Microbial Biodegradation and Bioremediation

TN

1

Microbial Bioremediation

A Potential Tool for Restoration of Contaminated Areas

Surajit Das and Hirak R. Dash,    Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela, Odisha, India

Due to rapid industrialization and large-scale anthropogenic activities, the pollution level is increasing at a rapid rate, which is a major concern. Though many remediation techniques are available, the use of microorganisms has many advantages like cost effectiveness, few or no by-products, reusability, and more. Microorganisms are readily available, rapidly characterized, highly diverse, omnipresent, and can use many noxious elements as their nutrient source. They can be applied in both in situ and ex situ conditions; in addition, many extreme environmental conditions can be cleaned by such entities. Most countries do not restrict industrialization in spite of increased pollution levels; however, these can be minimized using suitable remedial measures, particularly where microorganisms provide a useful tool for a better alternative. In this chapter, the role of microbes in bioremediation, their superiority over other mechanisms, and the implications of such tiny organisms for cleaning the environment will be discussed.

Keywords

Bioremediation; bacteria; genetic manipulation; extreme environment

1.1 Introduction

More than 1 billion people around the world cannot get healthy air to breathe; 3 million die annually due to air pollution (WHO, 2006). This century has witnessed the warmest quarter in recorded history (from September 2003 to November 2003). Globally, over 1 million seabirds and thousands of sea mammals are killed by pollution every year. Each year 1.2 trillion gallons of untreated sewage and industrial waste are dumped into waterways by the United States alone. More than 3 million children under the age of 5 die annually from various environmental pollutions (EPA, 2000). These statistics not only give us a clear understanding of the precarious conditions at present, but warn us to think today about a better tomorrow. An unprecedented increase in population, anthropogenic activities, and urbanization in the name of modernization have increased pollutants to critical levels. In day-to-day life, we use more than 60,000 chemicals in the form of fuels, consumer products, industrial solvents, drugs, pesticides, fertilizers, and food additives. Though industrialization is essential for the faster growth of the developing nations, its concomitant pollution level should not come without sustainable management. Hence, proper management policy, suitable remedial strategies, and sustainable utilization of resources without altering the natural ecosystem should be the prime aim of all researchers and decision-making bodies.

In this context, microorganisms play an important role in the maintenance and sustainability of any ecosystem as they are more capable of rapid adjustment towards environmental changes and deterioration. Microorganisms are considered to be the first life forms to have evolved; they are versatile and adaptive to various challenging environmental conditions. Microorganisms are omnipresent, and they impact the entire biosphere. They play a major role in regulating biogeochemical cycles, from extreme environmental conditions like frozen environments, acidic lakes, hydrothermal vents, bottoms of deep oceans, to the small intestines of animals (Seigle-Murandi et al., 1996). Microorganisms are responsible for carbon fixation, nitrogen fixation, methane metabolism, and sulfur metabolism, thus controlling the global biogeochemical cycling (Das et al., 2006). They produce diverse metabolic enzymes that can be employed for the safe removal of contaminants, which can be achieved either by direct destruction of the chemical or through transformation of the contaminants to a safer or lesser toxic intermediate (Dash and Das, 2012). Due to their versatility, microorganisms have provided a useful platform to be used for an enhanced model of bioremediation of heavy metals, hydrocarbons, polythenes, food wastes, greenhouse gases, etc. as discussed in the subsequent chapters.

1.2 Pollution: A Major Global Problem

The pollution crisis is a major problem all around the globe. It adversely affects millions of people every year, causing many health disorders and deaths. Although urban areas are usually more polluted than the countryside, pollution can also spread to remote places; for example, pesticides and other chemicals have been found in the Antarctic ice sheet. In the middle of the northern Pacific Ocean, a large collection of microscopic plastic particles are found in what is known as the Great Pacific Garbage Patch. Pollutants can be moved from one place to another through land, water, and the atmosphere, worsening the situation day by day (Figure 1.1). Air and water currents carry pollution, ocean currents and migrating fish carry marine pollutants far and wide, and the wind can pick up and scatter radioactive material accidentally released from a nuclear reactor or smoke from a factory from one country into another (Doney et al., 2012). Thus, pollution does not believe in the limitations of geographical boundaries.

Figure 1.1 Movement of pollutants through land, water, and atmosphere.

There are many major pollution-causing agents found around the globe, i.e., oil spills, fertilizers, garbage, sewage disposals, toxic chemicals. All of them contribute to global pollution in the form of soil, air, water, and marine environmental pollution. Fifty-three chemicals have been identified by United States Environmental Protection Agency as persistent, bioaccumulative, and toxic among 87,000 commercial chemicals (USEPA, 2000). Contamination may be defined as the presence of elevated concentrations of substances in the environment which may or may not be harmful for society. However, pollution is the deliberate introduction of the noxious elements by human beings into the environment, resulting in a toxic effect. These contaminants may be produced due to various natural as well as anthropogenic activities like the large-scale manufacturing of chemicals as well as their processing and handling. The pollution level has increased at a rapid pace throughout the globe and is a major concern for developed as well as developing nations. A comparative detail of air pollution in terms of CO2 emission and generation of municipal waste has been given in Table 1.1.

Table 1.1

A Comparative Account of CO2 Emission and Generation of Municipal Waste Throughout the Globe in Subsequent Years

Source: OECD Factbook, 2013; http://dx.doi.org/10.1787/factbook-2013-en.

Though the statistical data varies greatly among countries within years, most of the pollutants possess high residence time and may travel long distances. Subsequently, they transform to become highly toxic chemicals, thus worsening the situation. As they don’t obey geographical boundaries, pollutants can travel easily across long distances, as in the case of the occurrence of xenobiotics in Arctic regions where no immediate sources of pollution-causing agents are found. Thus, pollution has become a global problem and it should be taken care of irrespective of political borders and geographical boundaries.

