Bioremediation.

Bioremediation and Biodegradation.

The application of bioremediation as a biotechnological process involving microorganisms has become a crescent study field in microbiology, because of its increasing potential of solving the dangers of many pollutants through biodegradation. Microorganisms might be considered excellent pollutant removal tools in soil, water, and sediments, mostly due to their advantage over other bioremediation procedures. Moreover, bioremediation using biodegradation represents a high impact strategy, but still a low cost way tool of removing pollutants, hence a very viable process to be applied. The principles of bioremediation are based on natural attenuation, bioaugmentation and biostimulation. There are three types.

The simplest method of bioremediation is natural attenuation, in which soils are only monitored for variations in pollution concentrations to ensure that the pollutant transformation is active. Bioaugmentation is usually applied in cases where natural active microbial communities are present in low quantities or even absent, wherein the addition of contaminant degrading organisms can accelerate the transformation rates. In such cases, the adaptation of exogenous strains that exert highly efficient activities for pollutant transformation to new environments is a key challenge in implementation. The capacity of a microbial population to degrade pollutants can be enhanced also by stimulation of the indigenous microorganisms by addition of nutrients or electron acceptors.

Natural attenuation

Natural attenuation or bioattenuation is the reduction of contaminant concentrations in the environment through biological processes (aerobic and anaerobic biodegradation, plant and animal uptake), physical phenomena (advection, dispersion, dilution, diffusion, volatilization, sorption/desorption), and chemical reactions (ion exchange, complexation, abiotic transformation). Terms such as intrinsic remediation or biotransformation are included within the more general natural attenuation definition. Although, one of the most important components of natural attenuation is biodegradation, the change in form of compounds carried out by living creatures such as microorganisms. Under the right conditions, microorganisms can cause or assist chemical reactions that change the form of the contaminants so that little or no health risk remains. Natural attenuation occurs at most polluted sites. However, the right conditions must exist underground to clean sites properly. If not, cleanup will not be quick enough or complete enough. Scientists monitor these conditions to make sure natural attenuation is working. This is called monitored natural attenuation or (MNA). So, Monitored natural attenuation is a technique used to monitor or test the progress of natural attenuation processes that can degrade contaminants in soil and groundwater. It may be used with other remediation processes as a finishing option or as the only remediation process if the rate of contaminant degradation is fast enough to protect human health and the environment. Natural processes can then mitigate the remaining amount of pollution; regular monitoring of the soil and groundwater can verify those reductions.

When the environment is polluted with chemicals, nature can work in four ways to clean it up:

• Tiny bugs or microbes that live in soil and groundwater use some chemicals for food. When they completely digest the chemicals, they can change them into water and harmless gases.
• Chemicals can stick or sorb to soil, which holds them in place. This does not clean up the chemicals, but it can keep them from polluting groundwater and leaving the site.
• As pollution moves through soil and groundwater, it can mix with clean water. This reduces or dilutes the pollution.
• Some chemicals, like oil and solvents, can evaporate, which means they change from liquids to gases within the soil. If these gases escape to the air at the ground surface, sunlight may destroy them.

If the natural attenuation is not quick enough or complete enough, bioremediation will be enhanced either by biostimulation or bioaugmentation.

Biostimulation

Biostimulation involving the addition of soil nutrients, trace minerals, electron acceptors, or electron donors enhances the biotransformation of a wide range of soil contaminants. There are many examples of biostimulation of pollutants biodegradation by indigenous microorganisms. Trichloroethene and perchloroethene are reported to be completely converted to ethane by microorganisms in a short span of time with the addition of lactate during biostimulation. Electron shuttles, such as humic substances (HS), may play a significant stimulation role in the anaerobic biotransformation of organic pollutants through enhancing the electron transfer speed. Anthraquinone-2,6-disulfonate (AQDS) from the category of HS can serve as an electron shuttle to promote the reduction of iron oxides and transformation of chlorinated organic contaminants . Chen et al. reported that the biostimulation of indigenous microbial communities by the addition of lactate and AQDS led to the enhanced rates of Pentachlorophenol PCP dechlorination by the dechlorinating and ironreducing bacteria in soils.

