BSc 2nd Year Microbiology Bioremediation Notes Study Material
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BSc Microbiology Bioremediation Notes Study Material
What is Bioremediation?
Bioremediation is “the use of living organisms (primarily microorganisms) to degrade environmental pollutants or to prevent pollution through waste treatment.” Bioremediation is emerging as most ideal alternative technology for removing pollutants from the environment, restoring contaminated sites, and preventing further pollution. This environment friendly technology is expanding range of organisms to be used to clean up pollution, and forms a vital component of the so-called green movement of maintaining the nature’s overall ecological balance, an issue at present being top priority of environmental awareness and public policy.(BSc Microbiology Bioremediation Notes Study Material)
Need and Scope of Bioremediation
Application of bioremediation – the use of living organisms to degrade environmental pollutants or to treat waste streams to control pollution – is expanding world over. The OECD (Organisation for Economic Cooperation and Development) in a report entitled Biotechnology for a Clean Environment, issued in 1994 estimated that world market potential for all environmental biotechnologies would nearly double in the 1990s, rising from $40 billion in 1990 to $75 billion by 2000. Bioremediation is needed because certain chemicals accumulate in the environment to levels that threaten human health or environmental quality. It was in fact in mid 1960s, when DDT accumulation changed the old belief that serious efforts were begun to exploit the natural capacity of microorganism to degrade complex compounds. OECD has been sponsoring meetings of scientists and government representatives from U.S.A., Canada, Japan and West European countries since 1991 to consider the environmental applications of biotechnology.(Microbiology Bioremediation Notes)
At such meetings, besides other areas, representatives have examined the state of the art of bioremediation and how governments could extend aid to its research and development. At a recent meeting of OECD workshop, held in November 1994 in Tokyo, participants recognised that bioremediation can have local, regional, and global applications and that both indigenous and gentically- engineered microbes may play important roles.
Environmental Applications of Bioremediation
Research projects are being modified to expand the range of microorganisms used for bioremediation. There is search for naturally occurring microbes that have better pollutant degradation kinetics, attack a wider range of pollutant compounds, and do so over a wider range of microbial growth conditions. There is also search for microbes that could grow under extreme environmental conditions, such as tolerance to organic solvents, growth under extremely alkaline substrates, or high temperatures. This information would widen the scope of bioremediation even to nonaqueous pollutants and those found in the environmental conditions unfavourable to growth of most microorganisms.
Researchers have also been using genetic engineering to develop new microbial strains with novel biodegradative capabilities. For instance, modified microbes may be produced through genetic coding for the attack of complex chlorinated hydrocarbons such as dioxins which are nondegradable by naturally occurring microorganisms. Adding genes that code for enzymes that breakdown toxic chemicals to microbes able to survive and grow in much disturbed and harsh environments would greatly extend the range of compounds that might be treated with bioremediation. For example, in Japan a research team has already isolated a species of Pseudomonas that can grow in solvents containing more than 50% toluene, a condition that kills most organisms through disruption of cell membrane. Adding appropriate genes for catabolic enzymes to this strain has great potential for expanding the range of bioconversions into nonaqueous solvents.(BSc Microbiology Bioremediation Study Material)
Three different foci of R & D for bioremediation research are emerging worldwide. While Europeans are expanding their traditional waste and water treatment systems to cope with specific chemical pollutants, the U.S.A focuses on site-specific cleanup of soil and water contaminated with petroleum and xenobiotics, and the Japanese take aim at global environmental problems. Each of the three emerging technologies of bioremediation in environmental cleanup will be considered in somewhat detail below.(BSc Microbiology Bioremediation Notes)
[I] European upgrading of traditional waste and water treatment systems
Several European countries, particularly Germany, the Netherlands, Belgium, Austria and Italy have programmes to foster R&D in environmental biotechnology. German government agencies promote technology transfer and co-operative research between government-sponsored research institutes and private industry. In the Netherlands, the most developed country in environmental biotechnology, the government supports an innovative programme in bioremediation R&D and subsidises industries participating in the programme. For example, long back in 1978, the Dutch Nuisance & Air Pollution Act took advantage of the development of biofilters, a modification of traditional trickling filters, permitting installation of these biofilters to meet air quality standards. Following are examples of the developments made in the waste and water systems in some European countries.
1. Biogas (energy) from solid wastes and refuse. It could be recognised by some newly developed European waste treatment systems that some microbial degradation or transformation occur only in the absence of oxygen, and such a process could be important in environmental biotechnology. Under anaerobic conditions, microorganisms growing on wastes can sometimes produce valuable fuels. The dry anaerobic composting (Dranco) process converts the organic fraction of biodegradable organic solid waste and refuse into energy in the form of biogas (methane and carbon dioxide) and a humus-like material. The biogas is produced by a consortium of anaerobic bacteria that includes methanogens (methane-producing archaebacteria). Full-scale installations using Dranco process are in operation at Brecht, Belgium and Salzburg, Austria. The systems treat source-separated plant residue and paper from municipal waste.
In Salzburg, Austria both anaerobic and aerobic microbes are used. The first phase of treatment, anaerobic digestion takes place in a large tank lasting for about three weeks. The digested material then moves for aerobic maturation which takes about two weeks. The process is shown in Figure. The facility built by Organic Waste System of Ghent, Belgium can treat 20,000 tons of biowaste every year. Such contained aerobic and anaerobic treatment systems replace the traditional landfills for disposal of solid wastes. These new systems produce biogas that is about 55% methane, which is burnt to produce electricity.