1.3 Current Remediation Practices

Removal of pollutants from contaminated sites is a great challenge for environmental management. Various processes based upon ion exchange resins or biosorbents have been employed for the removal of toxic chemicals, but these are sensitive to environmental conditions (Wang and Chen, 2009). The most promising conventional techniques for remediation involve chemical precipitation, conventional coagulation, adsorption by activated carbons, adsorption by natural materials, ion exchange, and reverse osmosis (USEPA, 2007). In the conventional coagulation process, coagulants such as aluminum sulfate, iron salt, and lime are employed for the removal of toxic elements. This process can be applied when pollutants are present at high concentrations in the waste materials. The main shortcoming of this procedure is that it does not reach desired levels of precipitation (Henneberry et al., 2011). The process based on adsorption by activated carbon is highly efficient and the water can be reused after regeneration. However, among the disadvantages of this process are the facts that the sorption reaction is slow, it is suitable for low pollutant concentrations only, and the regeneration costs are also much higher (Mohan et al., 2001). Adsorption processes using natural waste materials are more eco-friendly and economical since natural waste materials are used as adsorbents and these processes produce fewer secondary effluents, but the adsorption reaction is slow (Ramadevi and Srinivasan, 2005). The ion exchange processes are based on various ion exchange resins. The major advantages of these processes are that they are insensitive to variability, they can achieve zero level of contaminants, and the resins can be reused after regeneration (Chiarle et al., 2000).

Cellulose acetate or aromatic polyamide membranes are used for the reverse osmosis process at very high pressure, which results in the solvent being forced out through the membrane to the dilute solution (Zhu and Elimelech, 1997). Alternatively, low-cost biological processes such as phytoremediation have also been employed to remediate metal-contaminated sites (Narang et al., 2011). Use of bacteria for metal removal from contaminated sites is also a promising technology. However, the cons for the bacterial-based or plant-based processes may include production of large volumes of pollutant-loaded biomass, the disposal of which is problematic. Thus, in the current scenario, biological methods, i.e., bacterial mediated bioremediation, have the upper hand in terms of sustainability, cost effectiveness, and easy applicability in situ.

1.4 Characteristics of Microorganisms Suitable for Remediation

There are many characteristic features of bacteria which make them suitable for application in bioremediation practices. In this chapter, the unique characteristics of bacteria have been described, with marine bacteria considered as a model microorganism. The marine environment is the largest habitat on Earth, accounting for more than 90% of total biosphere volume; the microorganisms present in the marine environment are responsible for more than 50% of the global primary production and nutrient cycling (Lauro et al., 2009). These marine bacteria can be isolated from the marine water, sediments, and mangroves associated with marine habitats, the normal flora of the marine organisms, and deep-sea hydrothermal vents. They usually require sodium and potassium ions for their growth and to maintain osmotic balance of their cytoplasm (MacLeod and Onofrey, 1957). This requirement for the Na+ ion is an exclusive feature of marine bacteria which is attributed to the production of indole from tryptophan (Pratt and Happold, 1960), oxidation of L-arabinose, mannitol, and lactose (Rhodes and Payne, 1962), as well as transport of substrates into the cell (Hase et al., 2001). Other physical characters imputed to marine bacteria include facultative psychrophilicity (Bedford, 1933), higher tolerance to pressure than their terrestrial counterparts (Zobell and Morita, 1957), the capacity to survive in seawater, mostly Gram-negative rods (Buck, 1982), and motile spore formers (Buerger et al., 2012) which distinguishes them from terrestrial bacteria. β-aminoglutaric acid or β-glutamate, which is rare in nature, is present in higher amounts in marine sediments and is utilized by the marine bacteria as osmolytes. Some of the thermophilic marine bacteria isolated from the deep-sea hydrothermal vents are also capable of producing nitrogen. The most unique feature of a photosynthetic marine bacterial genome is the presence of rhodopsin, which contains 2197 genes. In addition to that, marine cyanobacteria also harbor a similar pattern of gene contents which are correlated with their isolation sources. The sole cause behind the diverse genetic level in marine microbes is due to the acquisition of an alternative mechanism for obtaining carbon and energy. Copiotrophic marine habitats have higher genetic potential to sense, undergo transduction, and integrate extracellular stimuli. These characteristics are likely to be crucial for their ability to fine-tune and rapidly respond to changing environmental conditions like sudden nutrient influx or depletion.

1.5 Adaptation in Extreme Environmental Conditions

The vast diversity of microorganisms is significant to the functional role they play in their normal habitat. They respond very quickly to changing environmental patterns which makes them ideal for potential bioremediation and bioindicator purposes. There are various changes that occur periodically in the environment which include variation in temperature and pH of the surrounding environment, changing patterns of light, sea level rise, tropical storms, and terrestrial inputs. In marine environments, microorganisms get continuous exposure to changes in oceanic temperature; however, the level of exposure varies in different microbial niches. Some groups of microorganisms overcome this problem by shifting their physical locations beneath sediments or by symbiosis with other organisms, a trait mostly found in pathogenic microorganisms. Other reported mechanisms of adaptation towards elevated temperature in seawater are chemotaxis and adhesion to a β-galactoside receptor in the coral mucus (Banin et al., 2001), penetration into epidermal cells, differentiation into a viable-but-not-culturable state, intracellular multiplication, production of toxins that inhibit photosynthesis, and production of superoxide dismutase to protect the pathogen from oxidative stress. Though ocean acidification is mainly caused by accumulation of CO2 gas in marine environments, pH has not gone below 6.0. Bacteria are more adapted to this variation of pH conditions. However, Takeuchi et al. (1997) showed that the oceanic pH will very soon go below 6.0 and may reach 5.5, which will create a serious problem. Furthermore, rainfall and river flooding add pollutants and xenobiotics into the seawater, which has the potential to alter the microbial community’s structure and function. However, bacteria adapt to such situations by changing their pattern of growth rates, gene expression, and physiological or enzymatic activities, and undergoing changes in intimate or symbiotic associations with other organisms. Some groups of bacteria have also been reported to develop many unique mechanisms like the synthesis of bioactive compounds, biofilm formation in the environment, and production of biosurfactants when they are exposed to extremes in pressure, temperature, and salinity or depletion of micronutrients (Mangwani et al., 2012; Mangwani et al., 2014).