Among various nutrient media, glycerol appeared to show the most favorable metabolic characteristics against phenol toxicity on the indigenous Rhizobium Ralstonia taiwanensis, leading to better degradation efficiency of the toxic pollutant. Liliane et al. observed that biostimulation was more efficient when compared to natural attenuation of biodiesel in contaminated soils. However, the comparative study of Bento et al. revealed that bioaugmentation showed the greatest degradation potential and natural attenuation was more effective than biostimulation of soils contaminated with diesel oil. Results obtained by Yu et al. indicate that autochthonous microbes may interact and even compete with the enriched consortium during polycyclic aromatic hydrocarbons biodegradation and the natural attenuation appeared to be the most appropriate way to remedy fluorene and phenanthrene contaminated mangrove sediments while biostimulation was more capable to degrade pyrene contaminated sediments.

Biostimulation involves the modification of the environment to stimulate existing bacteria capable of bioremediation. This can be done by addition of various forms of rate limiting nutrients and electron acceptors, such as phosphorus, nitrogen, oxygen, or carbon (e.g. in the form of molasses). Additives are usually added to the subsurface through injection wells, although injection well technology for biostimulation purposes is still emerging. Removal of the contaminated material is also an option, albeit an expensive one. Biostimulation can be enhanced by bioaugmentation. This process, overall, is referred to as bioremediation and is an EPA-approved method for reversing the presence of oil or gas spills.

The primary advantage of biostimulation is that bioremediation will be undertaken by already present native microorganisms that are well-suited to the subsurface environment, and are well distributed spatially within the subsurface. The primary disadvantage is that the delivery of additives in a manner that allows the additives to be readily available to subsurface microorganisms is based on the local geology of the subsurface. Tight, impermeable subsurface lithology (tight clays or other fine-grained material) make it difficult to spread additives throughout the affected area. Fractures in the subsurface create preferential pathways in the subsurface which additives preferentially follow, preventing even distribution of additives.

Investigations to determine subsurface characteristics (such as natural groundwater velocity during ambient conditions, hydraulic conductivity of the subsurface, and lithology of the subsurface) are important in developing a successful biostimulation system. In addition, a pilot-scale study of the potential biostimulation system should be undertaken prior to full-scale design and implementation.

However, some biostimulative agents may be used in chaotic surfaces such as open water and sand so long as they are [oleophilic], meaning that they bond exclusively to hydrocarbons, and basically sink in the water column, bonding to oil, where they then float to the water’s surface, exposing the hydrocarbon to more abundant sunlight and oxygen where greater micro-organic aerobic activity can be encouraged. Some consumer-targeted biostimulants bond possess this quality, others do not.

With the introduction of therapeutic lasers, biostimulation also refers to the application of photon energy to injured tissue, in order to achieve a stimulatory and regenerative effect at the molecular level.

Bioaugmentation

We can define bioaugmentation as the technique for improvement of the capacity of a contaminated matrix (soil or other biotope) to remove pollution by the introduction of specific competent strains or consortia of microorganisms. The basic premise for this intervention is that the metabolic capacities of the indigenous microbial community already present in the biotope slated for cleanup will be increased by an exogenously enhanced genetic diversity, thus leading to a wider repertoire of productive biodegradation reactions. Moreover, genetically engineered microorganisms (GEMs) exhibiting enhanced degradative capabilities encompassing a wide range of aromatic hydrocarbons have also potential for soil bioaugmentation. It is thought that bioaugmentation approach should be applied when the biostimulation and bioattenuation have failed.

Many studies have shown that both abiotic and biotic factors influence the effectiveness of bioaugmentation, the most important abiotic factors are temperature, moisture, pH and organic matter content, however, aeration, nutrient content and soil type also determine the efficiency of bioaugmentation. Biotic factors, including competition between indigenous and exogenous microorganisms for limited carbon sources as well as antagonistic interactions and predation by protozoa and bacteriophages, also play essential roles in the final results of bioaugmentation.