2. Removal of inorganic compounds. Some wastewater systems, originally designed only to remove organic compounds from water aerobically to reduce BOD, have been modified to include anaerobic zones to remove inorganic compounds as well. One such system, a pilot-scaled fluidized bed reactor that removes nitrate from water, has been tested at the municipal water treatment facility in Blankaart, Belgium. The removal of nitrate from wastewater helps prevent eutrophication of the waterways receiving the treated water. At Blankaart, nitrate concentration was reduced from 75.0 mg per L to 0.1 mg per L when water was passed through bioreactor. The reactor contains the methylotrophic bacteria, such as us methylotrophus that carry out denitrification. Methanol is first added to bioreactor to support the growth of methylotrophs and later removed by trickling filters and granular activated carbon filters before the water is discharged for use. The bacteria convert nitrate to nitrite and then molecular nitrogen, which is released into the atmosphere.(BSc Microbiology Bioremediation Notes)
3. Removal of toxic chemicals from industrial wastewater. New waste treatment systems can also maintain populations of desired microorganisms that can biodegrade toxic compounds like hydrocarbons chlorinated solvents found in industrial plant wastewater. These compounds generally escape biodegradation in traditional wastewater at systems. Bacteria like Pseudomonas cepacia are able to biodegrade chlorinated hydrocarbons present in the effluents of pesticide industries manufacturing DDT, heptachlor, chlordane etc. In Matera, Italy, the wastewater is treated anaerobically in a bioreactor in a factory that produces epoxy resins from epichlorohydrin and phenolics. Besides alkaline hydrolysis and subsequent chloride removal, effluents containing unreacted toxic compounds are also treated with specific microbes. These acclimatised anaerobic microorganisms in the waste treatment digester are able to tolerate the concentration of epichlorohydrin and glycidol in the waste stream, and degrade these and other chlorinated organics so that the detoxified water can be released safely into the environment.(BSc Microbiology Bioremediation Notes)
Textile and dye industries in Hong Kong are using the bacterium, Acetobacter liquefaciens S-1 to treat their wastewater. This bacterium is able to consume bright coloured azo dyes in culture.(BSc Microbiology Bioremediation Notes)
4. Biological gas treatment systems. Traditional water treatment systems, particularly aerobic trickling filters, have been modified to treat air pollutants. In bioscrubbers and biotrickling filters, multiple microbial communities grow on solid surfaces to produce multilayered complexes called biofilms. When gas streams (coming out of water treatment plant) containing organic pollutants are passed through these systems, the pollutants are degraded. Several companies in the Netherlands and Germany have taken the lead in developing such biological gas treatment systems. Since 1978 when Nuisance & Air Pollution Act was enacted in the Netherlands more than 200 biofilters have been installed and this biotechnology is now in widespread use in that country to remove organic contaminants from air. Biofitration has been used since 1989 to treat gases given off by soybean toasters in Hengelo, the Netherlands.
The gases pass through a column packed with a proprietary solid support. A biofilm growing on this support biodegrades more than 95% of the organic compounds in the gases. This biofilter, covering an area of 240 sq. meters can process 300 cubic meters of gas per cu m of packing per hour. A cattle feed extrusion plant in Zwolle, the Netherlands, also uses biofiltration to remove more than 99% of the foul gases from its emissions. The facility handles 600 cu m of gas per cu m of packing per hour.(BSc Microbiology Bioremediation Notes)
Air treatment bioreactors are in use in the Netherlands to remove formaldehyde from air released from plywood production facilities and phenols from resin producers. Similar biofiltration systems are being tested to remove solvents from indoor air at point production facilities. Some fungi are being exploited in biofilters for the treatment of volatile organic compounds in air. Some fungi like Candida tropicalis are able to assimilate styrene – a fragrant liquid unsaturated hydrocarbon used chiefly in making synthetic rubber resins and plastics and in improving drying oils. In laboratory tests, such styrene – assimilating fungi are grown on a ceramic support. The mycelium of the fungi gives the biofilters a large surface area and greater capacity o eliminate pollutant than the conventional compost biofilters. In addition to styrene the biofilters can remove toluene, xylenes, alfa – methylstyrene and propene from test gases. The technique is being scaled up and marketed by TNO Institute for Experimental Sciences in Delft, the Netherlands in collaboration with four Dutch companies.
A German animal rendering plant treats 214,000 cu m of gas per hour using a biofilter filled with peat and heather that has a volume of 3,240 cu m. Odours are reduced 94 to 99%. A ceramics factory in Southern Germany uses biofilters to remove more than 99% of the ethanol and isopropyl alcohol released into the air from drying ceramics. The alcohols (90% ethanol and 10% isopropyl alcohol) are released at a concentration of 230 ppm organic carbon and a flow rate of 30,000 cu m per hour. A simple biofilter with a volume of 200 cu m removes them.
[II] American focus on site-specific cleanup
In the United States, bioremediation is mostly being used to clean up sites contaminated by toxic chemical spills or polluted from disposal of chemical wastes (primarily mixtures of nonaqueous chemicals). Federal and State Governments have enforced legally the cleanup of several highly contaminated sites that pose threats to human health, particularly where the pollutants are gradually seeping through soil into aquifers used for drinking water. The so-called superfund sites – the sites of highest priority in the list of EPA (The Environmental Protection Agency) – includes more than 1200 locations. Superfund sites contaminated with multiple pollutants and leaking underground storage tanks, numbering in the hundreds of thousands are being cleaned up through bioremediation with federal funding of about $20 million since 1990.