Any microorganism to be used for bioremediation practices has to possess the resistant genotype for the particular pollutant. Apart from that, microorganisms possess certain unique characteristics which make them more suitable for bioremediation practices. The bacteria used in bioremediation practices should possess metal-processing features as described in Figure 1.2. These processes include the uptake and reflux of contaminating metals; their bioabsorption, intracellular assimilation, immobilization, complexion, and precipitation; and their release (Stelting et al., 2010).

Figure 1.2 Metal-processing features of bacteria required to utilize in bioremediation.

1.6 Applications of Bacteria for Bioremediation

The use of bacteria for biodegradation of various natural and synthetic substances, and thereby reducing the level of hazards, is increasingly drawing attention. Bacteria possess a wide variety of bioremediation potentials which are beneficial from both an environmental and an economic standpoint. Bioremediation and biotransformation methods have been employed to tap the naturally occurring metabolic ability of microorganisms to degrade, transform, or accumulate toxic compounds including hydrocarbons, heterocyclic compounds, pharmaceutical substances, radionuclides, and toxic metals (Karigar and Rao, 2011). The goal in bioremediation is to stimulate microorganisms with nutrients and other chemicals that enable them to destroy the contaminants. The bioremediation systems in operation today rely on microorganisms native to the contaminated sites, encouraging them to work by supplying them with optimum levels of nutrients and other chemicals essential for their metabolism. However, researchers are still investigating ways to augment contaminated sites with non-native microbes as well as genetically engineered microbes suited for degrading the contaminants of concern at particular sites.

Microorganisms gain energy by catalyzing energy producing chemical reactions that involve breaking chemical reactions, thus transferring electrons away from the contaminant. The energy gained from these electron transfers is then invested, along with some electrons and carbon from the contaminant, to produce more cells. There are at least five critical factors that should be considered while evaluating the microbial bioremediation for site cleanup. These factors include:

1. Magnitude, toxicity, and mobility of contaminants: The site should be properly investigated and characterized to determine the (i) horizontal and vertical extent of contamination; (ii) the kinds and concentrations of contaminants at the site; (iii) the likely mobility of contaminants in future, which depends on the geological characteristics of the site.

2. Proximity of human and environmental receptors: Whether bioremediation is an appropriate cleanup remedy for any site is dependent upon the rate and extent of contaminant degradation.

3. Degradability of contaminants: The degradability of a compound is dependent on the occurrence of the compound in nature. In some instances, compounds with a high molecular weight, particularly those having complex ring structures and halogen substituents, degrade more slowly than simpler straight chain hydrocarbons or low molecular weight compounds. Thus, the rate and extent to which the compound is metabolized in the environment is determined by the availability of electron acceptors and other nutrients.

4. Planned site use: The critical factor deciding the appropriateness of bioremediation is the rate and extent of contaminant degradation.

5. Ability to properly monitor: There are inherent uncertainties in the use of bioremediation for contaminated soils and aquifers due to physical, chemical, and biological heterogeneities of the contaminated matrix.

1.6.1 Removal of Heavy Metals

Heavy metal pollution caused by various natural and anthropogenic activities is one of the most important environmental concerns. Though various physical and chemical methods have been proposed to remove such hazardous metals from the environment, they are least successful in terms of cost effectiveness, limitations, and generation of harmful substances (Wuana and Okieimen, 2011). Microorganisms solve these problems as they do not produce any by-products, and they are highly efficient even at low metal concentrations. Vibrio harveyi, a normal inhabitant of the saline environment, is reported to possess the potential for bioaccumulation of cadmium up to 23.3 mg Cd²+/g of dry cells. In line with that, Canstein et al. (2002) reported a consortium of marine bacteria to efficiently remove mercury in a bioreactor in a disturbance-independent mechanism. A new combination of genetic systems in bacteria for the potential degradation of phenol and heavy metals was also described. Bacteria also possess the properties of chelation of heavy metals, thus removing them from the contaminated environment by the secretion of exopolysaccharides which have been evident from the reports of a marine bacterium Enterobacter cloaceae (Iyer et al., 2005). This bacterium has been reported to chelate up to 65% of cadmium, 20% copper, and 8% cobalt at 100 mg/L of metal concentration. In line with that, certain purple nonsulfur bacterial isolates, e.g., Rhodobium marinum and Rhodobacter sphaeroides, have also been found to possess the potential of removing heavy metals like copper, zinc, cadmium, and lead from the contaminated environments either by biosorption or biotransformation. Thus, the bacteria have been designated for assessing pollution through their tolerance and biosorption of heavy metals. However, the genetic mechanisms of bioremediation towards toxic metals have been reduced for a smaller number of bacteria (Table 1.2).

