Bioremediation can be defined as any process that uses microorganisms or their enzymes to return an environment altered by contaminants to its original condition. There are a number of advantages to bioremediation, which can be employed in areas that cannot be reached easily without excavation. It is well documented that the presence of metals in the soil impacts both the physiology and the ecology of microorganisms by inhibiting a broad range of microbial processes, including methane metabolism, growth, and nitrogen and sulfur conversion. It is known that toxic metal cations can substitute for essential physiological cations within enzymes in organisms, rendering them nonfunctional. Metals also tend to impose oxidative stresses on microorganisms. The extent in which metals tend to inhibit the biodegradation of organic compounds is directly related to the metal speciation - the physical or chemical form of the metal species in the soil, which also governs its toxicity to microorganisms, and therefore its impact on the remediation technique. The physical and chemical state of a metal species can also be influenced by environmental conditions such as the pH, the ionic strength of the water phase, and soil properties, which include ion exchange capacity, clay type and content, and organic matter content. Indeed, when dealing with the remediation of soil contaminated with organic compounds, one must also account for the bioavailability of heavy metals.
Generally, bioremediation technologies are performed either in situ or ex situ. In situ bioremediation involves treating the contaminated material at the site, while ex situ bioremediation involves the removal of the contaminated material to be treated elsewhere. Some examples of in situ bioremediation technologies are composting, bioventing, bioaugmentation and biostimulation, and ex situ soil bioremediation technologies include soil biopiles, landfarming, and bioreactors (Obed and Kenneth 2002).
In situ bioremediation is defined as treating a soil pollutant without removing the contaminated soil. Because these types of technologies usually do not require the excavation of the contaminated soil, they are less expensive, create less dust, and release of reduced amounts of volatile contaminants. Some in situ technologies are discussed below.
Compost is the decomposed remnants of organic materials (those with plant and animal origins). Compost is used in gardening and agriculture, where it is mixed in with the soil. It improves soil structure, increases the amount of organic matter, and provides nutrients. Compost is a common name for humus, which results from the decomposition of organic matter. In the presence of large amounts of organic matter, heavy metals are immobilized and do not enter the food chain. Microbes perform most of the decomposition, although larger creatures such as worms and ants also contribute to the process. Decomposition occurs naturally in all but the most hostile environments, such as landfills or extremely arid deserts, which prevent the microbes and other decomposers from thriving.
Composting is the controlled decomposition of organic matter. Rather than allowing nature to take its slow course, a composter provides an optimal environment in which decomposers can thrive. To encourage the most active microbes, the compost pile needs an appropriate mix of the following ingredients: carbon, nitrogen, oxygen (air), and water. Decomposition happens even in the absence of some of these ingredients, but not nearly as quickly and not nearly as pleasantly (for example, the plastic bag of vegetables in your refrigerator is decomposed by microbes, but the absence of air encourages anaerobic microbes that produce disagreeable odors). The most effective decomposers are bacteria and other microorganisms. Also important are fungi, molds, protozoa, and actinomycetes, which are bacteria that look like fungi or mold and often appear as white filaments in decomposing organic matter. At a macroscopic level, earthworms, ants, snails, slugs, millipedes, sow bugs, springtails, and other organisms consume and break down the organic matter. Centipedes and other predators feed upon these decomposers.
The most rapid composting occurs with the ideal ratio (by dry chemical weight) of carbon to nitrogen, which ranges from 25:1 to 30:1. In other words, the ingredients placed in the pile should contain 30 times as much carbon as nitrogen. Since, grass clippings average about 19:1 and dry autumn leaves average about 55:1, mixing equal parts of these by volume approximates the ideal ratio. Commercial-grade composting operations pay strict attention to this ratio. For backyard composters, however, charts of the carbon-to-nitrogen ratios of various ingredients and the calculations required to obtain the ideal mixture can be intimidating, so many rules of thumb exist to guide composters to approximate this mixture. High-carbon sources provide the cellulose needed by the composting bacteria for conversion to sugars and heat. High-nitrogen sources provide the most concentrated protein, which allow the compost bacteria to thrive.
To perform bioremediation using composting, the compost is mixed with the contaminated soil along with a bulking agent such as straw, hay, or corncobs to make it easier to deliver optimum levels of air and water to the microorganisms. The most common designs are static pile composting, mechanically agitated composting, and window composting. In static pile composting, the contaminated soil is placed into piles and aerated with blowers or vacuum pumps. Mechanically agitated composting involves the placement of the contaminated soil in treatment vessels, where it is mixed to achieve aeration. In window composting, the soil is placed in long piles knows as windows and periodically mixed by tractors (Cunningham and Philip 2000). As stated before, the contaminated soil is mixed with a bulking agent or compost to enhance bacterial growth. A typical ratio of soil to compost is 75% contaminated soil to 25% compost. This ratio depends on the soil type and the characteristics and level of contamination. After mixing, the soil is covered to protect it from erosion and to maintain the proper moisture and temperature necessary for bacterial growth.
