alexa Bioremediation of Chromium in Pulp and Paper Processing Industrial Effluents by Indigenous Microbes | OMICS International
ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Bioremediation of Chromium in Pulp and Paper Processing Industrial Effluents by Indigenous Microbes

Biradar NV1, Sindagi AS2*, Reddy J3 and Ingalhalli SS1
1Department of Zoology, Science College, Dharwad -580001, Karnataka, India
2Department of Microbiology Karnataka Science College, Dharwad -580001, Karnataka, India
3Department of Botany, Sent Joseph’s Post Graduate and Research Centre, Bangalore-27, Karnataka, India
Corresponding Author : Sindagi AS
Department of Microbiology
Karnataka Science College
Dharwad -580001, Karnataka, India
Tel: 08050423977
E-mail: [email protected]
Received November 09, 2012; Accepted November 21, 2012; Published November 23, 2012
Citation: Biradar NV, Sindagi AS, Reddy J, Ingalhalli SS (2012) Bioremediation of Chromium in Pulp and Paper Processing Industrial Effluents by Indigenous Microbes. J Bioremed Biodeg 3:171. doi: 10.4172/2155-6199.1000171
Copyright: © 2012 Biradar NV, et al. This is an open-a ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

The present study was conducted with the goal of elimination of chromium toxicity in the pulp and paper processing industrial effluents. The physicochemical analysis of effluents showed that some of the parameters were exceeded and some were within the permissible limits as per the WHO standards. Based on the isolation, identification and biochemical characterization study the isolated strains (P1, P2, P3 and P4) were identified as Lactobacillus sps (P1) and Bacillus sps (P2, P3 and P4) according to Bergy’s Manual of Systematic Bacteriology. The MICs and MBCs study showed that all the bacterial isolates exhibited Cr tolerance for 1 ppm, 2 ppm, 5 ppm and 10 ppm. The bacterial isolates showed very high degree of resistance to the chromium. The growth studies showed that all bacterial strains exposed to high level of the chromium metal concentration in the environment which have adapted to this stress by developing various resistant mechanism. The rate of Cr (VI) reduction increased with decreasing concentration of Cr (VI) and increasing time interval for all the bacteria isolates. All the bacterial isolates showed the best chromate reduction for different concentration of Cr (VI).

Keywords
Indiscriminate; Anthropogenic; Persistence; Biomagnification; Contamination
Introduction
Metals when present in our body are capable of causing serious health problems, by interfering with our normal body functions. It plays an important role in glucose and cholesterol metabolism and is an essential element to man and animals but at higher levels is toxic to both [1]. Cr is one of the most widely used metals in industry, such as steel production, alloy preparation, wood preservation, leather tanning, metal corrosion, inhibitors, paints pigments, metal plating, tanning, electroplating, steel manufacture and other industrial applications. Cr (VI) is easily soluble and 100 fold more toxic than trivalent one. The Cr (VI) has been recognized as one of the most dangerous environmental pollutants due to its ability to cause mutations, irritations, corrosion of the skin and respiratory tract to most micro organisms and it also causes lung carcinoma in humans. The presence of Cr (VI) in the environment plays a selective pressure on microflora and possesses resistance to high levels while others are sensitive. The bacterial chromate resistance is generally combined to plasmids but it can also be coupled to chromosomal DNA.
Microorganisms can play an important role in the removal of Cr (VI) from the polluted sites. A wide variety of bacteria has been reported to reduce Cr (III) and Cr (VI) under aerobic and anaerobic conditions. Biotransformation of chromate by Chromium Resistant Bacteria (CRB) offers an economical as well as ecofriendly optimal for chromate detoxification and bioremediation. There is a need for innovative treatment technologies for the removal of heavy metal ions from waste water. Different microbes have been proposed to be efficient and economical alternative in removal of heavy metals and from water. In recent years, the use of biological methods particularly microbes to remove metallic ions from liquid effluents has received greater attention over the conventional physico- chemical methods [2].
The remediation of Cr contaminated sites poses a number of unique challenges technologies that are currently used to clean up heavy metal contaminated soils. Bioremediation is one of the promising technologies that are expected to play an important role in waste site clean up [3]. Metals accumulation in the environment as a result of industrial process arising as a matter of serious concern. Since microbes have developed survival strategies in metal polluted habitats, their different microbial detoxifying mechanisms such as bioaccumulation, biotransformation, bio-mineralization or biosorption can be applied either in situ or ex situ. Bacteria also make excellent biosorbents because of their high surface to volume ratios and a high content of potentially active chemisorption sites such as teichoic acid and in their cell walls. Biosorption of metals is one of the most promising technologies involved in the removal of toxic substances from industrial wastes and has been receiving a great deal of attention in recent years, only because of its potential application in industries Therefore this biological phenomenon can be affected by many chemical and physical variables such as pH, ionic strength biomass concentration and presence of different heavy metal solution [4].
