Biological treatment of contaminated soils and wastes is increasingly gaining acceptance. However, technological advances mostly take place in industrialized countries. Developing countries not only have limited access to these environmental technologies, but often lack an environmental regulatory framework as a driving force for the application of bioremediation.

Especially in the last 50 years of industrial development, the amount and variety of hazardous substances has increased drastically. There are an estimated 100,000 human-made chemicals in use, and hundreds of new ones are produced each year. Due to the increase in industrial and agricultural activities and exports of wastes, not only the traditional industrialized countries, but all nations are confronted with widespread soil pollution. A significant number of synthetic compounds, particularly those that are not related to natural ones, persist in the environment.
Essentially, there are three major categories of sites with polluted soils. (a) sites that have been polluted by either spillage or leakage during production, handling or use of industrial material. This includes activities to gain raw materials, such as mining and oil drilling; (b) locations that have been used as disposal sites for diverse waste; (c) farmlands that have been excessively exposed to pesticides.
Contaminated land sites are health hazards for human beings and thus are unsuitable for housing or agriculture. The downward migration of pollutants from the soil into the groundwater is especially problematic in developing countries where groundwater is often  directly used for drinking without any prior treatment.

Bioremediation principles
Biological cleaning procedures make use of the fact that most organic chemicals are subject to enzymatic attack of living organisms. These activities are summarized under the term biodegradation. However, the end products of these enzymatic processes might differ drastically. For instance, an organic substance might be mineralized (i.e. transformed to carbon dioxide and water). It might also be converted to a product that binds to natural materials in the soil, or to a toxic substance.
Bioremediation refers to the productive use of micro-organisms to remove or detoxify pollutants, usually as contaminants of soils, water or sediments that otherwise threaten public health. Bioremediation is not new. Micro-organisms have been used to remove organic matter and toxic chemicals from domestic and manufacturing waste discharge for many years. Indeed, micro-organisms are frequently the only means, biological or non-biological, to convert synthetic chemicals into inorganic compounds. What is new is the emergence of bioremediation as an industry that is driven by its particular usefulness for sites contaminated with petroleum hydrocarbons.

Different substances and approaches
In soil bioremediation, a general distinction is made between in situ treatments, i.e. on the contaminated site itself, and ex situ treatments, i.e. the soil has to be excavated and processed elsewhere (see box 1). Ex situ bioremediation covers a wide range of technologies, from relatively simple land farming to costly bioreactor treatments. The latter allows a more rigid control of the whole process and therefore bears the possibility to accelerate degradation. However, because the soil has to be removed, ex situ treatments are not always possible and generally more expensive.
Under field circumstances, micro-organism activity is often restricted by levels of nutrients and oxygen. In situ bioremediation therefore stimulates the indigenous microflora by supplementing the limiting factors, mainly by aeration and adding nitrogen and phosphate. However, the appropriate technology is determined by the type of contamination. For example, although in situ bioremediation mostly takes place under aerobic conditions, some hazardous substances are preferably degraded anaerobically (see box 2). The chlorinated organic solvent tetrachloroethylene (PCE), for example, is degradable in a two-step approach. Firstly, oxygen has to be removed so that PCE is transformed to dichloroethylene by anaerobic bacteria. Afterwards, the soil is aerated again and further degradation takes places by aerobic micro-organisms.
Whereas most degradable substances serve as a carbon and energy source for microbial growth, others do not. In this case, another substance needs to be added as external energy source. In the above mentioned degradation of PCE, for example, the anaerobic degradation stage is fuelled by methanol, whereas in the subsequent step aerobic bacteria acquire their energy from additional phenol.
Toxic metals, another class of hazardous substances, are not susceptible to bacterial degradation. In this case, bioremediation processes aim at sequestering the metals. As a result, metals become unavailable to biological processes and  no longer exert toxic effects.
At present, petroleum and petroleum-derived products still cause the most pervasive environmental contamination. Since they are generally susceptible to naturally occurring microbial activity, they have become a main target of bioremediation. Hydrocarbon-degrading micro-organisms are ubiquitous in most ecosystems. Over 30 different genera of oil-degrading bacteria and fungi have been identified for both in situ and ex situ bioremediation purposes.
Several geomorphological features of the site such as soil type, pH, organic matter content influence the clean-up. Finally, the future use of a site is of importance, because it determines the tolerance level of the pollution that may remain in the soil. Obviously, the criteria for a site projected for housing are stricter than for an industrial dump site.