Table 1.2

Catabolic Genes Reported in Bacteria Responsible for Bioremediation

1.6.2 Degradation of Polyaromatic Hydrocarbons and Other Recalcitrants

Polyaromatic hydrocarbons (PAHs) are ubiquitous in nature and are of great environmental concern due to their persistence, toxicity, mutagenicity, and carcinogenicity in nature. However, many marine bacteria have been reported to have the potential for bioremediation of the same in the process of metabolism to produce CO2 and metabolic intermediates, thus gaining energy and carbon for cell growth. The bioremediation potential of these bacteria can be increased, as was shown in an experiment by Latha and Lalithakumari (2001) when they transferred a catabolic plasmid of Pseudomonas putida containing hydrocarbon degradation genotype in a marine bacterium, which increased its efficiency. Some novel marine bacterial species like Cycloclasticus spirillensus, Lutibacterium anuloederans, and Neptunomonas naphthovorans have also been utilized in enhanced biodegradation of PAHs in a marine environment (Chung and King, 2001). Similarly, Achromobacter denitrificans, Bacillus cereus, Corynebacterium renale, Cyclotrophicus sp., Moraxella sp., Mycobacterium sp., Burkholderia cepacia, Pseudomonas fluorescens, Pseudomonas paucimobilis, P. putida, Brevundimonas vesicularis, Comamonas testosteroni, Rhodococcus sp., Streptomyces sp., and Vibrio sp. have been isolated from marine resources and are capable of degrading naphthalene by the process of mineralization. However, bacteria belonging to the genus Cycloclasticus play the major role in biodegradation of hydrocarbons. Bacterial isolates like Sphingomonas paucimobilis EPA505 have been found to utilize fluoranthene as their sole carbon source.

1.6.3 Petroleum and Diesel Biodegradation

Crude oil is the most important organic pollutant in the environment, as 1.7–8.8×10⁶ tonnes of petroleum hydrocarbons are being released to the marine and estuarine environments annually (McKew et al., 2007). These organic pollutants can be degraded by the oil-eating microbes present in the environment which are used for their carbon and energy source. Some of the important genera of bacteria that are capable of degrading oil include Acinetobacter, Marinococcus, Methylobacterium, Micrococcus, Nocardia, Planococcus, and Rhodococcus. In terms of commercial applications, a consortium has been developed by Deppe et al. (2005) by using Arctic bacteria like Agreia, Marinobacter, Pseudoalteromonas, Pseudomonas, Psychrobacter, and Shewanella for significant degradation of crude oil and its components. In addition to that, a more potent bacterium has been isolated from the Arabian Sea sediments capable of degrading oil by 39% in 8 days in laboratory conditions. Recently, bioaugmented and biostimulated products of marine bacteria have been reported for oil remediation in marine environments.

1.6.4 Degradation of Plastic

Several broad classes of plastic include polyethylene, polypropylene, polystyrene, polyethylene terephthalate, and polyvinyl chloride are used in environments for fishing, packing, etc. which ultimately pollute the environment. However, microorganisms can develop the mechanism to degrade the plastic to nontoxic forms. Recent findings showed that Rhodococcus ruber degrades 8% of dry weight of plastic in 30 days in concentrated liquid culture in vitro. Similarly, bacterial isolates belonging to genera Shewanella, Moritella, Psychrobacter, and Pseudomonas isolated from the deep seas of Japan possess the potential of degrading ε-caprolactone in an efficient manner. Some mangrove-associated bacterial species like Micrococcus, Moraxella, Pseudomonas, Streptococcus, and Staphylococcus were also found to degrade 20% of plastic (Kathiresan, 2003).

Besides their bioremediation function, bacteria have also been used for biosurfactant production from Acinetobacter anitratus, Bacillus pumilus, Bacillus subtilis, Myroides sp., Micrococcus luteus, and V. parahaemolyticus which may be utilized in the process of enhanced bioremediation.

1.7 Factors of Bioremediation

The optimization and control of bioremediation process is a complex system of many factors. These include the existence of a microbial population capable of degrading the pollutants, the availability of contaminants to the microbial population, and the environmental factors (i.e., type of soil, temperature, pH, and the presence of oxygen or other electron acceptors and nutrients) (Table 1.3). Microorganisms can be isolated from almost any environmental conditions. Microbes can adapt and grow at subzero temperatures as well as in extreme heat, desert conditions, water, and anaerobic conditions; with an excess of oxygen; in the presence of hazardous compounds; or on any waste stream. The main requirements are an energy source and a carbon source for microbes and other biological systems that can be used to degrade or remediate.

Table 1.3

Linked Factors of Efficient Bioremediation

Among the environmental factors, carbon is the most fundamental element of living forms and is needed in greater quantities than that of other elements. In addition to hydrogen, oxygen and nitrogen constitute about 95% of its weight. The type of bioremediation depends on the concentration of soil contaminants, presence and/or absence of other nutrients such as phosphorus and sulfur. For optimum microbial bioremediation, nutritional requirement of carbon to nitrogen ratio is 10:1 and carbon to phosphorus is 30:1.

For high concentrations of contaminants, the soil is agitated in a water solution containing an interface active agent to separate it from the soil. Then bioremediation is started to efficiently clean the soil. In the experimental stage, bioremediation alone has been able to turn contaminated soil into soil suited for landscaping. In less contaminated soils, contaminants can be treated using bioremediation alone. It may take 6 months to a year to purify soil containing 2% heavy oils, but at a concentration of 0.8%, the job can be done in about 1–2 months. Thus, this environmentally friendly method makes it possible to recycle and reuse soil without much effort. The optimum conditions of the environmental factors are provided in Table 1.4.

Table 1.4

Optimum Environmental Conditions Required for Microbial Activity

1.8 Microbial Bioremediation Strategies

1.8.1 In situ Bioremediation

In situ bioremediation is the application of a biological treatment to clean up hazardous compounds present in the environment. The optimization and control of microbial transformations of organic contaminants requires the integration of many scientific and engineering disciplines. Some of the in situ bioremediation practices have been discussed below.

Biosparging: This involves the injection of air under pressure below the water table to increase ground water oxygen concentrations and to enhance the rate of biological degradation of contaminants by naturally occurring bacteria. Biosparging increases the mixing in the saturated zone and thereby increases the contact between soil and ground water. The ease and low cost of installing small-diameter air injection points allow considerable flexibility in the design and construction of the system.

