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B. VINAY KUMAR AND V. VENKATA RAMANA. 1Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology,. J. N. T. University ...
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 15, No. (3) : 2013 : 585-588 © Global Science Publications ISSN-0972-3005

TREATMENT OF INDUSTRIAL EFFLUENTS BY A PHOTOTROPHIC PURPLE NON-SULFUR BACTERIUM RHODOPSEUDOMONAS PALUSTRIS JA190 B. VINAY KUMAR AND V. VENKATA RAMANA 1

Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J. N. T. University, Kukatpally, Hyderabad 500 085, India (Received 17 June, 2013; Accepted 15 July, 2013) Key words: Rhodopseudomonas palusrtis, COD, Purple non-sulfur bacteria.

Abstract - One strain of phototrophic purple non-sulfur bacterium belongs to the genus Rhodopseudomonas was enriched, isolated from the activated sludge of JETL ( Jeedimetla Effluent Treatment Limited) Hyderabad. Along with morphological, physiological characteristics, studies on the effect of augmentation with bacterial culture isolated in the anerobic treatment of industrial effluent, which has resulted in 88.7% more COD (chemical oxygen demand) reduction compared to control in 5 days, with a simultaneous increase in the biomass, indicated its significant role in anaerobic waste water treatment.

INTRODUCTION Most of the effluents that reach common effluent treatment plants come from various industries will contain several organic and inorganic constituents with less oxygen demand values and must be treated well before they let into the water bodies. Conventional and routine treatment processes are designed to isolate, alter, eradicate, and demolish undesirable substances present in industrial effluents. Most of the effluent treatment plants are indulged in aerobic treatment of the industrial effluents. Anaerobic processes are sometimes employed for the treatment of highstrength, readily degradable effluents. The primary advantage of the anaerobic process is low sludge production. In an attempt to explore and exploit phototrophic purple non-sufur bacteria which belongs to anoxygenic phototrophic bacteria (APB), in the treatment of industrial effluents, we could isolate and characterize few strains of phototrophic purple non-sulfur bacteria (PNSB) belonging to the genus Rhodopseudomonas, from the activated sludge of effluent treatment plant and were screened for COD reduction in the effluent collected from

equalization tank of a common effluent treatment plant. These bacteria have a capability of utilizing many numbers of organic compounds for their growth under photo and chemo heterotrophic conditions as monocultures (Vincenzini et al., 1982) or mixed culture (Balloni et al., 1980; Kobayashi, et al., 1974; Kobayashi, 1975a). Purple non-sulfur bacterial role also reported in treatment and utilization of sardine processing water (Azad et al., 2003) and in odorous swine wastewater treatment (Myung Kyum Kim et al., 2004). A reduction in BOD/ COD level of 20-99% has been reported (Sasaki et al., 1981; Mitsubishi 1985; Vatsala and Ramasamy 1989; Mochida et al., 1975; Mitsubishi Heavy Industries,Ltd, Japan ,1982) depending upon the waste and the organism used. Among few strains tested for effective growth under photohetrotrophic conditions, one strain JA190 which was isolated from the activated sludge process treatment unit exhibited 10-30% more growth compared to rest of the five strains isolated in the present study when grown in diluted effluent, was used for further studies on COD reduction and the same showed a maximum of 88.7 % COD reduction in 5 days, from the industrial effluent used in the study.

*Corresponding author’s email: [email protected], (Vinay Kumar Bathineni)

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MATERIALS AND METHODS Bacterial isolation Strains of phototrophic purple non-sulfur bacteria were isolated from the activated sludge process unit samples of JETL, Hyderabad, through enrichment studies by using Beibl and Pfennig’s media. All of them were purified by repeated streaking on the agar slants on same medium used for enrichment. The pure cultures were maintained in agar slants, stabs of Beibl and Pfennig’s media and in a lyophilized form at 4 oC. Bacterial identification Cell morphology (cell shape, cell division, cell size, flagella and visible internal or external structures) was observed by phase contrast microscopy (Olympus BH-2). In vivo absorption spectra were measured with a Spectronic Genesys 2 spectrophotometer in sucrose solution (Trüper and Pfennig, 1981). Media composition Media used for isolation and inoculum preparation is Beibl and Pfennig’s media (Biebl and Pfennig, 1981). The composition of the media (gL -1 ) is : KH2PO4 (0.5), MgSO4.7H2O (0.2), NaCl (0.4), NH4Cl (0.6), CaCl2.2H20 (0.05), Pyruvate (3.0), Yeast Extract (0.3), Ferric-Citrate solution 0.1%, w/v (5 mL), Micronutrient solution, SL7 (1 mL), SL7: (mgL-1): HCl (25%, v/v)-1 mL; ZnCl 2-70; MnCl2.4H2O-100; H3BO3-60; CoCl2.6H2O-200; CuCl2.H2O-20; NiCl2.6H2 O-20; NaMoO4.2H2O-40. pH was adjusted to 6.8-7.0 with 1 N NaOH/1 N HCl before autoclaving. Anaerobic treatment of industrial effluent The effluent sample was collected from equalization tank of a common effluent treatment plant. It was neutralized to pH 6.8 -7.0 and autoclaved at 121 oC for 15 minutes, in three 250 mL BOROSIL bottles. Two of them were aseptically inoculated with a 4-day old pure biomass of strain JA190 and activated sludge collected from the effluent treatment plant respectively. The other one was un-inoculated and was kept as control. The bottles were incubated in anaerobic light condition with a light intensity of 3000 lux without shaking. The same experiment was repeated for second batch; since the biomass gets acclimatized for using organic carbon in the effluent resulted in COD reduction.

