Physico-Chemical Analysis of Electroplating Industrial Effluents, Bioassay and Isolation of Nickel Resistant Bacterium

By N. Nagarajan¹, S. Sathyavathi², P. Gunasekaran² and P. Rajendran¹ *
November 2012

  1. Department of Advanced Zoology & Biotechnology, Vivekananda College, Tiruvedakam, Madurai – 625 214, Tamil Nadu, India
    *Corresponding Author
  2. Department of Genetics, School of Biological Sciences, Madurai Kamaraj University, Madurai – 625 021, Tamil Nadu, India
Abstract
Physico-chemical analysis of Electroplating industrial effluents in Madurai city, Tamil Nadu, India revealed the contents are beyond the threshold limits and nickel is prominent (687–5569 ppm). Bioassay studies such as Vigour index, Tolerance index and Percentage Phytotoxicity in cowpea, Vigna unguiculata confirmed that the effluents are highly toxic. Seventeen Colony Forming Units (CFU) were isolated after ten days from the effluent samples that were enriched with 10% nutrient broth. Two bacterial colonies showed hyper nickel tolerant capacity and the Minimal Inhibitory Concentration (MIC) is 1500 ppm. Nickel biosorption studies revealed that among them one has metal sorption potential. The objective of the current study is to use the hyper-nickel tolerant microbe for in situ and ex situ bioremediation.

Keywords: Bioremediation, Nickel, Electroplating Industrial Effluent, Biosorption

Introduction

Environmental contamination due to anthropogenic and natural sources is increasing day by day because of increase in population, industrialization and urbanization. The enigma for the public, scientists, academicians and politicians is how to tackle the contaminants that jeopardize the environment. The ideal solution for pollution abatement is Bioremediation, the most effective innovative technology to come along that uses biological systems for treatment of contaminants (Shukla et al., 2010). Recognizing the ability of microorganisms and biological materials to remove them from the polluted sites makes it cheaper method of heavy metal remediation. Microorganisms remove metals as a result of biosorption and bioaccumulation (Baigai, et al., 2012).Toxic metal classified as environmental pollution cannot be degraded. But their oxidation state can be changed to another less toxic state by microorganisms. Thus, bioremediation of heavy metal aims at sequestering the metals to make them unavailable to flow in the ecosystem or extract mobilizing them for reuse or safe disposal (Crawford, et al., 1995). Bioremediation using natural biomaterials is promising alternative to conventional methods (Kashefi et al., 2012).

Madurai, south Indian temple city, has a large number of electroplating industries and their effluents not only harm the environment but also have a negative effect on living organisms. The method used in handling and disposing the spent dye-bath varies within the individual industries due to their historic evolvement.Concentrated solutions such as spent plating baths, alkalies, static drag out solutions, and reject products may have concentrations of pollutants hundreds or thousands of times higher than the discharge limits of publically owned treatment works (POTW). Therefore it is important to develop low cost and friendly method for removal of toxic heavy metal ions from the wastewater. The wastewater treatment system in Indian industries is recommended to be essentially installed to meet the waste water discharge norms, but presently only 10% of the waste water generated is treated and the rest of untreated water is discharged as it is into nearby water bodies (Mehta and Bhardwaj, 2012).The use of  industrial effluents for irrigation has emerged in the recent past as an important way of utilizing waste water taking the advantage of the presence of considerable quantities of N,P,K and Ca alone with other essential elements (Niroula, 2003).Therefore, it is necessary to study the impact of these effluents on crop system before they are recommended for irrigation (Thamizhiniyan, et al 2009).

Hamza et al (2012) reported natural process employing microorganisms is considered to be very effective and environmental friendly method of decontamination. Heavy metals are non-degradable and must be reduced to acceptable limits before discharging into environment to avoid threats to living organisms (Alam et al., 2012; Shan et al., 2012). Use of microbial resources coupled to other modern techniques is one of the most promising and economical strategies for removing environmental pollutants (Chatterjee et al., 2008). Heavy industrialization, lack of safe procedures for and disposal of toxic substances have made the natural bioremediation process inadequate to reduce the quantity of toxic substances released into the nature. It has therefore become a necessity to augment the problem by the application of alternate bioremediation method (Sivasubramanian, 2010).

Objectives of the Study

The main objective of the current study is to analyse the constituents of different electroplating industrial effluents of Madurai city, assessing the toxicity, isolation of microbial strains from the effluents and develop in situ and ex situ bioremediation process for treatment. The other objective is to fabricate economically viable bioreactor for the small scale industries to treat the effluent before releasing into the environment and genetic improvement of the isolated microbial strain to remediate metals in the effluents after release from the industries.

Materials and Methods

Sample Collection and Preservation

Grab sampling, a multimedia sample collection technique (Butterfield, 2000) comprising discrete aliquots collected from one specific sampling location at a specific point of time was adopted in the present study. The grab sample provided a good representation of the concentrations of the contaminants present at the point from where the sample was collected. Four major electroplating industries, located at Jaihinapuram, Vilangudi and Kalavasal the most thickly populated areas of the city was identified for collection of samples. The concentrated effluent samples were batch–generated and the frequency of generation varies. The effluent was collected in polythene containers (35 L capacity) which were pretreated by soaking in 10% HNO3 for 48 h.  Sample was preserved by adding 5 ml of concentrated nitric acid per liter of sample and transported to the analytical laboratory. The containers were rinsed with the sample just before collection. A blank was also prepared with deionised water and preservative.

