**3. Types of organisms used in bioremediation**

Typically, bioremediation is based on the cometabolism action of one organism or a consortium of microorganisms [18]. In this process, the transformation of contaminants presents a little efficiency or no benefit to the cell, and therefore this process is described as nonbeneficial biotransformation [21, 22]. Several studies have shown that many organisms (prokaryotes and eukaryotes) have a natural capacity to biosorb toxic heavy metal ions [23]. Examples of microorganisms studied and strategically used in bioremediation treatments for heavy metals include the following: (1) bacteria: *Arthrobacter* spp. [24], *Pseudomonas veronii* [25], *Burkholde‐ ria* spp. [26], *Kocuria flava* [27], *Bacillus cereus* [28], and *Sporosarcina ginsengisoli* [29]; (2) fungi: *Penicillium canescens* [30], *Aspergillus versicolor* [31], and *Aspergillus fumigatus* [32]; (3) algae: *Cladophora fascicularis* [33], *Spirogyra* spp*.* and *Cladophora* spp. [34], and *Spirogyra* spp. and *Spirullina* spp*.* [35]; and (4) yeast: *Saccharomyces cerevisiae* [36] and *Candida utilis* [37].

Prokaryotes (bacteria and archaeans) are distinguished from eukaryotes (protists, plants, fungi, and animals). The cellular structure of eukaryotes is characterized by the presence of a nucleus and other membrane-enclosed organelles. Also, the ribosomes in prokaryotes are smaller (70S) than in eukaryotes (80S) [38]. The way in which microorganisms interact with heavy metal ions is partially dependent on whether they are eukaryotes or prokaryotes, wherein eukaryotes are more sensitive to metal toxicity than prokaryotes [12]. The possible modes of interaction are (a) active extrusion of metal, (b) intracellular chelation (in eukaryotes) by various metal-binding peptides, and (c) transformation into other chemical species with reduced toxicity. For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them to harmless products [39]. Bacteria and higher organ‐ isms have developed mechanisms associated with resistance to toxic metals and rendering them innocuous [20]. Several microbes, including aerobes, anaerobes, and fungi, are involved in the enzymatic degradation process. Most of bioremediation systems are run under aerobic conditions, but anaerobic conditions make it possible microbial organisms to degrade other‐ wise recalcitrant molecules [39].

Because several different types of pollutants can be present at a contaminated site, various types of microorganisms are required for effective remediation. Some types of microorganism are able to degrade petroleum hydrocarbons and use them as a source of carbon and energy. However, the choice of the organisms employed is variable, depending on the chemical nature of the polluting agents, and needs to be selected carefully as they only survive in the presence of a limited range of chemical contaminants. The efficiency of the degradation process is related to the potential of the particular microorganism to introduce molecular oxygen into the hydrocarbon and to generate the intermediates that subsequently enter the general energyyielding metabolic pathway of the cell. Some bacteria search the contaminant and move toward it because they flexibly exhibit the potential as a chemotactic response [40].

Numerous microorganisms can utilize oil as a source of food, and many of them produce potent surface-active compounds that can emulsify oil in water and facilitate its removal [21]. Bacteria that can degrade petroleum products include species of *Pseudomonas*, *Aeromonas*, *Moraxella*, *Beijerinckia*, *Flavobacteria*, *Chrobacteria*, *Nocardia*, *Corynebacteria*, *Modococci*, *Strepto‐* *myces*, *Bacilli*, *Arthrobacter*, *Aeromonas*, and cyanobacteria [40] and some yeasts [21]. For example, *Pseudomonas putida* MHF 7109 can be isolated from cow dung microbial consortia for the biodegradation of selected petroleum hydrocarbon compounds, such as benzene, toluene, and o-xylene (BTX) [23].

**3. Types of organisms used in bioremediation**

6 Advances in Bioremediation of Wastewater and Polluted Soil

wise recalcitrant molecules [39].

