Professional Documents
Culture Documents
4 JULY-2016
ABSTRACT- Microorganisms are the important factors in the degradation of the toxic substances in our environment.
Petrol and diesel oil is one of the complex mixtures which cannot be easily degraded. The Bacillus cereus was involved in
the degradation of oil during which the complex toxic substances were detoxified by the production of biosurfactants. In
our study we have identified that the biosurfactant producing Bacillus cereus have a high potential for hydrocarbon
degradation. The Bacillus cereus was isolated from hydrocarbon contaminated soil and identified based on morphology
and biochemical test according to the Bergeys manual of systematic bacteriology. The maximum hydrocarbon degrading
biosurfactant producing Bacillus cereus was obtained by qualitative and quantitative methods. In optimization studies, the
best results observed for Bacillus cereus were, Olive oil as the suitable carbon source, Sodium nitrate as the best Nitrogen
source and Optimum pH is 7 and Optimum temperature is 37C. The ability of these isolates to degrade hydrocarbons and
survive in the oil contaminated soil is attributed to the development of resistance by mutation on the plasmid. It is also
clearly evident that the specific gene was responsible for the production of biosurfactant and the degradation process.
According to the results from the present study the Bacillus cereus has high potential for hydrocarbon degradation and can
be used especially for Microbial Enhanced Oil Recovery and bioremediation of hydrocarbons in near future.
Key-words- Bacillus cereus, Biosurfactant, Hydrocarbon, Biodegradation, Plasmid DNA
-------------------------------------------------IJLSSR-----------------------------------------------
INTRODUCTION
India is suffering with ever increasing consequential Fuel and lubricating oil spills have become a major
environmental threats. Rapid industrialization can only environmental hazard to date. The contamination of the
move hand in hand with the efficient and optimum environment with petroleum hydrocarbons provides serious
feasibility of transport, which results in increased use of problems for many countries [5].
automobiles [1]. Diesel oil contains 2,000 to 4,000 Biodegradation is a biologically catalyzed reduction
hydrocarbons, a complex mixture of linear, branched and process of complex chemicals. This process is being
cyclic alkanes and aromatic compounds obtained from the performed by a variety of bacteria, fungi and yeast,
middle distillate fraction during petroleum separation transforms potentially toxic compounds into non-toxic
[2]
.The availability of nutrients, especially nitrogen and compounds to obtain energy and nutrients [6]. These
phosphorus significantly control microbial activities [3], and microorganisms are directly involved in biogeochemical
these nutrients are necessary to enhance the biodegradation cycling of many carbon sources, including petroleum
of oil pollutants [4]. hydrocarbons [7].
Biosurfactant are extracellular or membrane associated,
Access this article online heterogeneous group of low molecular weight surface
Quick Response Code: active compounds produce by different microorganism
Website: such as bacteria, fungi and yeast. On the basis of chemical
www.ijlssr.com composition they are categorized into glycolipids,
lipopeptides, phospholipids, neutral peptides, fatty acids
DOI: [8-9]
. They are mainly composed of hydrophilic and
10.21276/ijlssr.2016.2.4.4 hydrophobic moiety. The Hydrophilic moiety consists of
acids, peptides, mono, di, or polysaccharides while the
hydrophobic moiety consists of saturated or unsaturated
fatty acids. They have great potential to reduce surface and Serial dilutions were prepared and plated on Nutrient agar
interfacial tension of liquids, solid and gases, and enhance medium supplied with 80g/ml of cycloheximide and
their solubilisation in liquid solution. They have shown incubated at 30C for 2-3 days for the growth of the spore
good stability at extreme temperature, pH and salt forming bacterial colonies. The bacterial colonies were
concentration. Because of their unique characteristic and purified and sub-cultured on the same medium for further
better performance than synthetic surfactants, they have identification and biosurfactant production.
gained attention and importance in various fields such as
enhanced oil recovery, environmental bioremediation, food Identification of bacteria
processing and pharmaceuticals [10]. In addition to The preliminary identification of the isolates was done by
lubrication, fixing dyes, making emulsions, stabilizing the colony morphology, grams staining, motility and spore
dispersions, preventing foaming, the main application is in staining. The biochemical reactions include oxidase test,
the enhancement of oil recovery techniques, especially the catalase test, gelatin liquefaction, starch hydrolysis, lipid
trapped residual oil [11]. The oil bioremediation is due to hydrolysis, sugar fermentation tests, IMViC test, H2S and
their biodegradability and low critical micelle concentration Nitrate reduction test. The identification of the Bacillus
(CMC) [12-13]. Among them, lipopeptide biosurfactants cereus isolates was based on Bergy's Manual of
produced by Bacillus species are capable of generating the Determinative Bacteriology [21].
