Authors: Dharmendra kumar, ICAR-IARI, Division of Microbiology,
Polycyclic aromatic hydrocarbons (PAHs) are important pollutants found in air, soil and sediments. These compounds enter the environment in many ways. PAHs and their derivatives are widespread products of incomplete combustion of organic materials arising, in part, from natural combustion such as forest fires and volcanic eruptions, but for the most part by human activities. In recent decades the major source of PAH pollution is industrial production, transportation, refuse burning, gasification and plastic waste incineration.
The fate of polycyclic aromatic hydrocarbons in nature is of great environmental concern due to their toxic, mutagenic, and carcinogenic properties. For example,phenanthrene is known to be a human skin photosensitizer and mild allergen. It has also been found to be an inducer of sister chromatid exchanges and a potent inhibitor of gap junction intercellular communications. PAHs can sorb to organic-rich soils and sediments, accumulate in fish and other aquatic organisms, and may be transferred to humans through seafood consumption. Because many PAHs are so toxic there is interest in understanding the physicochemical processes and microbial degradation reactions that affect the mobility and fate of these compounds in groundwater and soil sediment system.The biodegradation of PAHs can be considered on one hand to be part of the normal processes of the carbon cycle, and on the other as the removal of man-made pollutants from the environment.
Physical and chemical properties of bioremediation:
Polycyclic aromatic hydrocarbons (PAHs) are a group of compounds containing carbon and hydrogen, composed of two or more fused aromatic rings in linear, angular, and cluster arrangements.
Many PAHs contain a “bay-region” and a “K-region”.The bay- and K-region epoxides, which can be formed metabolically, are highly reactive both chemically and biologically. Phenanthrene is the simplest aromatic hydrocarbon which contains these regions. The bay-region of phenanthrene is a sterically hindered area between carbon atoms 4 and 5 and the K-region is the 9, 10 double bond, which is the most oleinic aromatic double bond with high electron density
Bioremediation, which is also referred to as bioreclamation and biorestoration, can be described as ‘the process where by organic wastes are biologically degraded under controlled conditions to an innocuous state’.
The main principle of this technique is to remove pollutants from the natural environment and/or convert the pollutants to a less harmful product using the indigenous microbiological community of the contaminated environment. Bioremediation strategies are developed to promote the microbial metabolism of contaminants, by adjusting the water, air and nutrient supply.
This is accomplished by the biostimulation (the addition of a bulking agent such as wood chips and/or nutrients such as N/P/K) and bioaugmentation (often an inoculum of microorganisms with known pollutant transformation abilities) of the contaminated environment.
Bioremediation of PAH-contaminated soils, sediments,and water can be accomplished in a variety of ways, eg in situ treatment or ex-situ methods such as bio-piling and composting. Waste can also be treated in bioreactors, though this can be more costly than in situ technologies. It is important for bioremediation to be comparable in cost and success to physical and chemical treatments of contaminated land, such as landfilling, incineration and soil washing.
The applicability of bioremediation can be variable, but this is generally due to unfavourable site conditions, therefore a thorough understanding of site conditions will allow optimisation of bioremediation and subsequently more effective results. In commercial situations bioremediation of PAH-contaminated soils is not typically carried out when the site contains significant amounts of PAHs that have more than four rings as the low percentage removal of PAHs of this molecular weight and the time taken for successful reduction in PAH concentrations is not economically viable (Bio-Logic, personal communication).The method used is normally nutrient addition and aeration by frequent turning of contaminated soil. Total PAH levels during a bioremediation trial are generally reduced from approximately 3000mg to 1000mg total PAHs, per kg.
Bioremediation steps: Four component steps in the process are:
- The solubilization of the PAHs
- Their transport into the cell
- The expression of the degradative genes and
- The enzymatic breakdown of the PAHs
Factors like hydrophobicity, aqueous solubility and polarity have a large infl uence on the bioavailability of pollutants.Contaminated soils often contain a separate non-aqueous phase liquid (NAPL) that may be present as droplets or films on soil surfaces. Many pollutants, especially those that are hydrophobic, are virtually insoluble in water and remain adsorbed in the NAPL. PAHs have been found to persist in the NAPL due to their low water solubility and high octanol-water partition coeffi cients . However,the intracellular localization of the PAH degrading enzymes implies that the PAHs have to be solubilized and must enter into the cytoplasm before they can be metabolized. Therefore, for biodegradation to occur, bacteria must have access to the target compounds, either by dissolution of the target compounds in the aqueous phase or by adhesion of the bacteria directly to the NAPL-water interface.
Transport of PAHs into the cell:
Due to their low water solubility and high octanol-water partition coeffi cients, organic compounds such as PAHs tend to partition into cell wall structures. Such movement is generally brought about by passive transport down a concentration gradient from the environment into the cell. Such transport, however, depends on a number of factors, crucial amongst which are concentration and the bioavailability of contaminants from the surrounding medium.
