Research Article
Open Access
Effective Microbial Consortium of Bacteria Isolated from
Hydrocarbon Polluted Soils of Gujarat, India
Mandalaywala HP, Ratna Trivedi*
Department of Environmental Sciences, Shree Ramkrishna Institute of Computer Education & Applied Sciences
M.T.B. college campus, Athwalines, Surat-395001, Gujarat, India
*Corresponding author: Ratna Trivedi, Department of Environmental Sciences, Shree Ramkrishna Institute of Computer Education & Applied Sciences,
M.T.B. College campus, Athwalines, Surat-395001, Gujarat, India. E-mail:
@,
@
Received: February 03, 2016; Accepted: April 14, 2016; Published: April 20, 2016
Hydrocarbons are widely distributed in environment owing to
its extensive use as pesticides, petroleum products or other organic
compounds. Hydrocarbons being mutagenic and carcinogenic in
nature, it has led to various serious threats to living form. Pollution
can ensue due to accidental spillage or leakages while handling
and transportation of such compounds. Thus it becomes necessary
to ensure safe removal and disposal of pollutants, to avoid further
dispersal in different environmental layers. Bioremediation is one
such effective treatment methods which render the pollutants
harmless. Thus studying hydrocarbon utilizing bacteria becomes
an essential step to formulate an effective bioremediation process.
Hydrocarbon utilizing bacteria were isolated from a hydrocarbon
based polluted site and scrutinized by series of tests. Among the
isolates, five Pseudomonas species have been found to be effective in
hydrocarbon utilizing capability. The isolates were identified using
16 sRNA sequencing procedure.
Keywords: Hydrocarbons; Hydrocarbon utilizing bacteria;
Effective microbial consortium; 16s rRNA sequence
Introduction
Microorganisms play a vital role in maintaining a healthy
ecological balance. Their ability to transform and degrading
many types of pollutants have been widely recognized [1].
Several microorganisms may be involved in the reactions of
biogeochemical cycles and in some cases they are the only living
forms that are capable of degrading the complex elements of
nature and regenerating a form that can be consumed by other
organisms [2]. Hydrocarbons are organic compounds that lack
functional groups and thus make it a polar. They are chemically
less reactive at room temperature [3]; and insoluble in water. In
this study petroleum derivatives are taken into consideration
as hydrocarbon source. Various community play vital role in
degradation of hydrocarbon. Petroleum is a heterogeneous
mixture of hydrocarbons including aliphatic, alicyclic and
aromatic in varying concentration depending upon the origin
and nature. Henceforth, hydrocarbons term is used in general
for the ease of discussion here. Hydrocarbons differ in their
susceptibility to the microbial degradation, and are in general
ranked as follows:Linear alkanes > branched alkanes > small aromatics > cyclic
alkanes i.e., the simpler the structure, more the susceptibility
to microbial attack [4]. Hydrocarbons are hazardous to plants
and animals, are also known to be carcinogenic, mutagenic and
potent immuno-toxicants posing a serious threat to human and
animal health [4]. Thus incorporation of hydrocarbons in natural
environment is not desirable, makes its treatment and proper
disposal inevitable. Various important processes influence the
fate of hydrocarbons in nature, like sorption, volatilization,
a biotic transformation (chemical or photochemical), and
biotransformation. Since microorganisms play an important
role in biogeochemical cycles, the biotransformation is of major
concern. The other method mentioned either just fixes or
transports the contaminants or is not very effective in nature
[2]. Biotransformation is a natural process, which can be used
to solve environmental issues, the process then known as
Bioremediation.
Bioremediation and organisms degrading
hydrocarbons
Bioremediation can be defined as the use of living organisms
to detoxify or remove pollutants owing to their diverse metabolic
capabilities. The process is considered as non-invasive and cost
effective which makes it favorable to practice[4]. Bioremediation
is carried out by living forms. Among others, microorganisms
are also involved in biologically degrading complex matter
into simpler fragments, which renders it harmless and also
makes it available to other organisms for their consumption.
