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Microbial System For Environmental Management
Kannan.V, Centre for Advanced Studies in Botany
University of Madras, Guindy Campus Chennai - 600 025.
Email: kannanavo@yahoo.co.in
Population increase and industrial developments during the past two centuries resulted in an unprecedented increase in pollutants to an alarming level. Many kinds of xenobiotic compounds which differ greatly in their structure from natural organics are resistant to biodegradation while some others are degraded very slowly. Biomagnifications of some recalcitrant xenobiotics inflict serious ecological damage. The pesticides without which present day food production would not be possible have become an integral part of agriculture. But the persistent nature of these chemicals lead not only to serious soil pollution but also indirectly affect soil fertility due to their nontarget effect on microbes. Petroleum hydrocarbons though not xenobiotic, due to their large scale use and accidental spill, resulted in the damage of both water and terrestrial ecosystem all over the globe. Microorganisms have an extensive but finite capacity to recycle synthetic and natural organic molecules, as they are the conservators of our ecosystem since the origin of life on earth. Contamination of surface and ground water by pesticides has been a major environmental threat. Organophosphate pesticides are among the most widely used pesticides in non-crop areas as well as in food crops. Given the potential carcinogenic risk of these pesticides, there is a serious need to develop remediation process to eliminate or minimize contamination of surface and ground water. Biodegradation could be a reliable and cost-effective technique for pesticide abatement. Malathion-S-1, 2 bis (ethoxycarboxyl) ethyl O, O- dimethyl phosphorodithioate (M.F. C10H19O6PS2) is reported to affect central nervous system, immune system, adrenal glands, liver and blood. Number of bacteria capable of degrading malathion increased in the sediments with increasing frequency of application and increasing level of treatment. Fungal mediated slow malathion degradation has also been reported in Egypt. Our screening of Serratia marcescens isolated from decaying bone showed the capability of degradation of malathion by this organism under three assay conditions by changing the assay medium. Serratia marcescens showed good growth in both nutrient broth and minimal salts medium when amended with varying concentrations of malathion. Whole cells of Serratia marcescens when incubated with various concentrations of malathion amended in nutrient broth showed remarkable break down action on malathion during the three hours of incubation. However, the characteristic absorption of malathion in water at 210 nm shifted when amended with nutrient broth or mineral salts medium. With nutrient broth, the spectral analysis showed 2 characteristic absorption regions, one near 510-520 nm and the other at 320 nm. The absorption at 510-520 region characteristically declined with increase in incubation time while in contrast the absorption at 320 was gradually increased with the time of incubation and thus confirming the break down of malathion and the reduction in the concentration of malathion (Fig.1).
The assay with cells incubated in mineral salts medium amended with various concentrations of malathion, the break down of malathion was though once again observed, the specific absorption peak of malathion at 210 nm was not observed; instead, three specific absorption regions which extended from UV to visible region, the first one at the region of 230-240 nm, the second at 350 nm and third at 374 nm was observed. The spectrum obtained after 3 hr incubation, showed only 2 clear absorption regions, one at 230-235nm and other at 240-245 nm, with the total disappearance of absorption at 350 and 374 nm regions (Fig. 2). When the degradation assay was performed with distilled water as the medium, malathion exhibited its characteristic absorption region at 200-210 nm (Fig. 3). The degradation was clearly observed as decrease in specific absorption with increase in incubation period. The degradation capability varied between 9.9 to 12.6%, 4.33 to 6.74% and 3.33 to 7.73% hr-1 in nutrient broth, minimal salts medium and distilled water respectively and quantity of malathion degraded was 0.038 to 0.128, 0.013 to 0.081 and 0.012-0.116 mM hr-1mg protein-1 in nutrient broth, minimal salts medium and distilled water respectively during the experimental period (Table 1). The alteration of pH in the medium from 7.2 to 3.0 during the growth of the test organism (Fig. 4a) in malathion amended medium prove not only the breakdown of malathion but also the formation of acidic intermediates during the break down of malathion. Similarly, the pH of the assay mixtures also showed the decline of pH from 7.2 to 6.0-6.1 at the end of 3 hr (Fig. 4b) which further confirms the break down of malathion and the formation of acidic intermediates during the break down of malathion. Formation of monocarboxylic acid, hydrolytic acid products during break down of malathion has also been reported earlier. The TLC analysis of culture filtrate also confirms the breakdown of malathion by S. marcescens (Fig. 5). Thus, the present work showed that S. marcescens was able to break down malathion using hydrolytic process evidenced by the decrease in pH during growth as well as in whole cell assay, which seems to be different from that of organophosphorus hydrolase and thus the non-pathogenic strain of this bacterium could be a potential tool for cleaning contaminated environments.