Bioventing: Bioventing is a promising new technology that stimulates the natural in situ biodegradation of any aerobically degradable compounds by providing oxygen to the existing soil microorganisms. It uses low air flow rates to provide only enough oxygen to sustain microbial activity. Oxygen is most commonly supplied through direct air injection into residual contamination in soil by means of wells. Henceforth, the adsorbed fuel residuals are biodegraded and volatile compounds are also biodegraded as vapors move slowly through biologically active soil.

Bioaugmentation: Bioaugmentation is the introduction of a group of natural microbial strains or a genetically engineered strain to treat contaminated soil or water. Most commonly, it is used in municipal waste water treatment to restart activated sludge bioreactors. At sites where soil and ground water are contaminated with chlorinated ethanes, 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 nontoxic in nature.

Biopiling: This is a full-scale technology in which excavated soils are mixed with soil amendments, placed on a treatment area, and further bioremediated using forced aeration. The contaminants are reduced to carbon dioxide and water. A basic biopile system includes a treatment bed, an aeration system, an irrigation/nutrient system, and a leachate collection system. Moisture, heat, nutrients, oxygen, and pH are also controlled to enhance biodegradation. The irrigation/nutrient system is buried under the soil to pass air and nutrients either by vacuum or positive pressure.

1.8.2 Ex situ Bioremediation

The most common ex situ bioremediation practice involves composting, which is a process by which organic wastes are degraded by microorganisms, typically at elevated temperatures. A typical compost temperature is in the range of 55–65°C. The increased temperatures result from heat produced by microorganisms during the degradation of the organic material in the waste.

1.8.3 Bioreactors

Slurry or aquatic reactors are used for ex situ treatment of contaminated soil and water pumped up from a contaminated plume. Bioreactors involve the processing of contaminated solid material or water through an engineered containment system. The slurry reactor is a containment vessel and apparatus used creates a three-phase (solid, liquid, and gas) mixing condition. It increases the bioremediation rate of soil bound water soluble pollutants by increasing the bioavailability of the target contaminants to the biomass. The various advanced methods applied in bioremediation practices are described in Table 1.5.

Table 1.5

Various Developmental Methods Applied in Bioremediation

1.8.4 Alternative Bioremediation Technologies

Bioremediation technologies responding to the various contaminants may be divided into three discrete categories: (i) nutrient enrichment, (ii) seeding with naturally occurring microorganisms, and (iii) seeding with genetically engineered microorganisms (GEMs) (Table 1.6).

Table 1.6

Principal Features of Alternative Bioremediation Approach

1.8.5 Use of Microbial Consortia for Bioremediation

The use of microbial consortia instead of pure cultures has increased dramatically due to their known synergistic metabolism, which improves the efficiency of hydrocarbons and other chemical degradations. The biodegradation process becomes much more efficient and much faster with the use of consortia due to many factors: (i) the metabolic intermediate of one bacteria can be utilized by another for efficient degradation; (ii) the system becomes much faster from a bioremediation point of view; (iii) suitable trapping methods may be employed for efficient degradation, etc. The uses of some microbial consortia are shown in Table 1.7.

Table 1.7

Examples of Some Potential Microbial Consortia for Bioremediation

1.8.6 Improvement of the Strains by Genetic Manipulation for Enhanced Bioremediation

Microbial metabolic potential provides an effective mechanism for eliminating environmental pollutants. Anthropogenic pollution introduces some xenobiotic substances to which bacteria have not been exposed before. Upon exposure, resistant bacteria slowly change their metabolic pathway to survive with the stress. However, in order to increase the bioremediation potential and/or metabolic activity of any bacteria, insertion of certain functional genes into their genome is necessary. This phenomenon can be achieved by insertion of new genes into the genomic complexion, insertion of new plasmid, alteration of metabolic pathways like transport and chemotaxis, and most importantly, adaption of features towards the environmental conditions. Due to significant developments in the field of molecular microbiology and genetics, there are success stories of the development of genetically engineered microbes for bioremediation of toxic substances. However, limited reports are available to date for the genetic manipulation of marine bacteria to achieve the goal of enhanced bioremediation. Insertion of the bmtA gene coding for metallothionein into a suitable vector and its transformation into marine bacteria has been conducted and successfully employed in highly metal-contaminated environments. Similarly, Pseudoalteromonas haloplanktis, possessing a shuttle plasmid-encoding suppressor for amber mutation, has been used for genetic manipulation for bioremediation (Kivela et al., 2008). Bacteria possessing plasmid with a merA gene, responsible for converting the toxic form of mercury to the nontoxic form, may be transformed into marine bacteria for better applications in field conditions of bioremediation of mercury (Dash et al., 2013; Dash et al., 2014). Deinococcus radiodurans, the most radio-resistant organism, has been modified genetically to consume and digest toluene and the ionic form of mercury from nuclear wastes (Brim et al., 2000). A list of bacteria possessing genetic alterations in their genome to increase their bioremediation potential is given in Table 1.8.

