Towards Effective Bioremediation in Third World Countries

Fouad M. Qureshi a, Jameela Akhtar a, Uzma Badar a, Fehmida Fasim a, Nazia Jameel a, Salma Raihan a, Mah-e-Talat Hassan b, and Nuzhat Ahmed a*

a. Centre for Molecular Genetics, University of Karachi, Karachi 75270,
    Pakistan.
b. Environmental Technology, Vlaamse Instelling voor Technologisch
    Onderzoek (VITO), Boeretang 200, B-2400 Mol, Belgium.

Global state of pollution in soil, sediment and water has been well highlighted in literature and media for the past few decades and the need for prevention of pollution and the need to clean up already polluted areas is now felt more than ever. However, in practice very little of the set targets has been achieved. As a solution the bioremediation technologies have recently emerged as economically and practically viable options to conventional technologies. Bioremediation offers two main advantages that make it attractive. The first of the advantages being cost effectiveness provided the selected process functions with better efficiency than alternate conventional methods. The second one being, it’s practicality. For example, it is not practical to treat large areas of land (spread over hundreds of acres, perhaps) contaminated with hazardous organic pollutants by conventional technologies but it may be feasibly treated by application of microorganisms capable of degrading the pollutant (9).

Contaminated soil, sediment and water bodies usually contain pollutants that fall into two main categories; (a) organic- or (b) inorganic-compounds, that deserve different bioremediation strategies to be addressed. Organic pollutants may be eliminated by treating contaminate with microorganisms capable of degrading the compound. Inorganic pollutants may be removed by biological sequestration, which may depend on any of the phenomena of accumulation, precipitation, or solubilization.

Although, bioremediation is inexpensive, still its financial burden is too great to be borne by many Third World industries since; the local common-man cannot bear the necessary rise in price of their product. This fact is one of the major contributors to failure of many international treaties to control and reduce pollution. Therefore, it is necessary to generate more economical approaches to pollution control. It has been argued that technologies based on indigenous resources of the country, though they might not necessarily be most efficient but only reasonably efficient (17). A number of different bioremediation technologies being developed at the Centre for Molecular Genetics (CMG) are described as technologies arising from indigenous resources of a Third World country and their strategic synthesis to form different bioremediation regimes capable of treating different kinds of pollutants under a variety of environmental conditions.

Bioremediation for Inorganic Pollutants

Inorganic pollutants include heavy metals that are toxic to various physiological processes and in general life itself. However, the microorganisms have evolved complex mechanisms to counter the toxic effects of metals, which at many soil and water microhabitats are very significant components of the microorganism’s environment. Recently, biotechnologies have used microorganisms to control and clean-up heavy metal polluted wastes. These technologies exploit several mechanisms that microorganisms have evolved. This area is the focus of research activities at CMG and some achievements of this program are described below.

Metal Accumulation

Many bacteria (and other microorganisms and plants) posses the ability to sequester inorganic cations (especially metal ions) from their surroundings. If sequestration of ions depends on the phenomena of intracellular accumulation, it is referred to as bioaccumulation. If sequestration exploits the phenomena of adsorption to cell surface, it is termed biosorption. Both of these phenomena are generally referred to as accumulation.

A number of bacteria including Acinetobacter sp. CMG456 (1), Bacillus sp. CMG451, Pseudomonas sp. strains CMG454, CMG455, and CMG458 (6), Pseudomonas stutzeri CMG463 (7), Pseudomonas aeruginosa CMG156 (17), and a chromate sensitive mutant of CMG451 designated CMG460 (6) have been found to accumulate copper. CMG456 was able to produce its best biofilm on raching-rings as compared to coke or glass helices. Biofilms of CMG463 and CMG156 were obtained on foam cubes and PVC (polyvinyl chloride) cylinders respectively and were evaluated for removal of copper by lab-scale bioreactors. Both the biofilms efficiently removed copper from the synthetic liquid effluent (3,17).

