BIOREMEDIATION OF CHROMIUM CONTAINED SOIL BY PSEUDOMONAS FLURESCENS AND INDIGENOUS MICROORGANISMS:

Chromium is one of the toxic and hazardous pollutants in industrial wastewaters leading to soil contamination. In this study, the feasibility of remediating chromium contaminated soil using indigenous microorganisms and pseudomonas fluorescens was evaluated. The bioremediation of chromium-contaminated soil by indigenous microorganisms was found to be a promising solution and after bioremediation, the engineering properties of the soil were found to be improved. Introduction Chromium is an environmental concern as it is widely used in electroplating, leather tanning, metal finishing, pigment manufacture and nuclear weapons production. It is the second most common heavy metal contaminant in ground water, soil and sediments. Chromium occurs in aqueous system in trivalent and hexavalent forms. Trivalent Chromium (Cr3+) is considered to as an essential micronutrient for the maintenance of normal glucose, cholesterol and fatty acid metabolism. Hexavalent chromium (Cr6+) is toxic, carcinogenic, and mutagenic to animals as well as humans and is associated with decreased plant growth and changes in plant morphology. Tanneries are responsible the release of huge amount of chromium (as chromium sulphate), which has the tendency to convert into Cr6+ in the effluent. Field studies have established that biostimulation of indigenous bacteria is an effective means of removing environmental pollutants. The remediation of chromium contaminated sites poses a number of unique challenges. Many technologies are currently used to clean up heavy metal contaminated soils. The most commonly used ones are soil removal and land filling, stabilization/solidification, physico-chemical extraction, soil washing, flushing and phytoremediation. None of these techniques are completely accepted as the best treatment options because either these offer a temporary solution, or simply immobilize or are costly when applied to large areas. Bioremediation strategy is to detoxify Cr6+ in the soil matrix. Eliminating the toxicity of Cr6+ by its reduction to Cr6+ in the pH range of 6-9 severely restricts its ability to migrate to ground water. The bioremediation process generates no by products and can be tuned for the degree of contaminant removal desired. The key factors of the bioremediation process are the environmental conditions under which the remediation is carried out. Also, the process is dependant upon the nature of the soil and the pollutant. Hence in this study, an attempt was made to remediate the chromium contaminated soil by the indigenous microbes isolated from the soil contaminated by tannery effluent and a comparative study was conducted with pure culture of pseudomonas fluorescens. Optimization of parameters such as pH, moisture content, biomass concentration, carbon source, and contact time by both the cultures were studied with 100g of soil for 72h. After optimizing the operational parameters, reactor study was conducted with 2.5kg of soil for 120h by isolated mixed culture, engineering properties of soil (plastic limit, liquid limit, free swell index, 1

unconfined compression strength and permeability) and improvement on the same after bioremediation.

MATERIALS AND METHODS:

The chromium contaminated soil was collected from an industrial site located near Vellore, Tamil Nadu (India). The soil was stored at 4C in cooling system. The contaminated soil was brown in color with no specific odour. The effect of chromium contamination on engineering properties of the soil was studied by analyzing the engineering properties such as plastic limit, liquid limit, free swell index, unconfined compression strength and permeability. Both contaminated and uncontaminated soil samples were analyzed and the results were compared. All the engineering properties were analyzed as per IS 2720 and physico-chemical parameters were analyzed as per the standard methods. For weighing chemicals, Sartorius electronic weighing machine was used. Digital pH meter model D1-707 was used for the measurement of pH. SPEKOL 1200 UV-vis spectrophotometer was used to analyze the cell density at 440nm by direct photometric method. Inductive coupled Plasma equipment was used to analyze the chromium content at 540nm. The pH, moisture content and chromium content were found to be 8.5, 22.5% and 5.1mg/kg of soil respectively. The indigenous microbial community isolated from the contaminated soil by serial dilution method under controlled conditions. Pure culture of Pseudomonas fluorescens was procured from NCIM, Pune, India. Enrichment of both the cultures was carried out and growth of the cultures was observed by noticing the optical density at 440nm in the spectrophotometer. The chemicals require for the preparation of nutrient media and broth were obtained from Merck Chemicals and Excel Bio-sciences. Lab scale feasibility studies were conducted for a time duration of 72h and analyzing the samples at an interval of 24h for chromium content by both the cultures for optimizing the parameters in the range of pH 6.5, 8.4 and 10.5, moisture content 20, 25, and 30%, biomass concentration and carbon source in the range of 5, 10, and 15mL/100g of soil. Study on contact time was conducted for a time duration of 120h.

