Use of microorganisms (probiotic bacteria or friendly bacteria) for improving environmental conditions or restoration of contaminated sites is very well known.
Bioremediation has been defined as “a biological response to environmental abuse” (Hamer, 1993).
This definition serves to distinguish between the use of microorganisms to remediate contaminated sites and their application in biorecycle/bio-treatment processes designated to reduce inorgannic and organic contaminant emissions at source (Colleran,1997).
Bioremediation focuses on the former while it is recognized that at present biological treatment processes play a major role in preventing and reducing the organic and inorganic environmental contamination from the municipal, industrial and agricultural sectors. Bioremediation is based on the premise that naturally occurring bacteria in an impacted environment develop the means to degrade or tolerate the presence of organic contaminats. This indigenous activity can be considered passive or intrinsic bioremediation (Leavitt, 1997). Intrinsic bioremediation has been recognized most frequently for ground water (saturated systems) contaminated with hydrocarbons.
Sometime a more active remediation system is used. Semi- passive bioremedation systems are defined as those in-situ treatments that induce favourable conditions for accelerated biodegradation. The excellent remedial approach is aggressive bioremediation. It may be defined as engineered bioremediation to produce optimal treatment in both in-situ and ex-situ systems. Its advantage is much shorter treatment period, alleviating any construction limitations sooner than with passive system.
Microorganisms are able to degrade a chemical because they are using it as an energy source to promote their own growth. According to Glazer and Nikaido (1998), bioremedidation is defined as a spontaneous or managed process in which biological, especially microbiological, catalysis acts on pollutants and thereby remedies or eliminates environmental contamination. Bioremediation is currently being used to decrease the organic chemical waste content of ground water, soils, effluent from food processing industry and chemical plants, oily sludge from petroleum refineries and oil spills in the ocean.
Degradation of pesticides, insecticides and fungicides in-situ (in soil) by microbes Pseudomonas A3, P .putida, P .aeruginosa and Serratia marinorubra either singly or as a consortia has been successfully attempted. Some microbial strains such as S. marinorubra, Bacillus species YW and YDLK consortia have also been used for decolorization and degradation of industrial effluents and textile mill azo dyes and effluents. Heavy metal detoxification and biosorption by employing the bacterium bacillus species YW has been found to be effective in reducing hexavalent chromium to its non-toxic trivalent form and the chromate resistance and reduction was found to be plasmid mediated process (Lalithakumari, 2003).
The removal of less toxic trivalent chromim through biosorption using the EPS Azotobacter species as the biomatrix has also been successfully attempted. Bioremediation techniques fall into four categories — in situ treatment, composting, land farming, and above – ground reactors.
In-situ Bioremediation Techniques:
In-situ, in Latin, means “in the original place”. Thus, in-situ bioremediation means bioremediation based on the degradative activities of endogenous microbial populations. In other words, it relies on the indigenous microbial flora of subsurface soils and ground water. It depends on the premise that the microorganisms already present in a contaminated site have adapted to the organic chemical wastes found there and are able to degrade some or all of the components of these wastes.
The degradation by these so-called adapted microorganisms goes on until some nutrient or electron acceptor attains a limiting concentration. In most cases, oxygen level is the limiting factor, but phosphate and nitrate may also become limiting factor. The stimulation of natural biotransformation by nutrient addition (N, P) to the environment is called enhanced in-situ bioremediation. The addition of nutrients to contaminated soils and also to hydrocarbon- contaminated marine environments has been investigated.
One of the best examples of enhanced in-situ bioremediation was the clean-up of the Exxon Valdez oil spill (1989) in Alaska. The spill, about 11 million gallons of crude oil, severely affected 350 miles of shoreline in Bligh Reef in Prince William Sound. In this case, fertilizers were used to accelerate the removal of oil from the beaches, supplying extra nutrients that were in limiting concentrations.
A single application of inorganic fertilizer was shown to speed up the disappearance of oil by a factor of two to three over its rate of disappearance on untreated site. Samples of oil taken at the end of that time from surfaces of treated beaches showed changes in composition consistent with extensive biodegradation. Thus enhanced in-situ bioremediation offers several potential advantages in the elimination of hazardous wastes.