The combination of bioaugmentation and biostimulation might be a promising strategy to speed up bioremediation. Both indigenous and exogenous microorganisms could benefit from biostimulation by the addition of energy sources or electron acceptors. Bioaugmentation-assisted phytoextraction using PGPR or AMF is also a promising method for the cleaning-up of soils contaminated by metals.

Usually the steps involve studying the indigenous varieties present in the location to determine if biostimulation is possible. If the indigenous variety do not have the metabolic capability to perform the remediation process, exogenous varieties with such sophisticated pathways are introduced.

Bioaugmentation is commonly used in municipal wastewater treatment to restart activated sludge bioreactors. Most cultures available contain a research based consortium of Microbial cultures, containing all necessary microorganisms (B. licheniformis, B. thurengensis, P. polymyxa, B. sterothemophilus, Penicillium sp., Aspergillus sp., Flavobacterium, Arthrobacter, Pseudomonas, Streptomyces, Saccaromyces, Triphoderma, etc.). Whereas activated sludge systems are generally based on microorganisms like bacteria, protozoa, nematodes, rotifers and fungi capable to degrade bio degradable organic matter

At sites where soil and groundwater are contaminated with chlorinated ethenes, such as tetrachloroethylene and trichloroethylene, bioaugmentation is used to ensure that the in situ microorganisms can completely degrade these contaminants to ethylene and chloride, which are non-toxic. Bioaugmentation is typically only applicable to bioremediation of chlorinated ethenes, although there are emerging cultures with the potential to biodegrade other compounds including BTEX, chloroethanes, chloromethanes, and MTBE. The first reported application of bioaugmentation for chlorinated ethenes was at Kelly Air Force Base, TX (Major et al., 2002).

Bioaugmentation is typically performed in conjunction with the addition of electron donor (biostimulation) to achieve geochemical conditions in groundwater that favor the growth of the dechlorinating microorganisms in the bioaugmentation culture.

The latest technological advances in the bioaugmentation consist of genetic fingerprinting and molecular markers to determine the interactions between augmented organisms and native organisms (existing biomass). In addition, there are some prospects for genetically engineered microorganisms. These advances in genetic engineering would involve splicing genes from one organism into another the genes that code specificdegradative enzymes. A possible application would be in situations where the contaminent levels of the wastewater are below the levels needed for microorganism enzyme production. Genetic engineering could make it possible to alter the gene structure so the microorganisms produces the correct enzyme in the presence of some other contaminent already present in high concentration within the wastewater.

Factors affecting microbial degradation

Microorganisms can degrade numerous of organic pollutants owing to their metabolic machinery and to their capacity to adapt to inhospitable environments. Thus, microorganisms are major players in site remediation. However, their efficiency depends on many factors, including the chemical nature and the concentration of pollutants, their availability to microorganisms, and the physicochemical characteristics of the environment. So, factors that influence the rate of pollutants degradation by microorganisms are either related to the microorganisms and their nutritional requirements (biological factors) or associated to the environment (environmental factors).

Biological factors

A biotic factor is the metabolic ability of microorganisms. The biotic factors that affect the microbial degradation of organic compounds include direct inhibition of enzymatic activities and the proliferation processes of degrading microorganisms. This inhibition can occur for example if there is a competition between microorganisms for limited carbon sources, antagonistic interactions between microorganisms or the predation of microorganisms by protozoa and bacteriophages. The rate of contaminant degradation is often dependent on the concentration of the contaminant and the amount of “catalyst” present. In this context, the amount of “catalyst” represents the number of organisms able to metabolize the contaminant as well as the amount of enzymes(s) produced by each cell. Furthermore, the extent to which contaminants are metabolized is largely a function of the specific enzymes involved and their “affinity” for the contaminant and the availability of the contaminant. In addition, sufficient amounts of nutrients and oxygen must be available in a usable form and in proper proportions for unrestricted microbial growth to occur. Other factors that influence the rate of biodegradation by controlling the rates of enzyme catalyzed reactions are temperature, pH and moisture. Biological enzymes involved in the degradation pathway have an optimum temperature and will not have the same metabolic turnover for every temperature. Indeed, the rate of biodegradation is decreased by roughly one-half for each 10°C decrease in temperature. Biodegradation can occur under a wide-range of pH; however, a pH of 6.5 to 8.5 is generally optimal for biodegradation in most aquatic and terrestrial systems. Moisture influences the rate of contaminant metabolism because it influences the kind and amount of soluble materials that are available as well as the osmotic pressure and pH of terrestrial and aquatic systems.

