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Bioremediation of rubber processing industry effluent by Pseudomonas sp

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International Journal of Research in Environmental Science and Technology

Universal Research Publications. All rights reserved

ISSN 2249–9695
Original Article
Bioremediation of rubber processing industry effluent by Pseudomonas sp.
S. Shruthi, M.P. Raghavendra, H.S. Swarna Smitha and K. Girish*
Postgraduate Department of Microbiology, Maharani’s Science College for Women, JLB Road, Mysore – 570 005, Karnataka, India.
*Corresponding author: girishk77@yahoo.com
Received 07 April 2012; accepted 27 April 2012
Abstract
The present study was conducted to develop an ecofriendly process for the treatment of rubber processing industry effluent
using effective indigenous bacteria. Rubber processing industry consumes large volumes of water, chemicals and produces
enormous amounts of wastewater. The discharge of this wastewater to the environment without proper treatment causes serious
and prolong consequences. Economical and bio-friendly approaches are needed to remediate wastewater from rubber
processing industry. Hence, a study was conducted with the aim of treating and disposing of rubber processing industry
effluent that is rich in BOD, COD, PO43-, NH4 and solids. A bacterial strain was isolated from rubber processing industry
effluent and was identified as Pseudomonas sp. The efficiency of this bacterium to carry out bioremediation of rubber
processing industry effluent was studied. Bacterial treatment of the wastewater yielded substantial reduction in total solids
(73%), suspended solids (75.7%), dissolved solids (68.8%), BOD (72.1%), COD (79.4%), ammonia (71.3%) and phosphate
(68.8%) after 15 days of incubation. From the results of the present study it could be inferred that the isolated Pseudomonas sp.
strain could effectively be used for the treatment of effluents from rubber processing industry.
© 2011 Universal Research Publications. All rights reserved
Keywords: Rubber processing industry, Effluent, Bioremediation, Pseudomonas sp.

