Biotic degradation of organomercury and inorganic mercury takes place in a ..... three of the Pseudomonas isolates could grow in the presence of lO'Yo TeE.
Environmental Pollution Detection and Bioremediation by Marine Bacteria ON. Ramaiah, Jaysankar De & S. R. Iyer
INTRODUCTION
1\ A"icroorganisms, in particular bacteria, play far more important ecological roles in natural 1V knvironments than their small sizes would suggest The term environment for bacteria is everything that surro'mds and sustains them including air, plant, animal surfaces and interiors, soil, water and even the micrometer sized organic particles. Thus, from an ecological perspective, bacteria are integral parts of the organismic communities that interact variously with each other . and between other, higher -and lower- fomls of life. These interactions in any ecosystem are of greater significance both in terms of ecological dynamics and stability. Their synthetic and degradative functions are pivotal to the continuum of life processes on earth (Brock et al. 1994). Some microbes are autotrophic: capable of generating new organic matter. However, most microbes, in particular bacteria, are heterotrophic. Heterotrophic bacteria are largely responsible for biodegradation and recycling of organic matter. Thus, they are intimately involved in biogeochemical reactions of the key elements of living systems viz. C, H, N, 5, P, 0 in addition to a few trace elements. In sinlple terms, biogeochemical reactions are all those changes an element undergoes in its oxidation state as it moves through an ecosystem, in and out of organisms. Significantly, heterotrophic microorganisms are the ONLY agents capable of regenerating biologically essential elements in forms usable particularly, by photosynthesizers. Microbiology is a many faceted science. Its understanding is essential for a better appreciation of ecological and technological events to which microbes are intimately linked. Efficient and economically beneficial industrial applications of microbes have been possible by understanding the basic ecology, physiology, biology and biochemistry of microbes. Readers' attention is drawn • National Institute of Oceanography, Dona Paula, Goa 403 004, India.
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to several recent publications in arena of Microbiology and Biotechnology (Atlas el. aI., 1984; Dewain and Solomon, 1986; Labeda, 1990; Glazer and Nikaido, 1995; Hunter and Belt, 1996 and Cooksy, 1998) In this case study,we report the results of oUlfstudies on the usefulness of marine luminous bacteria in marine pollution pre-screerting, the direct viable counts of bacteria in sensing marine environmental pollution stress. In addition we have furnished a few of our results on the use of marine bacteria for bioremediation of oil, peSticide and several other pollutants.
Bioluminescent Marine Bacteria Bioluminescence, emission of light without heat, is a characteristic of many species of marine and estuarine bacteria whose taxonomy, ecology and phylogeny have been extensively studied for over several decades now (Velankar, 1955; Nealson and Hastings, 1979; Rupy, 1977; Ramaiah, 1989; Ramaiah, and Chandramohan, 1992; Ramaiah et. aI., 2000). There are nine species of luminous marine bacteria (Nealson and Hastings, 1992) which are phylogenetically closely knil. Bacterial luminescence is extremely sensitive to toxicants, carcinogens, genotoxics at their nano or picomolar concentrations (Ulitzl1r, 1986; Bulich, 1982; Stebbing, 1985, Ramaiah and Chandramohan 1993, 1994). Many researchers have investigated on certain ecological (Ruby and Nealson, 1976; Ramaiah and Chandramohan, 1988; Lapota el. aI., 1988 and Ruby el. aI., 1998), physiological (Gupta el. aI., 1985), bio.::hemical (Kurfurst el. aI., 1982), genetiC (Engebrecht and Silverman 1983; Nealson el. aI., 1993; Ruby, 1995 ,md Ramaiah el. aI., 2000) and biotechnological (Lee el. aI., 1982 and King et. aI., 1990) facets of luminous bacteria. Thu;;, as the biology, biochemistry, physiology and genetics of these bacteria have been quite well understood, the research and commercial applications of marine luminous bacteria and their luminescence have been continuing.
