INTRODUCTION:

In the past century pollution was a term used with reference to contamination of air, water and soil. It technically means addition of foreign material, or a change in the natural habitat which affects all living beings directly or indirectly. Earlier people used the word pollution mainly with reference to water pollution. It is only with the rise of industrial revolution that air pollution became common mans terminology. With the growing human population soil pollution has also gained its share of importance.

Photosynthetic bacteria like Rhodopseudomonas palustris is a purple, non-sulphur bacterium that are available as a by-product from agricultural wastes¹. The organism has a more easily digestible cell wall, and also they are rich in carotenoids, proteins, some biological cofactors and vitamins². Mass production of Rhodopseudomonas palustris is widespread and has found a lot of applications in aquaculture as compared to microalgae³.

It can grow not only in the dark (aerobically) but also anaerobically in the presence of light⁴.

Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of different stress in its environment and during metabolism. The oxidative stress and the involvement of sigma factors has been explored much of which will come to the limelight based on preliminary studies on bioremediation. Having already established the role of oxidative stress on increasing the susceptibility of bacteria to antibiotics⁵, the present work widens the scope of oxidative stress on bacteria.

MATERIALS AND METHODS:

The Rhodopseudomonas palustris culture from Sathyabama University culture collection centre was subcultured in 1 litre sterile Erlenmeyer flask containing PTBM (Phototropic Bacterial Medium). The following is the composition of PTBM (per litre), ammonium chloride - 1g, magnesium sulphate - 0.3g, calcium chloride - 0.2g, potassium hydrogen phosphate - 0.5g, sodium chloride - 0.5g, sodium succinate - 2g, yeast extract - 1.5g, disodium hydrogen phosphate - 0.3g, ferric-citrate (0.1% w/v) - 5mL; trace metal solution- 1 mL. Trace metal solution- [(per litre) - ZnCl₂ – 70mg; MnCl₂·4H₂O – 100mg; H₃BO₃- 60 mg; CoCl₂·6H₂O - 200 mg; CuCl₂·H₂O - 20 mg; NiCl₂·6H₂O – 20mg; Na₂MoO₄·2H₂O - 40 mg] at the pH = 8 ± 2. The medium was poured up to the brim and tightly capped, sealed with parafilm, and kept in incandescent illumination at 2400 lux at 32°C for 7 to 12 days and observed for brown / brownish-red colour⁶.

Culture for Bioremediation – stress induction:

The optimally grown culture of Rhodopseudomonas palustris were sub-cultured into 250ml airtight bottles (the culture was filled to the brim). The control culture was maintained as such while the culture to be stressed was prepared by adding 1ml of hydrogen peroxide to the 250ml bottle. Triplicates of control culture and stressed culture were made and maintained under light.

Protein estimation:

The control and stressed cultures were assayed for protein levels in the cells according to Lowry et al⁷.

Bioremediation:

Water samples from municipal waste drainage were collected from Chitlapakkam (were the drainage meets the local water body), Tamilnadu, India using sterile polycarbonate sampling bottles. The samples were added to the medium in the ratio of 1:9, the medium was poured up to the brim and capped tightly, sealed with parafilm, and kept in incandescent illumination at 2400 lux at 32 °C for 7 to 12 days.

Determination of physicochemical parameters

Pollution indicator parameters of importance like TDS (Total Dissolved Solids), BOD (Biological Oxygen Demand), COD (Chemical Oxygen Demand), nitrogen and sulphate were estimated. Standard estimation procedures prescribed by APHA⁸ (1998) were followed for all the parameters. To determine the total dissolved solids in the wastewater, standard procedures approved by AOAC⁹ (1998) were followed.

These parameters were assayed for untreated wastewater which would provide the baseline data that could be used to compare with the other samples. The parameters were also assayed for wastewater that was treated with Rhodopseudomonas palustris that was grown without stress and the stressed culture. The results were compared with the baseline data obtained from assaying the untreated water.

RESULTS AND DISCUSSION:

The Rhodopseudomonas palustris culture obtained was acclimatized to the laboratory conditions. It had adapted well and the growth was significant. The organism grew at a faster rate as indicated in the table 1. The colour change of the medium is an indicator of the organism occupying the entire medium and utilizing its resources. The control culture grew to its maximum stationary phase by day – 7. This is indicated by its characteristic red pigmentation present in the medium.