Bioremediation is achieved by existing metabolic potential of
indigenous microorganisms or by introducing microorganisms
through selection on basis of their catabolic activities or even by
introduction of genetically enhanced microorganisms which are
incorporated with such functions [1]. Its effectiveness depends
on the extent to which the microbial population or consortium
can be enriched and maintained in environment, when few
or no indigenous degradative microorganisms exists in the
contaminated area. Hydrocarbons are insoluble in water, thus
in order to consume hydrocarbon derivatives, microorganisms
need to emulsify it first in the solution or medium. For this, they
are known to produce surface active agents i.e., biosurfactants, thus rendering the hydrocarbons susceptible to biodegradation
[5].
Microbial community
Various microorganisms have hydrocarbon degradative
potential. They consume hydrocarbons either individually or
in consortia. Many aerobic bacterial genus viz., Pseudomonas,
Mycobacterium, Rodococcus, Arthobacter, Acinetobacter,
Nocardia and Bacillus are known for their degradative properties.
Some fungi also have the ability to degrade hydrocarbon
based pollutants like Planerochaete chrysosporium (White rot
fungus) is an example of ligninolytic fungi capable of degrading
polyaromatic hydrocarbons and other harmful environmental
pollutants. Cunninghamella echinulata and Mycorrhizal fungi
have also been used for the remediation of PHC-polluted soil [6].
Effective microbial consortium
The advantages of employing mixed cultures as opposed to
pure cultures in bioremediation have been well documented.
The degradative capacity of any microbial consortium is not
necessarily the result of merely adding together of the capacities
of the individual strains forming the association. It could be
attributed to the effects of synergistic interactions among
members of the association [7].
Mutagenesis to enhance degradation
Newly arisen mutations can have very different impacts on
the fitness of the organism, ranging from deleterious through
neutral to beneficial. However, they appear at very different rates
[8]. There are various ways to induce mutation. One such method
is use of UV radiation. UV mutagenesis has also come up as a tool, to enhance degradation by indigenous bacteria [9]. Indigenous
microorganisms play a major role in bioremediation, as they are
adapted to the extremities of the polluted site. They are able to
consume the pollutant as substrate and convert it to simpler
forms which are safe to handle by the environment. Studying
the characteristics of the polluted site and physiology of the
indigenous microbes can thus help to develop a relation between
the two;thus can give better understanding and insight on the
bioremediation processes. The phylogenetic study helps one to
infer the interrelationship between organisms. Thus phylogenetic
study of such indigenous microbes can help us understand the
relation between them better. And this can also help us to infer
the synergistic relationship that may prevail between them to
carry out the degradation process. The purpose of the study is
to collect the sample from hydrocarbon contaminated site, to
identify microbial communities and prepare microbial indices
and to determine relationship between physiological study in
community development.
Materials and Methods
Sample collection
Hydrocarbon based polluted samples were collected
from industrial area located near south Gujarat (Figure 1).
Physico-chemical & organic analysis of the sample
The collected samples were scrutinized for their physicchemical
& organic properties according to the standard
prescribed methods [10].
Enrichment: The samples were then enriched by suspending
in Mineral Salt Medium (MSM).
Figure 1: Showing Industrial area in South Gujarat region from where sample has been collected. (Courtesy: Google earth)
Microbial analysis: The samples after enrichment was
scrutinised further by inoculating in different mediums:
Mineral Salt Medium (MSM) and Tributyrin Agar medium
(TBA); in addition to petroleum derivatives so as to culture
only hydrocarbon degrading microbes [11]. Thus a synthetic
medium was formulated using hydrocarbon based substrate i.e.,
petroleum derivatives along with the hydrocarbon extracted
from the collected sample. Tributyrin Agar showed colonies
development within 48 hours as compared to more than 72
hours in the former, thus TBA was used further in the study.
Tests for hydrocarbon degrading activity: Series of
preliminary tests were carried out to check the hydrocarbon
degrading capacity of the isolates, viz., drop collapse test, oil
spread method, Blue plate agar, Haemolytic activity was checked
using blood agar and then confirmatory test-Phenol sulphuric
acid test [12].
Effects of various factors on degradation: Various factors
like temperature, pH, nitrogen sources and substrate sources and
concentration were tested for their effect on degradation rate.
