Environmental Applications of BioTechnology

S.B. Sullia

With industrialization and the extensive use of pesticides in agriculture, the pollution of the environment with man-made (synthetic) organic compounds has become a major problem. Many of these novel compounds introduced into nature are called as xenobiotics (xenos meaning foreign in Greek), and a large number of them are not easily degraded by the indigenous microflora and fauna. The list of xenobiotics is very long and some of them are directly applied to nature in the form of pesticides or fertilizers , some others are released as industrial waste products (effluents). Other than the above compunds, the xenobiotics would also include a wide variety of dumpings such as plastics, detergents and oil spills, either inadvertent or deliberate. One glaring example of deliberate oil spills is the massive oil slick that covered 700 square km of ocean surface during the 1991 Gulf war. The slick made history spilling more than 330 million gallons of oil, and killing several birds, fishes and other fauna and microflora. Oil slicks due to damage to ships carrying oil tankers has been occurring occasionally and these are cases of unintended damage to nature.

The chemical pollutants such as toxic pesticides are of two types, biodegradable and nonbiodegradable (recalcitrant). A biodegradable pesticide may be converted by microbial action into a nontoxic coumpounds within a few months whereas a recacitrant chemical may remain in nature for several years in the toxic form. The duration of persistence of some of the common pesticides is given in the Table below.
Persistence of some pesticides in the environment

Biodegradability or recalcitrance depends on the nature of the chemical molecule.Often a simple change in the substituents of a chemical molecule may make difference between recalcitrance and biodegradability. The herbicide 2,4-D (2,4 dichlorophenoxy acetic acid) is biodegraded within days but 2,4,5-T differs only by the addition of of chlorine molecule in the meta-position (Fig......). The additional substitution interferes with the hydroxylation and cleavage of the aromatic ring. Similarly methoxychlor is less persistent than DDT which has great stability.

Alkyl benzyl sulfonates

The laundry detergents are surfactants which attach themselves to the lipophilic droplets on fabrics (stains, fats etc) and remove them. Ordinary detergents like soap are poor
detergents as they produce deposit with minerals found in water. The new class of detergents which are far more efficient are the anionic, cationic and nonionic detergents. Alky benzyl sulfonates (ABS) are a group of chemicals used as anionic laundry detergents. The alkyl portion of the ABS molecule may be linear or branched(Fig.....). Nonlinear ABS molecules are superior as detergents but are serious pollutants as they are not easily degraded by microorganisms. They may cause pollution of groundwater on seepage, thus posing problems of drinking water pollution.

Biomagnification

The concentration of the xenobiotic in the environment when diluted may vary from ppm (parts per million) to ppb (parts per billion) levels, and at at still lower levels it may not have any effects. However, the compound may become progressively more concentrated in the body of certain animals as it moves up the food chain. The process is called biomagnification.

This was first discovered in California where a lake had been treated with the pesticide DDD (related to DDT) to kill some insects. Later on the fish that ate the phytoplankton containing DDD as well as the birds that ate the fishes started dying. The body fat of the birds contained 100,000 times higher concentration of DDD than the lake water or the phytoplankton. Many other such fat-soluble compounds get concentrated in the animal bodies. Prominent examples are pthalate esters and PCBs(Polychlorobiphenyls) used as pesticides.

Bioremediation

Bioremediation is a pollution control technology that uses biological systems to catalize the degradation or transformation of various toxic chemicals to less harmful forms . The general approaches to bioremediation are to enhance natural biodegradation by native organisms (intrinsic bioremediation), to carry out environmental modification by applying nutrients or aeration (biostimulation) or through addition of microorganisms (bioaugmentation). Unlike conventional technologies, bioremediation can be carried out on-site. Bioremediation is limited in the number of toxic materials it can handle (Hart, 1996), but where applicable, it is cost-effective (Atlas & Unterman, 1997?).