Pseudomonas sp. CMG58, Escherichia coli CMG59, and Morexella sp. CMG61 and their plasmid cured derivatives CMG58A, CMG59A, and CMG61A respectively have been found to accumulate nickel while Pseudomonas aeruginosa CMG64 has been found to accumulate cadmium. All of these strains have been evaluated for either nickel or cadmium removal by immobilizing biomass in bioreactors (2,18). Several biomass immobilization materials including raching-rings, wood-shavings, Cytodex, and Cytopore have been used for immobilization of CMG64 cells and Cytopre has been found to support most biomass per unit weight of material. The reason being that Cytodex, and Cytopore are porous and provide greater colonizable surface area per unit volume (or per unit weight). CMG64 has been found to remove cadmium by a precipitation mechanism that has been confirmed by transmission electron microscopy (4).

Metal Precipitation

Sequestration of cations may also be achieved by precipitation and subsequent biosorption of the precipitate. A bacterial strain CMG 480 identified only as ‘cucurbit yellow vine disease bacterium’ based on 16S rRNA homology (7) was isolated from tanneries in Karachi. CMG480 is capable of reducing Cr(VI) to Cr(III) followed by precipitation as CrPO4, yet it resists high concentration of Cr(VI) (1 mM). This dual property is a rare observation and it provides a great deal of robustness. Electron microscopy and energy dispersive X-ray microanalysis demonstrated extracellular deposition of the precipitate. Possibly, the mechanism involves taking-up of CrO42+ followed by reduction and exocellular deposition. Therefore making it very easy to retrieve the precipitate for recycling purposes. These observations make CMG480 a very promising candidate for bioremediation applications.

Metal Solubilization

Solubilization achieves the reverse of precipitation by converting precipitates to free ions. A recently isolated bacterium CMG823 solubilizes zinc oxide and zinc phosphate with concomitant increase in H+ ion concentration of the medium possibly due to assimilation of ammonia and production of 2-keto-gluconic acid which has been identified with GC MS (Fasim, F. et al., unpublished work).

Genetics of Heavy Metal Resistance

Seven genes for resistance to copper have been found in an Enterobacter sp. CMG457 isolated from polluted soil in Pakistan contained in the Pco operon (pcoABCDRSE). CMG457 resists high concentrations of Cu2+ ion up to 4 mM in Tris-minimal medium (Badar, U. et al. Unpublished work). Proteins PcoABCD form copper efflux system, PcoS and PcoR regulate operon by forming sensor/receiver system, PcoE is a periplasmic copper-binding protein (14,20,21).

Five genes, czrSRCBA, involved in Zn and Cd resistance (12), have been identified in Pseudomonas aeruginosa CMG103. The predicted gene products of czrCBA show a significant similarity to the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants.

Bioremediation for Organic Pollutants: Biodegradation

Hydrocarbons in the habitats of natural microflora are widely distributed, arising from sources like forest and prairie fires, combustion of fossil fuel, crude oil, coal liquefaction and gasification processes (11). On an evolutionary scale microorganisms have been exposed to hydrocarbons for eons and have evolved complex mechanisms for their metabolism. Further, the living organisms depend upon environmental sources of these compounds, as they are not synthesized.

Banking on the microorganism’s natural abilities to degrade various organic contaminants that are toxic in nature and are often recalcitrant, biotechnological solutions to eliminate such pollutants have emerged as an effective tool. CMG has recently started a program for the development of this important resource. Some of the program’s achievements are as follows.

Mononuclear Aromatic Hydrocarbons (MAHs)

Fifty-six bacterial cultures have been isolated coastal areas of Pakistan selected for their potential to degrade aromatic compounds. The isolated bacteria were challenged by varying concentrations of phenol, which while being a MAH is commonly found polluting sites of industrial and petroleum wastes in Pakistan. Their tolerance to phenol was found to be 5 mM, 10 mM, 15 mM, and 20 mM in 57%, 23%, 9%, and 5% of the isolates respectively. Considering the isolates being of marine origin, maintenance of culture and the tests were conducted in presence of 3% NaCl (w/v) in the medium. However, about 30%, 23%, and 29% of isolates were found to tolerate 5 mM, 15 mM, and 20 mM phenol in very low concentration of NaCl, respectively. This phenomenon demonstrated the synergy of inhibitory effects of phenol and NaCl.