The main objective of this study is physicochemical analysis of effluents, isolation, identification and biochemical characterization of Cr resistant bacteria, determination of MIC of bacterial isolates, determination of optimal growth conditions of bacterial isolates and growth studies of bacterial isolates at different concentrations of Cr for different time intervals versus absorbance at 600 nm to reduce the toxicity.
Materials and Methods
Sample collection
The effluent samples of pulp and paper industries situated near by Kali River at Dandeli (Karnataka) were collected at the point of outlet (P1) from industry and Kali River reaching point (P2) early in the morning 7.30 a.m. as per APHA (2) Standards. The samples were collected in well cleaned plastic containers for physico-chemical analysis and well sterilized borosil glass bottles covered with brown wrappers were used to collect samples for microbiological analysis.
Physico-chemical analysis of effluents
The samples of effluents were analysed for physico-chemical parameters. The parameters color, temperature, odour & DO fixation were observed during the collection of the samples prior to the other physico-chemical analysis. The other parameters like pH, conductivity, Total Dissolved Solvents (TDS), total alkalinity, total hardness, nitrate, chloride, sulphate, chromium, calcium, magnesium, COD & BOD were analysed as per the standard procedures prescribed by APHA [5].
Isolation of bacteria from effluents
The bacterial isolates of pulp and paper processing effluents were screened on Luria Bertani (LB) agar plates supplemented with Cr (VI) metal by the standard spread plate method [6]. The isolation was done by serial dilution, standard spread plate method and plates were incubated at 37°C for 24 to 48 hours. The colony characters of bacterial isolates like nutrition, colony color, temperature, growth of colony, shape of colony, margin, elevation, density and gram nature of cells were studied. These bacterial isolate were selected and used for further studies.
Identification and Characterization of the Bacterial Isolates
The selected bacterial isolates were grown on nutrient agar and nutrient broth. The isolated bacteria were identified based on the cellular morphology, growth condition, gram nature and motility. The bacterial isolates were biochemically analyzed for the activities of catalase, starch hydrolysis, utilization of glucose, maltose, mannose, sucrose, lactose, citrate utilization, nitrate reduction, indole test, H2S production and urease activity [Table 1]. The biochemical tests were used to identify the isolates according to Bergey’s Manual of Systematic Bacteriology [7].
Determination of Optimal Growth Conditions
The optimal growth conditions with reference to pH & temperature were determined. The bacterial isolates were grown in LB medium with different pH values (5,6,7 and 8) and incubation was carried out at temperature 4°C, 25°C, 30°C, 37°C and 40°C. The optical density of the log phase growing cultures conditions was noted at 450 nm to determine the growth [8].
Metal tolerance level of bacterial isolates (MICs and MBCs)
The bacterial isolates were tested to determine the MICs and MBCs for chromium. The experimental tubes were prepared by supplementing LB broth for different concentration of Cr (VI) 1 ppm to 10 ppm. One milliliter of test organism suspension was added to each tube. The tubes were incubated at 37°C for 24 hours and visual turbidity was noted. An aliquot of 0.1 ml of nonturbidal tubes was subcultured to agar for determining MBCs [9].
Remediation of Cr (VI) in Liquid Medium (LB)
The chromate reduction capability of bacterial isolates were investigated under aerobic conditions in Luria Bertani (LB) broth amended with various concentrations of chromium ranged from 1 ppm to 10 ppm at pH 7.0. LB broth was inoculated with 200 μl of pure culture, shaken at 150 rpm at 37°C for 48 hour. Cr (VI) added after 3 hr of incubation and measured at different time intervals (3 to 48 hours). Cells were centrifuged at 10,000 rpm for 10 min, then the supernatant was filtered through Whatmann No.1 filter paper and then the chromate reduction was quantified by measuring the decrease in absorbance at 382 nm using atomic absorption spectrophotometer [10].
Results and Discussions
Today indiscriminate and uncontrolled discharge of metal contaminated industrial effluents into the environment has become an issue of major concern. Heavy metals found in waste waters are harmful to the environment and their effects on biological systems are very severe. The main advantages of using bacterial Cr (VI) reductions are that it does not require high energy input nor toxic chemical reagents and the possibility of using non-hazardous strains [10].
The pollution of the environment with toxic heavy metals is spreading throughout the world along with industrial progress, microbes and microbial products of soluble and particulate forms of metals especially dilute external solutions. Microbe related technologies may provide an alternative or addition to conventional method of metal removal or metal recovery [8].
The uses of conventional technologies such as ion- exchange, chemical precipitation, reverse osmosis and evaporative recoveries for this purpose is often inefficient and is very expensive. The most reliable way is the biological treatment, in which microbes serves as efficient detoxifiers of pollutants. It is cost effective and highly suitable for reduction of pollutants in as effluent because microbes are capable of oxidising the organic and inorganic constituents [11]. The Cr-resistant bacterial isolates were obtained from effluents promising candidates for detoxification of Cr contaminated sites. The present innovate technology bioremediation by indigenous microbes can be employed to stop the water pollution at the Kali river Dandeli and to prevent chromium toxicity in the pulp and paper processing effluents. Further studies are needed to increase the biosorption capacities of biomass and develop appropriate technology applicable in the treatment of industrial wastewaters.