2.5 2 Absorbance 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5 Time in Days

Pseudomonas Fluroscens Isolated Mixed Culture

Fig.1: Optical density for Pseudomonas fluorescens and isolated mixed culture For Pseudomonas fluorescens, the optimum pH, moisture content, biomass concentration and carbon source were found as 6.5, 20%, 10mL and 10mL/100g respectively and for isolated mixed culture the optimum parameters were found as 8.4, 25%, 15mL and 15mL/100g respectively. After optimization of the parameters, 2.5kg of soil was conducted for 20 days by taking samples at various time intervals. Then the engineering properties were studied with remediate soil as per IS 2720.

RESULTS AND DISCUSSION:

Lab-scale feasibility studies: The effect of pH on chromium reduction of the soil was studied in the range 6.5, 8.4, and 10.5. The maximum chromium reduction was observed at pH 6.5 by pseudomonas fluorescens and at 8.4 by isolated mixed culture. The results are illustrated in fig2 . This might be due to the presence of variety of culture and the preferable pH for any biological system ranging from 6 to 8. Previous studies conducted in the pH range of 7 to 9 were found to give maximum efficiency. Paul and Henry were able to reduce 81% of Cr6+ to Cr3+ in a pH range of 7.5 to 9.5.

Fig.2: Effect of pH Wendy observed a maximum chromium reduction of 96% at pH of 8.1. Ramakrishna and Ligy in their studies observed an efficiency of 90% chromium reduction with pH 9. The effect of moisture content was studied with the range of 20,25 and 30%. The maximum efficiency of 61.1% by Pseudomonas fluorescens and 63.5% by isolated mixed culture were observed with moisture content of 20% and 25% respectively and the results are illustrated in Fig.3. Some of the previous studies conducted with the moisture content of 35%. Janet in their lab studies achieved an efficiency of 33% of chromium reduction in 21 days with a moisture content of 35% under aerobic condition. The effect of biomass concentration on bioremediation by both the cultures was studied.
64 % Reduction of Chromium 63 62 61 60 59 58 57 56 55 15 20 25 30 Moisture conte nt in % 35
Pseudomonas fluorescens Isolated mixed culture

Fig.3: Effect of moisture content

In this study, the biomass concentration dosages of 5, 10, and 15 mL of dosages and 60.3% by isolated mixed culture at a dosage of 15mL were observed and the results are indicated in Fig.4. This might be due to the more density and the variety of bacteria in the isolated mixed culture.
70 % Reduction of chromium 60 50 40 30 20 10 0 0 5 10 15 20 Biomass concentration in mL/100g of soil Pseudomonas fluorescens Isolated mixed culture

Fig.4: Effect of biomass concentration Leela and Ligy in their studies found that a spent media of 24h or 48h culture of bacteria containing metabolic products and other extra cellular products were responsible for chromium reduction, Also chromium reduction was associated with the viability of bacterial cells. The effect of carbon source was studied with range of dosages of 5, 10 and 15mL/100g of soil. In this study, molasses were taken as a carbon source as these are available in large quantity as a waste material. Maximum efficiency of 65.1% by Pseudomonas fluorescens with a carbon source dosages of 10mL and 67.5% by isolated mixed culture with a dosages of 15mL were observed. The result are given in Fig.5. The similar studies were also found to perform maximum efficiency with molasses as a carbon source. Jeysingh and Ligy observed a maximum efficiency of 33g with a optimum molasses dosage of 34mg/g of soil.