The extremely halophilic archaea, in particular, are well adapted to saturating NaCl concentrations and have a number of novel molecular characteristics, such as enzymes that function in saturated salts, purple membrane that allows phototrophic growth, sensory rhodopsins that mediate the photo tactic response, and gas vesicles that promote cell floatation. Their novel characteristics and capacity for large-scale culturing make halophiles potentially valuable for biotechnology. Many industrial processes also use salts and frequently release brine effluent into the environment. Halophiles are likely to be useful for bioremediation of contaminated hypersaline brine.
A moderately halophilic bacterium Planococcus halophilus was isolated from solar crystallization multi pond system from Kelambakkam near Chennai. The test organism Planococcus halophilus showed good growth on Nutrient Broth. When inoculated in minimal salts medium amended with kerosene and diesel (1%) the test organism showed its capability to utilize these hydrocarbons as sole carbon sources. Among the carbon sources tested the test organism showed good growth with kerosene compared to diesel and glucose as sole carbon sources. Thus the test organism was preferably utilizing kerosene, though diesel was also utilized for growth it was not to the extent as to that of kerosene. This indicates that the test organism is able to degrade kerosene well compared to diesel, which is well proved by the peak reduction of hydrocarbons recorded in the extracts of culture filtrate of the test organisms analyzed by gas chromatography (Fig. 6). As the test organism showed good degradation potential against kerosene, its ability to remediate refinery effluent was tested with effluents from CPCL, Chennai (Fig. 7). The characteristics of refinery effluent prior to treatment and after treatment with Planococcus halophilus is presented in Table 2. Biological treatment of refinery effluent with Planococcus halophilus for 4 days drastically reduced the oil and grease content of the refinery effluent. The reduction was as high as 91.2 % on the 4th day after treatment. Similarly more than 10 fold decrease was recorded in the total suspended solids. Further, the sulphide in effluent was also reduced up to 28% of that present prior to treatment with the test organism. The COD was at 121 mg/L and BOD was recorded as 14 mg/L after treatment with Planococcus halophilus that was very much below the limits of tolerance for wastewater. Though attempts were made to study the hydrocarbon degradation in hypersaline environments since 1978 no positive report is available on degradation of hydrocarbon by halophiles. Hydrocarbon-degrading moderate halophiles have been isolated from a variety of environments, including the Great Salt Lake and Antarctic saline lakes. A biofilm of a moderately halophilic bacterium isolated from a saltern at the Great Salt Lake, Utah had been utilized, for the treatment of hypersaline wastewaters containing phenol. Benzoate and other aromatic compounds were reported to be degraded by Halomonas halodurans by cleavage of aromatic rings. Moderate halophiles belonging to the family Halomonadaceae have been recently isolated from highly saline sites contaminated with the herbicide 2,4-dichlorophenoxyacetic acid. Recently Planococcus alkanocladisticus was also reported to degrade alkanes, which strongly support the present report. Biodegradative enzymes are often encoded on plasmids. However, most studies on plasmid-encoded pathways of hydrocarbon have been limited to members of the Pseudomonas sp. Chromosome encoded degradation is reported in Acinetobacter. Our observation of a single plasmid (Fig. 8) suggest that it may play a role in degradation of hydrocarbon since a plasmid less strain of Rhodococcus was demonstrated to be very slow in degrading alkanes.
