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Journal of Global Biosciences

ISSN 2320-1355
Volume 3, Number 6, 2014, pp. 935-940
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researchsubmission@hotmail.com

Research Paper
PHENOL DEGRADATION BY BACTERIA ISOLATED FROM COIR
RETTING BEDS OF SOUTH KERALA COAST
Reshma, J. K., V. Salom Gnana Thanga* and Anu Mathew
PG Department of Environmental Sciences,
All Saints College, Thiruvanathapuram
*Department of Environmental Sciences,
University of Kerala, Kariavattom.
Abstract

The coir-retting environment harbours a variety of potential microbes


which can degrade phenols. The growth and adaptability of these bacteria
in environment containing phenolic compounds have induced their
biodegradation potential. Studies were conducted to assess growth and
degradation of phenol (10 and 50 ppm) by 9 bacterial isolates under in
vitro conditions. The percentage degradation was derived from the
residual phenol concentration in batch culture experiments. All the 9
cultures showed increase in degradation percentage with period of
incubation. Aeromonas sp., Campylobacter sp., E. coli, Mesophilobacter sp.
and Brucella sp. showed more growth and degradation with 50 ppm of
phenol compared to other cultures. All the cultures showed 100 per cent
degradation with 10 ppm of phenol. After pilot-scale studies, these
cultures can be used for phenol degradation in phenol containing
wastewaters.
Key words: phenol, retting, coir-ret-liquor, biodegradation, bacterial
cultures.
INTRODUCTION

Coconut (Cocos nucifera) is cultivated in tropical countries. The fibrous mesocarp of coir
is used to make ropes. The waste of coir yarn industry (coir pith) gets accumulated in
large quantities making their disposal difficult, though it is used as soil conditioner [1].
Biological treatment of wastewaters containing organic pollutants has been universally
accepted as a cost-effective method for prevention of environmental pollution.
Microorganisms play a dominant role in the transformation of the otherwise stable endproducts. Microorganisms make use of organic pollutants for their growth and
functioning of cellular process by electron transport mechanisms. Phenol and its
derivatives are some of the major hazardous compounds in industrial wastewater, and
for this reason, biodegradation of phenol has attracted keen attention[2] and the present
study is an attempt to find out the most efficient bacterial isolates which can utilize

Journal of Global Biosciences


ISSN 2320-1355

Vol. 3(6), 2014 pp. 935-940

phenol as a carbon source and thereby reduce the phenol concentrations especially in
water bodies where the range of phenol concentration is well above the permissible
limits as exhibited by the water bodies in coir retting ground. Phenols and other
aromatics are discharged from a variety of industrial processes; the predominant being
coal refining, petroleum refining, phenol manufacture, pharmaceuticals etc. Many
studies conducted earlier demonstrate that microbes isolated from the retting areas
have a wide range of phenol degrading capacity.
Micro-organisms that degrade phenol were isolated as early as in 1908[3] and
biodetoxification of phenol was proven as a viable method for remediating phenol
containing wastewater. The process of coir retting, a major industry in the coastal
regions of Kerala, also releases huge quantities of phenolic compounds in the estuarine
system, where the coconut husk is steeped into water for liberating the fibers from
husk. In this paper, biodegradation potential of phenol degrading bacterial strains was
tested under in vitro conditions with two different concentrations of phenol.
MATERIALS AND METHODS
Culture conditions
The bacterial strains which used for the study were isolated from coir-ret-liquor and
tolerant to high concentration of phenol. They were identified as Micrococcus sp.,
Moraxella sp., Pseudomonas sp. Brucella sp, Escherichia coli Campylobacter sp.,
Aeromonas sp., Vibrio sp., and Mesophilobacter sp. based on biochemical
characterization. These may be functionally dominant microflora and hence, these
cultures were used for further degradation studies.
Growth of bacterial cultures in different concentrations of phenol
Growth of the bacterial isolates were carried out for a period of four days, in minimal
media containing (gL-1)KH2PO4 (0.17), Na2HPO4(0.98), MgSO4(4.87), (NH4)2SO4(0.10),
FeSO4(0.05), CaCO3(0.20), ZnSO4(0.08), CuSO4(0.016), CoSO4(0.015), and H3BO4(0.006),
containing two different concentrations of phenol (10 and 50 ppm). The first sample
(time zero) was taken immediately after substrate addition for each isolate and growth
was recorded in terms of optical density (ODA600). The samples were incubated at room
temperature and 1ml aliquots of the isolates were taken each day at 24 h interval for a
period of four days, in order to monitor the OD600. Two sets of control were maintained.
One set of broth added with respective cultures without phenol and another set of broth
with phenol without culture. The experiment was done in triplicate.
Biodegradation potential of the isolated bacterial cultures
The residual phenol in the sample was determined by Bray et al. [4]. To, 1 ml of sample
(crude culture extract), 2 ml of 20% Na2CO3 and 1 ml of properly diluted FolinCiocalteaeu reagent were added. The tubes were placed in boiling water bath for 1
minute. Blanks were also treated similarly. Absorbance was measured at 725 nm. The
amount of residual phenol in the sample was measured using a standard graph
prepared from catechol.
RESULTS AND DISCUSSION
Biodegradation of phenol by nine pure cultures were tested in two concentrations of
phenol viz., 10 and 50 ppm at different time intervals from 24th to 96th hour of
incubation. Based on the residual phenol concentration, the percentage degradation
was derived. Complete degradation was achieved by all bacterial isolates except Vibrio
sp. with the lesser concentration (10 ppm of phenol). However with 50 ppm, complete
degradation was not observed with any of the culture studied. Neverthless,