Different bioremediation technologies Bioremediation can be broadly subdivided into five approaches: Ex situ treatments: Contaminated soil is excavated and treated at another site * Bioreactors. Liquids, vapours, or solids in a slurry phase are treated in a reactor. Microbes can be of natural origin, cultivated or even genetically engineered. Processes can be monitored, regulated and modelled mathematically very precisely. * Solid-phase technologies. Contaminated soils are excavated, placed in a containment system through which water and nutrients percolate. This is particularly useful for petroleum-contaminated soils. * Composting. In this variation of solid-phase treatment, large amounts of degradable organic matter are added to a contaminated material. The process itself usually consists of a aerobic incubation for several weeks or months. * Land farming. Contaminated sludge, soils or sediments are spread on fields and cultivated in the same way as a farmer might plough and fertilize agricultural land. This is an inexpensive manner to clean up petroleum-contaminated soil by microbial activity. Although its application is restricted to readily degradable material, leaching into groundwater is still a threat. In situ treatments: The treatment of the contaminated soil takes place at the site of the contamination * In situ bioremediation. Most in situ processes involve the stimulation of indigenous microbial populations (e.g. by adding nutrients or aeration). Since the soil is not removed this method is relatively cost effective. However, precise control of the biological processes is problematic.

 
Limitations and alternatives
Non-biological treatments of waste such as landfill, chemical extraction, electro-reclamation and thermal treatment (e.g. incineration) still form the majority of the techniques applied. With the exception of landfill, the physical-chemical processes on which they are based are fast and controllable, but have a high energy demand. In contrast, bioremediation approaches are less input-demanding. However, the degradation of a contaminant in the soil is not a linear process over time. Instead, degradation processes slow down with the declining concentration of pollution that remains in the soil. As a consequence, bioremediation is time-consuming and not able to clean the soil 100 per cent. This holds especially true for bioremediation by land farming and for in situ treatments, which could take several months to years. The application of bioremediation is also hampered by restricted knowledge of microbial physiology and processes, which sometimes result in unexpected and unwanted toxic by-products. Furthermore, some synthetic compounds such as polychlorinated biphenyls (PCBs), highly substituted nitro compounds and polyaromatic hydrocarbons (PAHs) have proven to be very recalcitrant to microbial attack. For soils contaminated with these substances, incineration or chemical treatment have the advantage over bioremediation.

Genetically engineered micro-organisms
Since its beginning, genetic engineering has claimed to be able to construct tailor made micro-organisms with improved degrading capabilities for toxic substances. Indeed, the landmark patent for a genetically modified organism (GMO), filed in the USA by A.M. Chakrabarty in 1971, was for a bacterium with hydrocarbon degrading abilities. However, little progress has been made in developing robust strains of organisms for in situ use. Of the at least 29 approved field tests of recombinant bacteria carried out worldwide since 1986, only one was for bioremediation purposes.
The application in the environment of GMOs for bioremediation is restricted by two contradictory ecological factors. On the one hand, specifically-designed organisms lack the evolution from which naturally occurring organisms have benefited for thousands of years. As a result, the latter can often cope better with changing environmental conditions such as changes in temperature, substrate or waste concentrations. GMOs usually lose the competition for survival with naturally occurring organisms. This fragility of the GMOs restricts their life-span in the environment and can be desirable in cases when they are no longer needed for bioremediation.
On the other hand, when exposed to the contaminating substances they are supposed to degrade, GMOs show a higher viability than naturally occurring bacteria. Under these circumstances, artificially designed micro-organisms profit from their tailored enzymatic equipment. There are concerns about the negative effect of these GMOs on the complex and delicate microbial ecosystems by competition or the exchange of genetic material in the soils to which they are applied. Even more worrisome is their potential effect outside the treatment area. While recombinant strains may appear harmless in the laboratory, it is virtually impossible to assess their impact in the field.
Consequently, in countries with a biosafety regulatory framework, the deliberate release of GMOs for bioremediation purposes is subject to regulation.
On a laboratory scale, the development of GMOs for degrading particular toxic substances, such as toluene, PCBs, and benzoates, shows some progress. When restricted to contained use in bioreactors, GMOs could be used for the treatment of industrial discharges which are reasonably well-defined and selected. However, under field conditions, in situ bioremediation techniques remain more promising than the application of GMOs. An example is the stimulation of indigenous bacterial communities by nutrient or oxygen supply.