It was observed that of the isolates subjected to toluene and xylene stress (also MAHs), 39% and 5% were able to utilize toluene and xylene as sole source of carbon, respectively. While only 9% were found to be able to tolerate the synergistic inhibitory effects of toluene and xylene together and utilize them as carbon source (5). The significance of this observation is not yet understood. Pseudomonas aeruginosa CMG556 has been found to secrete a white substance that collects at air—liquid interface in response to xylene-stress that has been found to be fatty acid by GC-MS. It is not known whether the fatty acid promotes solubilization of xylene in aqueous medium or whether it complexes with xylene in some unknown manner. (Akhtar, J. et al., unpublished work). Production of membrane trans-fatty acids in response to MAH stress has been reported earlier in literature by several workers (16) but the disassociation of fatty acid from cell has not been found before(13). The evidence of dissociation or secretion comes from the fact that no viable bacteria could be recovered from fatty acid phase whereas the aqueous phase was found to be teeming with viable bacteria.

Polynuclear Aromatic Hydrocarbons (PAHs)

A soil bacterial isolate Pseudomonas aeruginosa CMG154 has been found to utilize a PAH belonging to pyrethroid group of pesticides called cypermethrin ((R,S)-alpha-cyano-3-phenoxybenzyl (1RS)-cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-carboxylate) as sole source of carbon. This pseudomonad also utilizes endosulfan (a hexachlorinated pesticide) and an organophosphate pesticide O,S-dimethyl phosphor amidothioate as carbon source in addition to a number of MAHs such as benzoic acid, benzaldehyde, benzyl alcohol, m-xylene, p-xylene, toluene, m-toluate, and p-toluate. Some of the breakdown products of cypermethrin have been discovered and tentatively identified by thin layer chromatography (Qureshi, F.M. et al., unpublished work). Confirmation of metabolite identity by mass spectroscopy, infrared spectroscopy and nuclear magnetic resonance spectroscopy is under way.

Towards Effective Bioremediation—Synthesis

The remarkable ability of microorganisms to exchange genetic material across very unrelated individuals allows them to rapidly adapt to the changing environment. This fact and the Darwinian-evolution — ‘survival of the fittest’— have resulted in a very diverse collection of microorganisms that can be found in almost all kinds of extreme environments from the chills of Antarctica to the super-heated marine thermal vents, from dryness of the deserts to the wets of oceans, from brines to acidic mine streams, from the heights of clouds to depths of sea-basins, etc. Based on such observations the concept of a living Earth has begun to gain popularity.

From the above it is evident that microorganisms posses great potential to be exploited for bioremediation of wastes or contaminated environments. Metal accumulating bacteria can be utilized for removing metals from industrial wastes, mining wastes, or metal contaminated streams (8,17,22-24). Such technologies also provide the facility to recover the metals from bacteria, purified and recycled. Metal precipitating bacteria can be used in a similar manner to remove metals but provide added benefit of relative ease of metal recovery since it is not bound to the cell. Bacteria can also be used for reduction of sulfates to sulfides, which also precipitate out of solution in addition to many other applications beyond the scope of this text. Metal solubilization is very important since it provides a mechanism for bringing bound-metals into solution as free ions (10) that can then be taken up by metal-accumulating bacteria. This demonstrates the importance strategic use of consortium of different microorganisms to achieve difficult bioremediation goals.

Organic compounds are present widely in soils and are very important for its fertility. However, they can be a problem when present in significantly large amounts as compared to the norms in nature, such as accidents, spills, seepage, etc. On the other hand, man-made or xenobiotic compounds always disrupt the balance of nature and result in disastrous ecological consequences when out of control. Fortunately, nature has provided us a large resource in the form microbes that be effectively and efficiently used for biodegradation of xenobiotics (9,11,19). It is also possible to artificially enhance degradation in a manner similar to natural selection as described above (9).

However, sometimes target sites contain other contaminants (such as metals) that can be detrimental to the microorganism or the biodegradation process. This situation can be remedied by the use of transgenic bacteria that can be constructed by introducing metal-resistance genes into biodegrading microorganisms or vice versa (15). Co-contamination situations can also be remedied by strategic use of microbial-consortia, as explained before.

Finally, financially constrained countries like in the Third World are often unable to take effective countermeasures against pollution due to economic infeasibilities of imported technology. Industries in such countries are often concerned that the rise in price of their products would adversely affect their sales. It is proposed that this problem can be addressed by the use of indigenously developed technologies and natural resource for bioremediation. It has been argued that indigenously produced technologies with efficiencies reasonable enough to allow effective and economical operation provide a practical solution for Third World countries (17).

References

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