Introduction

There has been an emergence of interest in the use of biological systems and bioremediation to degrade, modify, move, transform or sequester environmental contaminants. Traditional methods of remediation include chemical and physical processing and are often plagued with problems such as the creation of toxic bi-products and environmental damage. Bioremediation of a site offers the advantages of lower costs, environmental soundness, insitu or exsitu solutions, higher aesthetics, and increased public acceptance. The following will focus on how microorganisms can be used and manipulated to remediate various sites of environmental contaminants.

Bioremediation

Definition

Bioremediation is the use of microbes, fungi or plants to remove toxic contaminants from a site. This can be accomplished in situ or ex situ. In situ bioremediation involves remediating the contaminant at the site of contamination. In situ remediation requires extensive monitoring to ensure the success of the remediation effort and to control environmental impacts. Ex situ remediation involves removing the contaminants from the site and treating them at a processing location in a bioreactor. While this method may be more financially and environmentally costly, it allows more control over the remediation process. This includes setting growth conditions in the reactor and choosing foreign or engineered microbes that may be more efficient at remediating than native organisms.

Advantages

Interest in bioremediation has increased in recent years as it is seen as a more environmentally-friendly method of dealing with anthropogenic contaminants. Additionally, bioremediation offers a cost-effective strategy to remove dispersed contaminants from an area using processes that capitalize on natural processes. This results in less danger to the environment and the workers. Lastly, bioremediation generally results in the complete illumination of the contaminant without the production of harmful intermediates or biproducts.

Disadvantages

Bioremediation has not completely replaced traditional, more harmful, methods due to some disadvantages. Firstly, and most obviously, bioremediation takes a long time. The process relies on the metabolism and, usually, the growth of bacteria and so it is limited by their lifecycles. Also, bacteria may use a different substrate for nutrition and will not move to the contaminant as an energy or carbon source until their preferred substrate is depleted. Additionally, there are some types of contaminants that are not suitable for bioremediation. Site conditions (soil pH, soil type, moisture level, nutrient availability, etc.), contamination concentration, bioavailability of contaminants and the presence of a mixture of contaminants also affect the use bioremediation. However, these latter limitations can easily be overcome if the use of a bioreactor and/or a mixture of microorganisms or remediation processes are available options. Lastly, there is still a small fear about introducing foreign organisms into a site and engineering organisms for this purpose. However, as will be seen throughout this document, these fears are quickly being put to rest as the techniques in bioremediation are perfected and optimized.

Types of remediation

Remediation is accomplished by intrinsic remediation (natural attenuation), bioaugmentation or biostimulation. Intrinsic remediation only occurs in situ and involves letting the site make use of natural flora to remediate itself over time. While eventually successfully, this process takes a long time and the removal of contaminants is generally time sensitive to avoid further contamination. In order to increase the rate of remediation, bioaugmentation and biostimulation may be employed. Bioaugmentation involves the introduction of remediating species to a site whereas biostimulation involves providing the ideal growth conditions for native microbes to flourish and remediate the site. Methods of biostimulation include aeration, nutrient addition, and moisture balancing. Biostimulation can also involve increasing the bioavailability of the contaminant by addition or surfactants. These additional steps ensure an optimized system and efficient remediation.

Various microbial processes are exploited to remediate contaminants from sites. As such, many different methods can be employed to ensure the overall decontamination of the site. This is important because most sites are contaminated with numerous toxins or contain toxins that require multiple steps to degrade. Additionally, many contaminants such as PCBs require both an aerobic and anaerobic degredation step. Bioremediation processes generally used both in situ and ex situ include; biosorption, biocontainment, sequestration, bioaccumulation, biovolatilization, etc. Any unique metabolic pathway or cell surface characteristic can potentially be used in a remediation system.

Microorganisms

Sources

There are three possible sources of microbes for use in bioremediation. Organisms may be mined, engineered or augmented in situ. Mining for microbes for remediation purposes involves isolating organisms from a site with similar contamination characteristics to the potential remediation site. The premise is that organisms that live, and thrive, in contaminated areas have adapted special abilities to live in the presence of the contaminant. In addition to living in the presence of the toxin, many have developed a use for the contaminant and are thus potential candidates for bioremediation. It has been found that the majority of bacteria isolated this way are generally from common soil genera such as Bacillus, Pseudomonas and Xanthomonas. Once these bacteria that can survive among a specific toxin have been identified, they can be enriched in media containing the contaminant to further select for bacteria that can degrade the toxin. This process is known as enrichment. Enrichment is a common step in any bioremediation process to ensure the optimization of the system. (LI ET AL) Species such as Xanthomonas, Bacillus and Hyphomicrobium have been isolated from soil at a site near Zibo City, China. This soil is heavily polluted with hydrocarbons at concentrations up to 200 g per kg of dry soil. The isolated indigenous bacteria were enriched using a BAC (Biological Activated Carbon) system which has an activated carbon apparatus for attachment of bacteria and media and feeds the culture hydrocarbons as a sole nutrient source. The effluent from the column was used to inoculate a sample of the contaminated soil and the bioremediation efficiency showed marked improvement over the control, pre-enrichment, culture. Using this mining and enrichment process allows for the selection of the most efficient microbes to use to remediate a specific contaminant. These enriched populations can also serve as a source of genes for the genetic engineering of organisms for bioremediation, as discussed below.

Additionally, it has been shown that degrading organisms can be forced to increase their degredation ability through specialized enrichment. (EXAMPLE FROM MY STUFF)

It is also possible that remediation organisms exist at the contaminated site itself. If these organisms can successfully compete with nonbiodegredation organisms and thrive, then these organisms can be stimulated by altering environmental conditions to ensure their success in remediation. These microbes may also be isolated and enriched for use in a bioreactor. It is always preferred to use native organisms whenever possible to limit the impact on the ecosystem at the site.

Lastly, and more recently, bacteria have been genetically engineered for the purpose of bioremediation. These engineering strategies involve the insertion of remediation-associated genes to increase or impart the ability to remediate contaminants. The use of an organism in bioremediation can be improved by changing/adding the substrate it uses or increasing the efficiency of a pathway involved in remediation. Pseudomonas putida is a common soil bacteria that is used in bioremediation. It is a preferred organism as it has a natural ability to degrade contaminants such as styrene and naphthalene and is considered to be safe (i.e. not a potential pathogen). Genes have been introduced into this organism to allow it to use different substrates and thereby increase the types of contaminants it can degrade. Toluene dioxygenase (TDO) catalyzes asymmetric cis dihydroxylations of aromatic compound leading to the clearing of contamination by aromatic hydrocarbons. Inserting these genes into P. putida, the organism could use benzene, toluene and chlorobenzene as a sole carbon source. To further increase efficiency, TDO genes can be transformed into a strain harbouring the vgb gene encoding Vitreoscilla hemoglobin protein (VHb) which enhances oxygen microbial utilization rate under low dissolved oxygen concentration (a limiting factor in this reaction in bioremediation). These transformations are just two examples of the extensive manipulations possible to optimize bioremediation.

It is also possible to confer an ability to bioremediate to an organism. This is useful as these traits can be developed in well-described, safe and culturable organisms such as E. coli.

Genes for use in engineering come from a variety of sources including bacteria with a natural ability to degrade certain contaminants. New genes are discovered all the time and with the