Effective microbial consortium: Microbial consortium
was formulated from the isolates, to evaluate the degradation
properties of individual isolates as compared to different
consortium so formulated.
Mutagenesis: Induced mutation was carried out by exposing
24 hours old culture to UV radiation for 30 mins. The degradation
carried out by mutated strain was compared to that of carried out
by wild type strain [9].
Result and Discussion
Analysis of samples
Sample collected from polluted sites were characterized
for their physical, chemical and organic properties. The results
obtained are as mentioned in the tables below (Table 1; Figure
2(a,b,c,d,e)).
These samples were suspended in MSM media, as mentioned
above for enrichment of indigenous microorganisms. Figure 3
shows the enrichment flasks.
Then the medium was streaked on MSM and TBA, with
petrol spread on top so as to grow only hydrocarbon consuming
colonies. In table 2&3, the results of microbial community count
and calculation of microbial diversity Indices is shown.
Only bacterial community was observed in the sample, no
fungal or actinomycete growth was observed throughout the
study. Bacterial colonies so formed were checked for varies
characteristics, viz., Gram’s reaction, motility, Capsule formation and so on. 11 isolates were obtained and were studied further.
The results are mentioned in the table 4.
Morphological characterization of isolates
Out of 11 above mentioned isolates, 8 isolates viz.,
A,B,C,D,F,G,H & K were found to be Gram Negative. The remaining
three isolates; E,I&J were found to be gram variable. Their
microscopic study showed that all of the isolates were short rods.
The isolates were checked for capsule formation by carrying out
capsule staining method: Maneval's method of capsule staining.
It was found that isolates were non- capsulated. Motility was
checked by standard method, which indicated that all isolates
were motile.
Oil spreading assay
This method was developed by Morikawa. It is based on the
oil displacement activity [12]. All the isolates were assessed by
this method. But none of the isolates gave positive result.
Drop collapse method
This assay relies on the destabilization of liquids by
secondary metabolites produced by hydrocarbon consuming
microorganisms, i.e., Biosurfactants [12]. None of the isolates
gave positive result in this method.
Blue agar plate
This method was developed by [13]. The microbes of interest
are inoculated on a light blue mineral salt agar plate containing
cationic surfactant Cetyl-Trimethyl-Ammonium Bromide (CTAB)
and the basic dye methylene blue. If microbes are able to degrade
hydrocarbons, they secret anionic surfactants which form a dark
blue, insoluble ion pair with cetyl-trimethyl-ammonium bromide
and methylene blue. The positive isolates will thus form dark
blue coloured colonies [12]. Isolates A, C, D, F, G & H gave positive
results in this test. Other colonies showed negative results
(Figure 4).
Blood agar haemolysis
This method was developed by [14]; and is based on the fact
that hydrocarbon degrading microbes are able to haemolyse the
red blood; observe for zone of hemolysis [12]. Isolates A, C, D, F &
G showed zone of hemolysis (Figure 5; Table 5).
Confirmatory test
Phenol- sulphuric acid test: The positive isolates were
further scrutinized by confirmatory test [12] (Figure 6). Only
positive results are shown here. Isolates A, C, D, F& G showed
positive results in this test. Isolate H gave positive result only with
blue agar plate; still it was checked further for the hydrocarbon
degrading property.
Table 1: Result of physical analysis of sample.
Parameters |
Sample-A |
Sample-B |
Sample-C |
Sample-D |
Physical state |
Viscous Liquid |
Viscous Liquid |
Viscous Liquid |
Solid |
Appearance |
Dark Brown |
Dark Brown |
Dark Brown |
Black |
Flammability |
Flammable |
Flammable |
Slight Flammable |
Flammable |
Figure 2: Results of Physical, chemical and organic analysis of sample. a: Physical Analysis : pH (10% Solution). b: Physical Analysis: Loss on Drying.
c: Chem. Analysis: SO3-2 and Phosphate (P). d: Chem. Analysis: Chloride (%) & Sulphur (%). Org. Analysis: Ash content (%), Annealing loss (%)
and Oil & grease (%). f: Org. Analysis: Calorific Values Gross and net (Kcal/ gm).
Table 2: Microbial Community.