Compost remediation is known to give fast clean-up results, taking weeks rather than the months needed for other approaches. Allen (1992) revealed that considerable alleviation of hazardous wastes or contaminated plants, soils, and sediments was possible through composting. Compostable substrates (feedstocks) contain metabolizable carbon, which enhance microbial diversity and activity during composting and promote the degradation of xenobiotic organic compounds such as pesticides, PAHs, and PCBs. Metallic pollutants are not degraded during composting but may be converted into organic species that are less bioavailable. Recalcitrant materials, such as organochlorines, may not undergo degradation in composts or in soils, and the effects of forming organic complexes with metallic pollutants may be nonpermanent or short-lived. Ultimately, composting degrades the pollutants to innocuous levels or binds them into innocuous compounds, and has substantial potential for the remediation of polluted materials.
Bioventing is a remediation technique that involves the introduction of oxygen into the contaminated soil through injection wells in order to stimulate the growth of indigenous and exogenous microorganisms. This technique is mostly used at sites where contamination consists of light petroleum products. This is due to the fact that light products are more easily biodegraded than heavier petroleum products. As can be seen in Fig. 19.2, bioventing is a soil vapor extraction (SVE) technique in which oxygen is injected into the soil to stimulate the growth of indigenous bacteria as well as the aerobic biodegradation of contaminants. Although the injected airflow is carefully controlled to minimize volatilization, the resulting vapor by-products from the
Atmospheric Vapor Discharge Treatment . (if needed)
Atmospheric Vapor Discharge Treatment . (if needed)
Vapor Phase Adsorbed Phase
Fig. 19.2 Bioventing (from FRTR 2000)
Vapor Phase Adsorbed Phase
Optional Depending in the Site Conditions
Fig. 19.2 Bioventing (from FRTR 2000)
biodegradation of pollutants are extracted by means of extraction wells. This vapor is then treated and atmospherically discharged. Nutrients can also be added to the soil to stimulate the growth and metabolism of the indigenous species. Although bioventing is an effective remediation technique, it cannot be used in sites where the depth to the groundwater table is less than three meters. This is because groundwater upwelling can occur within bioventing wells under vacuum pressures, provoking the elimination of vacuum-induced soil vapor flow (USEPA 2003).
Bioaugmentation refers to the use of a microbial strain that occurs naturally in the contaminated soil or the introduction of a genetically engineered variant in order to achieve soil bioremediation. Usually, the first step involves studying the indigenous varieties present in the contaminated soil. If the indigenous varieties do not have the metabolic machinery to perform the remediation process, exogenous varieties (or enzymes) that do have it are introduced. This process is usually used to remove by-products of raw materials and waste. Bacteria are the agents most commonly used in this degradation process.
Biostimulation involves the introduction of nutrients or substrates such as fertilizers, to stimulate the growth and metabolism of the indigenous species performing the biodegradation of the pollutant. Substrates containing nitrogen and phosphorus are the most popular of these stimulants due to their electron-accepting capabilities.
One of the main advantages of ex situ bioremediation is that it requires less time than in situ treatment. Another advantage is the certainty over the outcome of the treatment due to the ability to uniformly screen, homogenize, and mix the soil. These factors have made this one of the most commonly used treatment approaches. However, ex situ bioremediation involves the excavation of the contaminated soil and its subsequent treatment elsewhere, which makes it less cost-effective. Ex situ treatment technologies include slurry-phase bioremediation and solid-phase bioremediation.
Slurry-phase bioremediation, also known as the bioreactor method, is a controlled treatment that involves excavating the contaminated soil, mixing it with water, and
placing it in a bioreactor. Figure 19.3 shows a typical bioreactor system. As shown in the figure, the method involves processing the soil to achieve a low viscosity. This processing involves the separation of stones and rubbles from the contaminated soil. Next, the soil is mixed with a predetermined amount of water to form the slurry. The concentration of water added depends on the concentration of pollutants, the rate of biodegradation, and the physical nature of the soil (USEPA 2003). When this is done, the soil is removed and dried using pressure filters, vacuum filters, or centrifuges. The final procedure is the disposition of the soil and the further treatment of the resulting fluids.
Solid-Phase Bioremediation is an ex situ technology in which the contaminated soil is excavated and placed in piles. Bacterial growth is stimulated through a network of pipes that are distributed throughout the piles. By pulling air through the pipes, ventilation is provided for microbial respiration. Moisture is introduced by spraying the soil with water. Solid-phase systems require a large amount of space, and the clean up requires more time than slurry-phase processes (USEPA 2001). Some solid-phase treatment processes include land farming, soil biopiles, and composting.