Physico-Chemical Analysis of Effluent Samples

The effluent samples were analyzed using APHA (1980) procedure and the metal content was analysed using Perkin - Elmer 2380 atomic absorption spectrophotometer.

Bioassay Studies

Certified, cowpea (Vigna unguiculata C152), seeds were purchased from Farm Aid Corporation, Madurai. Vigour index (Vi) studies with different concentrations of Ni in electroplating effluent were performed following the procedures of Abdul Baki (1953). The seeds were dressed with 0.1% mercuric chloride for 3 min and thoroughly washed with distilled water. Filter paper soaked in 10 ml of chosen dilutions of electroplating effluent was placed in sterilized corning Petri plates (13 cm) and 10 randomly selected seeds were placed over it. The seeds were irrigated with equal quantity of different effluent samples and the seeds irrigated with distilled water were taken as control. The Petri plates were kept at indoor laboratory conditions under diffused light at 28 ± 2°C. Germination counts were taken after 48 hrs and the shoot and root lengths were measured after seven days. The experimental data were analyzed statistically (Fisher, et al., 1949). The vigour index (Abdul Baki, 1973), Tolerance index (Turner, et al., 1972) and Percentage of Phytotoxicity (Chou, et al., 1976) are calculated as follows:

Vigour index:
(Mean Root Length + Mean Shoot Length) × Germination (%)
Tolerance index:
Mean length of longest root in treatment  ⁄  Mean length of longest root in control
Percentage of Phytotoxicity:
Radical length of control − Radical length of test  ⁄  Radical length of control × 100

Isolation of Microbes from Electroplating Effluents

Serial dilution technique was used to score individual colony forming units (CFU) from the electroplating industrial effluent samples enriched with 10% nutrient broth kept in shaker for ten days. They were transferred to nutrient broth and incubated at 37° C for 24 hours. Individual colonies were isolated using streak plate technique and the procedure was repeated thrice for purification. Pure colonies were stored on agar slants and kept at -4° C and used for further experiments.

Minimal Inhibitory Concentration (MIC) Assay

Minimal inhibitory concentration (MIC) assay was done to test the Ni toxicity in microbes as explained by Bansal et al. (2000). Nickel sulfate (NiSO4.6H2O) was incorporated in the nutrient medium in 2-fold serial dilution to obtain final concentrations of 500, 1000, 1250, 1500, 1625 and 1750 ppm respectively. Colony forming units isolated from the electroplating effluent was patched on nutrient medium amended with different Ni concentrations and incubated at 37°C. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the metal ion that caused visible suppression of bacterial growth.

Screening of Metal Accumulating Bacterium

The heavy metal resistant isolates were seeded in nutrient agar plates containing metals and incubated at 37°C for 48 hrs.  The culture plates with bacterial colonies were exposed to hydrogen sulfide (resulting from the reaction of ferric sulfide with 0.1 M chlorohydric acid).

Results and Discussion

The physico chemical analysis of effluent samples from the selected industries reveal that certain parameters were higher than the permissible limits of CPHEEO(1993)/CPCB( 2000). Nickel, the prominent heavy metal in the effluent samples is several times higher than permissible levels (Figure 1 and Table 1). The mobilization of heavy metals into the biosphere by human activity has become an important process in the geochemical cycling of these metals. This is acutely evident in urban areas where various stationary and mobile sources release large quantities of heavy metals into the atmosphere and soil, exceeding the natural emission rates (Costa, 1999).

Plate 1 · Growth pattern of Ni sorbing bacterium
at different Ni concentrations
Plate 1

Regulation, handling and bioremediation of hazardous material require an assessment of the risk to some living species other than human being or assessment of hazard to the entire ecosystem (Issazadeh et al., 2012). Various concentrations of untreated effluents produced inhibition in seed germination and seedling growth parameters, length of root, length of shoot, number of lateral roots in cowpea.

The toxic effect of electroplating effluent on vigour index, phytotoxicity and tolerance of cowpea is shown in Figure 2, 3 and 4, respectively. The results shows that cowpea germination is not affected at lower concentration of electroplating effluent (0.01%). However in higher concentrations the growth parameters were affected revealing their toxicity. Germination failure and significant decrease in vigour index (Vi) exposed to electroplating effluent may be attributed to dissolved solids and other inorganic constituents. Malini shetty and Somashekar (1998) observed similar growth inhibition of Phaseolus aureus exposed to industrial effluent that contained high concentrations of dissolved solids. These results are in agreement with the observations in Vigna mungo and Vigna radiata exposed to different concentrations of distillery and textile mill effluents (Kannabiran and Pragasam, 1993)

Microbial population in metal polluted environment contains microorganisms which have adapted to toxic concentration of heavy metal and become metal resistant. These microorganisms can be used to remove heavy metals from the environment by various approaches like bioaccumulation and bioadsorption, oxidation and reduction, methylation and demethylation (Bolty and Gorby, 1995). The microbe based approach for removal and recovery of toxic metals from industrial effluents can be economical and more efficient in comparison to physicochemical methods for heavy metal removal (Gadd, 1992).

