Lecture No.

11
NON-MONETARY BIO-REMEDIATION MEASURES FOR ECOSYSTEM
PROTECTION
It has only been recongnised quite recently that biological systems, primarily of
microbial origin could prove

potential means of degrading some such complex

environmental pollutants as well as preventing pollution through waste treatment (solid

as well as liquid). Such recent environmental applications of biotechnology emerged into
a new area of research and development, bioremediation.
What is Bioremediation?
Bioremediation is the use of living organisms (primarily micro-organisms) 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 organism to be used to clean up
pollution, and forms a vital component of the so-called green movement of maintaining
the natures overall ecological balance, an issue at present being top priority of
environmental awareness and public policy.
Need and Scope of Bioremediation
Application of bioremediation- the use of living organism 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.
Threaten human health or environmental quality
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.

At a recent meeting of OECD workshop, held in November 1994 in Tokyo,

participants recognized that bioremediation can have local, regional

and global

applications and that both indigenous and genetically- engineered microbes may play
important roles.
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.
Researchers have also been using genetic engineering to develop new microbial
strains with novel biodegrative capabilities. For instance, modified microbes may be
produced through genetic coding for the attack of complex chlorinated hydrocarbons
such as dioxins which are non-degradable 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 distruption of cell membrane. Adding appropriate genes
for catabolic enzymes to this strain has great potential for expanding the range of
bioconversions into nonaqueous solvents.
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.

[I] European upgrading of traditional waste and water treatment systems.

European countries, particularly Germany, the Netherlands, Belgium, Austria and
Italy have programmes to foster R &D in environmental biotechnology 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.
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 that includes
methanogens (methane- producing archaebacteria.
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 than moves for aerobic maturation which takes about two weeks.
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 entrophication 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
methylophilus 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 and then to molecular nitrogen, which is released into the atmosphere.
3. Removal of toxic chemicals from industrial wastewater. New waste treatment
systems can also maintain populations of desired specific and chlorinated solvents found
in industrial plant wastewater. These compounds generally escape biodegradation in
traditional wastewater treatment systems. Bacteria like Pseudomonas cepacia are able to
biodegrade chlorinated hydrocarbons present in the effluents of pesticide industries
manufacturing DDT, heptachlor, chlordane etc.
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 (agar plate containing 100 ppm methyl red) in culture.
4. Biological gas treatment systems (biofilters, biofilms, bioscrubbers, biotrickling filers).
Tradition 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 complexed called biofilms.
When gas streams (coming out of water treatment plant) containing organic pollutants
are passed through these systems, the pollutants are degraded.
Netherlands and Germany have taken the lead
Pollution Act was enacted in

1978 when Nuisance & Air

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.
Air treatment bioreactors are in use in the Netherlands to remove formaldehyde
from air released from phywood production facilities and phenols from resin producers.
Similar biofltration systems are being tested to remove solvents from indoor air at point
production facilities. Some fungi are heing exploited in bioflters 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- assimilatingfungi 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 hiofilters. Biofiltrs can remove toluence, xylenes, a
methylstyrene and propene from test gases.
A ceramics factory in Sounthern Germany uses biofilters to remove more than
99% of the ethanol and isopropyl alcol released into the air from drying ceramics.
II] American focus on site- specific cleanup
In the United States, bioremediation is mostly being used to clean up sites
contaminated by toxic 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
Production Agency).
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 the 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 environment quality.
Bioremediation appears to be an attractive 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.
Several large chemical producers like Dupont, and other manufactures like
General Electric and General Mortors are seeking way to clean up trichloreothylene
(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.
Complex organic pollutants including petroleum products in oil spills.

Heavy fossil fuels, related compounds as well as chlorinated compounds polluite

sites both, in water and land.
a) Bioremediation of sites in water. Pollutants are often mixtures of complex chemicals.
Crude oil, for exam-le contains thousands of hydrocarbons with different structure:
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 in organics 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 coastline killing fish and birds.. In January 1969 occurred the
Second major oil spill of the coast of Santa Barbara in U.S.A. Discharging oil at the rate
of 1000 gallons per hours. 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.
Spills can be dealt with more easily if they are confided to a small area on the
water surface. For this mechanical booms or barriers are spread around an oil slick to
check its progress and prevent it from hitting the shoreline. The spilled oil can also be
treated with dispersants, which are sprayed from 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 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 polyurethane is the most promising.
By for the safest way of treating an oil slick is biremediation- the use of biological
agents for degrading oil. These microbial surfactants are sprayed from 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 spills.
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.

Monitoring the persistence and activity of specific microorganisms.

A novel method has been developed using reporter genes. These genes produce
and easily monitored effect when microbial activities are occurring at the site
Some examples of bioremediation
Bioremediation through simple addition of nitrogen-containing fertilizers 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.
b) Bioremediation of contaminated sites on land.
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 if there is enough dissolved oxygen in the
groundwater.
Genetically engineered bacteria like Pseudomonas fluorescens strain HK 44 are being
used to treat naphathalene- contaminated soil

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.
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. Radioactive
metals as uranium and thorium are removed by Rhizopus arrhizus, and penicillium
chrysogenum can accumulate radium. The yeast, Saccharomyces Thiobacillus
thiooxidans bring about bioleaching of zinc. The bacteria, like from sulphide rocks. Fungi
belonging to thegenera, thrichoderma, 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.
Green plants are also able to remove heavy metals from contaminated soil and
groundwater. Some strains of Brassica juncea accumulate heavy metals like chromium
when growing in metal-contaminated soil. Such a process of toxic removal by using
plants is also called phytoremediation.
II] Japanese global applications of bioremediation technology
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.
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
nor contribute to global climate change.

3. Biodegradable plastics. Biodegradable plastics made of microbially produced

polyhydroxybutyrate have already been developed and being marketed by ICI in
the U.K.
4. Biodesulphuisation. Japanese have also developed microbial systems to remove
inorganic sulphur from industrial air emissions.
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 on 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.
If current trends continue, the Metherlands and Germany are likely to become worldwide
leaders in biotreatmetns of wastes by the end of the century.
Phytoremediatin Biotechnology of Clenaing 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 know, Different bacteria; fungi (yeasts
dimorphic as well as filamentous ) such as Spices 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 vasucular plants, some aquatic weeds such as species of Salvinia lemna, Azolla,
Eichhormia; sedges and even tree species are also known to tolerate, uptake and even
accumulate heavy metals and other toxicants in their cells.
The process of recovery of hazardous substances from soil or groundwater contaminated
with municipal or industrial wastes. Etc. by using plants is called phytoremediation.
1] Recovery of heavy metals from soil
Indian mustard (Brassica juncea) can accumulate heavy metals when grown in
chromumy- 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.
For instance , a variety of tree, Sebertia acumumata (Sapotaceae), a native of New
Caledonia accumulates an astonishing 20 20% of its body dry weighy of nickel. The
plants bleeds a bluish green latex (sap).