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Journal of Global Biosciences


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Vol. 3(6), 2014 pp. 935-940

Mesophilobacter sp. and Campylobacter sp. could degrade about 99 percent of phenol
followed by Aeromonas sp. which recorded 96 per cent removal. However, very less
degradation was observed with Moraxella at higher concentration. Abd-El Haleem et al.
[5] studied the effects of mixed nitrogen sources on bioremediation of phenol by
immobilized Acinetobacter sp. strain W-17 and found that complete phenol (500 mg/l)
degradation was achieved after incubation for 120 h. Ke et al. [6] also reported 100%
degradation of phenol (1260 ppm) by microbial consortia in a UASB reactor.
Almost all cultures showed a uniform increase in percentage degradation with
incubation period. Aeromonas sp., Campylobacter sp., E. coli, Mesophilobacter sp. and
Brucella sp. showed more or less similar degradation percentage in both lower and
higher concentrations of phenol. In the remaining four bacterial cultures i.e. Micrococcus
sp., Pseudomonas sp., Moraxella sp. and Vibrio sp., degradation was higher at lower
phenol concentration as shown in Figure 1-9. In contrast, Mahiudddin et al [7] showed
that Pseudomonas fluorescens completely degraded phenol up to 600 ppm in 24 hours
and corresponding bacterial cell growth (i.e., OD at 660 nm) was 0.873 at 24 hours
from the initial cell density 0.110. After 24 hours the cell density was recorded to be
decreased with time due to absence of carbon source. Their studies further revealed
Pseudomonas fluorescens PU1 also completely degraded 800 ppm and 1000 ppm of
phenol in 72 hours whereas it degrades about 99 percent of phenol in 48 hours as well.
The highest cell growth was found at 72 hours for 800 ppm and 1000 ppm of phenol
as 0.999 and 1.055, respectively. Joseph and Chandrika [8] studied biodegradation of
phenol (100 to 800 ppm) using several bacterial cultures. They also observed that the
rate of biodegradation reduced at higher concentrations of phenol. Similar results were
also reported by Nisha and Meera[9] in a study on bioremediation of phenol
contaminated soil. The phenol-degrading strains convert a considerable portion of
substrate carbon to biomass.