Seventeen Colony Forming Units (CFU) were isolated from the selected industrial effluent samples. Minimal Inhibitory Concentration (MIC) test results indicate among the isolates two of them are hyper metal tolerant strains and are capable of living in 1500ppm nickel concentration. The ability of the microorganisms to grow and survive under high metal concentration is attributed to stress induced selection of these microbes in particular environments (Murthy et al., 2012). Among the two hyper nickel tolerant bacterial colonies one strain showed black colouration (Plate 1).

Black colour of the bacterial colony is because of the formation of metal sulfide indicating presence of biosorbed nickel from the medium (Plate 1). It may be due to metal accumulation in side the bacterium by metal sequesterization or metal adsorption outside the cell wall (Pumpel, 1995). The actual metal biosorption mechanism has yet to be studied. Reddy (2003) reported that the general growth inhibition trend in bacteria against metals is in the order Ni >Cu>Co. This bacterium can be utilized for in situ and ex situ bioremediation of the electroplating effluents.

Table 1 · Physico-Chemical Parameters of the Electroplating Effluent Sample
Parameters CPHEEO/CPCB
(Permissible level)
Electroplating Industries
I II III IV
1 Appearance   Light Green Light Green Light Green Light Green
2 Odour unobjectionable Objectionable Objectionable Objectionable Objectionable
3 Turbidity NT units 2.5 NTU 10 4.9 7.2 46.7 2.7
4 Total Dissolved Solids 500 5726 4939 13107 20309
5 Electrical Conductivity mic.mho/cm 8676 7463 19859 30771
6 pH 6.5 7.1 7.1 5.7 7.1
7 Alkalanity pH as Ca Co3 200 178 1.45 69 448
8 Total Hardness as Ca Co2 200 899 768 1495 2727
9 Nickel 3.0 687 5569 2172 3566
10 Aluminium - 9.039 13.41 10.24 4.741
11 Boron 2.0 112.5 212.4 303.1 34.06
12 Cadmium 1.0 0.003 -0.006 -0.013 -0.022
13 Cobalt - 0.264 0.551 0.739 1.145
14 Chromium 2.0 0.443 70.06 0.841 0.136
15 Copper 3.0 0.493 2.967 1.977 0.489
16 Iron 0.3 2.187 69.3 14.74 1.909
17 Magnesium 0.4 28.59 67.72 36.89 49.26
18 Manganese - 1.91 17.81 2.059 1.999
19 Lead 0.05 0.216 0.674 0.339 0.326
20 Zinc 5.0 11.39 106.7 17.58 18.18
21 Calcium as Ca 70 237 203 395 720
22 Sodium - NT NT NT NT
23 Potassium - NT NT NT NT
24 Ammonia as NH2 - 0 0 0 0
25 Nitrite as NO3 - 0 0 0 0
26 Nitrate as NO2 45 20 19 24 23
27 Chloride as Cl 200 545 576 1364 6313
28 Fluoride as F 1 1 0.8 1.2 1.3
39 Sulphate as SO4 200 3013 2380 7520 5933
30 Phosphate as PO4 - 0 0 0 0
31 Tidy's as O2 - 19 20 35 34
(Metal contents -mg/l)
CPHEEO – Central public health environment engineering organization
CPCB –Central pollution control board

Figure 1 · Percentage increase of metals in the effluents than the threshold level
Figure 1

Figure 2 · Effect of industrial effluent on Vigour index of Vigna unguiculata
Figure 2

Figure 3 · Effect of industrial effluent on % Phytotoxicity of Vigna unguiculata
Figure 3

Figure 4 · Effect of industrial effluent on tolerance index of Vigna unguiculata
Figure 4

Plate 2 · Growth of cowpea seeds on different Electroplating effluent concentrations (0, 0.01, 0.1, l, 5, 10 & 100%)
Plate 2

Conclusion

Physiochemical analysis of the effluent samples and phytotoxic studies on Vigna unguiculata indicates that the effluent samples are potential of pollutants and toxic to plants. Isolation of hyper nickel tolerant bacterial strains from the effluent indicates the possibilities of using them for bioremediation of nickel from the effluent. Further characterization and genetic identification with exploring nickel sorption potentials of the organisms will help to developing appropriate in situ and ex situ bioremediation strategies for effluent treatment.

Acknowledgement

The work was financially supported by Department of Science and Technology (DST) and Tamil Nadu State Council for Science and Technology (TNSCST). The authors 1 &2 thank DST for the JRF. The authors also thank Department of Genetics, Madurai Kamaraj University (MKU) and Department of Advanced Zoology and Biotechnology and the Management of Vivekananda College for providing laboratory facilities.

Reference

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