Sample |
Bacteria (CFU/ml) |
Colonies |
A |
1.2 x 105 |
3 |
B |
1.1x 105 |
2 |
C |
1.1x105 |
2 |
D |
1.3x105 |
4 |
Table 3: Microbial diversity Indices (Using Past-3 Software).
Index |
Bacterial diversity |
Taxa_S |
4 |
Individuals |
4 |
Dominance_D |
0.2512 |
Simpson_1-D |
0.7488 |
Shannon_H |
1.384 |
Evenness_e^H/S |
0.9975 |
Brillouin |
0.6034 |
Menhinick |
1.845 |
Margalef |
2.164 |
Equitability_J |
0.9982 |
Fisher_alpha |
12.73 |
Berger-Parker |
0.2128 |
Chao-1 |
4 |
Results of effects of various factors on degradation
Nitrogen Sources
The effect of different nitrogen sources on the degradation
capacity was also studied. The nitrogen sources taken into
consideration were peptone, Sodium Nitrate (NaNO3), potassium
Figure 3: Samples A, B, C & D suspended in MSM media.
Figure 4: Blue Agar Plate.
Figure 5: Blood Agar Plate.
Nitrate (KNO3), Calcium Nitrate Tetra Hydrate (Ca(NO3)2. 4H2O),
Ammonium Sulphate (NH4SO4), Ammonium Chloride (NH4Cl) and
NH4NO3). Among these, peptone gave better results (Figure 7a).
Here a too exceptionally high result with isolate G was observed
with Ammonium Sulphate was due to pigment formation.
Different substrate concentration
A synthetic medium was prepared by using petroleum
derivatives. The concentration and type of petroleum derivative
is varied to study its effect on the growth and degradative
properties with varying substrates Figure 7b to 7g. shows
results of different concentration of petrol, kerosene, diesel and combinations of these petroleum derivatives at different
concentration.
Degradation by formulated consortium
Formulated consortium was studied for their degradative
properties, and the result of the same are as shown in figure
7h & figure 7i. In Figure 7h, OD of consortium formulated by
combination of two isolates: Consortium I and by combination
of three isolates: Consortium II. The relative population size of
the isolates for these consortiums were kept equal i.e., in 1:1
ratio. The results of consortium I as been shown in Figure 7h;
combination of CF and CG gave highest OD among others. And
in figure 7i, OD of combination of Consortium II has been shown.
Among which ACF showed maximum OD i.e., 0.4, which is higher
than CF & CG as well which is nearly 0.25 (Figure 8).
Enhancing degradative property by mutation
The hydrocarbon degrading property of isolates was seen to
be improved when isolates were mutated by UV radiation. The
figure below shows that mutation enhanced the degradative
properties of the isolates as compared to that of wild type strains
(Figure 9).
Identification of microorganisms
Isolated microorganisms has been identified by 16s RNA gene
sequencing (Saffron’s Gene Laboratory) and further submitted to
NCBI. The submitted sequences got accession numbers as below
(Table 6):
Figure 6: Phenol – Sulphuric Acid test (Confirmatory test).
Table 4: Characterization of isolates.
Isolate |
Gram’s Reaction |
Capsule formation |
Motility |
Oil Spreading Assay |
Drop Collapse |
Blue Agar Plate |
Haemolytic reaction
|
Confirmatory test
(Phenol- Sulphuric Acid test) |
A |
Negative |
No |
Yes |
No |
No |
Yes |
Yes: ß |
Yes |
B |
Negative |
No |
Yes |
No |
No |
No |
No |
No |
C |
Negative |
No |
Yes |
No |
No |
Yes |
Yes: ß |
Yes |
D |
Negative |
No |
Yes |
No |
No |
Yes |
Yes: ß |
Yes |
E |
Variable |
No |
Yes |
No |
No |
No |
No |
No |
F |
Negative |
No |
Yes |
No |
No |
Yes |
Yes : ß |
Yes |
G |
Negative |
No |
Yes |
No |
No |
Yes |
Yes: ß |
Yes |
H |
Negative |
No |
Yes |
No |
No |
Yes |
No |
No |
I |
Variable |
No |
No |
No |
No |
No |
No |
No |
J |
Variable |
No |
No |
No |
No |
No |
No |
No |
K |
Negative |
No |
No |
No |
No |
No |
No |
No |
Table 5: Diameter of zone of haemolysis.