Soil bioremediation for different substances
Contamination	Volatility	Biodegredability		Solubility in water	In situ possibilities
		Aerobic	Anaerobic
Hydrocarbons
Gasoline	+	+	-	+	yes
Kerosine	±	+	-	+	yes
Gasoil	-	±	-	-*	yes
Domestic fuel	-	±	-	-*	yes
Lubricants	-	-	-	-	no
PAH
light (2-3 rings)	±	+	-	±*	yes
heavy (4-5 rings)	-	-	-	-*	no
Chlorinated Hydricarbons
Aliphatic (per, tri)	+	-	+	+	yes
Chlorobenzene	+	+	-	+	yes
Pesticides	-	±	-	-	no
PCB	-	-	-	-	no
Heavy metals	-	-	-	±	yes
Aromatics (BTEX)	+	+	±	+	yes
PAH: Polyaromatic Hydorcarbon; Pesticides: Organochloro-pesticides (e.g. DDT); PCB: Polychlorinated Biphenyl; BTEX: Benzene, Toluene, Ethylbenyene, Xylene  *Solubility can be enhanced by detergents (for hydrocarbons) or by acidification (heavy metals)  Source: J. P. Okx, L. Hordijk and A. Stein (1996), "Managing Soil Remediation Problems." Environmental Science & Pollution Research, 3 (4), pp. 229-235.

 
Bioremediation in the South
At present, bioremediation is mostly applied in industrialized countries. However, its basic principles are of universal validity. What is more, in many developing countries, a warm climate and high humidity are naturally occurring stimulants for microbial remediation processes. The application of land farming or extensive in situ techniques, which stimulate the natural degradation capacity of the soil, could be useful where time and space are no limiting factors. Basic remediation techniques are relatively cheap and do not need extensive training and controls.
At present, there are several efforts in bioremediation in the South, especially in oil producing developing countries. In Brazil, for instance, a project of the Federal University of Santa Catarina and Petrobrás, the Brazilian national oil company, addresses the degradation of benzene, toluene, ethylbenzene and xylene (BTEX) from gasoline spills. Brazilian gasoline is mixed with ethanol, which hampers the degradation of the BTEX compounds because bacteria prefer degradation of ethanol to BTEX. Therefore, a new approach called phytoremediation is now under development, which uses plants for bioremediation of gasoline spillage. The Brazilian Environmental Institute (IBAMA) and federal environmental agencies have tried to regulate the use and application of bioremediation. However, they have not yet reached a definitive framework.
Another example are Nigeria’ s environmental problems caused by petrochemical activities for the last thirty years. The Nigerian state oil industry is engaged in joint ventures with several multinational oil companies, including Shell (UK/the Netherlands), Chevron (USA) and Elf (France). In the past few years they have been heavily criticized for environmental pollution. In 1996, the Nigerian Department of Petroleum Resources (DPR) adopted guidelines and standards for environmental protection for its oil industry. DPR commissioned a Nigerian company for the clean-up and the costs should be shouldered by the polluters. In Nigeria, the expertise needed to develop its own technologies is not available at the moment. Also, the Nigerian private company’ s access to foreign bioremediation technologies is limited by budget constraints.

Environmental standards as incentives
The clean-up of contaminated sites, whether by biological or conventional technology, is only one out of three possible solutions. The second, which is the most widespread and in the short-term cheaper alternative, is simply to ignore the pollution. The third, and probably the most cost-effective alternative in the long term, is to prevent pollution beforehand, for instance by an environmentally friendlier method of production. In principle, biological remediation technologies are applicable here as well.
Developing countries are confronted with the existence of a worldwide network of waste trade, mainly from the North to the South. This problem was addressed by the Basel Convention on the Transboundary Movement of Hazardous Wastes (Basel Convention), a legally binding international treaty, ratified by 117 nations. Since January 1998, a ban has been imposed on all exports of hazardous wastes for final disposal or recycling from the industrialized North to developing countries. However, the waste that has already reached the South has remained an environmental problem for the recipients.