Environmental factors

Soil type and soil organic matter content affect the potential for adsorption of an organic compound to the surface of a solid. Absorption is an analogous process wherein a contaminant penetrates into the bulk mass of the soil matrix. Both adsorption and absorption reduce the availability of the contaminant to most microorganisms and the rate at which the chemical is metabolized is proportionately reduced. Variations in porosity of the unsaturated and saturated zones of the aquifer matrix may influence the movement of fluids and contaminant migration in groundwater. The ability of the matrix to transmit gases, such as oxygen, methane and carbon dioxide, is reduced in fine grained sediments and also when soils become more saturated with water. This can affect the rate and type of biodegradation taking place [126].The oxidation-reduction potential of a soil provides a measurement of the electron density of the system. Biological energy is obtained from the oxidation of compounds in which electrons are transferred to various more oxidized compounds referred to as electron acceptors. A low electron density (Eh greater than 50 mV) indicates oxidizing, aerobic conditions, whereas high electron density (Eh less than 50 mV) indicates reducing, anaerobic conditions

Degradation by genetically engineered microorganisms (GEM)

As mentioned above, bioaugmentation and biostimulation are methods that can be applied to accelerate the recovery of polluted sites. In the late 1970s and early 1980s, bacterial genes encoding catabolic enzymes for recalcitrant compounds started to be cloned and characterized. Soon, many microbiologists and molecular biologists realized the potential of genetic engineering for addressing biodegradation. A genetically engineered microorganism (GEM) or modified microorganism (GMM) is a microorganism whose genetic material has been altered using genetic engineering techniques inspired by natural genetic exchange between microorganisms. These techniques are generally known as recombinant DNA technology. Genetically engineered microorganisms (GEMs) have shown potential for bioremediation of soil, groundwater and activated sludge, exhibiting the enhanced degrading capabilities of a wide range of chemical contaminants. As soon as the prospect of releasing genetically modified microorganisms for bioremediation became a reality, much of the research effort in the field was aimed at biosafety and risk assessment.

There are at least four principal approaches to GEM development for bioremediation application. These include:

• Modification of enzyme specificity and affinity;
• Pathway construction and regulation;
• Bioprocess development, monitoring and control;
• Bioaffinity bioreporter sensor applications for chemical sensing, toxicity reduction and end point analysis.

Genetically engineered microorganisms

Molecular biology offers the tools to optimize the biodegradative capacities of microorganisms, accelerate the evolution of “new” activities, and construct totally “new” pathways through the assemblage of catabolic segments from different microbes.

Genes responsible for degradation of environmental pollutants, for example, toluene, chlorobenzene acids, and other halogenated pesticides and toxic wastes have been identified. For every compound, one separate plasmid is required. It is not like that one plasmid can degrade all the toxic compounds of different groups. The plasmids are grouped into four categories:

• OCT plasmid which degrades, octane, hexane and decane;
• XYL plasmid which degrades xylene and toluenes,
• CAM plasmid that decompose camphor and
• NAH plasmid which degrades naphthalene.

The potential for creating, through genetic manipulation, microbial strains able to degrade a variety of different types of hydrocarbons has been demonstrated by Friello et al. [131]. They successfully produced a multiplasmid-containing Pseudomonas strain capable of oxidizing aliphatic, aromatic, terpenic and polyaromatic hydrocarbons.