80 % Reduction of chromium 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Molasses dosage in mL/100g of soil Control Pseudomonas fluorescens Isolated mixed culture

Fig.5: Effect of carbon source Ramakrishna and Ligy in their studies achieved a maximum of 95% chromium reduction with a dosage of 10mL of molasses and peptone. The effect of contact time was studied at pH 8.4, optimum moisture content biomass concentration and carbon source. The experiment was conducted for 120h and a sample were analyzed for chromium content at every 24h. The effect of time on degradation is indicated in Fig.6. The maximum efficiecy of 66.2% by Pseudomonas fluorescens and 69.9% by isolated mixed culture for a period of 5 days were observed. Few studies conducted for the similar range of contact time were found to give lesser efficiency than this study. Janet attained a maximum chromium reduction of 33% in 21 days under a optimum conditions. Syed and wasay found a maximum chromium reduction of 37% in clay loam soil using Aspergillus niger with a content of 20 to 25 days.

BIOREMEMEDIATION OF CHROMIUM CONTAMINATED SOIL

Reactor study on reduction of chromium by isolated mixed culture From the lab scale feasibility studies, it was found that, the maximum efficiency of chromium reduction was performed by isolated mixed culture. Hence, a reactor study was conducted by isolated mixed culture on chromium reduction for 21 days. A plexi glass reactor was fabricated with dimensions 0.60.30.3 m and filled with 2.5kg of soil. The study was carried out with the optimum conditions arrived in the lab scale feasibility studies. A maximum chromium content of 71.7% was observed by isolated mixed culture in 21 days, which is indicated in Fig.7. Ramakrishna and Ligy attained a maximum chromium reduction of 80% in the first 8h. Oliver and Kieft found a maximum of 100% efficiency in a thick vadose zone with longer contact time. Paul and Henry observed a maximum of 81% efficiency in 147h with shaken culture of Pseudomonas fluorescens LB 300.

Effect of chromium contamination on soil properties The effects of chromium contamination on the soil properties such as liquid limit, plastic limit, free swell index, unconfined compression strength and permeability were studied by analyzing uncontaminated and contaminated soil samples as per IS 2720. No change was observed in free swell index whereas all the other properties were found to be affected. Permeability is the most affected property with a percentage decrease of 45.15%. Liquid limit and plastic limit were found to be reduced by 12.5 and 10.25% respectively and the compression strength was reduced by 16%. The soil sample after bioremediation was taken and studied for its engineering properties. All the soil properties were analyzed as per IS 2720 and are presented in Table 1. From the results it was observed that the engineering properties were better improved by the bioremediation of the chromium contaminated soil. The liquid limit was improved by 10% and the plastic limit by 7%. Also, the permeability was improved by 32% and the compression strength by 11% which makes the soil to sustain the load without getting sheared. From the studies, it was evident that the engineering properties which were affected by chromium contamination could be improved through bioremediation. However, the performance of the bioremediation is dependent upon the nature, and the extent of the contaminated soil, availability of microorganisms and the various environmental conditions that support the bioremediation process.

SL.NO
1 2 3 4 5

Properties
Liquid limit Plastic limit Free swell Index Permeability Unconfined compression strength

Uncontaminated soil
32% 39% Nil 4x102 cm/s 2.102kg/sq.cm

Contaminated Soil
28% 35% 4% 7.29x102 cm/s 1.765 kg/sq.cm

Remediated soil
31.20% 37.50% 4% 5.9x102 cm/s 1.873 kg/sq.cm

CONCLUSION:
The present study focuses the ability of an indigenous microbial community isolated from chromium contaminated site to biotransform chromium (VI). Investigation of effect of chromium contamination on soil properties was conducted and found that chromium contamination effects engineering properties significantly. The chromium (VI) reduction capacity of the pure culture of Pseudomonas fluorescens and isolated mixed microbial culture was compared. Though no significant difference was observed in the chromium reduction by both the cultures, isolated mixed culture was found to be the more suitable one. The performance of bioremediation process in improving the engineering properties was also elucidated. The bioremediation technique adopted in this study was land 7

farming. This technique could be easy and suitable for practical field applications as it involved usual agriculture equipment and practices. Beyond the lab scale study, pilot study on bioremediation of chromium contaminated soil may be carried out before field application.