Table 1. Observation of colour change in PTBM (Control culture)

S.No.	Days	Colour change in the medium
1	Day – 1	Golden yellow
2	Day – 2	Light green
3	Day – 3	Green
4	Day – 4	Light Orange
5	Day – 5	Brown
6	Day – 6	Brick red
7	Day – 7	Red

When the culture was subjected to oxidative stress using hydrogen peroxide, growth was affected initially as the organism had to adapt to the stressed environment. Going by its reputation it can be seen that the organism was able to adapt well and grew at the same rate once it got over the initial acclimatization period. This is evident from the colour change that is presented in table 2.

Table 2. Observation of colour change in PTBM (Stressed culture)

S.No.	Days	Colour change in the medium
1	Day – 1 to 3	Golden yellow
2	Day – 4 and Day – 5	Light green
3	Day – 6	Green
4	Day – 7	Light Orange
5	Day – 8	Brown
6	Day – 9	Brick red
7	Day – 10	Red

Stress induction was confirmed by assaying the level of protein in control and stressed culture. The organism upon stress would have a change in its proteome. The level of certain proteins can be upregulated resulting in higher concentration, or downregulated leading to a decrease in protein levels. It is also possible for stress to induce novel protein synthesis, called as stress proteins, which might lead to an increase in protein concentration. From table 3, it is observed that the level of protein has increased significantly when the organism was subjected to oxidative stress. The increase in protein levels may be due to increase in regular proteins being produced in higher concentration or due to induction of novel protein synthesis.

Table 3. Protein levels in control and stressed Rhodopseudomonas palustris.

	Control Rhodopseudomonas palustris	Stressed Rhodopseudomonas palustris
Protein (µg/mL)	653 ± 12	712 ± 11

The changes in physicochemical parameters of municipal wastewater due to treatement with Rhodopseudomonas palustris in comparison with untreated wastewater is presented in table 4.

There is a significant decrease in total dissolved solids indicated bioremediation activity performed by the organism. The bacteria upon stress is much more capable of reducing the TDS compared to control culture. A near 50% reduction in nitrogen levels by the stressed organism indicate that it prevents microbial growth drastically and thereby helps in the cleanup process. A 1/3^rd reduction in sulphate levels also helps in reducing the toxicity of the wastewater.

Biological Oxygen demand (BOD) is the measure of the oxygen required by microorganisms whilst breaking down organic matter. While Chemical Oxygen Demand (COD) is the measure of amount of oxygen required by both potassium dichromate and concentrated sulphuric acid to breakdown both organic and inorganic matters. BOD and COD concentrations of the wastewater were measured, as the two were important in unit process design¹⁰.

Upon oxidative stress, Rhodopseudomonas palustris significantly reduced the BOD by 2/3^rd the original level proving to be much more effective that its control. A 90% reduction in COD by the organism under stress indicates that oxidative stress has a positive effect in increasing the bioremediation property of Rhodopseudomonas palustris.

Oxidative stress induces synthesis of enzymes to counteract its toxic effects¹¹. Taking into account the increase in protein level, it can be proposed that certain silent genes are activated that result in novel protein production. These novel proteins in turn make the organism much more competent and increase their bioremediation ability.

CONCLUSION:

Rhodopseudomonas palustris is an organism with a potential in the bioremediation of wastewaters. Oxidative stress on this organism has a significant effect on the organism and based on the levels of protein it is presumed to synthesize novel proteins (stress proteins) when subjected to oxidative stress. These proteins in turn play a vital role in increasing the organism’s bioremediation potential which would be an appreciable ability for an environmentalist.

ACKNOWLEDGEMENT:

The authors would like to thank the management of Sathyabama University for their support in bringing out this work. The organizers of NASHE 2016 also thanked for providing us a platform to present this work.

REFERENCES:

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9. AOAC. Official methods of analysis of the Association of Official Analytical Chemist. Alexandria, VA: Association of Official Analytical Chemists. 1998; 432-444.

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11. Narendrakumar G, Ramesh Kumar. V. A Comparative Study of the Levels of Antioxidant Scavenger Enzymes – Catalase and Superoxide Dismutase in Escherichia coli and Pseudomonas aeruginosa during Exogenous Hydrogen Peroxide Stress. Journal of Pharmacy Research 5(8); 2012: 4021-4023.

12. Received on 16.03.2016 Modified on 04.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 345-347

DOI: 10.5958/0974-360X.2016.00061.5

S. No.	Parameter	Untreated wastewater (mg/L)	Wastewater treated with control culture (mg/L)	Wastewater treated with stressed culture (mg/L)
1	Total dissolved solids	1170	970	854
2	Nitrogen	150	110	94
3	Sulphate	90	74	63
4	BOD	373	133	102
5	COD	1350	350	150