Biodegradation, mineralization, bioremediation, biodterioration, biotransformation, bioaccumulation and biosorption are some terms with minor subtle differences but often overlappingly used. Biodegradation is the general term used for all biologically mediated breakdown of chemical compounds and complete biodegradation leads to mineralization. Biotransformation is a step in the biochemical pathway which leads to the conversion of a molecule(precursor) into a product. A series of such steps are required for a biochemical pathway. In environmental terms, it is importance whether the product is less harmful or not( Bennett & Faison, 1997). Biodeterioration refers usually to the breakdown of economically useful compounds but often the term has been used to refer to the degradation of normally resistant substances such as metals, plastics, drugs, cosmetics, painting, sculpture, wood products and equipment (Rose, 1981). Bioremediation refers to the use of biological systems to degrade toxic compounds in the environment. Bioaccumulation or biosorption is the accumulation of the toxic compounds inside the cell without any degradation of the toxic molecule. This method can be effective in aquatic environments where the organisms can be removed after being loaded with the toxic substance.

The fungi are unique among microorganisms in that they secrete a variety of extracellular enzymes. The decomposition of lignocellulose is rated as the most important degradative event in the carbon cycle of earth (Bennett & Faison, 1997). Enormous literature exists on the role of fungi in the carbon and nitrogen cycles of nature (Frankland et al., 1982; Cooke & Rayner, 1984; Carroll & wicklow,1992). The role of fungi in the degradation of complex carbon compounds such as starch, cellulose, pectin, lignin, lignocellulose, inulin, xylan, araban etc. is well known. Trichoderma reesei is known to possess the complete set of enzymes required to breakdown cellulose to glucose. Degradation of lignocellulose is the characteristic of several basidiomycetous fungi.

Fungi in bioremediation

Fungi are good in the accumulation of heavy metals such as cadmium, copper, mercury, lead and zinc. Systems using Rhizopus arrhizus have been developed for treating uranium and thorium (Teen-Seers et al., 1984).

The ability of fungi to transform a wide variety of hazardous chemicals has aroused interest in using them in bioremediation. (Alexander, 1994).The white rot fungi are unique among eukaryotes for having evolved nonspecific methods for the degradation of lignin; curiously they do not use lignin as a carbon source for their growth (Kirk et al., 1976). Lignin degradation is, therefore, essentially a secondary metabolic process, not required for the main growth process. Lamar et al. (1993) compared the abilities of three lignin-degrading fungi, Phanerochaete chrysosporium, P. sordida and Tramates hirsuta to degrade PCP (Pentachlorophenyl) and creosote in soil. Inoculation of soil with 10% (wt/wt) Phanerochaete sordida resulted in the greatest decrease of PCP and creosote. P. sordida was also most useful in the degradation of PAHs (Polycyclic aromatic hydrocarbons) from soil. Davis et al. (1993) showed that P. sordida was capable of degrading efficiently the three ring PAHs, but less efficiently the four-ring PAHs.

Phanerochaete chrysosporium has been shown to degrade a number of toxic xenobiotics such as aromatic hydrocarbons ( Benzo alpha pyrene, Phenanthrene, Pyrene) chlorinated organics (Alkyl halide insecticides,Chloroanilines, DDT, Pentachlorophenols, Trichlorophenol, Polychlorinated biphenyls, Trichlorophenoxyacetic acid), nitrogen aromatics ( 2,4-Dinitrotoluene, 2,4,6-Trinitrotoluene-TNT) and several miscellaneous compounds such as sulfonated azodyes. Several enzymes which are released such as laccases, polyphenol oxidases, lignin peroxidases etc. play a role in the degradative process. In addition, a variety of intracellular enzymes such as reductases, methyl transferases and cytochrome oxygenases are known to play a role in xenobiotic degradation (Barr & Aust, 1994).

Phanerochaete chrysosporium has been shown to effect the bioleaching of organic dyes ( Nigam et al. ,1995). Pauli ollikka et al. (1993) have also shown the decolorization of azo-triphenyl methane dyes by lignin peroxidase produced by P. chrysosporium. Sami and Radhaune (1995) have demonstrated the role of lignin peroxidase and manganese peroxidase from P. chrysosporium in the decolorization of olive mill waste water. The work carried out in our laboratory ( Asoka, Manjunath & Sullia, 2000; Asoka, Geetha & Sullia,2002 ) has shown that Phanerochaete chrysosporium and microbial consortia were effective in color removal from textile dye effluents The fungus caused 80% decolorization in broth containing 2.5% of effluent. There was reduction in BOD and COD values. A local isolate of Fusarium sp. caused various degrees of decolorization ranging from 35 to 85 %.