Direct Viable Counts as Sensors of Environmental Pollution Stress Various anthropogenic activities and some natural processes, bring about adverse environmental conditions that affect pllysiological responses such as viability (Byrd el. aI., 1991; Colwell el. aI., 1985; Oliver el. aI., 1995 and Naganumo, 1996), metabolism, resting (dormant) stages and death (Amy and Morita, 1983) among microorganisms in situ. Through relevant bacteriological parameters, these resportses can be understood and thus are useful for monitoring environmental quality. Among the maIlY methods available for microbiologists, acridine orange direct counts (AODC) and direct viable counts (DVe) are widely employed 10 study total and viable fractions of bacteria in environmental samples. The technique of DVC, originally developed by Kogure et. al. (1979) is very useful for determining metabolically active pacterial cells and heterotrophic potential of microbial assemblages in the marine environment (Kogure el. aI., 1987; Walker and Keevil, 1993; Joux and LeParon, 1997 and Ramaiah el. aI., 2(02). Besides, it is easy to perform yielding highly reproducible resuhs (Nilsson et. at, 1991; Wiechert et. at, 1992 and Ravel el. aI., 1995). lis applications to study metabolic activities of bacteria in coastal marine regimes subjected to anthropogenic alterations were studied for the first time by Ramaiah el. al. (2002). The changes in DVC in ecological situations with gluts of extraneous organic maller input (e.g. through domestic sewage) on the one hand and, a plethora of xenobiotic toxicants (effluents of certain industries) on the other, were systematically analyzed and several laboratory experiments were done with an idea of evaluating the reliability of DVC for sensing the pollution
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stress in the marine environment. Actually these rationales evolv~d because the DYC recorded for the first time from the Indian coasts, were less than 20% of AODC from a polluted zone than those from a currently pollution free location in Gulf of Kuchch where DYC were ca. 35% of the AODC during the 1997 pre-monsoon months. Following this observation, it was planned to examine the fraction of DYC in samples originating from polluted, moderately polluied and relatively unpolluted coastlll zones. From the extensive field and laboratory analyses, we report on the reliability of Dye for sensing risks in the marine environment caused by industrial activities.
Lipid Hydrolyzing Marine Bacteria Oxidation and hydrolysis of lipids by heterotrophic bacteria lead to production of several intermediate molecules that are important in the formation of acetyl-CoA (Brock et. a!., 1994) and in the nutrition and growth of many microbial communities (Harris et. a!., 1991 and Chrost and Gajewski, 1995). Marine bacteria1lipases are of great importance in the catabolic breakdown of varieties of lipid containing organic molecules and recycling of all biologically essential elements present in such complex compounds. Many recent studies highlight the involvement of bacterial lipases in the digestion of food in many marine animal guts (Mymes et. a!., 1994; Pollak and Montgomery, 1994 and Donachie et. a!., 1995). Apparently, degradation of natural hydrocarbons by marine bacteria is dependent on their capacity to emulsify and solubilize lipids. In addition, most bacteria capable of breaking down lipids possess degradative properties useful in mitigating problems associated with marine oil spills (Ewell et. a!., 1996 and Martinez et. a!., 1996). Although the growth of all bacteria including lipolytic ones is greatly influenced by environmental parameters (e.g., pH, temperature and salinity), physiological studies on growth of lipid degrading marine bacteria under varying conditions of these parameters are not available. A selection of bacterial isolates capable of high lipolytic activity would prove very useful in dealing with organic waste treatment including oil pollution, not only for their individual potential but also as suitable candidates in consortia to be constructed in marine pollution bioremediation (Ewell et. al., 1996). Further, from the available literature, it is also apparent that few studies have measured marine bacterial lipid hydrolysis rates. The present work was therefore aimed at finding out the optimal growth conditions for select strains of lipase positive bacteria, their rates of Tween-SO and tributyrin hydrolysis and also, their ability to breakdown and grow solely on a selection of crude and refined petroleum hydrocarbons.