The decision to clean up a contamination largely depends on the political will and whether the costs involved are feasible. Only if these two factors are met, environmental regulations have a chance to be enforced.
Notwithstanding the high costs, government decisions in industrialized countries have proven to be the driving force in creating a demand for clean-up technologies. For instance, in 1980 the US government issued the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA), usually referred to as the "Superfund Act". Under the guidance of the US Environmental Protection Agency (EPA), the Superfund sets standards for abandoned hazardous waste sites, administers liability for the contamination, and creates a trust fund out of tax payments by the chemical and petroleum industry for cases in which the responsible persons cannot be identified. When the EPA identifies a polluter, it negotiates the cost for a clean-up process. In case the responsible party refuses to pay, Superfund advances the expenses and then attempts to get the money back through legal action.
More than 75,000 sites in the USA had been identified originally as contaminated land environments. A more recent estimation, however, identified an additional 37,000 sites in need for remediation. The estimated costs of the entire clean-up operation is about US$ 1,100 billion. In Europe, approximately 495,000 sites have been classified as contaminated, with remediation costs between US$ 280 and US$ 1,000 billion.
Given these large financial sums, cost efficiency is a crucial argument when deciding on a certain remediation technology. The costs per cubic meter for soil bioremediation range between US$ 52 and 131. This is far below the US$ 327 to 1046 in case of incineration, and US$ 196 to 327 per cubic metre for landfilling. Although the economic advantages are striking, there is still less investment into bioremediation as compared to other environmental techniques, such as waste water treatment or air pollution control. Nevertheless, the global remediation/clean-up market is estimated at US$ 10 to 15 billion annually, with a growth rate of 10 to 15 per cent per year.

Future developments
The importance of environmental legislation for industrialized countries is addressed by the Organisation for Economic Co-operation and Development (OECD). It advises its member countries to implement environmental standards and legislation as a "pacer for environmental biotechnology". The idea is to create the demand for new products and services and thereby initiate a new market. This strategy seems to have worked for countries such as Denmark, Germany and the Netherlands. In these countries with an early environmental legislation, a high- technology environmental biotechnology industry has developed. Since these companies are competitive on an international scale, the initialization of a demand for environmental services and goods has spun off into an export oriented industry.
The effect of environmental standards and regulatory frameworks in developing countries is considerably different. Environmental considerations are often interpreted as secondary to other goals such as economic development. In this view the differences in environmental standards reflect the stage of industrial development. However, developing countries that are willing to implement a more rigid environmental legislation are confronted with several implications. For instance, low or missing environmental standards in developing countries still attract transnational industries to locate environmentally unsustainable activities in the South. On the other hand, to comply with standards from industrialized countries, developing countries often have to rely on technologies as developed by and under control of the industrialized nations. Although it is highly recommended for developing countries to enforce an environmental regulation, it should be clear that it will not have the same consequences as for industrialized countries. It is obvious that these regulations will not be able to stimulate the development of a highly competetive environmental industry, because this road is already blocked by the technologically more advanced developed countries. However, given the political will to enforce environmental standards, they could help to build the capacity for adapted solutions. Many technologies, for instance in situ bioremediation, can be applied effectively at relatively low costs and are not necessarily dependent on technology developed in industrialized countries.
Volker Lehmann

Editor Biotechnology and Development Monitor

Sources
M. Alexander (1994), Biodegradation and Bioremediation. San Diego, USA: Academic Press.

R.L. Crawford and D.L. Crawford (eds.) (1996), Bioremediation: Principles and applications. Cambridge. UK: Cambridge University Press.

OECD (1994), Biotechnology for a Clean Environment. Paris, France: OECD Publications.

A.D. Little International Inc. (1994), Seeking Market Opportunities for Dutch Environmental Technologies. Report on behalf of Dutch Ministry of Economic Affairs. Den Haag, the Netherlands.

Personal communications with J. Okx and W. Visser (TAUW Milieu, the Netherlands), M. Pieters (Environmental Consultant, the Netherlands), H. X. Corseuil (Federal University of Santa Catarina, Brazil).