1. Introduction disposed, can severely damage the environment. The high

The rapid industrialization is accompanied by
both direct and indirect adverse effect on environment. The
rate of contamination of natural water bodies increases with
increased industrialization (Senthil et al., 2012). Rubber
processing industry is one of the most important industries.
Rubber generally occurs in plants as milky white latex but the
chemical composition however, varies from species to
species. Chemically, rubber is a polyterpene consisting of a
long chain (500 – 5000) of isoprene units joined together end
to end to form giant molecules called polymers which are
coiled up like tiny springs (Iyagba et al., 2008). Raw material
products from natural rubber processing sector provide huge
benefits to human beings as they are exploited to manufacture
many kinds of important rubber goods (Anon, 2007). Rubber
processing industry usually generates large quantities of
wastewater containing high concentration of organic matter,
suspended solids and nitrogen (Rakkoed et al., 1999).
Wastewater from the rubber industry has high concentrations
of gaseous and liquid effluent that, if not properly treated and
International Journal of Research in Environmental Science and Technology 2012; 2(2): 27-30
27
level of phosphate and ammonia in rubber effluent makes it a
good medium for algal growth and can result in
eutrophication of surface waters if discharged without proper
treatment (Iyagba et al., 2008).
Several methods have been described in the
scientific literatures for industrial effluent treatment, however
in recent years microbes have been drawing tremendous
attention due to their ability to degrade waste materials and
thereby improving water quality (Boominathan et al., 2007).
Effluents from rubber processing industry were reported to
support microbial growth by a number of workers (Atagana
et al., 1999 a & b; Iyagba et al., 2008; Senthil et al., 2012).
The objective of this study was to isolate bacteria from rubber
industry effluent and evaluate their remediation potential for
use in the biotreatment of rubber industry wastewater.
2. Materials and methods
2.1. Sample collection
Rubber effluent samples were collected from a
rubber factory at Mysore. For microbiological analysis,
samples were collected in 500 ml sterile bottles. Clean plastic operating at 120 rpm. 1.0 ml of this was transferred
containers rinsed several times with the sample were used for aseptically to a second flask containing inorganic salts
physicochemical determinations. The wastewater sample medium. After this solution became turbid, the culture was
used for dissolved oxygen (DO) and biological oxygen transferred to third flask and incubated. This culture was used
demand (BOD) determinations were collected directly into for biodegradation studies.
dark DO bottles and were added some drops of manganous 2.6. Confirmation of biodegradation
sulphate solution to fix the dissolve oxygen. The samples Various parameters such as biological oxygen
were stored at a temperature of 40C until required (usually demand (BOD), chemical oxygen demand (COD), total
between 24 and 48 h). The effluents were collected from five suspended solids (TSS), total dissolved solids (TDS), total
different points on the processing - A = aeration tank; B = solids (TS), ammonia (NH4+) and phosphate (PO43-) were
settling tank; C = backwashed effluent; D = holding tank; E = assayed using standard protocols (APHA, 1995). The effluent
sludge. was inoculated with 1% inoculum and incubated for 15 days
2.2. Isolation of Pseudomonas sp. and the estimation was done at the interval of five days.
The collected samples were brought to the lab 3. Results
and stored in a refrigerator to arrest the microbial growth. 3.1. Isolation and Identification of Pseudomonas sp.
They were brought back to room temperature prior to the Yellow-greenish colonies were found on
conduction of experiment. A Pseudomonas selective isolation Pseudomonas selective isolation agar medium. Colonies
agar (Krueger and Sheikh, 1987) was used to isolate were round to slightly irregular, smooth, and 0.5 to 2 mm in
Pseudomonas sp., from effluent. It consisted of 350 µg of diameter. Gram’s staining revealed negative rods (straight or
nitrofurantoin per ml and 2 µg of crystal violet per ml in a slightly curved) – a characteristic of Pseudomonas species.
nutrient agar base. Sterile media was prepared and Cells exhibited motility with a polar flagellum. The
approximately 15ml was poured into each sterilized Petri identification was confirmed through the results of number of
plates, and allowed to solidify. 0.1ml of sample was biochemical tests as mentioned in table 1. Pseudomonas sp.,
inoculated on solidified agar media by the method of spread was isolated from the effluents collected from all the five
plating using sterile spreader. These Petri plates were different points of the processing viz., A = aeration tank; B =
incubated in an incubator for 24 - 48hours. The Petri plates settling tank; C = backwashed effluent; D = holding tank; E =
were observed after incubation for various bacterial colonies. sludge.
The colony characteristics such as Gram’s reaction, colour, 3.2. Biological oxygen Demand (BOD) and Chemical
margin, elevation were noted. Oxygen Demand (COD)
2.3. Identification of Pseudomonas sp. A high BOD and COD removal efficiency was
The culture was identified by standard observed on treatment of effluent with indigenous
procedures such as morphology, and biochemical Pseudomonas sp. The overall efficiencies of BOD and COD
characteristics as given by Bergey's Manual of Determinative removal by the biological treatment were found to be 72.1%
Bacteriology (Buchanan and Gibbons, 1984). The and 79.4% respectively in comparison to the initial levels
physiological and biochemical tests were conducted (Table 2).
following the methods as described by Cappuccino and 3.3. TDS, TSS, Ammonia and Phosphate
Sherman (1999) to identify the bacteria. TDS, TSS, TS and NH4+ and PO43- values were
2.4. Pure culture also reduced after treatment, signifying the degradation of
To prepare inoculum, the culture was grown at toxic solid components in the effluent by the isolate.
30°C on a rotary shaker operating at approximately 120 rpm Reduction in ammonia and phosphate levels indicates that the
in the inorganic-salts medium amended with 10% effluent bacterium degrades organic and inorganic constituents. TDS
and harvested in early stationary phase by centrifugation at (68.8%), TSS (75.7%), ammonia (71.3%) and phosphate
4°C. The pellets were washed, the cells were resuspended in (68.8%) reduction were observed after treatment of the
buffer, and the entire centrifugation and washing procedure effluent by the bacterium. The pH of the effluent changed
was repeated. The final pellet was resuspended in inorganic from acidic to alkaline resulting in reduction of acidity of the
salts solution. The bacterial growth was observed. effluent (Table 2).
2.5. Acclimatization of Pseudomonas sp. 4. Discussion
The bacterium was acclimatized by growing it The increasing global concern on the
and serially transferring in minimal organic salts medium environment demands that wastes should be properly
amended with 10% of rubber processing industry effluent. managed in order to minimize and possibly eliminate their
The minimal medium used in degradation studies contained potential harm to public health and the environment (Asia
(mg / ml) KH2PO4 – 0.675; Na2HPO4 – 5.455; NH4NO3 – and Akporhonor, 2007). It is well known that wastewater
0.25; MgSO4 – 0.2; Ca (NO3)2 – 0.1; and 1 ml mineral from industries contain suspended solids, reduced oxygen,
solution. Bacterium was cultured in a 500-ml flask with inorganic compounds. In order to minimize the
100ml/flask medium at 30oC for 24 h on a rotary shaker environmental and health hazards, these pollutants need to be
International Journal of Research in Environmental Science and Technology 2012; 2(2): 27-30
28
 Table 1. Characteristics of bacteria used for identification.
Sl. No. Characteristics Pseudomonas sp.
1. Cell shape Rod shaped
2. Gram staining Gram negative
3. Colony on media Yellow-greenish colonies, round to slightly irregular, smooth, and 0.5 to 2 mm in diameter
4. Motility motile with a polar flagellum
5. Spore --
6. Catalase +
7. Oxidase +
8. Starch hydrolysis --
9. Fermentation No acid, No gas
10. Indole test --
11. Methyl red test --
12. Voges–Proskauer test --
13. Citrate utilization test +
14. H2S production test --
15. Urea hydrolysis --
16. Nitrate reduction --
17. Gelatin Hydrolysis +