The higher are the concentration and bioavailability of a PAH, the higher is its transport into the cell.While some bacteria can utilize this transport to achieve rapid degradation of PAHs, with concomitant growth,the rapid accumulation of some more toxic PAHs may lead to disruption of the cell membrane or inhibition of the membrane proteins etc., ultimately causing cell death. At low PAH concentrations and low bio-availability the passive diffusion process above may not function, potentially decreasing the efficiency of the degrading microorganism and therefore the bioremediation process. However some bacteria seem to have adapted to low bio-availability and low concentrations of PAHs.
Expression of the degradative genes
Once the PAHs have entered into the cell, the next step is the transcription of the degradative genes to produce the required enzymes. Generally the degradative genes have been found to be inducible, being expressed under certain conditions only. The inducer molecule is often the pathway substrate and/or a pathway intermediate but some structural analogues of the natural effectors (gratuitous inducers) can also induce the pathway even if they are not themselves substrates for the corresponding catabolic enzymes. The regulator may act as a transcriptional activator in the presence of the inducer or as a transcriptional repressor in the absence of the inducer.
Bacterial metabolism of PAHs:
The principal mechanism for the aerobic bacterial metabolism of PAHs is the initial oxidation of the benzene ring by the action of dioxygenase enzymes to form cis-dihydrodiols. These dihydrodiols are dehydrogenated to form dihydroxylated intermediates, which can then be further metabolised via catechols to carbon dioxide and water. There is a large diversity of bacteria that are able to oxidise naphthalene using dioxygenase enzymes,including organisms from the genus Pseudomonas and Rhodococcus. A few bacteria are also capable of oxidising PAHs by the action of the cytochrome P450 monoxygenase enzyme to form trans-dihydrodiols such as Mycobacterium sp.24 Rockne and colleagues reported the ability of marine methanotrophs in degrading PAHs via the action of the methane monoxygenase gene.
Fungal metabolism of PAHs:
There are two main types of fungal metabolism of PAHs; these are mediated by the non-ligninolytic and ligninolytic fungi (also known as the white-rot fungi). The majority of fungi are non-ligninolytic, as they do not grow on wood, and therefore have no need for the lignin peroxidase enzymes that are produced by the ligninolytic fungi. However, many ligninolytic fungi such as Phanerochaete chrysosporium and Pleurotus ostreatus can produce both non-ligninolytic and ligninolytic type enzymes, but it is unclear to what degree each enzyme contributes to the breakdown of the PAH molecule
Anaerobic metabolism of PAHs:
PAHs are a common contaminant of anaerobic environments such as aquifers and marine. Bioremediation of polycyclic aromatic hydrocarbons sediments.8–10, Even aerobic environments such as contaminated soils, sediments and groundwater can develop anaerobic zones.This is due to the organic contaminant stimulating the in situ microbial community, resulting in the depletion of molecular oxygen during aerobic respiration. This oxygen is not replenished at the same rate as its depletion, which results in the formation of anaerobic zones proximal to the contaminant source.
APPROACHES TO THE BIOREMEDIATION OF
Treatment of soils and sediments
Soils and sediments can be treated for PAH contamination both byin situ and ex situ methods. Landfarming is an in situ treatment for soils, which focusses upon stimulating the indigenous microorganisms in the soil by providing nutrients, water and oxygen. For example, a pilot-scale landfarming treatment of PAH-contaminated soil from a woodtreatment facility was achieved by biostimulation of the soil with water, ground rice hulls (as a bulking agent), and pelletised dried blood (as a nitrogen source) and bioaugmentation of the microbial community with an inoculum of Pseudomonas aeruginosa (strain 64).
Treatment of waters
As with soils and sediments, contaminated groundwater can be remediated both in situ and ex situ to the contaminated site. However, it is often not feasible to remediate contaminated groundwater ex situ due to the costs involved with abstraction and shipping of the contaminated water, and the fact that much of the contamination will be sorbed within the aquifer. Therefore, in situ treatment of aquifers can be accomplished by the biostimulation, and possibly the bioaugmentation of the indigenous aquifer community.
The persistence and toxicity problems associated with PAHs in the environment have resulted in a large amount of laboratory-based work that has concentrated on the ability of a variety of microbes (fungi and bacteria) to transform these complex aromatic molecules. The pathways of aerobic PAH transformation have been established and it is known that many environments contain microbes capable of reducing PAH concentrations. These factors have led to an interest in the potential use of microbes to remediate PAH-contaminated soils and more recent work has established that it is possible to use microbial-based processes to remediate PAH-contaminated soil.
These processes,eg land-farming and biopiling, are effective on shallow contamination but when PAH contamination is at depth then the use of bioremediation becomes more problematical. However, a recent field study has shown that bioremediation of contaminated aquifers is possible by the introduction of aeration to the subsurface. In addition, the potential of the biodegradation of PAHs under anaerobic conditions is promising, allowing further advances for the in situ treatment of the contaminated subsurface.
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