Extremely halophilic bacteria were originally of interest because of the striking changes they caused in the landscape by imparting various shades of red to natural salterns, spoiled foods and discoloured hides. Halophiles produce a variety of red pigments, which impart color to the environment in which they are found. They are named as bacterioruberin, ß-bacterioruberin and or bacterioruberin. Due to their specific role as immune modulators and prophylactic action against cancers, these protein molecules gain importance in recent research activities. The biotechnological uses of bacterial pigments are only poorly understood. Nubel et al., have used carotenoid as a character to describe the diversity of oxygenic phototrophic microorganisms. The pigments produced by the red extremophilic organism comprise phytoene, ß-carotene, lycopene and derivatives of bacterioruberin. Halococcus sp. was isolated from the santerns of Kelambakkam near Chennai. Experiments conducted to find out the optimum temperature for growth and pigment production showed interesting observations. Halococcus sp, the test organism showed red pigment production only at 30 C. The pigment produced at temperatures 18 C, 38 C and 56 C was not red but yellow. Further the test organism though showed scanty growth at 4 C, the pigment produced was again not red, but yellow (Fig. 9). The experiment conducted to prove the essentiality of oxygen for growth and pigment production showed that the shaker grown condition was more preferable to the test organism than the static condition. Here again the red pigment production was observed only under shaker grown condition and under static condition Halococcus sp test the organism produced only a yellow pigment (Fig. 10). Further the experiment on the response of the test organism against different hydrocarbons showed interesting results. The growth of the test organism was drastically inhibited by toluene and benzene. Though low to moderate level of growth was observed in chloroform and xylene amended medium, the test organism did not produce the red pigment. Surfactant like SDS amended medium also did not support the red pigment production. Once again a yellow pigment was produced with absorption maximum at 376 nm and thus a pigment entirely different from red pigment was produced by the test organism. The test organism produces 2 pigments, a red pigment, which is always produced at optimal growth conditions, and the second yellow pigment which is produced only under unfavorable conditions. But these 2 pigments are not produced at the same time which is proved by the spectral analysis of the pigments. Members of Halobacteriaceae possess C50 carotenoids of the bacterioruberin group. The role of this pigment in protection against the harmful intensities of sunlight to which the cells are exposed in their natural environment was already shown by Dundos and Largens. In the present study wherever the conditions of growth becomes unfavorable, i.e. lower or higher pH, lower or higher temperature, lower or higher salinity and lower oxygen far from optimum growth of the organism resulted in the loss of formation of red pigment i.e. bacterioruberin and an alternate pigment normally yellow in color was formed. The red pigment fraction in halophilic bacteria is mainly due to the C50 - carotenoids (bacterioruberin). The test organism at favorable conditions produced the red pigment known as -bacterioruberin having absorption maxima at 464nm, 494-496nm and 528nm and a yellow pigment with absorption maximum at 376 nm during unfavorable conditions. Similar absorption maxima values were also reported for -bacterioruberin from Halobacterium salinarum. -bacterioruberin of the test organism Halococcus sp is distinctly different from the new red pigment of Salinibacter reported by Anton et al. Further Oren also reported the absorption maxima of archael bacterioruberin as 470nm, 496nm and 530nm and thus the red pigment produced in the test organism, Halococcus sp can be clearly identified as bacterioruberin. Based on the absorption spectrum it is possible to identify that it could be the new kind of pigment -carotene or -carotein as reported by Baxter and could be the third rhodopsin like pigment observed in Halobacterium halobium. Thus it can be concluded that the test organism Halococcus sp. is highly responsive to environmental conditions, which is exhibited by its growth and pigment production. The production of two kinds of pigments, red -bacterioruberin only at optimal growth conditions and an the yellow pigment, -carotene or -carotein only at unfavorable growth conditions, shows some close relationship of the red pigment with growth and metabolism of the test organism. It is acting not only as a protecting agent against harmful radiations of sun light but also as modulator of growth and metabolism of the test organism. Since the test organisms produces the yellow pigment in the presence of hydrocarbons in the medium. This organism Halococcus sp can be used as indicator organism to evaluate hydrocarbon contaminated in the environment Thus the microbial system can be efficiently used for sustainable environment management of healthy ecosystem.
About the Author:

Dr.V.Kannan is a Faculty in Centre for Advanced Studies in Botany, University of Madras. He is actively engaging both teaching as well as research for more than 2 decades. He is working on the emerging research areas of environmental microbiology and bioremediation by using microbial and plant systems. Currently working on the microbial diversity and biotechnological applications of halophilic enzymes and the uses of osmoregulants as protein and enzyme stabilizers.

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