The Federal government supported research on the potential for bioremediation of sites contaminated with heavy fossil fuels, creosote, munitions (such as TNT and other nitroaromatics), and chlorinated compounds, among others. This funding comes particularly from Departments of Energy and Defense to clean up contaminated sites on federal lands. Several companies have also taken up bioremediation as cost-effective measure of restoring environmental quality.
Bioremediation appears to be an attactive alternative to the physical removal and subsequent disposal/destruction of pollutants. The cost of moving and incinerating of polluted matters is at least 10 times that of biological treatment, especially if the latter is carried out in situ (in situ bioremediation). Several large chemical producers like DuPont, and other manufacturers like General Electric and General Mortors are seeking ways to clean up trichloroethylene (TCE) and polychlorinated biphenyls (PCBs). TCE, once a widely used cleaning solvent, is also the most prevalent ground water pollutant of interest to the Departments of Energy and Defense.(BSc Microbiology Bioremediation Notes Study Material)
1. Complex organic pollutants including petroleum products in oil spills. Heavy fossil fuels, related compounds as well as chlorinated compounds pollute sites both, in water and land.(BSc Microbiology Bioremediation Notes)
(a) Bioremediation of sites in water. Pollutants are often mixtures of complex chemicals. Crude oil, for example contains thousands of hydrocarbons with different structures; refined oils have hundreds of different components: PCBs have dozens of congeners; and some pollutants are undefined combinations of oils, pesticides, other organic compounds and inorganic such as heavy metals.
Oil spills on ocean waters caused alarming threats to biodiversity and human health. The world first woke up to the disaster of an oil spill when on 18 March 1967 a Liberian tanker, Terry Canyon, ran ground on the southwest coast of Great Britain, near the entrance to the English Channel, spilling 60,000 tons of crude oil into the sea. Oil splattered on to 160 km. of a coastline killing fish and birds. In January 1969 occurred the Second major oil spill off the coast of Santa Barbara in U.S.A. discharging oil at the rate of 1000 gallons per hour. In 1978, Amoco Cadiz disaster dumped 68 million gallons of oil along the French coast.
On March 24, 1989 the Supertanker Exxon Valdez belonging to the Exxon Corporation, plied on to a reef off the coasts of Alaska, U.S.A. spilling over 11 million gallons of oil into the clean waters of Alaska’s Prince William Sound. As the oil hit 1930 km of shoreline, 100,000 seabirds died including 150 rare species of bald eagles. Many dead seals sank to the bottom of the ocean and at least 1000 sea otters perished. Indian coasts have also suffered from tanker disasters. In July, 1973, 3000 tons of oil washed on to the Gujarat coast when an oil tanker, Cosmos Pioneer ran aground. In 1974, an American oil tanker, Transhuron collided with one of the atolls of the Laccadives, spilling 5000 tons of special furance oil. In June 1989, a Maltese tanker, M.T. Puppy collided with a British vessel, spilling over 5500 tons of furnace oil into the open seas off Bombay.
The massive oil slick in the Gulf in 1991 has been the largest so far, spreading over 700 sq. km. This oil slick made history, spilling more than 330 million gallons of oil, roughly 30 times more than the quantity spilled by the Exxon Valdez on the shores of Alaska. The oil slick stretched over an area of more than 80 km long and 20 km wide moving south at a speed of 20 km a day. It hit Sandi, Bahrain, Qatar and the U.A.E. shores.(Microbiology Bioremediation Notes Study Material)
Spills can be dealt with more easily if they are confined to a small area on the water surface. For this mechanical booms or barriers are sp around an oil slick to check its progress and prevent it from hitting shoreline. The spilled oil can also be treated with dispersants, which are sprayed form aircrafts or ships. Dispersants cause the oil to spread farther and disperse in a way similar to the manner soap removes oil from our hands, allowing the oil to be emulsified and washed away with water. A dispersant contains a surfactant, a solvent, and a stabiliser. Absorbents are also used to facilitate the cleanup of oil spills. Natural materials like peat moss, straw, sawdust and pine bark can be used. Synthetic absorbents include polyethylene, polystyrene, polypropylene, and polyurethane. Of all these, polyurethane is the most promising.(Microbiology Bioremediation Notes)
By for the safest way of treating an oil slick is bioremediation- the use of biological agents for degrading oil. These microbial surfactants are sprayed form the air. They mix with the oil, emulsify it and disperse it throughout the water body so thinly that it no longer remains hazardous. Bacteria and yeasts can grow on several fractions of hydrocarbons as heptane, decane, hexadecane etc. However, not every clean-up method can clear up all oil Clean-up methods are chosen on a case-by-case basis. Professor Ananda M. Chakrabarty a hydrocarbon biotechnologist, working at the University of Illinois Medical Centre, Chicago, U.S.A has developed many a new strain of oil- eating bacteria. He could develop a very efficient oil-eating “superbug” using species of Pseudomonas through recombinant DNA technology.(BSc Microbiology Bioremediation Notes)
Indigenous versus introduced microorganisms. Several hundred U.S. companies sell microorganisms for environmental cleanup. But many of these microbial products are however, of dubious value and are little more than snake oil. Often, naturally occurring (indigenous) microbes at a contaminated site are already biodegrading the pollutant, and addition of introduced microbial products is of no help. Most cultures sold to biodegrade hydrocarbons in contaminated soil and water do not enhance the rates of biodegradation above those carried by indigenous microbes. Efforts have, therefore been made to modify the environment of the given site to enhance the activity of naturally occurring microorganisms, present already at the contaminated site.