Table 1.8

List of Some Engineered Bacteria Developed for Enhanced Bioremediation

1.9 Pros and Cons of Using Bacteria in Bioremediation

Bacteria are found in a wide range of environmental conditions, from the sea floor to fish stomachs, and develop unique mechanisms of resistance in adverse and diverse conditions. Thus, there is ample opportunity to employ them as potential bioremediating agents. When a bacterium utilizes the contaminant as its food source, its number increases rapidly in the contaminated environments; on subsequent decontamination, the number decreases to produce harmless biomass. The process is cost effective in comparison to chemical processes and can be carried out onsite. Utilization of bacteria in bioremediation is highly specific; hence, the chance of forming harmful by-products is lower, which is the major advantage of utilizing these isolates. However, there are some disadvantages in the process of using bacteria. In the case of mixed contaminants, finding a suitable consortium becomes difficult. In the same case, the process is time consuming, sometimes taking years to finish. Though there are fewer chances of forming by-products, in some cases, lethal by-products may form. After the process is over, the bacterial biomass is degraded, and the serious problem of biofouling may occur. Another problem associated with the use of recombinant strains is the instability of the cloned genes in the contaminated environment due to change of habitat. These problems persist not only with the marine microorganisms but also with bacterial entities isolated from any environments. However, when the potential of the microorganisms is concerned in bioremediation, marine bacteria have been proven to be the most valuable and efficient candidates.

1.10 Conclusion and Future Prospects

One of the major problems that the twenty-first century is facing is environmental pollution, and research communities are paying close attention. The global requirement for a solution to this problem includes various remediation aspects, but bioremediation is one step ahead of all these due to its many advantages over other remediation protocols. Among various microorganisms employed in bioremediation, marine bacteria can adapt quickly to rapidly changing, noxious environments and may potentially be utilized to solve the problem by remediating toxic materials. Though many studies have been conducted and a large number of marine microbial entities have been discovered so far, the microbial diversity from different marine habitats is yet to be explored. Hence, by combining their molecular aspects with metabolic approaches, the microbial diversity of the oceanic environment can be explored. The treatment of environmental pollution by employing microorganisms is a promising technology; however, various genetic approaches to optimizing enzyme production, metabolic pathways, and growth conditions will be highly useful to meet the demand.

Acknowledgments

The research at the Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela, Odisha, India is supported by research grants from the Department of Biotechnology, the Ministry of Science and Technology, the Government of India, and the Indian Council of Agricultural Research.

References

1. Angelim AL, Costa SP, Farias BCS, Aquino LF, Melo VMM. An innovative bioremediation strategy using a bacterial consortium entrapped in chitosan beads. J Environ Manage. 2013;127:10–17.

2. Antizar-Ladislao B, Spanova K, Beck AJ, Russell NJ. Microbial community structure changes during bioremediation of PAHs in an aged coal-tar contaminated soil by in-vessel composting. Int Biodeterior Biodegradation. 2008;61:357–364.

3. Balba MT, Al-Awadhi N, Al-Daher R. Bioremediation of oil contaminated soil: microbiological methods for feasibility assessment and field evaluation. J Microbiol Met. 1998;32:155–164.

4. Banin E, Khare SK, Naider F, Rosenberg E. Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of zooxanthellae. Appl Environ Microbiol. 2001;67:1536–1541.

5. Bedford RH. Marine bacteria of the northern Pacific Ocean The temperature range of growth. Contrib Cancer Biol Fish. 1933;8:433–438.

6. Behkish A, Lemoine R, Sehabiague L, Oukaci R, Morsi BI. Gas holdup and bubble size behavior in a large-scale slurry bubble column reactor operating with an organic liquid under elevated pressures and temperatures. Chem Eng J. 2007;128:69–84.

7. Brim H, McFarlan SC, Fredrickson JK, et al. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments. Nat Biotechnol. 2000;18:85–90.

8. Buck JD. Nonstaining (KOH) method for determination of Gram reactions of marine bacteria. Appl Environ Microbiol. 1982;44:992–993.

9. Buerger S, Spoering A, Gavrish E, Leslin C, Ling L, Epstein SS. Microbial scout hypothesis and microbial discovery. Appl Environ Microbiol. 2012;78:3229–3233.

10. Canstein H, Kelly S, Li Y, Wagner-Dobler I. Species diversity improves the efficiency of mercury-reducing biofilms under changing environmental conditions. Appl Environ Microbiol. 2002;68:2829–2837.

11. Chiarle S, Ratto M, Rovatti M. Mercury removal from water by ion exchange resins adsorption. Water Res. 2000;34:2971–2978.

12. Chung WK, King GM. Isolation, characterization, and polyaromatic hydrocarbon degradation potential of aerobic bacteria from marine macro-faunal burrow sediments and description of Lutibacterium anuloederans gen nov., sp nov., and Cycloclasticus spirillensus sp nov. Appl Environ Microbiol. 2001;67:5585–5592.

13. Czyz A, Jasiecki J, Bogdan A, Szpilewska H, Grzyn GW. Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments. Appl Environ Microbiol. 2000;66:599–605.

14. Das S, Lyla PS, Khan SA. Marine microbial diversity and ecology: present status and future perspectives. Curr Sci. 2006;90(10):1325–1335.

15. Dash HR, Das S. Bioremediation of mercury and importance of bacterial mer genes. Int Biodeterior Biodegradation. 2012;75:207–213.

16. Dash HR, Mangwani N, Chakraborty J, Kumari S, Das S. Marine bacteria: potential candidates for enhanced bioremediation. Appl Microbiol Biotechnol. 2013;97:561–571.

17. Dash HR, Mangwani N, Das S. Characterization and potential application in mercury bioremediation of highly mercury resistant marine bacterium Bacillus thuringiensis PW-05. Environ Sci Pollut Res. 2014;21:2642–2653.

18. De J, Ramaiah N, Vardanyan L. Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury. Mar Biotechnol. 2008;10:471–477.

19. Deppe U, Richnow HH, Michaelis W, Antranikian G. Degradation of crude oil by an arctic microbial consortium. Extremophiles. 2005;9:461–470.

20. Doney SC, Ruckelshaus M, Duffy JE, et al. Climate change impacts on marine ecosystems. Ann Rev Mar Sci. 2012;4:11–37.