Besides Pseudomonas species, HCl and Raulstonia species have also been successfully used for oil degredation. Most of the Psedumonas species strains harboured a catabolic plasmid, which encodes the genes for hydrocarbon degradation. The biotransformation of this plasmid to various bacteria in natural soil and marine water has been carried out, indicating the horizointal transfer of catabolic genes from one bacterium to another, paving way to create “Superbugs” for bioremediation in differing and metamorphosing ecosystems.
Bio-venting is one of the most widely used methods of remediating soils contaminated by petroleum hydrocarbons. Bio-inventing supplies air to an unsaturated soil zone by using a combination of pumps and blowers that apply a vacuum to the target area, while continuously injecting low volumes of air.
In-situ bioremediation under anaerobic conditions may also be enhanced by providing electron acceptors such as sulphate or nitrate. However, in-situ methanogenic bio-remediation has not, so far, been investigated thoroughly, although it is recognised that degradation of organic pollutants in anaerobic microniches in soil, sediment and groundwater environments contributes significantly to overall in-situ bioremediation rates.
Ex-situ Bioremediation:
Ex-situ bioremediation techniques are usually aerobic and involve treatment of contaminated soils or sediments using solid or slurry-phase systems. Solid-phase systems include compost heaps, landfarming (soil-treatment units) and engineered biopiles.
Composting:
Compost is a mixture of soil, partially decayed plants, and sometimes manure and commercial fertilizer. It is very rich in microorganisms. It has long been used by farmers and gardners to make soils more fertile and to improve crop yields. Compost heaps consist of contaminated soil or sediment supplemented with composting material in order to enhance water and air-holding capacity and improve physical handling properties.
Composting techniques are used for remediation of highly contaminated sites and have proved successful for military sites contaminated with explosives such as 2, 4, 6-trinitrotoluene (TNT), hexahydro-1, 3, 5-trinitro-l, 3, 5-triazine (RDX) and N- methyl-1, 2, 4, 6-tetranitroaniline (Tetry1).
Biodegradation or biotransformation of over 90% of these explosives was achieved within 80 days in a compost pile maintained at 55°C. After 150 days, a starting concentration of 18,000 mg of explosives per kg of soil was reduced to 74 mg per kg.
Landfarming:
Landfarming is used to dispose of oily sludges from petroleum refinery operations. The oily sludge from refinery wastes is mixed with soil and subjected to enhanced in-situ bioremediation. The sludges may be pretreated or not. Biological pretreatment of refinery effluents partially mineralizes the organic waste components; the residual solid waste (sludge) then has a high content of aromatic hydrocarbon compounds. In contrast, untreated settled solids, such as the solids from tank bottoms, contain high amounts of aliphatic hydrocarbons and silt.
The terrain of a landfarm must be flat to minimize runoff; the soil should be light and loamy for proper aeration; and a clay layer should underlie the porous surface soil to reduce the possibility of groundwater contamination through seepage. The optimal temperature range for biodegradation is 20-30°C. Inorganic fetilizer is applied to the site to provide fixed nitrogen, and phosphate and Ca CO3 are added to raise the pH of the soil-waste mixture to about 7.8. For untreated sludge, maximal oil biodegradation rates in soil are achieved at a hydrocarbon load of 5-10% by weight – that is , 100-200 metric tons of hydrocarbon per ha. In such landfarms, about 50-70% of the applied organic waste is degraded before the next batch of sludge is applied.
However, a disadvantage of land farming is that the process is quite slow. Moreover, the heavy metal constituents of the sludge gradually accumulate in the landform soil. As a result, a plot of land used intensively as a landform cannot be used later for grazing livestock or growing crops.
Above-ground Bioreactors:
The above-ground bioreactors are based on the same technology as fermenters. There are four common types of bioreactors – the stirred- tank reactor, the bubble column, the air lift, and the packed bed (or fixed bed). They are used for the treatment of either excavated soil or groundwater containing high levels of contaminants. Contaminated soil is mixed with water and introduced into the reactor as a slurry.