Aeromonas

Micrococcus
10
ppm
24

48 72
Hours

96

Fig: 1 Phenol Degradation by Micrcoccus

150
100
50
0

% degradation

% degradation

150
100
50
0

10
ppm
24

24

48 72
Hours

96

Pseudomonas
% degradation

% degradation

10
ppm

96

Fig: 2 Phenol Degradation by Aeromonas

Campylobacter
150
100
50
0

48 72
Hours

150
100
50
0

10
ppm
24

48 72
Hours

96

Fig:3 Phenol Degradation by Campylobacter Fig:4 Phenol Degradation by Pseudomonas

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Journal of Global Biosciences


ISSN 2320-1355

Vol. 3(6), 2014 pp. 935-940

Moraxella
150
100
50
0

% degradation

% degradation

150
100
50
0

Mesophilobacter
10
ppm

24

48 72
Hours

96

Fig:5 Phenol Degradation by Moraxella

10
ppm
24

Brucella
10
ppm

72

96

% degradation

% degradation

100
80
60
40
20
0
48

96

Fig:6 Phenol Degradation by Mesophilobacter

Vibrio

24

48 72
Hours

150
100
50
0

Hours

10
ppm
24

48

72

96

Hours

Fig: 7 Phenol Degradation by Vibrio

Fig: 8 Phenol Degradation by Brucella

% degradation

E.coli
150
100

10
ppm

50
0
24

48
72
Hours

96

Fig: 9 Phenol Degradation by E.coli


Growth of the cultures also correlated with the degradation pattern. The former five
cultures which showed increased degradation in 50 ppm phenol also was found to have
increased growth in the same concentration (OD600 96h = >0.3), while the cultures
which showed lesser degradation had less growth at 50 ppm of phenol. The increase in
growth with degradation confirms the utilization of phenol as carbon source by the
degrading microbes as shown in Figure 10. Mohite et al [10] studied the growth of
bacteria and phenol concentration in media and showed the inverse proportion with
each other. The decrease in phenol concentration accompanied with increase in
biomass.

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Journal of Global Biosciences


ISSN 2320-1355

Vol. 3(6), 2014 pp. 935-940

OD- 600 nm

Growth rate of the bacteria during 96


hours
0.4
0.3
10 ppm
0.2
50 ppm
0.1
0

Fig: 10- Growth rate of different strains in phenol enriched cultures during 96 hours
Regina et al. [11] studied the behavioral patterns of hydrocarbon-utilizing bacteria in
media containing different concentrations of crude oil. They showed that lower doses of
crude oil were more highly utilized than higher doses. Neumann et al. [12] adapted
Pseudomonas strains to high concentrations of phenol (1000 mgL-1) and further
biodegradation was carried out at a concentration of 500 mgL-1. They opined that the
cultures could grow by utilizing phenol as a sole source of carbon and energy. The
concentration of phenol and presence of halogenated substrates seem to play a crucial
role on degradation shown in our study and also reported by others. Lin et al [13]
concluded from their study that high concentrations of phenol are usually inhibitory to
growth of organisms
CONCLUSION
Bacterial strains isolated from coir-ret-liquor showed tolerance to high phenol
concentration. This clearly reveals the adaptability of the cultures to survive in such
unique ecosystems. It is evident that coir-retting environment harbours a variety of
potential microbes which can degrade phenols. The growth and adaptability of these
bacteria in environment containing phenolic compounds have induced their
biodegradation potential. The cultures developed from the coir-ret-liquor have proved
to perform well in the biodegradation of phenolic compounds. Further research should
be done with emphasis on large-scale pilot studies to evaluate the effectiveness of these
phenol degrading microbes as suggested by Agarry and Durojaiye [14] and if feasible, it
will be a possible solution for treating wastewater containing phenolic and related
pollutants.
ACKNOWLEDGEMENT
The authors are extremely thankful to the financial support provided by University
Grants Commission for carrying out this study.
REFERENCES
1. Christopher, P.A., Viswajith, V., Prabha, S., Sundhar, K., Malliga, P. (2007). Effect of
coir pith based cyanobacterial basal and foliar biofertilizer on Basella rubra L., Acta
agriculturae Slovenica, 89: 59-63.

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Journal of Global Biosciences


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Vol. 3(6), 2014 pp. 935-940

2. Shingler, V. (1996). Molecular and regulatory check points in phenol degradation by


Pseudomonas sp. CF 600. In: Molecular Biology of Pseudomonas. (Nakazawa T,
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from an industrial effluent, African J. of Biotechnol., 7(13):22322238.
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