low interfacial tension between the hydrocarbon and
aqueous phases required to mobilize the entrapped oil [14]. Cultivation media for biosurfactant production
One of the most important bacteria producing biosurfactant Mineral Salt Broth (MSB, pH 7.2) composed of (g/l)
is Bacillus spp. which is mainly used for biotechnological KH2PO4 0.5g, K2HPO4 1g, NaNO3 4g, MgSO4.7H2O 0.5g,
applications [15]. Bacillus licheniformis produce a variety of KCl 0.1g, FeSO4.7H2O 0.01g, Yeast extract 0.01g, Carbon
products such as extracellular enzymes, biosurfactants, source (2%), Crude oil, Trace element 0.05 ml, 2ml
biopesticides and are ecofriendly. They are used for contained (g/l) NaMo4 0.1g, MnCl2 1.75g, ZnSO4.7H2O
enhanced oil recovery and inhibition of sulfate reducing 3.1g, CuSO4.5H2O 2g, H3BO3 1.5g for biosurfactant
bacteria. The biosurfactants produced from Bacillus spp. production [22-24].
possess the property of functionality under extreme
conditions of pH, temperature, salinity and in addition are Screening of biosurfactant producing Bacillus
from renewable resources and are ecofriendly [16-18]. Since cereus
the lipopeptide biosurfactants produced by Bacillus spp. The bacterial strains maintained on nutrient agar at 4C was
have high ability to reduce the surface tension, they find inoculated in 50 ml of seed medium (Nutrient broth) in a
wide use in the oil industry and bioremediation [19]. The aim 100 ml Erlenmeyer flask and incubated in a rotary shaker at
of our research was to investigate the biosurfactant 120 rpm at 30C. After growth for 18 hrs, standard
producing ability of Bacillus cereus for hydrocarbon inoculum 5% (v/v) was used in the fermentation medium
degradation in oil contaminated soil samples in and around which is equivalent to 1x108 CFU/ml. For crude oil
the Erode district, Tamil Nadu, India. biodegradation mineral salt broth supplied with 2% crude
oil was used as the sole carbon and energy source. The
MATERIALS AND METHODS media inoculated with 5% (v/v) inoculum of the isolate was
Soil samples incubated at 30C on a rotary shaker at 120 rpm for 7 days
In this study totally twenty soil samples were collected to [25]
. The culture was centrifuged (Eppendorf cooling
isolate the hydrocarbon degrading bacteria Bacillus cereus centrifuge) at 6000 rpm for 15 minutes at 4C and
from oil contaminated soil. The soil samples were supernatant was collected for extracellular biosurfactant
extending from the ground surface to a depth of 10-20 cm assay by different methods.
from petroleum contaminated areas near petrol station
refining areas in Erode. Samples were then transported to Biosurfactant assay
laboratory under sterile conditions. The research work was
carried out during the period of January 2014 to May 2014 Drop collapse method
in department of microbiology lab in Vivekanandha The slightly modified drop-collapse test was performed [26].
College of Arts and Sciences for Women (Autonomous), In this method, the supernatant from each bacterial isolate
Tamilnadu (India). was placed onto a glass slide. Then, petrol and diesel was
added onto the surface of the supernatant. If the drop of oil
Isolation of bacteria on the supernatant became flat in 1 min after adding the oil,
One gram of soil sample was suspended in 10ml of normal the result was taken to be positive. If the drop remained
saline to make tenfold serial dilution of the sample for the beaded, the result was scored as negative indicating the
isolation of bacteria. The dilution factor is expressed in lack of biosurfactant production.
colony forming unit per ml (CFU/ml) [20]. The suspension
was heated at 70C for 15 minutes to reduce any vegetative
bacterial cells and facilitate the isolation of Bacillus sp.
http://ijlssr.com IJLSSR 2015 All rights are reserved Page 325
Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 4
tension between the liquid drop and the hydrophobic Degradation of hydrocarbon
surface whereas with non biosurfactant producing culture The optical density (OD) reading of biodegrading activity
the drops remained stable [16]. of each isolate on hydrocarbon (Petrol and diesel) based on
the turbidity of MSM broth at regular intervals of 4 days
gives the degradative activity on hydrocarbons by bacteria.