Analytical methods The culture was centrifuged at 10,000 rpm for 10 minutes, washed twice with saline and oven dried at 102 o C for 24 h (Sawada et al.1997) for determination of dry biomass at regular intervals. COD, BOD and other parameters were analyzed according to Standard Method (APHA, 2000). For bacterial chlorophyll-a estimation, culture pellet was suspended in methanol and centrifuged. The bacterial chlorophyll extracted into the supernatant was analyzed (Cohen-bazire et al., 1957). The extinction coefficient of bacterial chlorophyll-a of the extract was determined at 775 nm in a spectronic GENESIS 2 spectrophotometer and the B,Chl-a content was calculated according to the following formula: BChl-a (mg 100 mL-1 ) = O.D775 x 2.19 RESULTS AND DISCUSSION Isolation of Rhodopseudomonas palustris JA190 Activated sludge sample was collected from JETL and inoculated in the Beibl and Pfennig’s medium to enrich the purple sulfur and non-sulfur bacteria resulted in the isolation and purification of Rhodopseudomonas palutris strain JA190, a purple non-sulfur bacterial isolate. Reduction in COD The COD reduction in second batch experiment was comparatively faster than the first batch as the biomass was acclimatized to utilize organic carbon present in the effluent. The initial COD of the effluent sample was 16000 mgL -1 , which was reduced to 1800 mgL-1, i,e 88.7 % COD reduction in 5 days by JA190 was observed. All the experiments were done with one fold dilution of the effluent sample. The COD reduction in un-inoculated bottle (control), activated sludge inoculated bottle, strain JA190 inoculated bottle are 7.5%, 37.5% and 88.7% respectively. The results indicated that strain JA190 is 51.2% more efficient than activated sludge (JETL), in COD reduction under anaerobic light condition. The COD reduction of uninoculated bottle was 7.5%. These results are nearly similar to that of Myung Kyun Kim et al. (2004) during Odorous swine wastewater treatment by purple non-sulfur bacteria, Rhodopseudomonas palustris, isolated from eutrophicated ponds and Azad et al. (2003) during Rhodovulum sulfidophilum in the treatment and utilization of sardine processing wastewater.

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Table 1. Physico-chemical characteristics of industrial effluent used in the study Parameter

Before treatment (mgL-1)

After treatment (mgL-1)

COD (mgL-1) Total dissolved solids Total suspended solids Volatile organic acids Total nitrogen Sulfates Phosphates pH

16000 + 25 8800 + 10 900 +10 208 + 2 250 + 4 450 + 3 30 + 1 7.0 + 0.5

1800 +8 6400 +15 700 +10 48 + 2 125 + 2 300 + 2 15+ 1 7.0 + 0.2

Supplementary Fig. 1. Whole-cell absorption spectrum of strain JA190T

Fig. 1. COD reduction (mgL-1) of effluent with respect to time, un-inoculated (), inoculated with activated sludge () and strain JA190 inoculated ()

Increase in the biomass & bacterial chlorophyll-a During the whole experiment, increase in the biomass and bacterial chlorophyll-a content was estimated (Fig. 2). Initial biomass and bacterial chlorophyll of strain JA190 are 0.8 (gL-1), 0.04 (mg mL-1) which are increased to 7.0 (gL-1) and 0.48 (mg mL-1) in 5 days, respectively. Removal of organic acids, phosphates, total nitrogen, sulfates, sodium, potassium and chlorides Reduction of organic acids (77%), phosphates (50%), total nitrogen (50%), sulfates (33%), sodium (22%), potassium (19%) and chlorides (15%) from the effluent sample was observed within 5 days of second batch experiment under photoheterotrophic conditions.

CONCLUSION Industrial effluents are major pollutants which contain various chemical constituents with high COD values which have to be treated well before disposing it to the environment. Groups of conventional treatment processes are designed to separate, modify, remove, and destroy undesirable substances carried by effluent from industrial sources. A strain of phototrophic purple non-sulfur bacterium Rhodopseudomonas palutris JA190 which was isolated from the effluent was when employed for anaerobic treatment showed 51.2% more efficient than the conventional process and can be exploited for treating industrial effluents effectively. ACKNOWLEDGEMENTS

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Fig. 2. Increase of biomass ( gL-1) () and bacteriocholrophyll-a (mgL-1) was absent in control throughout the experiment.

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