Pseudomonas putida that contained the XYL and NAH plasmid as well as a hybrid plasmid derived by recombinating parts of CAM and OCT developed by conjugation could degrade camphor, octane, salicylate, and naphthalene and could grew rapidly on crude oil because it was capable of metabolizing hydrocarbons more efficiently than any other single plasmid.

This product of genetic engineering was called as superbug (oil eating bug). The plasmids of P. putida degrading various chemical compounds are TOL (for toluene and xylene), RA500 (for 3, 5-xylene) pAC 25 (for 3-cne chlorobenxoate), pKF439 (for salicylate toluene). Plasmid WWO of P. putida is one member of a set of plasmids now termed as TOL plasmid. It was the first living being to be the subject of an intellectual property case. At that point, it seemed that molecular techniques, either through plasmid breeding or sheer genetic engineering, could rapidly produce microbes with higher catalytic abilities, able to basically degrade any environmental pollutant.

The application of genetic engineering for heavy metals removal has aroused great interest. For example, Alcaligenes eutrophus AE104 (pEBZ141) was used for chromium removal from industrial wastewater and the recombinant photosynthetic bacterium, Rhodopseudomonas palustris, was constructed to simultaneously express mercury transport system and metallothionein for Hg2+ removal from heavy metal wastewater.

For polychlorinated biphenyls degradation, chromosomally located PCB catabolic genes of R. eutropha A5, Achromobacter sp. LBS1C1, and A. denitrificans JB1 were transferred into a heavy metal resistant strain R. eutropha CH34 through natural conjugation.

Genetic engineering of endophytic and rhizospheric bacteria for use in plant associated degradation of toxic compounds in soil is considered one of the most promising new technologies for remediation of contaminated environmental sites. To select a suitable strain for gene recombination and inoculation into the rhizosphere, there are three criteria that has been recommended: first, the strain should be stable after cloning and the target gene should have a high expression, second, the strain should be tolerant or insensitive to the contaminant; and third, some strains can survive only in several specific plant rhizosphere. Many bacteria in the rhizosphere show only limited ability in degrading organic pollutants. With the development of molecular biology, the genetically engineered rhizobacteria with the contaminant-degrading gene are constructed to conduct the rhizoremediation. Examples about the molecular mechanisms involved in the degradation of some pollutants such as trichloroethylene (TCE) and PCBs has been studied.

For heavy metals, Sriprang et al. introduced Arabidopsis thaliana gene for phytochelatin synthase (PCS; PCSAt) into Mesorhizobium huakuii subsp. rengei strain B3 and then established the symbiosis between M. huakuii subsp. rengei strain B3 and Astragalus sinicus. The gene was expressed to produce phytochelatins and accumulate Cd²⁺, under the control of bacteroid-specific promoter, the nifH gene.

Finally, the use of GEM strains as an inoculum during seeding would preclude the problems associated with competition between strains in a mixed culture. However, there is considerable controversy surrounding the release of such genetically engineered microorganisms into the environment, and field testing of these organisms must therefore be delayed until the issues of safety and the potential for ecological damage are resolved Reports on the degradation of environmental pollutants by genetically engineered microorganisms are focused on genetically engineered bacteria using different genetic engineering technologies: Pathway modification, modification of substrate specificity by Comamonas testosteroni VP44. Obstacles associated with the use of GEM in bioremediation applications

While genetic engineering has produced numerous strains able to degrade otherwise intractable pollutants in a Petri dish or in a bioreactor, the practical translation of this research into actual in situ bioremediation practices has been quite scanty. One major issue in this respect is the growing realization that the strains and bacterial species that most frequently appear in traditional enrichment procedures are not the ones performing the bulk of biodegradation in natural niches and may not even be any good as bioremediation mediators. The use of stable isotope probing (SIP) and equivalent methods in microbial ecology have revealed that Pseudomonas, Rhodococcus, and the typical aerobic fast growers that are widely favored as hosts of biodegradation related recombinant genes are far less significant under natural conditions. Furthermore, using fast-growers as agents for biodegradation is the inevitable buildup of unwelcome biomass. As an alternative, the optimal clean-up agent would be the one that displays a maximum catalytic ability with a minimum of cell mass. The expression of biodegradation genes can be artificially uncoupled from growth with the use of stationary-phase promoters or starvation promoters. In addition, the recent advances in the area of recombinant DNA technologies have paved the way for conceptualizing “suicidal genetically engineered microorganisms” (S-GEMS) to minimize such anticipated hazards and to achieve efficient and safer decontamination of polluted sites. In some cases, whether the introduced bacterium is recombinant or not makes little difference, because the problem is that of implantation of foreign microbes in an unfamiliar territory. The introduction of bacterial biomass in an existing niche may create a palatable niche for protozoa that prevents the bacterial population to grow beyond a certain level. Ingenious approaches have been developed to circumvent this problem, including encapsulation of the inoculum in a polymeric matrix or protection in plastic tubing.