Monitoring the persistence and activity of specific microorganisms. It is necessary to maintain specific, complex microbial populations having necessary biodegradative capacities in order to treat the pollutants. It is difficult to ensure the persistence of these specific populations. A novel method has been developed using reporter genes. These genes produce an easily monitored effect when microbial activities are occurring at the site. The method has been developed and demostrated by Gary S. Sayler at the University of Tennessee, Knoxville, U.S.A. Sayler uses expression of the lux gene, which codes for bioluminescence, to monitor degradation. The fermenter vessel glows from light emitted by the aerobic bacterium, Pseudomonas fluorescens strain HK44 when naphthalene is present as the carbon and energy source and oxygen as the electron acceptor, expressing biodegradative activity of the bacterium (naphthalene degradation). Strain HK44 was engineered to contain both, the nah gene, which code for an enzyme that catabolines napthalene, and the lux gene for bioluminescence; both genes being under the control of the same promoter.
Some examples of bioremediation. The largest bioremediation project in the US for the treatment of the Exxon Valdez Alaskan oil spill – highlights the benefits of environmental modification approach to bioremedation. Here site cleanup is achieved by enhancing the activity of naturally occurring oil- eating microbes through modification of the environment. A massive cleanup involving 11000 workers attempted to remove oil from more than 1000 miles of shoreline. Physical cleanup of the oil, using high-pressure water to wash the rocks, cost Exxon over $ 1 million per day and the treatment was slow and also lest subsurface oil that recontaminated shorelines.(BSc Microbiology Bioremediation Notes)
Bioremediation through simple addition of nitrogen-containing fertilisers to the contaminated shorelines, stimulated the metabolism of indigenous hydrogen-degrading microorganisms and degraded both surface and subsurface oil three to five times faster than occurred at untreated test sites. Natural hydrocarbon degrading microbes were already abundant in the water of Prince William Sound and tidal flux aerated the shorelines making bioremediation a viable treatment. The cost of remediating hundreds of miles of contaminated shoreline was less than $ 1 million and could complete within a couple of year contrary to more than a decade in un-fertilised condition. Though rates of hydrocarbon degradation were stimulated, it did not lead to complete removal of the hydrocarbons. Residual hydrocarbons were contained in asphalt-like materials that are insoluble in water and should affect biodiversity.(BSc Microbiology Bioremediation Notes)
(b) Bioremediation of contaminated sites on land. Petroleum hydrocarbons can also be successfully removed from land-based sites using environment moderation, as demonstrated more than 20 years ago in tests on the cleanup of a gasoline-contaminated aquifer in Amber, Philadelphia. In 1972 in this test, hydrocarbons were removed by adding fertilisers and using forced aeration to stimulate the growth of indigenous hydrocarbon-utilising microorganisms in the groundwater.(BSc Microbiology Bioremediation Notes Study Material)
Soil microbes in aquifers are responsible for a significant portion of the degradation of aromatic compounds when land sites are contaminated. These microbes degrade benzene, toluene, ethyl benzene, and xylenes. Most subsoil indigenous microbes biodegrade low levels of these compounds it there is enough dissolved oxygen in the groundwater.
Leaking underground storage tanks in the U.S. have contaminated soil and groundwater with low molecular weight aromatics that can threaten human health, when these hydrocarbons get into drinking water supplies. Bioremediation can treat both soil and groundwater in one step. The cleanup of pollutants from leaking underground storage tanks at a bus maintenance and fueling facility in Denver is an example of such a treatment. Gasoline, diesel fuel, and lubricating oil contaminated the soil and underlying groundwater near the leaking tanks. Beginning in 1993, the polluting hydrocarbons were removed by air sparging (physical transfer to the atmosphere) and bioventing in which the contaminated water was pumped to the surface and reinjected. Oxygen introduced into the water in this way supported the biodegradative metabolism of indigenous microorganisms.(BSc Microbiology Bioremediation Notes Study Material)
The dissolved concentration in the petroleum-contaminated groundwater was as high as 2.8 mg per L and natural bacteria are able to consume fuel residues at a rate of about 4.2 g of hydrocarbon degradation per L of water per year. In 1993, the Air Force completed a full-scale soil bloventing project to clean up a 27000- gal jet-fuel spill at Hill Air Force Base in Utah. During this 18 month project, jet-fuel residues in soil were reduced from an average total petroleum hydrocarbon concentration of about 900 mg per kg to less than 10 mg per kg. In this process 60% of the hydrocarbons removed from the soil volatilised directly into the atmosphere and remaining 40% was biodegraded to carbon dioxide and water.
Bioreactors are being tested by the Department of Energy at its Savannah River site in South Carolina as well as by companies such as Envirogen in Lawrenceville, N.J. to treat volatile compounds. TCE and other organic contaminants can be removed from soil and groundwater by simultaneous use of in situ thermal catalytic oxidation and bioremediation. In such a faster cleanup of contaminants, concentrations of volatile organics can be quickly reduced from the soil above the water table by vacuum extraction. These contaminants are then converted to carbon dioxide and hydrochloric acid by catalytic oxidation treatment at the surface.(BSc Microbiology Bioremediation Notes Study Material)
The Savannah River system uses a proprietary-metal catalyst and operates at about 425°C. Bioremediation accomplished by encouraging indigenous methane-oxidising microorganisms, removes contaminants from water-saturated soils as well as much of the residual contamination after volatilisation. Methane, oxygen, and nutrient gases that supply phosphorus and nitrogen are pumped into the contaminated soil to encourage indigenous microbes which convert the contaminants to carbon dioxide hydrochloric acid through the action of the enzyme, methane monooxygenase.(BSc Microbiology Bioremediation Notes)
The system can degrade more than 250 organic compounds, reducing TCE concentration to less than 5 ppb-low enough to meet drinking water standards.