Among the fungal systems, Phanerochaete chrysosporium is emerging as the model system for bioremediation. The basidiomycetous fungus Pleurotus ostreatus has been shown to produce an extracellular hydrogen peroxide dependent lignolytic enzyme which removes the color due to remozol brilliant blue. Oxidative enzymes play a very major role in biodegradation. Other fungi which can be used in bioremediation are obviously the members of Zygomycetes e.g., the mcoraceous fungi and the arbuscular mycorrhizal fungi. Aquatic fungi and anaerobic fungi are the other candidates for bioremediation.

Among other fungi used in bioremediation, the yeasts, e.g., Candida tropicalis, Saccharomyces cerevisiae, S. carlbergensis and Candida utilis are important in clearing industrial effluents of unwanted chemicals. Agaricus bisporus and Lentinus oloides are important in lignocellulose decomposition. Corius versicolor is important in cleaning up pulp and paper mill wastes. Consortia of fungi and bacteria (usually uncharacterised) are used in composting, the most useful waste disposal practice. Phenolic azo dyes have been shown to be oxidized by the enzyme laccase produced by Pyricularia oryzae( Chivukula and Renganathan, 1995).

Bacteria in Bioremediation

Several bacteria have been found to be good degraders of toxic pesticides such as halocarbons. Some sulfate reducing bacteria transform tetrachloroethane to cis-1,2-dichloroethene by anarobic dehalogenation of halocarbons. Methanogenic bacterial consortium has been shown to degrade perchloroethene. Mono and dichlorobenzenes are degraded aerobically by various Pseudomonas and Alcaligenes strains. Pentachlorobenzenes(PCBs) are degraded by strains of Acinetobacter and Alcaligenes the same way as Phanerochaete chrysosporium, the fungus.

Several soil-inhabiting bacteria have been reported to degrade chlorophenols under both aerobic and anaerobic conditions. Pentachlorophenol is degraded by a monoxygenase enzyme which removes chlorine from the molecule making it nontoxic, and this enzyme is found in some soil bacteria.

Nitroaromatics are highly recalictrant because of the strong aromatic rings. Under anaerobic and microaerophilic conditions, the nitro groups of trinitrotoluene (TNT) can be reduced to amino groups but each subsequent step is slower.

Petroleum products contain a mixture of several hydrocarbons which are difficult to degrade by any one bacterium. Short-chain alkanes are toxic to many microorganisms and are difficult to degrade. Intermediate chain length (C10-C24) are degraded most rapidly. Very long chain alkanes become increasingly resistant to biodegradation. Monoxygenases and dioxygenases are the enzymes involved in the degradation ofalkanes. The aromatic hydrocarbons present in petroleum are also difficult to degrade. Some aromatic compunds such as benzene and toluene can be degraded by bacteria, especially species of Pseudomonas.

Biodegradation of oil spills is a major problem because it usually occurs in marine water surface and seeding with bacteria becomes difficult. Besides, there is no single baterium that can degrade all the components of the oils which are petroleum products. The genetically engineered strain of Pseudomonas putida has been reported by Anand Chakrabarty, an Indian born scientist working in USA which can degrade more than 3 to 4 components of petroleum. Other bacteria used in the treatment of oil spills are strains of Alcaligenes eutropus, Rhodococcus sp., Bacillus sp. and several unidentified bacteria. There are, however, the problems of production, storage and transport of large quantities of seeding cultures, and often mixtures of cultures are required. Nutrients like nitrogen and phosphate enhance the potential of microorganisms for biodegradation. The oleophilic fertilizer Inipol EAP-22 is used extensively.

Bacteria such as Pseudomonas and Bacillus have been shown to degrade the azo- or reactive dyes from textile industry effluents. The process is often referred to as biobleaching. The bacteria are often used in consortia for biobleaching (Ashoka et al.,2002).

Future Outlook

Whenever bioremediation figures as the topic of discussion, bacterial agents come into focus and fungi are much less studied. One should realize, however, the greater potential of fungi by virtue of their aggressive growth, greater biomass production and extensive hyphal reach in soil. More research will be focused in future on using the diverse fungal flora for bioremediation. The work on the microbial diversity in the electroplating industrial effluents is going on in our laboratory. There are several promising fungi that can degrade zinc, cyanide and chromium in these effluents.