The efficacy of a desired in-situ catalytic activity (biodegradation or otherwise) depends first on its presence in the target site. One key enzyme may not be there, or it may preexist in the site but not be manifested. Alternatively, it can be hosted by just a very minor part of the whole microbial population, so that its factual expression in the site might not be significant .

A field release of P. fluorescens HK44 for bioremediation application has been successfully conducted on moderately large-scale and controlled field condition. However, the future application of genetically engineered bacteria for pollution remediation will not be free from the risks associated with their release in the environment. The future risk regarding use of other engineered bacteria is still unclear. Therefore, the future perspectives of engineered bacterial strains under the field conditions will be the focus of review, which may help us to assess the obstacles related with application of genetically engineered bacteria in environmental bioremediation.

The major problem encountered in successful bioremediation technology pertains to hostile field conditions for the engineered microbes. Besides, the molecular applications are mainly confined to only few well characterized bacteria such as E. coli, P. putida, B. subtilis, etc. Other bacterial strains need to be tried for developing the engineered microbes. The specific characteristic of open biotechnological applications has clearly necessitated the development of engineered bacterial strains to meet the new challenges. The main concern is to construct GE bacteria for field release in bioremediation with an adequate degree of environmental certainty. Efforts should be made to examine the performance of engineered bacteria in terms of their survival, potential of horizontal gene transfer, which may affect the indigenous microflora within a complex environmental situation. Often the novel scientific researches always give rise to still more fascinating questions pertaining to public concern. In the majority cases, the bacteria designed for bioremediation processes have been designed for specific purpose under the laboratory conditions, ignoring the field requirement and other complex situations. However, there is no evidence that the deliberate release of GE bacteria for bioremediation has caused a measurable adverse impact on the natural microbial community. At least the overstated idea of risk appraisal has fuelled so much debate and triggered so many research efforts, which have immensely contributed a lot in the field of environmental microbiology. However, survival of the GE bacteria in complex environmental situations is still a big question, needs to be addressed in the light of latest findings.

Conclusion

Microbial activities are very important for the renewal of our environment and maintenance of the global carbon cycle. These activities are included in the term biodegradation. Amid the substances that can be degraded or transformed by microorganisms are a huge number of synthetic compounds and other chemicals having ecotoxicological effects like hydrocarbons and heavy metals. However, in most cases this statement concerns potential degradabilities which were estimated in the laboratory by using selected cultures and under ideal growth conditions. Due to a whole range of factors: competition with microorganisms, insufficient supply with essential substrates, unfavorable external conditions (aeration, moisture, pH, temperature), and low bioavailability of the pollutant, biodegradation in natural conditions is lesser. So, environmental biotechnology has the objective of tackling and solving these problems so as to permit the use of microorganisms in bioremediation technologies. For this reason, it is necessary to support the activities of the indigenous microorganisms in polluted biotopes and to enhance their degradative abilities by bioaugmentation or biostimulation. Genetic engineering is also used to improve the biodegradation capabilities of microorganisms. Nevertheless, there are many risks associated to the use of GEM in the field. Whether or not such approaches are ultimately successful in bioremediation of pollutants may make a difference in our ability to reduce wastes, eliminate industrial pollution, and enjoy a more sustainable future.