Genetically – engineered bacteria like Pseudomonas fluorescens strain HK44 are being used to treat naphthalene- contaminated soil in field tests at Qak Ridge National Laboratory, E.Tennessee, U.S.A. In the tests, underground steel tanks, called lysimeters surround a central sampling core. Each lysimeter is about 8 feet in diameter and 10 feet deep.(BSc Microbiology Bioremediation Notes)
It is fitted with a cover, sampling ports, and sprinklers to stimulate rainfall. The Iysimeters are loaded with contaminated soil and Pseudomonas fluorescens HK44 and monitored for the parameters such as colonisation, gene transfer, bioluminescence, and naphthalene degradation. Bioremediation has been only occasionally used in Europe to clean up sites contaminated with petroleum spills. The situation may change over times as Eastern European countries are beginning to face such problems severely soon. Many sites in Czech Republic, Lithuania, Latvia, Ukraine and Russia are at least as polluted as the super fund sites in U.S.A.(Microbiology Bioremediation Notes Study Material)
2. Heavy metal-polluted sites. Besides organic compounds bioremediation can be used to treat sites contaminated with heavy metals or radionuclides. Microbes-algae, bacteria and fungi as well as higher plants have capabilities to uptake these pollutants. After uptake, these either accumulate or are assimilated by them. Accumulated heavy metals are recovered for recycling or disposal. For example, Zooglea ramigera adsorbs copper and cadmium up to the levels of 300 and 100 mg of metal per g dry wt. respectively. Pseudomonas putida, Arthrobacter viscous and Citrobacter spp remove several toxic heavy metals from industrial effluents.(BSc Microbiology Bioremediation Notes Study Material)
Radioactive metals as uranium and thorium are removed by Rhizopus arrhizus, and Penicillium chrysogenum can accumulate radium. The yeast, Saccharomyces cerevisiae accumulates uranium from dilute solution. The bacteira, like Thiobacillus thiooxidans bring about bioleaching of zinc, cobalt and nickel from sulphide rocks. Fungi belonging to the genera, Trichoderma, Aspergillus. Aureobasidium, Ophiostoma and Rhodotorula are shown to have biosorption ability of heavy metals and these seem to play important role in detoxification of industrial effluents. The Royal Dutch Shell uses sulphate-reducing bacteria to immobilise metals at an old zinc refining site at Budelco, the Netherlands.
The groundwater at this site was heavily contaminated with zinc and cadmium. For bioremediation, groundwater is pumped through a bioreactor to which ethanol, ammonia and phosphate are added to support the growth of sulphate-reducing bacteria. These bacteria convert the sulphate in the groundwater to hydrogen sulphide, which reacts with heavy metals to form insoluble metal sulphides. A flocculent is used to retain the precipitated sulphides.
Green plants are also able to remove heavy metals from contaminated and groundwater. Some strains of Brassica juncea accumulate heavy metals like chromium when growing in metal-contaminated soils. Tests are being made at Rutgers University Centre for Agricultural Molecular Biology on modified Brassica juncea growing in chromium-contaminated soil. Small amount of chromium was taken up by the plants and removed from the soil. Some plants can also accumulate lead. The plants can then be harvested and the metals recovered for recycling or disposal. The use of these plants is being developed by Burt D. Ensley at Phytotech, Corp., Monmouth junction, NJ, U.S.A. in conjunction with scientists at Rutgers University, New Brunswick, N.J., U.S.A. Such a process of toxic removal by using plants is also called phytoremediation.
Some of the limitations of bioremediation can be overcome through genetic engineering. One can produce strains of microbes that are resistant to heavy metals, have greater degradative capabilities and able to grow in more than one kind of environments. The first patent for a genetically engineered organism was granted in the U.S. in 1981 for a bacterium that degrades hydrocarbons. This has never been a candidate for cleaning up oil spills, as it was engineered to degrade low molecular weight hydrocarbons such as toluene and octane that evaporate rapidly. Efforts to engineer genetically modified microbes that can degrade specific pollutants in situ have yet to meet with great success. In most countries environmental regulations greatly restrict the release of these microbes into the environment for in situ bioremediation. The real benefits of genetically modified microbes for bioremediation are likely to be in contained bioreactors.
[III] Japanese global applications of bioremediation technology
In Japan, academic, industrial and government research is coordinated by the Ministry of International Trade & Industry (MITI). Developing long-term global applications of environmental biotechnology is one of the R&D priorities of this agency. Japanese bioremediation research focuses on ways to reverse environmental degradation in the 21st century. The research agenda of MITI places great emphasis on global problems such as climate change and desertification rather than on site- specific cleanup.