The result demonstrated that Bacillus cereus have the
greatest ability to degrade petrol and diesel. Our results
showed that all the Bacillus cereus isolates maximally
utilized the hydrocarbon substrates (Petrol and diesel) when
supplied as the sole source of carbon and energy although,
the level of utilization differs from one to another (due to
differences in their growth) and from one hydrocarbon
substrate to the other, due to the obvious differences in their
molecular sizes. The degrading capabilities on different
hydrocarbons revealed that the Bacillus cereus isolated
from the soil were able to degrade hydrocarbons. The cells
were able to multiply within the days of study, indicating
that they were able to degrade and utilize the hydrocarbons
Figure 3: Drop collapse method- Control, Collapsed
for their growth and development; hence the concomitant
droplets 1, 2, 3 -Positive
increase in the concentration of the broth (turbidity) was
observed. It was also noticed that as the incubation hours
Chemotaxis
increased there was gradual increase in the concentration of
In this study all the isolates show positive chemotaxis to
the broth, and degradation of hydrocarbons. Bioremediation
petroleum oil. Recent results with the biodegradation of
is proposed as an alternative to various physicochemical
hydrocarbon have shown the potential of chemotaxis to
treatments at many hydrocarbon contaminated sites [33].
enhance biodegradation in laboratory-scale
Extensive hydrocarbon exploration activities often result in
microorganisms. Indeed chemotaxis have already been
the pollution of the environment, which could lead to
studied in bacteria able to degrade a wide variety of organic
disastrous consequences for the biotic and abiotic
pollutants such as naphthalene, BTEX (Benzene, Toluene,
components of the ecosystems if not restored. Remediation
Ethylbenzene, and Xylene), and pesticides. The similar
of hydrocarbon contaminated system could be achieved by
result was showed toluene-degrading bacteria are
either physicochemical or biological methods [34].
chemotactic towards the environmental pollutants, benzene,
Surfactants and biosurfactants can increase the pseudo
toluene, and trichloroethylene [31]. In addition, the
solubility of petroleum components in water. Moreover,
chemotaxis of Ralstonia to herbicide [32]. Bacterial
biosurfactants can be as effective as synthetic chemical
chemotaxis towards environmental pollutants have
surfactants due to their high specificity and their
important role in bioremediation (Figure 4).
biodegradability.
Optimization Parameters
Inoculum size
The results showed that, the utilization of hydrocarbon
gradually increased with the growth of Bacillus cereus in
different volume of inoculum. It was determined by the
optical density value of Bacillus cereus culture. Totally four
different inoculum sizes such as 250 l, 500 l,750 l and
1000 l were analyzed in different incubation time intervals
such as 24hrs, 48hrs, 72hrs and 96hrs and the O.D. values
were noted. The 750 l of inoculam size favored the
growth of Bacillus cereus and the gradual increase of
Bacillus cereus cells to degrade the oil in medium at
different time intervals. The O.D. values of 0.048, 0.090,
0.112 and 0.148 were also recorded (Table 1). It was also
noticed that as the incubation time was extended, bacterial
Figure 4: Chemotaxis activity of Bacillus cereus towards concentration increased. Low inoculum size required
oil droplets longer time for cells to multiply and produce the desired
effect [35]. A small amount of inoculum can lead to
insufficient number of microbial cells and a reduced
amount of the secreted enzymes, while a much higher
http://ijlssr.com IJLSSR 2015 All rights are reserved Page 328
Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 4
inoculum could lead to or cause a lack of oxygen and cultures grown at 32C and 42C. At 37C it produced the
depletion of nutrients in the culture media [36]. highest biosurfactant production of 1.233 O.D. representing
a reduction in surface tension. When the incubation
Table 1 Degradation of Hydrocarbon using various temperature increased to 42C, bacterial growth and
inoculum size of Bacillus cereus biosurfactant production were significantly decreased,
indicating that the biosurfactant produced by Bacillus
Inoculum OD Reading (nm)/ cereus was temperature dependent (Figure 6).