Typically, bioremediation is based on the cometabolism action of one organism or a consortium of microorganisms [18]. In this process, the transformation of contaminants presents a little efficiency or no benefit to the cell, and therefore this process is described as nonbeneficial biotransformation [21, 22]. Several studies have shown that many organisms (prokaryotes and eukaryotes) have a natural capacity to biosorb toxic heavy metal ions [23]. Examples of microorganisms studied and strategically used in bioremediation treatments for heavy metals include the following: (1) bacteria: *Arthrobacter* spp. [24], *Pseudomonas veronii* [25], *Burkholde‐ ria* spp. [26], *Kocuria flava* [27], *Bacillus cereus* [28], and *Sporosarcina ginsengisoli* [29]; (2) fungi: *Penicillium canescens* [30], *Aspergillus versicolor* [31], and *Aspergillus fumigatus* [32]; (3) algae: *Cladophora fascicularis* [33], *Spirogyra* spp*.* and *Cladophora* spp. [34], and *Spirogyra* spp. and

*Spirullina* spp*.* [35]; and (4) yeast: *Saccharomyces cerevisiae* [36] and *Candida utilis* [37].

Prokaryotes (bacteria and archaeans) are distinguished from eukaryotes (protists, plants, fungi, and animals). The cellular structure of eukaryotes is characterized by the presence of a nucleus and other membrane-enclosed organelles. Also, the ribosomes in prokaryotes are smaller (70S) than in eukaryotes (80S) [38]. The way in which microorganisms interact with heavy metal ions is partially dependent on whether they are eukaryotes or prokaryotes, wherein eukaryotes are more sensitive to metal toxicity than prokaryotes [12]. The possible modes of interaction are (a) active extrusion of metal, (b) intracellular chelation (in eukaryotes) by various metal-binding peptides, and (c) transformation into other chemical species with reduced toxicity. For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them to harmless products [39]. Bacteria and higher organ‐ isms have developed mechanisms associated with resistance to toxic metals and rendering them innocuous [20]. Several microbes, including aerobes, anaerobes, and fungi, are involved in the enzymatic degradation process. Most of bioremediation systems are run under aerobic conditions, but anaerobic conditions make it possible microbial organisms to degrade other‐

Because several different types of pollutants can be present at a contaminated site, various types of microorganisms are required for effective remediation. Some types of microorganism are able to degrade petroleum hydrocarbons and use them as a source of carbon and energy. However, the choice of the organisms employed is variable, depending on the chemical nature of the polluting agents, and needs to be selected carefully as they only survive in the presence of a limited range of chemical contaminants. The efficiency of the degradation process is related to the potential of the particular microorganism to introduce molecular oxygen into the hydrocarbon and to generate the intermediates that subsequently enter the general energyyielding metabolic pathway of the cell. Some bacteria search the contaminant and move toward

Numerous microorganisms can utilize oil as a source of food, and many of them produce potent surface-active compounds that can emulsify oil in water and facilitate its removal [21]. Bacteria that can degrade petroleum products include species of *Pseudomonas*, *Aeromonas*, *Moraxella*, *Beijerinckia*, *Flavobacteria*, *Chrobacteria*, *Nocardia*, *Corynebacteria*, *Modococci*, *Strepto‐*

it because they flexibly exhibit the potential as a chemotactic response [40].

The application of biotechnology to the treatment of heavy metals is a relatively new subject. A better understanding of the processes through which microorganisms capture heavy metals, particularly the metabolism and detoxification pathways, has been accumulated. It can help the solution with maximum efficiency in dealing with environmental problems associated with heavy metal contamination [41]. The changes arising from the biotechnological approach include bioleaching, bioextraction, biosorption, bioencapsulation, and bioremediation [42]. In this regard, genetic engineering is a fundamental approach to modulate the metabolic pathways of these microorganisms and to inhibit the toxic the action of the metals by the modulated activity. The modified microorganisms can change the inorganic form into the organic form by some reactions, for instance, by transforming the metals through oxidation– reduction reductions, thus increasing the solubility.