Japanese are therefore concentrating on much broader issues. Following are the ways in which they plan to handle environmental problems through bioremediation at global scale.(BSc Microbiology Bioremediation Notes)
1. Replacement for petrochemicals (hydrogen fuel). There has been a search for the microbes that produce some substances that can act as replacement for petrochemicals. Biotechnology research for producing hydrogen for use as automotive fuel that will not contribute to global climate change is of particular interest. One thrust of the work of Japanese researchers is use of microbes to produce such fuels and materials (as hydrogen) that do not emit carbon dioxide and thus reducing air pollution Researchers at several institutions including the National Institute of Bioscience and Human Technology and the National Institute for Resource and the Environment, both in Tsukuba, are seeking hydrogen-producing algae as sources of this clean-burning fuel.
2. Reversal of global warming. Even more far-reaching is the Japanese effort to reverse global warming. Besides looking for hydrogen and other fuels that would not contribute to global climate change there are also simultaneous efforts to develop bioremediation system to remove carbon dioxide already emitted into the atmosphere from the burning of carbon-based fossil fuels. This removal of the “extra” carbon dioxide polluting the atmosphere due to fossil fuel burning would be an extremely dramatic demonstration of bioremediation. The “extra” carbon dioxide contributes to global warming. Microbes can remove enough of this greenhouse gas and some of them convert carbon dioxide to various organic compounds.(BSc Microbiology Bioremediation Notes Study Material)
The recombinant DNA technology should be of much use in such efforts to produce new strains of microbes that would theoretically do so more efficiently At the Marine Biotechnology Laboratory at Kamaishi, Japan algae have been isolated that convert carbon dioxide to carbohydrates 10 times faster at 40°C than terrestrial green plants. Such algae could be grown in bioreactors near power plants where they would remove carbon dioxide from the air as it is released by fossil fuel burning at power plant. However, the organic compounds produced by assimilation of this greenhouse gas are likely to be subsequently biodegraded to carbon dioxide and water releasing this gas back to air.
A successful bioremediation programme using such algae and other microbes should therefore depend on finding such microbes that produce lignin or other polymeric compounds resistant to biodegradation as the original fossil fuels were. Such conversions would immobilise carbon, reducing the build up of carbon dioxide in the atmosphere.(Bioremediation Notes Study Material)
Japanese researchers have been growing some algae in bioreactors at the Marine Biotechnology Laboratory, Kamaishi. The alga, Chlorococcum littorale, isolated from Kamaishi Bay and grown successfully in laboratory could tolerate high levels of carbon dioxide, converting it to polysaccharides. Another alga being studied in the same laboratory, Prasinococcus capsulatus produces large amounts of extracellular mucilaginous polysaccharide. This alga is being studied for its potential to remove carbon dioxide from the atmosphere and for commercial applications of the polysaccharide.
Another group of microbes, the marine foraminifera, can also convert carbon from carbon dioxide into calcium carbonate. This process, occurring in coral reef formation, removes carbon from the usual biogeochemical carbon cycle that circulates carbon between organic compounds and carbon dioxide. Such a treatment would consume enormous quantities of calcium, creating its own impact on the environment. Despite these limitations the efforts may succeed in attacking global pollution problems. Such Japanese strategies if developed would raise the need for international understanding in the uses of biotechnology.
3. Biodegradable plastics. Biodegradable plastics made of microbially produced polyhydroxybutyrate have already been developed and being marketed by ICI in the U.K. These ICI’s biodegradable plastics are used worldwide in trash bags that biodegrade in landfills, In Japan and elsewhere studies are being made on microorganisms that produce such biodegradable polymers that form solid wastes. Researchers under the guidance of Yoshiharu Doi in the polymer chemistry division of the Institute of Physical and Chemical Research (the Riken Institute) in Saitama have isolated a strain of the bacterium, Alcaligenes eutrophus that produces a copolyester of 3- and 4- hydroxybutyrate. The cells of this bacterium produces 80% of their biomass as this polymer, when grown on 1,4- butanediol or y-butyrolactone.(Bioremediation Notes Study Material)
4. Biodesulphurisation, Japanese have also developed microbial systems to remove inorganic sulphur from industrial air emissions. The Bio-SR system developed by Dowa Mining has been put to commercial use. Industrial exhaust gas containing hydrogen sulphide passes through an acidic ferric sulphate solution which oxidises the sulphide to elemental sulphur while reducing the ferric ion to ferrous ion.(BSc Microbiology Bioremediation Notes Study Material)
An iron- and sulphur-oxidising bacterium, Thiobacillus ferrooxidans regenerates the ferric sulphate solution. The bacterium is grown in a bioreactor at a pH between 2 and 3. At a commercial facility of NKK Corporation, Tokyo, concentrations of hydrogen sulphide in an effluent gas are reduced from a range of 400 to 2000 ppm to less than 10 ppm. Thus 99% of the sulphide that would have otherwise gone to the atmosphere is captured as sulphur.
In the U.S. also, the Energy Biosystems in Woodlands, Texas have hel developing similar systems to remove organic sulphur-containing compoul from fossil fuels. The bacterium Rhodococcus biodegrade dibenzothiophenes and other organosulphur compounds in the fuel Biodesulphurisation produces a cleaner fuel that does not produce sulphu oxide emissions when it burns. The process may be used to produce low-sulphur diesel fuel that can meet the sulphur dioxide air emission standards of the Clean Air Act.