S. No size ( l ) Incubation (Hrs)
24 48 72 96
Optimum pH
Degradation of Hydrocarbon at different pH by Bacillus
cereus showed the production of biosurfactant at a wide
range of pH from 5.0 to 9.0 in different incubation time
intervals such as 24 hrs, 48 hrs, 72 hrs and 96 hrs as shown
in Figure 5. The pH 7.0 was selected as the best pH for With the increase in enzymatic activity of mesophilic and
biosurfactant production because it produced the highest thermophilic microorganisms associated with increasing
surface tension reduction. It was also observed that as the temperatures, it is expected that biodegradation rates can be
incubation time was extended the biodegradation was at the enhanced to a certain extent, typically in the range of 30C
maximum. At an acidic pH (5.0) and extreme alkaline pH to 40C. Above 40C, the membrane toxicity of
(9.0), these isolates produced lower levels of biosurfactant. hydrocarbons is increased, thus hindering biodegradation
The pH range of the experimental samples (6 to 7)
[39]
.
observed in this study is within the favourable range for
biodegradation of oil in polluted soil. Similar observations Carbon source
have been documented [37-38]. The ability of Bacillus cereus to utilize various types of
carbon sources for biosurfactant production was tested with
Olive oil, Almond oil and Palm oil at 0.7, 1.0, 2.0 ml. All
oils were gradually degraded showing increased O.D.
values at different incubation hours (24 hrs, 48 hrs, 72 hrs
and 96 hrs) at optimized temperature 37C and pH 7.
Among these carbon sources tested, Olive oil has highly
favoured the Bacillus cereus growth in concentration 0.7 ml
with the O.D. value of 1.325 in 96hrs was shown in (Figure
7). Increase in the concentration of olive oil has not shown
maximum growth. The appropriate reason for this needs
further investigation. The next carbon source which
supported maximum growth was Palm oil at 1 ml
concentration on 96 hrs incubation. From the results it is
evident that almond oil supported least growth.
Temperature
Degradation of hydrocarbon by Bacillus cereus at different
temperatures at 32C, 37C and 42C at various time
intervals such as 24 hrs, 48 hrs, 72 hrs and 96 hrs at pH 7.0
was studied. The Bacillus cereus grown in MSM broth at
pH 7.0 produced maximum biosurfactant when incubated at
temperature 37C, which was significantly different from
http://ijlssr.com IJLSSR 2015 All rights are reserved Page 329
Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 4
and Cardoso V.L: Biosurfactant production by Pseudomonas ecosystem in petroleum microbiology, edited by R.M Atlas,
aeruginosa grown in residual soybean oil. Appl. Biochem. Macmillan, New York. 435-4730:1984.
Biotechnol, 2009; 52:156-168. [40] Rodrigues, L. Banat, J.M. Teixeira,J. and Oliveira,R:
[25] Maliji D., Olama Z. and Holail H. Environmental studies on Biosurfactants: potential applications in medicine,
the microbial degradation of oil hydrocarbons and its Advance access publication, 2006; (9).
application in Lebanese oil polluted coastal and marine [41] Smith GP, Press B, Eberiel D, McCarthy SP, Gross, RA
ecosystem. Int. J. Curr. Microbiol Appl. Sci., 2013; 2(6): Kaplan L: An accelerated in laboratory test to evaluate the
1-18. degradation of plastics in landfill environments. Polymers.
[26] Plaza, G.A., I. Zjawiony and I.M. Banat: Use of different Materials Science & Engineering, 1990; 63: 862866.
methods for detection of thermophilic [42] Danbara,H., H. Arima, T. Baba, T. Matano, MYamaguchiand
biosurfactant-producing bacteria from T. Kikuchi, Concentration of trace elements in grass on
hydrocarbon-contaminated and bioremediated soils. J. Shinshu high land area Proceed.Int.Grass. Cong. Aug, 1985;
Petrol. Sci. Eng., 50: 71-77 2006. 24-31, Kyoto, Japan.
[27] Grimm AC, Harwood CS: Chemotaxis of Pseudomonas sp.
To the polyaromatic hydrocarbon Naphthalene. Appl. Source of Financial Support: Nil
Environ. Microbiol.1997; 63: 4111-4115.
[28] Shahaby A.F: Assessment Mixed Culture of Actinomyces and Conflict of interest: Nil
Sacchromyces for biodegradation of Complex Mineral Oil
hydrocarbon. Int. J. Curr. Microbiol. Appl. Sci., 2014; 4:
401-414.
[29] Sepahy A.A., Assadi M.M., Saggadian V. and Noohi A.
Production of biosurfactant from Iranian oil fields by
isolated Bacillus. International J. Environ. Sci. Technol.,
2005; 1(4): 287-293.
[30] Ramos J.L., Duque E., Ramos-Gonzalez M.I: Survival in
soils of an herbicide-resistant Pseudomonas putida strain
bearing a recombinant TOL plasmid. Appl. Environ.
Microbiol, 1991; 57: 260266.
[31] Parales R, Ditty JL, Harwood C: Toluene-degrading bacteria
are chemotactic towards the environmental pollutants. Appl.
Environ. Microbiol, 2000; 66: 4098-4104.