Future work will be more fucused on the biotechnological aspects. It may be possible to clone the highly efficient degradative enzyme producing genes into bacteria and conversely, baterial genes can be transferred to fungi which are suitable. The high surface-to-cell ratio of filamentous fungi makes them better degraders under certain niches like contaminated soils. Fungi have been shown to even solubilize partially coal, a highly polymeric substance more complex than lignin. There is no doubt, therefore, regarding fungi being harnessed more and more in environmental bioremediation work in future.

Literature cited

Alexander, M 1994. Biodegradation and Bioremediation. Acad. Press, San Diego, Calif.
Asoka, C., Manjunath, K. and Sullia, S.B. 2000. Biological treatment of combined textile dye effluent. In : Ecology of Fungi, D.J.Bhat and S. Raghukumar (eds.), 63-67.

Asoka, C. , Geetha, M.S. and Sullia, S.B. 2002. Bioleaching of composite textile dye effluent using bacterial consortia. Asian Jr. Microbial Biotech. Env. Sci.,4:65-68.

Atlas, R.M. and Unterman, R. 1999. Bioremediation. In: Industrial Microbiology & Biotechnology, 2nd Ed. , ASM Press, Washington, 666-681

Barr, D.P. and Aust, S.D. 1994. Mechanisms the white rot fungi use to degrade pollutants. Env. Sci. Technol., 28: 79A-87A.

Bennett, J.W. and Faison, B.D. 1997. Use of Fungi in Biodegradation. In: Environmental Microbiology, ASM Press, Washington.

Carroll, G.C. and Wicklow, D.T. 1992. The Fungal Community. Its Organization and Role in the Ecosystem. 2nd ed. Marcel Dekker Inc., N.Y.

Chivukula, M. and Renganathan, V. 1995. Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Appl. Environ. Microbiol., 61:4374-4377.

Cooke, R.C. and Rayner, A.D.M. 1984. Ecology of Saprophytic Fungi. Longman, London.
Davis, M.W., Glaser, J.A., Evans, J.W. and Lamar, R.T. 1993. Field evaluation of lignin degrading fungu Phanerochaete sordida to treat creosote contaminated soil. Environ. Sci. Technol., 27: 2572-76.

Frankland, J.C., Hedger, N.H. and Swift, J.J.(Eds.) 1982. Decomposer Basidiomycetes. Their Biology and Ecology. Cambridge Univ. Press, Cambridge.

Kirk, T.K., Connors, W.J. and Zeikus, J.G. 1976. Requirement of growth substrate during lignin degradation by two wood rotting fungi., Appl. Eviron. Microbiol., 32: 192-194.

Lamar, R.T. Evans, J.W. and Glaser, J. 1993. Solid phase treatment of pentachlorophenol-contaminated soil using lignin degrading fungi. Environ. Sci. Technol., 27: 2566-71.

Nigam, P., Banat, I.M., McMullan, G., Dalel Singh, and Marchant, R. 1995. Microbial degradation of textile effluent containing Azo, Diazo and reactive dyes by aerobic and anaerobic bacterial and fungal cultures. 36th Annu. Conf, AMI, Hisar, 37-38.

Pauli Ollikka, Kirsi Alhonmaki, Veli-Matti Leppanen, Tuomo Glumoff, Timo Raigola and Hari Suominen 1993. Decolorization of Azo Triphenyl Methane Heterocyclic and Polymeric dyes by lignin peroxidase, isoenzymes from Phanerochaete chrysosporium. Appl. Environ. Microbiol., 59: 4010-4016.

Rose, A.H. 1981. Microbial Biodeterioration. Vol 6, Economic Microbiology. Academic Press, London.

Sami and Radhaune 1995. Role of lignin peroxidase and manganese peroxidase from Phanerochaete chrysosporium in the decolorization of olive mill waste water. Appl. Envron. Microbiol., 61: 1098-1103.

Professor S B Sullia, Department of Microbiology & Biotechnology, Bangalore University, Bangalore- 560 056