21. Hase CC, Fedorova ND, Galperin MY, Dibrov PA. Sodium ion cycle in bacterial pathogens: evidence from cross-genome comparisons. Microbiol Mol Biol Rev. 2001;65:353–370.

22. Henneberry YK, Kraus TEC, Fleck JA, Krabbenhoft DP, Bachand PM, Horwath WR. Removal of inorganic mercury and methylmercury from surface waters following coagulation of dissolved organic matter with metalbased salts. Sci Total Environ. 2011;409:631–637.

23. Iyer A, Mody K, Jha B. Biosorption of heavy metals by a marine bacterium. Mar Pollut Bull. 2005;50:340–343.

24. Karigar CS, Rao SS. Role of microbial enzymes in the bioremediation of pollutants: a review. Enzyme Res In: http://dx.doi.org/doi:10.4061/2011/805187; 2011.

25. Kasai Y, Kishira H, Harayama S. Bacteria belonging to the genus Cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment. Appl Environ Microbiol. 2002;68:5625–5633.

26. Kathiresan K. Polythene and plastics-degrading microbes from the mangrove soil. Rev Biol Trop. 2003;51:629–634.

27. Kivela HM, Madonna S, Krupovic M, Tutino ML, Bamford JKH. Genetics for Pseudoalteromonas provides tools to manipulate marine bacterial virus PM2. J Bacteriol. 2008;190:1298–1307.

28. Kumamaru T, Suenaga H, Mitsuoka M, Furukawa K. Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase. Nat Biotechnol. 1998;16:663–666.

29. Latha K, Lalithakumari D. Transfer and expression of a hydrocarbon degrading plasmid pHCL from Pseudomonas putida to marine bacteria. World J Microbiol Biotechnol. 2001;17:523–528.

30. Lauro FM, McDougald D, Thomas T, et al. The genomic basis of trophic strategy in marine bacteria. PNAS. 2009;106:15527–15533.

31. MacLeod RA, Onofrey E. Nutrition and metabolism of marine bacteria III The relation of sodium and potassium to growth. J Cell Comp Physiol. 1957;50:389–401.

32. Mangwani N, Dash HR, Chauhan A, Das S. Bacterial quorum sensing: functional features and potential applications in biotechnology. J Mol Microbiol Biotechnol. 2012;22:215–227.

33. Mangwani N, Shukla SK, Rao TS, Das S. Calcium mediated modulation of Pseudomonas mendocina NR802 biofilm influences the phenanthrene degradation. Colloids Surf B. 2014;114:301–309.

34. McKew BA, Coulon F, Osborn AM, Timmis KN, McGenity TJ. Determining the identity and roles of oil-metabolizing marine bacteria from the Thames estuary. Environ Microbiol. 2007;9:165–176.

35. Mohan D, Gupta VK, Srivastava SK, Chander S. Kinetics of mercury adsorption from wastewater using activated carbon derived from fertilizer waste. Colloids Surf A Physicochem Eng Asp. 2001;177:169–181.

36. Mukred AM, Hamid AA, Hamzah A, Yusoff WMW. Development of three bacteria consortium for the bioremediation of crude petroleum-oil in contaminated water. J Biol Sci. 2008;8:73–79.

37. Narang U, Bhardwaj R, Garg SK, Thukral AK. Phytoremediation of mercury using Eichhornia crassipes (Mart.) Solms. Int J Environ Waste Manage. 2011;8:92–105.

38. Natrajan KA. Microbial aspects of acid mine drainage and its bioremediation. Trans Nonferr Met Soc China. 2008;18:1352–1360.

39. Niu G, Zhang J, Zhao S, Liu H, Boon N, Zhou N. Bioaugmentation of a 4-chloronitrobenzene contaminated soil with Pseudomonas putida ZWL73. Environ Pollut. 2009;57:763–771.

40. OECD. OECD Factbook 2013: Economic, Environmental and Social Statistics OECD Publishing In: http://dx.doi.org/doi:10.1787/factbook-2013-en; 2013.

41. Pratt D, Happold FC. Requirements for indole production by cells and extracts of a marine bacterium. J Bacteriol. 1960;80:232–236.

42. Rahman KSM, Rahman TJ, Kourkoutas Y, Petsas I, Marchant R, Banat IM. Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients. Bioresour Technol. 2003;90:159–168.

43. Ramadevi A, Srinivasan K. Agricultural solid waste for the removal of inorganics: adsorption of mercury (II) from aqueous solution by Tamarind nut carbon. Indian J Chem Technol. 2005;12:407–412.

44. Rhodes ME, Payne WJ. Further observations on effects of cations on enzyme induction in marine bacteria. Antonie Van Leeuwenhoek. 1962;28:302–314.

45. Sei K, Nakao M, Mori KMI, Kohno T, Fujita M. Design of PCR primers and a gene probe for extensive detection of poly (3-hydroxybutyrate) (PHB)-degrading bacteria possessing fibronectin type III linker type-PHB depolymerases. Appl Microbiol Biotechnol. 2001;55:801–806.

46. Seigle-Murandi F, Guiraud P, Croize J, Falsen E, Eriksson KL. Bacteria are omnipresent on Phanerochaete chrysosporium Burdsall. Appl Environ Microbiol. 1996;62:2477–2481.

47. Sharma A, Rehman MB. Laboratory scale bioremediation of diesel hydrocarbon in soil by indigenous bacterial consortium. Indian J Exp Biol. 2009;47:766–769.

48. Stelting, S., Burns, R.G., Sunna, A., Visnovsky, G., Bunt, C., 2010. Immobilization of Pseudomonas sp. strain ADP: a stable inoculant for the bioremediation of atrazine. In: 19th World Congress of Soil Science, Soil Solutions for a Changing World, Brisbane, Australia, August 1–6, 2010.