Besides the increase of the solubility by microorganisms modifying microorganisms to increase their resistance through factors involving the solubility of heavy metals, their interaction with other factors (e.g., complexation reactions, changes in pH, sorption, precipitation, bioaccumu‐ lation, and encapsulation) can result in increased solubility or render the heavy metals inert in the environment [18]. Genetic engineering can be applied to modify the microorganisms and achieve interesting features such as accelerated growth, tolerance to extreme environ‐ mental conditions and pH variations, and low cost cultivation. Recent studies have demon‐ strated the ability of certain fungi (e.g., *Aspergillus* and *Penicillium*) and some yeasts (e.g., *S. cerevisiae*) to remove heavy metals from certain environments. The species *Escherichia coli*, *Bacillus subtilis*, *Saccharomyces boulardii*, *Enterococcus faecium*, and *Staphylococcus aureus* have also been used for the removal of heavy metals from water bodies [43]. The process of metal accumulation on the cell surface is dependent on the metabolic activity of the microorganism as well as the characteristic of cell surface, and it is known as bioaccumulation [44]. It has been noted that metal ions interact with the proteins necessary for the proper functioning of the cell structure, affecting its metabolic functions. Genetic engineering, which allows the improve‐ ment of the metabolic structure of microorganisms, enables the high accumulation of metals or reduces the toxicity of metals, thereby promoting the decontamination of water bodies.

Many papers on bioremediation with wild or genetic modified microorganisms have been published over the years. Figure 3 shows the data obtained from a search of the web covering a period of 20 years (1995–2014), which deal with the development of methodologies for the decontamination of environments containing various heavy metals.

With the recent advances in genetic engineering, it is now relatively easy to construct geneti‐ cally engineered microorganisms (GEMs) through reshuffling the genes, promoters, etc., and this can enhance their performance *in situ*. Several GEMs have been successfully constructed and experimentally tested for efficient bioremediation under laboratory conditions [45]. Recombinant DNA techniques can be used to enhance the ability of an organism to metabolize a xenobiotic through the detection of genes associated with degradation, transforming them

**Figure 3.** Scientific publications on bioremediation using microorganisms.

into appropriate bioremediation agents. Recombinant DNA technology explores the use of different approaches including PCR, antisense RNA technique, and site-directed mutagenesis.

Engineered strains of *Deinococcus geothermalis* have been developed for the bioremediation of environments containing mixed radioactive waste at high temperatures. Recombinant strain of *Acenitobacter baumanii* was found to enhance degradation rates at sites contaminated with crude oil [45]. In the presence of metals, some higher organisms produce cysteine-rich peptides, such as glutathione (GSH), phytochelatins (PCs), and metallothioneins (MTs), which can bind and sequester metal ions in biologically inactive forms. The overexpression of MTs in re‐ combinant bacterial cells resulted in enhanced metal accumulation, thus offering a promising strategy for the development of microbial-based biosorbents [12].

Recent studies show that certain GEMs have increased ability to metabolize specific chemicals such as hydrocarbons and pesticides [12, 23].

Genetic engineering techniques and studies on the metabolic potential of microorganisms have allowed the design of genetically modified microorganisms capable of degrading specific contaminants. This approach offers an opportunity to create an artificial combination of genes that do not exist together in nature. The most commonly used techniques include engineering with single genes or operons, pathway construction, and alternation of the sequences of existing genes [22]. Genetic and biochemical techniques, such as PCR, *in situ* hybridization, and use of antibodies, can also contribute greatly to our knowledge regarding the potential activity of the microorganisms present at polluted sites. DNA tests can indicate the presence of particular microbes potentially involved in biodegradation, and the use of enzyme-specific antibodies can reveal the induction of catabolic enzymes. Changes in the composition of bacterial populations may be observed during treatment, and differences can be noted in comparison with nonpolluted sites. DNA probes targeting specific genetic sequences, i.e., the genes responsible for the degradation ability of the microorganism, can be used to characterize a contaminated site throughout the bioremediation program, to determine the overall com‐ munity structure and catabolic activity [46].

The first two genetically modified bacterial strains were *Pseudomonas aeruginosa* (NRRL B-5472) and *P. putida* (NRRL B-5473), and these contained genes for naphthalene, salicylate, and camphor degradation. *Pseudomonas fluorescens* HK44, which can degrade naphthalene, represents the first example of a microorganism genetically engineered for bioremediation purposes [22]. The associated research demonstrated that the genes responsible for the naphthalene degradation pathway were arranged under a common promoter, which resulted in the simultaneous degradation of naphthalene [22]. Other authors have shown that some bacteria, such as *Geobacter metallireducens*, can remove uranium from drainage waters in mining operations and from contaminated groundwater [21].