Physico-Chemical analysis of effluents
The physico-chemical parameters of pulp and paper processing effluents (P1, P2) were analyzed and recorded in table 2. The results showed that some of the parameters exceeded and some were within the safe land of WHO standards. The physico-chemical parameters which exceeded the safe limit of WHO standards influences larger on the aquatic system and their reproductivity.
Isolation of bacteria from effluent
The bacterial isolates of pulp and paper processing effluents were screened on Luria Bertani Agar (LBA) by spread plate method. The colony characters of bacterial isolates (P1, P2, P3, and P4) were illustrated in table 3. The isolates were selected randomly, purified and preferred on nutrient agar for further studies.
Identification and characterization of bacterial isolates
The bacterial isolates were obtained from the pulp and paper processing effluents. The biochemical characteristics of the 4 bacterial isolates were illustrated in table 4. Based on the biochemical parameters and morphological characters the isolated bacterial strains were identified as Lactobacillus sps (P1) and Bacillus sps (P2, P3, P4), according to Bergey’s Manual of Systematic Bacteriology [10].
Determination of Optimal Growth Conditions
The results of optimal growth conditions of all the 4 bacterial isolates (P1, P2, P3, P4) were carried at different temperature (4°C, 25°C, 30ºC, 37ºC and 40ºC) and pH (5, 6, 7, and 8) were represented in table 5. The bacterial isolates showed optimum growth at 37ºC for pH 7.0.
Metal-tolerance levels of bacterial isolates (MICs & MBCs)
The MICs & MBCs of the 4 bacterial isolates were carried out in the Luria Bertani Broth. The MICs & MBCs results of Cr resistant bacterial isolates were represented in table 6. The MICs and MBCs results were obtained as 10 ppm for all the bacterial isolates of pulp and paper processing effluents. All the bacterial strains exhibited Cr tolerance from 1 to 10 ppm. The MICs and MBCs values varying concentrations from 1 to 10 ppm. The P1, P2, P3, and P4 bacterial isolates showed very high degree of resistance to chromium. All bacterial strains were exposed to high level of Cr metal in the environment have adapted to this stress by developing various resistant mechanism.
Remediation of Cr (VI) in liquid medium (LB Broth)
The Cr (VI) reduction as well as growth studies of bacterial isolates was monitored with different concentrations ranged from 1 to 10 ppm of Cr (VI) at 37ºC for 48 hrs in aerobic condition under shaking at 150 rpm. The rate of Cr (VI) reduction increased with decreasing concentrations of Cr (VI) and increasing the time intervals. All the bacterial strains showed the best chromate reduction for different concentrations of Cr (VI). The bacterial growth was also monitored at different concentrations of Cr (VI) 1, 2, 5, and 10 ppm. All bacterial isolates showed the varying growth for different concentrations of Cr (VI) in the Luria broth and was measured at O.D 640 nm. The chromate reduction was measured absorbance at 382 nm for all the bacterial strains at different concentrations of Cr (VI). The results of chromate reduction and bacterial growth were illustrated in table 7a-7d and table 8a-8d.
The high Cr (VI) reduction was observed at time interval 48 hours and absorbance was high compared to the other. The P1 and P2 bacterial isolates showed the high absorbance compared to the other two bacterial isolates (P3 and P4). The lower the time interval lower Cr (VI) reduction was observed. The higher the time interval higher the reduction was observed. All the bacterial isolates showed optimal growth at different concentrations of Cr (VI). The increase in the growth of bacterial isolates was observed at increased time interval at 48 hrs of time interval.
Conclusion
From the above discussion we concluded that 4 bacterial isolates namely Lactobacillus sps and Bacillus sps are resistant to Cr with different potentialities to remediate or to reduce toxic Cr (VI) to Cr (III). The results of the present study thus provide some of the interesting observations about the new bacterial strains which were isolated from the pulp and paper processing effluents which can tolerate the Cr (VI). It was also proved that potential of Lactobacillus sps, and Bacillus sps for reduction of Cr from effluents. However practical applications of these organisms to the treatment of Cr-containing waste water and effluents need further studies.
Acknowledgement
Authors are grateful to Vision Group on Science and Technology, Government of Karnataka, Bangalore, for granting the sanction for SPiCE project work at our college. We also thankful to Principal, HOD and Co-ordinator, Department of Microbiology, Karnataka Science College, Dharwad for his kind co-operation and encouragement throughout the work.
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Tables and Figures at a glance

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Table 1 Table 2 Table 3 Table 4 Table 5
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Table 6 Table 7a Table 7b Table 7c Table 7d
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Table 8a Table 8b Table 8c Table 8d
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