Tablei2. Physico-chemical and organic characteristics of the in pollutant degradation. Many researchers have employed
effluent before and after treatment (after 15 days of Incubation) Pseudomonas sp., for the treatment of industrial effluents. It
After treatment can degrade hydrocarbons, phenols, celluloses, tannins,
Before Percentage
Parameters with
treatment reduction polyvinyls, pesticides, cyanides, etc. (Anandapandian et al.,
Pseudomonas sp.
2004).
pH 5.7 ± 0.30 7.5 ± 0.26 -- The BOD and COD of the effluents, depicted in
TDS (mg/l) 2240 ± 3.4 700 ± 2.5 68.8% Table 2, showed that all the effluents had higher BOD and
COD values than the permissible limits. Wastes containing
TSS (mg/l) 3512 ± 4.8 855 ± 2.7 75.7%
high BOD and COD are responsible for a heavy depletion of
TS (mg/l) 5752 1555 73.0% oxygen levels in the particular sector of the stream or soil
Ammonia (mg/l) 94 ± 3.0 27 ± 2.2 71.3% (Tariq et al., 2006). Therefore, these effluents needed further
elimination of BOD and COD through proper treatment
Phosphate (mg/l) 48 ± 2.0 15 ± 2.2 68.8%
methods before discharge. In the present study a successful
BOD (mg/l) 1340 ± 2.0 374 ± 1.7 72.1% reduction of both BOD and COD of effluent from rubber
COD (mg/l) 2834 ± 1.9 585 ± 2.0 79.4% processing industry was observed to a level enough to make
Values are the mean of five replicates ± SE
the effluent ready to be discharged into the environment, by
treating with Pseudomonas sp., inoculum. %BOD and
brought down to permissible limits for safe disposal of waste %COD reduction were in the range 70-80%, which is a
water (Manju et al., 1998). Rubber processing industries significant reduction. High BOD and COD reduction in the
consume large volume of water and chemical for processing treatment of textile effluent by Pseudomonas species was
of rubber. A considerable amount of wastewater is generated, reported (Ashok Kumar et al., 2011; Telke et al., 2012).
which is very toxic and contain strong colour, a large amount Pseudomonas sp., isolated from sugar industry effluent
of suspended solids, a highly fluctuating pH, high reduced the BOD, COD, toxicity of industrial effluents of
temperature, COD, BOD, etc. (Asia and Akporhonor, 2007; pharmaceutical, sugar and dairy industries (Anandapandian et
Mohammadi et al., 2010). Because of these characteristics, al., 2004). Significant decrease in BOD and COD indicate the
treatment of rubber wastewater is an essential requirement reduction of pollutants level in the effluent (Sangita et al.,
before it is being disposed to natural water system (Atagana, 2012).
1999a & b; Iyagba et al., 2008). A high organic load in the effluent which was
The objective of this study was to remediate evident by high TS, TSS, and TDS of the untreated effluent
rubber processing industry effluent by indigenous bacterial was higher than the limit. Rubber industry effluent which is
treatment. Effluents from rubber processing industries have rich in NH4+ and PO43- is good medium for algal growth and
been known to harbor many microorganisms (Atagana et al., if discharged without treatment into surface waters can result
1999 a & b; Iyagba et al., 2008; Senthil et al., 2012). The in eutrophication (Ye et al., 1988). Therefore any treatment
Pseudomonas sp., was isolated from the effluent. procedure employed should effectively reduce the level or
Pseudomonas sp., is the most common bacterium employed eliminate NH4+ and PO43- compounds in the effluent so that it
International Journal of Research in Environmental Science and Technology 2012; 2(2): 27-30
29
becomes non-toxic to the environment on discharge. In the
present study significant decrease was observed in the
ammonium and phosphorus levels of the effluent on
biological treatment with Pseudomonas sp., thereby reducing
the above mentioned risk. Similarly in the present study,
biological treatment of effluent with bacterium reduced the
levels of total suspended solids (TSS) and total dissolve
solids (TDS). Microbial treatment is known to reduce the
levels of total suspended solids (TSS) and total dissolve
solids (TDS) of industrial effluents (Monica et al., 2011).
Pseudomonas sp., was found efficient to degrade the tannic
components and make the tannery effluent non-toxic that can
be reused. The strain was isolated from the tannery effluent
only (Arun et al., 2004).
The results of this study revealed that the
Pseudomonas sp., isolated from the effluent is efficient
enough to degrade the toxic components in the rubber
processing industry effluent and therefore could effectively
be used for the treatment of effluents from rubber processing
industry. This biodegradation study certainly will be helpful
to some extent for making a pollution free environment.
Acknowledgements
The authors thank University Grants Commission (UGC),
New Delhi, India for financial support (MRP(S) – 277/08-09/
KAMY004/ UGC- SWRO Dt: 30-Mar-09).
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