Japan’s official policy for the development of its biochemical industry since 1990 has required the industry to work to preserve the global environment, treat wastes, and conserve natural resources. The policy calls for the biochemical industry to use the sophisticated functions of microorganisms that make them friendly to the environment. Under this! policy MITI has initiated research projects at several national laboratories to work in collaboration with industry and Universities. These projects include work to develop biotechnology systems to combat global environmental changes.(BSc Microbiology Bioremediation Notes)
5. Reversal of desert formation. At present, 4.5 million sq km (35% of the total land area of the planet) is threatened by desertification due to loss of water from various soils. Efforts are being made to develop microbes that could help reverse desert formation. Such biological systems produce products like biopolymers that retain water reversing the phenomenon on large landscapes. In Japan, a MITI – supported programme initiated the use of the bacterium, Alcaligenes latus to produce a superbioabsorbent, a polysaccharide composed of glucose and glucuronic acid that can absorb and hold more than a thousand times its own weight in water. This ability is yet to be tested in the field.
The above account shows that bioremediation has promising future with several potential applications to clean up the polluted environments and treat wastes. Compared to physical cleanup methods, in situ bioremediation is much less expensive and causes less environmental perturbation. It may alter manufacturing practices, fuel production and composition, the way of individual’s disposal of wases, cost of products, and environmental quality.
However, employing bioremediation effectively in many applications requires both further R&D and clarification of government policies, particularly in release of genetically- engineered organisms, a controversial issue, scientifically as well as politically. Most European countries as well as the U.S. and Japan have regulations that ban the deliberate release of genetically engineered microorganisms. However, it may be possible to use them in contained bioreactors. Most successful applications of bioremediation such as the treatment of the shorelines contaminated by the Exxon Valdez oil spill, have relied on indigenous microorganisms and simple environmental modifications such as nutrient applications and aeration. The current and emerging applications of bioremediation are different in Europe and Japan than they are in U.S.A. Research in U.S.A. promises to contribute to the development of cost-effective solutions for bioremediation of contaminated sites. This new biotechnology will enable contaminated soils and waters to be cleaned up for reuse.
In several European countries existing biological waste treatment technologies are to be extended to new areas for removal of harmful toxicants from air and industrial waste streams. If current trends continue, the Netherlands and Germany are likely to become worldwide leaders in biotreatment of wastes by the end of the century.(BSc Microbiology Bioremediation Notes Study Material)
Japanese research could aptly combine a quest for improved global environmental quality with a drive for energy self-sufficiency. Their efforts to reduce global warming, if successful, will have far-reaching effects. Japanese programmes to replace petroleum with biologically produced hydrogen and to replace gasoline- powered automobiles with cars that burn hydrogen would greatly reduce future buildup of carbon dioxide in the atmosphere. A change to hydrogen fuel produced by microbes would be a major new use of bioremediation for pollution prevention that would revolutionise not only the petrochemical and automotive industries but the worldwide economy.
Phytoremediation – Biotechnology of Cleaning Up the Environment By Plants
The ability of microorganisms to uptake and accumulate heavy metals such as Co, Cd, Zn, Mn, Cu, Pb, Ni, Hg, Ag, etc. is very well known. Different bacteria; fungi (yeasts, dimorphic as well as filamentous) such as species of Saccharomyces, Rhodotorula, Aureobasidium, Ophiostoma, Aspergillus, Rhizopus and Trichoderma: some algae, and diatoms such as Thalassiosira pseudonana have such an ability and being studied for their biotechnological potential as agents of effluent detoxification.
Among vascular plants, some aquatic weeds such as species of Salvinia, Lemna. Azolla, Eichhornia; sedges and even tree species are also known to tolerate, uptake and even accumulate heavy metals and other toxicants in their cells. Besides microorganisms plants are also being studied for their potential of environmental cleanup. Green plants are not only the lungs of nature with unique ability of purifying impure air by photosynthesis releasing oxygen to sustain aerobic life in the biosphere, but it has also been only quite recently demonstrated that they could also be very useful in cleaning up the hazardous waste sites. Vegetation can filter contaminated runoff and may someday be used in the treatment of hazardous industrial and other waste sites.
Though phytoremediation has a long history but its industrial application is quite recent. Plants are being tested for their ability to clean up contaminated soil, and even genetically engineered varieties are on the horizon. “The process of recovery of hazardous substances from soil or groundwater contaminated with municipal or industrial wastes etc. by using plants is called phytoremediation.” Some examples of experimental field tests on removal of toxicants from soil and industrial wastes by using plant systems are given below.
[I] Recovery of heavy metals from soil
It has already been briefly described earlier under bioremediation that green plants such as specific strains of Indian mustard (Brassica juncea) can accumulate heavy metals when grown in chromium-contaminated soils. Modified strains of this plant have been shown to accumulate up to 40% of their biomass as heavy metals, such as lead and chromium. Researchers in the State of New Jersey in U.S.A. carried out field trials in 1994 and demonstrated that the plants could be safely grown in chromium contaminated soil at a site adjacent to Liberty State Park, N. J. A small amount of chromium was taken up by the plants and removed from the soil. The plants were then harvested and the metal recovered for recycling or disposal. The use of these Indian mustard plants is being developed by Burt D. Ensley at Phytotech. Corporation, Manmouth Junction, N.J., U.S.A. in collaboration with Ilya Raskin and coworkers at Rutgers University, New Brunswick, N.J., U.S.A. Tests to optimise metal uptake by such modified Brassicas are already in progress at these research centres. It is quite possible that in near future such plant systems could be effectively used for cleanup of metal contaminated soils at various sites.