Mercury Resistant Bacteria Pollution of environment with heavy metals i.e. mercury, lead or cadmium has dramatically increased in many countries. Ground and wastewater are often considerably loaded with these unwanted toxic metals. Conventional methods to eliminate unwanted metals comprise precipitation, ion exchange, extraction and some other procedures are not always satisfactory under aspects of environment protection and with regard to costs, selectivity and sensitivity (Swain et. a!., 1992). Chemicals including organo-mercury compounds, lead, arsenic and dichloro diphenyl trichloroethane (DDT) were historically used as antifoulants. Among the many heavy metals, mercury has an affinity to bind to organics forming generally recalcitrant and highly toxic organomercurial complexes in marine sediments (Gerlach, 1981; Misra, 1992) upon constanl leaching and anthropogenic inputs.
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The total amount of PCBs produced worldwide is estimated to be 1.2 million tons (Furukawa et. aI., 2(00) and in many cases exceed the permissible limil of 5 ~g g-I (w) in edible fishes according to the Unit~ States FDA guideline (Connolly and Thomann, 1992). Of these PCBs, almost the entire amount is retained in coastal sediments and open ocean waters, clearly indicating that the open ocean water serves as a vast reservoir and final source of PCBs (Sarkar, 1994). Aquatic use of organotin compounds, which are toxic to both prokaryotes and eukaryotes, in particular their incorporation as tributyltin biocides in controlled release points on ships pose
the greatest immediate impact upon harbour and coastal aquatic biota. Removal of toxic organotin compounds from the ocean is very important from a global environmental viewpoint (Fukagawa el. aI., 1994). Microorganisms in the marine environment are suggested to undergo selection pressure in the presence of toxic pollutants and develop resistance (Nies, 1999). In recent years a large number of microorganisms have been isolated, which are able to degrade compounds previously considered to be non-degradable. This suggests that under the selective pressure of environmental pollution, a microbial potential for the degradation of recalcitrant xenobiotics is developing and can be harnessed for pollutant removal by biotechnological processes. Taking into account that only a small portion of bacteria can presently be isolated by conventional methods, it is evident that nature harbours a significant resolJrce useful in biotechnological applications. There is considerable evidence about mercury resistance among common microbial species (Pahan el. aI., 1990; Barbieri et. aI., 1996; Canstein et. aI., 1999 and Macaladyet. aI. 2000). The toxicity of a compound depends ultimately on its concentration, exposure duration and its bioavailability. Thus the bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. Biotic degradation of organomercury and inorganic mercury takes place in a number of forms. The most thoroughly researched one involves mercury resistance in bacteria possessing genes of the mer operon (Robinson, 1984 and HOOman and Brown, 1997). This capacity appears Widespread in nature and has been found for both gram- negative and gram- positive bacteria and under aerobic and anaerobic conditions. Bacteria showing resistance to methyl mercury has been reported to posses resistance to TBT (Fukagawa et. aI., 1994). Barkay (1987) had reported the inorganic mercury stress increased mercury resistant bacteria but they did not check the relationship between methyl mercury and other heavy metals. To examine the bioremediation potential of marine bacteria, we carried out several field Scurveys and obtained bacterial strains with unusual resistance to mercury.
MATERIALS AND METHODS Assays with Luminous Marine Bacteria In order to adapt tropical species of luminescent bacteria in pollution pre-screening efforts, we conducted various experiments with a variety of environmental strains of luminous bacterial species viz., Vibrio harveyi, V. fischeri and Photobacteriltm leiognathi. Details of methods followed are available in Ramaiah (1989) and in Ramaiah and Chandramohan (1993, 1994).
Experiments were carried out in order to observe the behaviour of bacterial luminescence when different chemicals were added in small volumes at very low concentrations available in Table 1.
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Luminous bacterial strains of the species V.harveyi, V. fischeri alld P. leiogllathi were used for the study of light output kinetics with a view to find out whether there is any species specific behaviour of light output. These 'cultures were grown in seawater complete liquid medium containing 50 mM Tris and incubated at 28° C for 24 hours. Aliquots of 50, 100 or 200 1'1 of the cultures were pipetted out into sterile mini vials of luminometer, The contents in the vial were all