Landfarming, also known as land treatment, is a bioremediation technique that involves excavating the contaminated soil and spreading it on a thin surface. Biodegradation of pollutants is stimulated aerobically by tilling or plowing the soil. Nutrients and minerals are also added to promote the growth of the indigenous species. Figure 19.4 is a schematic representation of the landfarming system. According to US Environmental Protection Agency report on underground storage tanks (USEPA 2003), before the remediation can take place, the site must be prepared by clearing and grading the soil, installing leachate collection and treatment systems, and building vapor treatment facilities. Also, the report states that if the soil is contaminated to a depth of less than three feet then there is no need for excavation. As can be seen in the figure, soil moisture is controlled by periodically sprinkling the soil with water, and erosion is controlled by erecting barriers or terraces around the contaminated soil. Sprinkling with water also minimizes the dust created while tilling the soil to promote aeration.
Soil biopiles, also known as biocells, is a biodegradation technique used for the remediation of excavated soil contaminated with petroleum products. This technology involves the accumulation of contaminated soil into piles and the stimulation of microbial activity either aerobically or through the addition of nutrients, minerals, or moisture.
Fig. 19.5 Biopile system (from FRTR 2000)
The biopiles are typically between three and ten feet high. This technique is similar to the landfarm method due to the fact that it also uses oxygen as a way to stimulate bacterial growth. However, while tilling or plowing is used to aerate land farms, biopiles are aerated by injecting the air through perforated piping placed throughout the pile (USEPA 2003). A schematic of this technology can be seen in Fig. 19.5. As can be seen in the figure, the contaminated soil is piled up to a depth of a few feet and then the piping is laid down. The next load of contaminated soil is then added. This process continues until the desired pile height is achieved. The soil is usually mixed with a bulking agent (straw) to improve aeration and thus enhance the growth of the microbial population. Since air is also injected into the soil there is also the possibility of the evaporation or volatilization of contaminants. To counter this problem, the system also incorporates the monitoring and containment of soil vapors.
19.5 Mechanism of the Remediation of Heavy Metal Contaminated Soil Using Microbes
Land pollution in the tropics is caused by industrial activities such as mining, refining, and electroplating. Due to the acidity of the soils in this region, microbes can easily mobilize metals that cause serious ecological risks. Therefore, the methylation, complexation, and changes in valence state of heavy metals are currently being studied in order to reduce the mobility and bioavailability of metals (Natural and Accelerated Bioremediation Research NABIR 2003). There is a great interest in microorganisms that can transform and/or remove metal contaminants. The remediation of metal contaminated soils often involves five general approaches: metal isolation, immobilization, mobilization, physical separation, or extraction (Evanko Cynthia and Dzombak 1997). Bioremediation processes involve immobilization or mobilization. A combination of these approaches is often used by industry to treat metal-contaminated sites; combining approaches can be more cost-effective than using just one.
The ability of a microorganism to survive and grow in a metal-contaminated habitat can depend on genetic and/or physiological adaptation. Such physiological changes in the cells of microbes reduce the rate of metal uptake and intracellular metal toxicity, while genetic changes result in the reduced intracellular and extracellular concentrations of the toxic metal species. Tolerance of heavy metals can result from intrinsic properties of the organism, such as the possession of extracellular mucilage or polysaccharides or an impermeable cell wall. A good example is provided by fungi that can grow in saturated CuSO4 (about 1.3 M) and very high concentrations of other heavy metals. Such solutions are very acidic, and these organisms are actually sensitive to submillimolar levels at close to neutral pH. Although fungal abundance and species diversity were found to be reduced in Zn-polluted soil, there was little difference in Zn tolerance between fungi isolated from control or polluted sites, and most achieved 50% growth at 700 |M Zn2+. Bdellospora, Verticillium, and Paecilomyces sp. were Zn tolerant at the control site, while Aureobasidium and Penicillium sp. were Zn tolerant at the polluted site (Gadd 1990). Similar findings were obtained from Cu, Ni, Fe, and Co-polluted soils, where fungal populations were not significantly different at polluted or control sites and tolerance was displayed by fungi towards heavy metals. Penicillium sp. comprised 60% of the tolerant isolates, followed by Trichoderma, Rhodotorula, Oidiodendron, Mortierella, and Mucor sp. (Freedman and Hutchinson 1980). Four filamentous fungi were found to be able to remove significant amounts of nickel from a 10 mM Ni solution according to the following order: Fusarium solani > Papulaspora sepedonoides > Mucor racemosus > Aspergillus flavus (Saxena et al. 2006). The interactions between microorganisms and metals can be divided into six distinct processes (Mohapatra 2006): (1) intracellular accumulation; (2) cell wall associated metal interactions; (3) metal siderophores; (4) extracellular mobilization/immobilization of metals by bacterial metabolites; (5) extracellular polymer-metal interactions, and; (6) transformation and volatilization of metals.
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