Another such study is being made by Scott Cunningham, a senior researcher at DuPont. He has already undertaken field tests for such study on plants to remove lead contamination from DuPont facilities. They include manufacturing sites for alkyl lead, Remington lead-based bullets and lead-based explosives as well as lead paint and lead pipes. According to him lead is “sticky” and difficult to coax into a plant, and “using plants as waste treatment systems is in its infancy”. Experimental possibilities, according to Cunningham, include genetically engineering the plants to include genes for enzymes from microbes or animal livers to things that breakdown molecular bonds. The lead uptake in the field tests conducted by him has not reached amount that would promise a site cleanup. He hopes to achieve 20 tons of plants per acre with 1 to 3% metal content. He claimed to have got there. But for the time being, the plants are being used “as a guardian, for in situ stabilization, so that the site looks like a field, not a concrete block”.(BSc Microbiology Bioremediation Notes Study Material)
While microorganisms breakdown the organic bonds, the plants themselves take up the metals through their root system and sequester the contaminants in their cells. Useful plants can be found growing on ore outcroppings or contaminated areas. One variety of plant came from the lot of a convenience store. For instance, a variety of tree, Sebertia acuminate, a native of New Caledonia accumulates an astonishing 20-25% of its body dry weight of nickel. The plant bleeds a bluish green latex (sap).(Bioremediation Notes Study Material)
[II] Treatment of municipal wastewater and industrial wastes
Plants as such may not be able to sequester toxic pollutants from wastes. Roots and rhizomes of some plants provide an ideal habitat for the growth of different kinds of microorganisms, chiefly bacteria and fungi. These microbes are very effective in remediation of contaminated wastewater and industrial wastes containing chlorides, bromides, sulphates etc. The microbes remain active even in extreme conditions through frosts and surface freezing and the hot summer, as well as highly extreme pH and anaerobic reducing conditions. Each plant on earth harbours its own microbes around the roots and the microbes are able to adapt themselves with growing roots.(Bioremediation Notes Study Material)
In U.S.A. suitable species of plants are being grown in artificially designed experimental marsh systems to study the potential of plants in treatment of wastewater. One such plant’s wastewater system, the newest tourist attraction is the one designed for treatment of wastewater of Albemarle’s two bromine plants in Magnolia. At Albemarle’s Magnolia South Bromine Facility in Arkansas, tourists do not come to see the chemicals being manufactured but for this wastewater treatment system using marshy plants. The South unit of wastewater system is a 54-acre artificial marsh populated by sedge plants such as Scirpus lacustris, maiden cane and Typha latifolia. Each marsh unit is 2000 feet long, 180 feet wide and 12 to 18 inches deep with 40,000 plants. A 30 acre storm water impoundment basin is used for heavy rainfall. The marsh designed by a former National Aeronautics & Specs Administration (NASA), U.S.A. scientist treats the facility’s rainwater runoff and noncontact process water.
The marsh does what a mechanical wastewater treatment plant would do, but with a smaller outlay of less than $ 2 million and for lower operating costs. A mechanical system would have required as much electricity as an entire town, whereas the marsh requires only monitoring equipment demanding as much energy as a single hair dryer. Bill Wolverton, former NASA research Head and founder of Wolverton Environmental Services Picayune. Missouri. U.S.A. designed this Albemarle’s two marsh systems. He also helped build a rock reed filter marsh at Degussa’s Theodore, The Alaska, U.S.A. plant in the late 1980s. Wolverton, who retired from NASA in 1989 designs plant systems for treatment of indoor air, municipal wastewater and industrial waste.
This technology is being used in biotreatment of wastes. Wolverton has also been consultant to Biosphere 2 earlier in which people were sealed into a closed environment. Phytoremediation is most exciting and challenging in indoor air treatment dealing with problems of sick building syndrome, and in the industrial area. Albemarle’s first marsh at the South plant has been filtering and cleaning rainwater runoff and noncontact process water since 1993. The system was the cheapest alternative for dealing with increasing demands of the Clean Water Act in U.S.A. Based on the success of the South plant, a second unit was built in the beginning of 1995 at Albemarle’s Magnolia West Facility This second unit, about one-fourth the size of the first one, started operating in early October in 1995.
Each of the marsh systems consists of three areas. Runoff and cooling water enter a lagoon, where pH and temperature fluctuations are equalized. The lagoon releases water to two parallel marshes. The storm water impoundment basin is used for heavy rainfall. The marshes are the heart of the biotreatment process of phytoremediation. The plants, bulrush (Scirpus spp), maiden cane, and cattails (Typha spp), create the environment for the cleanup process, whereas the microorganisms, chiefly bacteria living on roots and rhizomes of these sedges breakdown the contaminants (chlorides, sulphates, bromides etc) entering the system. The new marsh is beginning to! establish itself with natural selection of the most ideal strains of microorganisms to attack the pollutants. It is in the developmental stage at present and its effectiveness is likely to increase with time. The marsh is engineered to act as a natural marsh designed with deep holes and shallow areas.(Bioremediation Notes Study Material)
The system can handle daily loads ranging from thousands to several millions gallons of water. You cannot design a mechanical system that can fluctuate so much. It can handle a serious accidental spill. If a tank truck spilled on site, the marsh would treat. The only problem about these natural systems is that we don’t have knobs to shut things off. Albemarle intends to set up similar facilities in Baton Rouge, Houston and Orangeburg, S.C. in U.S.A., and in Feluy (Belgium) and Thann (France).(BSc Microbiology Bioremediation Notes Study Material)
BSc 2nd Year Microbiology Bioremediation Notes Study Material