49. Sutiknowati LI. Hydrocarbon degrading bacteria: isolation and identification. Makara Sains. 2007;11:98–103.

50. Takeuchi K, Fujioka Y, Kawasaki Y, Shirayama Y. Impacts of high concentrations of CO2 on marine organisms: a modification of CO2 ocean sequestration Energy. Convers Mgmt. 1997;38:S337–S341.

51. US Environmental Protection Agency, 2000. Marine Litter—Trash That Kills. Available from: <http://www.epa.gov/owow/oceans/debris/toolkit/files/trash_that_kills508.pdf>.

52. U.S. EPA. Treatment Technologies for Mercury in Soil, Waste, and Water Washington, DC: Office of Superfund Remediation and Technology Innovation; 2007; 20460.

53. Wang J, Chen C. Biosorbents for heavy metals removal and their future. Biotechnol Adv. 2009;27:195–226.

54. WHO. Air Quality Guidelines for Particulate Matter, Ozone, Nitrogen Dioxide and Sulfur Dioxide-Global Update 2005 Geneva: World Health Organization; 2006.

55. Winter RB, Yen K, Ensley BD. Efficient degradation of trichloroethylene by a recombinant Escherichia coli. Biotechnol. 1989;7:282–285.

56. Wuana RA, Okieimen FE. Heavy metals in contaminated soils: a review of sources, chemistry, risks and best available strategies for remediation. ISRN Ecol In: http://dx.doi.org/doi:10.5402/2011/402647; 2011.

57. Zhu X, Elimelech M. Colloidal fouling of reverse osmosis membranes: measurements and fouling mechanisms. Environ Sci Technol. 1997;31:3654–3662.

58. Zobell CE, Morita RY. Barophilic bacteria in some deep sea sediments. J Bacteriol. 1957;73:563–568.

2

Heavy Metals and Hydrocarbons

Adverse Effects and Mechanism of Toxicity

Surajit Das, Ritu Raj, Neelam Mangwani, Hirak R. Dash and Jaya Chakraborty,    Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela, Odisha, India

Chemicals are a part of our daily lives; all living and inanimate matter is composed of many chemicals and every manufactured product involves the use of chemicals. When properly used, these chemicals significantly contribute to the improvement of our quality of life, health, and well-being. When improperly managed, however, certain chemicals are highly hazardous and can negatively affect health as well as the environment. The most important environmental pollutants in areas with high anthropogenic pressure include heavy metals such as cadmium, copper, lead, chromium, and mercury. Their presence in the environment, even in trace amounts, can cause serious problems for all organisms. Though heavy metals are the natural constituents of the Earth’s crust, indiscriminate human activities alter their biochemical balance and geochemical cycle. In any mode of entry into the human body, either through inhalation or ingestion, the heavy metals present serious threats to the organism. Prolonged exposure to these toxic metals creates deleterious health effects in humans. Another major group of pollutants includes polyaromatic hydrocarbons, which are formed due to incomplete burning of coal, oil, gas, garbage, or other organic substances and may cause many health hazards in each and every part of the human body. This chapter reviews the toxic effects of many heavy metals and hydrocarbons on human beings as well as the mechanisms of their toxicity.

Keywords

Heavy metals; PAHs; health hazards; toxicity; environmental contamination; anthropogenic activities

2.1 Introduction

Rapid industrialization, population growth, and complete disregard for environmental health have led to global environmental pollution. The release of pollutants into the environment may occur accidentally or due to anthropogenic activities which ultimately results in soil, water, and air pollution, leading to many health hazards. From the galaxy of chemicals, heavy metals and polycyclic aromatic hydrocarbons (PAHs) are two of the major environmental pollutants (Dash et al., 2013). From the environment, these chemicals enter into the food chain. The flow of chemicals through the lower constituents of the food chain to different tropic levels imparts risk to the ecosystem, as chemicals tend to bioaccumulate and can be transferred from one food chain to another. Pollutants have been detected in various food chains where the results are usually detrimental to microorganisms, plants, animals, and humans alike (Vinodhini and Narayanan, 2008).

Contamination of the soil, water, and air with organic compounds and heavy metals has drawn much public concern as many of them are toxic and mutagenic as well as carcinogenic. Out of these, PAHs are considered to be the most acute toxic component; they are associated with many chronic diseases and disrupt the natural equilibrium between living species and their natural environment (Mangwani et al., 2014). In earlier days, heavy metals were present naturally in soils as natural components; however, currently their presence in the environment has been increased due to human activities. This is a widespread problem around the globe in areas where excessive concentration of heavy metals such as As, Cd, Hg, Pb, Cr, and Se can be found in soil. Contamination of soils is one of the most critical environmental problems as the contaminants infiltrate deep into the layer of underground water and pollute the ground water as well as the surface water (Perfus-Barbeoch et al., 2002).

Contamination of the environment with hydrocarbons and toxic metals constitutes a nuisance to the environment due to their persistent nature and tendency to spread into ground as well as surface water. There are around 30 chemical elements which play a pivotal role in various biochemical and physiological mechanisms in living organisms and are recognized as the essential elements of life. As the concentrations of toxic metals and metalloids have largely increased due to anthropogenic activities, they can disturb important biochemical processes, thus constituting an important threat to the health of plants and animals (Da Silva and Williams, 2001). Living organisms absorb these elements from soils, sediments, and water by contact with their external surfaces through ingestion and through inhalation of airborne particles as well as vaporized metals. These excess heavy metals aid neurological depositions, enhancing oxidative damage—the key component for chronic inflammatory disease as well as a suggested initiator of cancer. Further, potential pathological roles of metal ions are emerging in the form of premature aging and many other characteristic features of a wide range of diseases (Chowdhury and Chandra, 1986; Duruibe et al., 2007). In this chapter, the adverse effects of heavy metals and PAHs on the ecosystem and human health have been discussed in