Granulated charcoal, plastic spheres, glass beads, or diatomaceous earth provide a large surface area for microbial growth in bioreactors. The large surface area of the microbial biofilm that forms on such supports leads to a rapid rate of biodegradation. The microbial inoculum may be taken from activated sludge from a sewage treatment plant, an indigenous population at the contaminated site, or from a pure culture of appropriate microbes.
Such bioreactors can be used in series to accomplish different kinds of degaradation. For example, the first reactor can be operated in an anaerobic mode and its effluent transferred to a second reactor operated in an aerobic mode. Mineralization requires aerobic conditions whereas bio-transformations such as de-halogenations of certain compounds require anaerobic conditions.
Environmental Factors Influencing in-situ Bioremediation:
Both the rate and the extent of microbial remediation of organic contaminates in-situ are affected by a number of environmental factors, some of which may be mainpulated whereas others are difficult to modify within the contaminated site. Important environmental factors which affect in-situ bioremediation are:
PH:
Most laboratory based bioremediation studied have been carried out in about neutral pH since the majority of bacteria show optimal growth at this pH range. In many studies, adjustment of pH enhances the rate of biodegradation.
Temperature:
Temperature directly affects the metabolism and growth of bacteria. The vast majority of in-situ bioremediation applications have been carried out under mesophilic conditions (between 20 and 40°C). However, the thermophilic or thermotrophic species are capable of degrading a diverse range of organic compounds in wastes or wastewaters at higher temperature (60-70°C). A variety of techniques have been utilized to increase the temperature in in-situ soil remediation applications. All these studies indicated that even modest increase in temperature may significantly increase bioremediation rates.
Water content:
Water content in soils or sediments in an important factor affecting bioremediation rates. Microbes generally require water activity values (aw) of 0.9-1.0 in order to metabolize and grow. The majority of bacteria grow optimally at aw values in the upper limit of this range.
Soil type:
In general, in-situ bioremediation rates are enhanced when the soil is granular or porous. In-situ degradation rates are slowed down under unfavourable geological characteristics which include low permeability of soil, faractured rock, and water-logged or arid conditions.
Nutrient availability:
In almost all cases nutrient supplementation (addition of N,P) significantly increases bioremediation rates. It has shown very good results in hydrocarbon bioremediation of soils and ground waters when N and P levels have been shown to be limiting.
External Electron availability:
Many in-situ bioremediation techniques use some provision of oxygen supply to enhance aerobic respiratory breakdown of organic contaminats. Addition of hydrogen peroxide is used to introduce oxygen. Hydrogen peroxide is about 7 times more soluble in water than oxygen and its decomposition in soil yields 0.5 mol of O2 per mol of H2O2 introduced to contaminated site (2H2O2 -> 2H2O + O2).
Bioavailability of organic pollutants:
This is an important factor governing the rate of in- situ bioremediation. Improvements in bioremediation rates have been achieved by the addition of bio-surfactants or synthetic detergents to the contaminated zone. Addition of biodegradable solvents, which assist in desorption and dissolution rates also exhibits increase in the biodegradation of the adsorbed pollutants (e.g. adsorption of PAHs by soil particles)
Cometabolism:
It is a process whereby microbes involved in the metabolism of a growth promoting substrate also transform other organic contaminants (cosubstrates) that are not growth supporting if supplied as sole carbon and energy source. Cometabolic transformation of organic pollutants is an important process in both aerobic and anaerobic environments. Bacterial transformation of DDT, PCBs provide examples of both aerobic and anaerobic cometabolic biodegradation. Provision of a readily metabolizable substrate may also promote pollutant transformation by enhancing the growth of associated microbes involved in the overall microbial activity.
Gene Expression:
The ability of indigenous microorganisms to degrade organic pollutants is dependent on expression of the genes encoding the required uptake and degradative enzyme systems. If the bioavailable concentration of a pollutant is too low, the expression of inducible operons may not occur.