Colony |
Isolate A |
Isolate C |
Isolate D |
Isolate F |
Isolate G |
Zone diameter |
1 mm |
2 mm |
2 mm |
1 mm |
1 mm |
Table 6:
Sample |
Isolate |
Strain |
Accession number |
A |
Isolate A |
Pseudomonas aeruginosa strain RRLP1 |
KU314415 |
Isolate G |
Pseudomonas aeruginosa RRLP1 |
KU314419 |
B |
Isolate C |
Pseudomonas aeruginosa Strain AS-1 |
KU314416 |
Isolate H |
Pseudomonas aeruginosa Strain JQ-41 |
KU314420 |
C |
Isolate D |
Pseudomonas aeruginosa Strain SI5(1)3 |
KU314417 |
D |
Isolate F |
Pseudomonas spp. Strain 14-1 |
KU314418 |
Phylogenetic study
Phylogram was prepared in MEGA 7 software Figure 10.
Conclusion
The samples extracted from the hydrocarbon based waste
matter showed majorly Gram negative to gram variable
microorganisms. Out of eleven, six Pseudomonas species
were isolated with Hydrocarbon degrading properties. These
dominated Pseudomonas species were identified by 16s RNA
sequence analysis. The Pseudomonas species identified are
Isolate A: Pseudomonas aeruginosa strain RRLP1, Isolate C:
Pseudomonas aeruginosa Strain AS-1, Isolate D: Pseudomonas
aeruginosa Strain SI5 (1)3, Isolate F: Pseudomonas spp. Strain
14-1, Isolate G: Pseudomonas aeruginosa RRLP1, Isolate H:
Pseudomonas aeruginosa Strain JQ-41. On assessing effects of
various parameters on their hydrocarbon degradation properties,
it was found that at the isolates were able to grow under various
physical conditions since their origin physical condition was also
extreme. It was observed that, at temperature 30°C, consistent
growth was observed as compared to 37°C, which showed varied
growth response. When isolates were exposed to varied pH
values, they gave growth in pH range of 4.5 to pH 9.0. However, at pH 4 no growth was observed with any isolate and at pH 9,
comparatively less growth was observed considering lesser OD
values. Consistent growth was seen in pH range of 5.5 to 6.5. It
was also observed that isolates were able to grow in different N
sources but among those, peptone gave consistent results. And
in carbon concentration, petrol 0.005% gave consistently good
growth than Kerosene and diesel at different concentrations.
High growth among the isolates was observed by isolate D:
Pseudomonas aeruginosa Strain SI5(1)3, at petrol concentration
of 0.005% (v/v), with peptone as Nitrogen source, with pH of 6.0,
when incubated at temperature 30°C for 72 hours. And isolate
F: Pseudomonas spp. Strain 14-1 was able to show pigmentation
in different physical conditions which was not observed in any
other isolate. The study represented that Pseudomonas species
isolated from hydrocarbon contaminated waste matter were
able to degrade various petroleum derivatives at different
concentration and also under the influence of various physical
conditions. Moreover different strains of Pseudomonas should
be further studied for their hydrocarbon degradative properties
at large scale. This work can be helpful to design consortia to
biodegrade hydrocarbon pollution in natural environment and
to have more insight in this area of research which needs more
attention considering the prevailing pollution rate.

Figure 7: Results of effects of different parameters (a, c, d, f, g, h and i are the isolates as mentioned below). a: Effect of different Nitrogen (N)
source. b: Effect of different Petrol concentration. c: Effect of Different Kerosene Concentration. d: Effect of Different Diesel Concentration. e: Effect of
Petrol+Diesel in different conc. f: Effect of Petrol+Kerosene in different conc. g: Effect of Diesel+kerosene different conc. h: Degradation by consortium
prepared by combining two isolates. i: Degradation by consortium prepared by combining three isolates.
Figure 8: Comparison between individual isolate and consortium.
Figure 9: Effect of Mutation on cell growth.
Figure 10: Phylogram of the isolates.
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