(JQ963329) which is an oil-degrading bacterium isolated from
petroleum polluted soils in Kharg Island, Iran (Fig. 1). Earlier, some
of the P. stutzeri strains were reported to show 99% identity with P.
chloritidismutans, which is a perchlorate reducing bacterium
(Cladera and Lalucat, 2006). Occurrence of P. stutzeri was reported
in western Canadian subsurface coalbeds (Penner et al., 2010), in
Ishikari coal elds, Japan (Shimizu et al., 2007) and in the Sydney
coal basin in Australia (Li et al., 2008) suggesting its ubiquitous
occurrence and role in the coalbeds. This strain, designated as P.
stutzeri-BHU, when inoculated in the mineral salts medium with

217

coal as sole source of carbon and energy it failed to grow (data


not shown). However, it was able to solubilize coal in YMB supplemented with coal as the culture supernatant showed signicantly
higher absorbance at 450 nm (A450nm = 0.50) than the uninoculated
medium (A450nm = 0.016). It was reported earlier that a wide range
of organisms growing on rich media generate alkaline metabolic
products, leading to ionization of acidic groups in low-rank coal
and thereby rendering the coal humates water-soluble (Quigley
et al., 1989a,b). After 7 days of incubation the culture in YMB
amended with coal (treated) became highly viscous when compared to that without coal (control) which might be due to the
emulsication of coal in the medium.
3.2. P. stutzeri-BHU produces a surface active compound in response to
coal
Whether the viscous material produced by P. stutzeri-BHU in
the coal amended YMB was a biosurfactant was analyzed by measuring the surface tension of the medium after suitable dilution.
Surface tension of the 1:2 diluted culture supernatant was reduced
to 47 mN/m compared to the surface tension of 1:1 diluted YMB
medium without coal (control), which was reduced to 58 mN/m.
Triton X 100 used as positive control reduced the surface tension
of water to 36 mN/m. Evaluation of the emulsifying ability of the
cell free culture supernatant of coal amended culture showed
100% emulsication index whereas control, Tween 80, SDS and Triton X 100 showed 15%, 74%, 66% and 66% emulsication indices,
respectively (Fig. 2a). Further, cell free culture supernatant of coal
amended culture fully emulsied (E24 = 100%) the entire hydrocarbons used for the test (Fig. 2b). The ability of the P. stutzeri-BHU to
show lipase activity on tributyrin agar also indicated its ability to
produce biosurfactant. In addition, production of lipase, reduction
in the surface tension of the medium and emulsication of
different hydrocarbons by the culture supernatant indicated that
P. stutzeri-BHU strain isolated from the Jharia coalbed produced
copious amounts of biosurfactants in YMB supplemented with coal
powder. P. aeruginosa, isolated from the formation water of Venezuelan oil eld, produced biosurfactant with glucose as sole carbon
source. However, the addition of crude oil enhanced the production of biosurfactant (Rocha et al., 1992).
3.3. Biosurfactant produced by P. stutzeri-BHU is a rhamnolipid
The ability of the supernatant from coal amended culture to
cause heamolysis of erythrocytes and to inhibit the growth of B.
subtilis indicated that the surface active compound produced by
P. stutzeri might be a rhamnolipid. Detection of greater amount
of rhamnose in the supernatant of the coal amended culture
(YMB + coal) compared to the control (YMB) indicated that coal
was able to induce rhamnolipid production by P. stutzeri-BHU
(Fig. 3a). Further characterization of the puried biosurfactant by
FTIR revealed its molecular composition. A broad band observed
at 3416 cm1 corresponds to OH stretching vibrations of hydroxyl
groups. The strong adsorption around 2924 cm1 is expected to be
symmetric stretch of CH2 and CH3 groups of aliphatic chain. The
corresponding symmetric stretch is seen at 2853 cm1. The symmetric stretch around 1467 cm1 indicates the presence of ester
carbonyl groups (C@O in COOH) of the biosurfactant. The CO
stretching bands at (13001000 cm1) 1024 cm1 and 1086 cm1
conrms the presence of bonds formed between carbon atoms
and hydroxyl groups in the chemical structure of rhamnose ring.
Bands in the range of 35002700 cm1, 17201680 cm1 and
950900 cm1 indicate the presence of an ester compound; all
these peaks are characteristic of the rhamnolipid nature of the coal
induced biosurfactant. Members of the genus Pseudomonas are
known to degrade hydrocarbons and produces rhamnolipid-type

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D.N. Singh, A.K. Tripathi / Bioresource Technology 128 (2013) 215221

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.

Maximum production of biosurfactant was observed after seven


days when the culture reached stationary phase of growth (data
not shown). Different hydrocarbons induced production of biosurfactant differentially, maximum production occurring in the presence of raw coal followed by oxidized coal, olive oil, soyabean
oil, kerosene and diesel, and minimum production by naphthalene
(Fig. 3a). All other hydrocarbons produced signicantly lower
amount of the biosurfactant when compared to coal (raw and oxidized coal). When equal amount of rhamnolipid (1 mg/ml) was taken from each treatment, only the supernatant amended with coal
(oxidized and raw coal) showed maximum emulsication ability of
75%. This reects that the minimum active dose of the biosurfactant was the least in case of coal induced biosurfactant as compared to the biosurfactant produced in response to other
hydrocarbons. Since the minimum amount of rhamnolipid required for the formation of emulsion was the least for the coal induced biosurfactant (Sriram et al., 2011b) the rhamnolipid
biosurfactant produced by P. stutzeri-BHU seems to be coal specic.
When different types of coal were used as hydrocarbons, lignite
coal caused maximum production of rhamnolipid followed by
bituminous and anthracite coal (Fig. 3b). Similarly, when the three
types of coal were used for testing coal solubilization ability of
P. stutzeri-BHU, lignite was maximally solubilized (Fig. 3c). Earlier,
biosurfactants produced by Penicillum decumbens and Pseudomonas
uorescens were shown to play important role in the degradation of
lignite coal and in altering the properties of coal (Breckenridge and
Polman, 1994; Yuan et al., 2006). Production of surfactants by microbes is one of the simplest methods for achieving coal solubilization. Hydrophobic ends of the biosurfactants interact with the
hydrophobic components of the coal due to which hydrophilic
heads of biosurfactants are exposed to the aqueous environment
rendering biosurfactant-coal complex more hydrophilic than the
raw coal (Yin et al., 2011). This change improves the contact

D.N. Singh, A.K. Tripathi / Bioresource Technology 128 (2013) 215221

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

Fig. 3. (a) Production of biosurfactant by Pseudomonas stutzeri in response to


different hydrocarbons (1% w/v, v/v); (b) production of biosurfactant by Pseudomonas stutzeri in response to different ranks of coal and (c) solubilization of
different ranks of coal by Pseudomonas stutzeri. Error bars indicates SD.

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,

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D.N. Singh, A.K. Tripathi / Bioresource Technology 128 (2013) 215221

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.

Fig. 5. Heavy metal sensitivity of biosurfactant producing Pseudomonas stutzeri


evaluated by measuring the zone of inhibition by the lter disks soaked in 1 M
solution of each metal.

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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Fig. 4. Determination of stability of coal induced biosurfactant produced by


Pseudomonas stutzeri at (a) different temperatures (b) pH and (c) salt concentration.
Error bars indicates SD.

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