Bioremediation, Cleaning up

#### **Chapter 4**

## Bioremediation of Petroleum-Contaminated Soil

*Raman Kumar Ravi, Shalini Gupta and Reeta Verma*

#### **Abstract**

Petroleum is not only an important energy resource to boost economic development but also a major pollutant source of soil. Petroleum toxicity can cause an adverse impact on the environment, as well as has negative effects for both animals and humans due to its carcinogenic nature. Therefore, its removal from the environment becomes a matter of concern. Although a lot of techniques are in use for remediation of petroleum-contaminated soil, exploitation of fungal ability provides a sustainable solution for this due to their ability to survive in harsh environmental conditions. Mycoremediation is the bioremediation technique employed for the removal of toxic compounds using fungal biomass. The fungi have been proved as a potential biomass degrader for complex organic compounds, resulting in the production of versatile extracellular enzymes. In this chapter, we have highlighted the basic concept of mycoremediation, the enzymatic system involved in the degradation process, the mechanism of fungal degradation, and factors affecting the degradation process. The chapter also provides useful insight for greater future understanding and improvement of the technique towards solving the problem of petroleum-contaminated soils.

**Keywords:** bioremediation, mycoremediation, petroleum contaminated soil, enzymes, toxicity, degradation

#### **1. Introduction**

Petroleum is a vital resource that is dominating the world economy [1]. The petroleum is composed of a complex mixture of aromatic, aliphatic, heterocyclic hydrocarbons, asphaltenes, and non-hydrocarbon compounds, of which 60–90% are biodegradable [2]. In the past few decades, with the development of the petroleum industries, related activities like exploration, transportation, management, or storage and refining of hydrocarbons have caused contamination of soil environment and posed a serious global problem [3]. In view of the high toxicity, carcinogenicity, mutagenicity, and teratogenicity nature of petroleum compounds, their bioaccumulation in the food chain will interfere with the biochemical and physiological processes that directly or indirectly lead to human health [4, 5]. The petroleum contamination may induce oxidative stress. They may cause alteration in the soil's chemical composition, its properties, and low nutrient availability, which leads to inhibition of seed germination. The petroleum compounds may cause various harmful effects on plants such as reduction of photosynthetic pigments, slowdown of nutrient assimilation, inhibition of root growth, foliar deformation and tissue necrosis, as well as destroy biological membranes, disturb the signaling

of metabolic pathways, and disrupting plant roots architecture [6–9]. The hydrocarbons with low-molecular weight can penetrate the plant cells causing plant death. Besides this, many petroleum compounds and their derivatives are carcinogens. It is also reported that petroleum contamination can cause the depression of the nervous system, narcosis, and irritation of the mucous membranes of the eyes in humans [10–13]. Therefore, nowadays, petroleum contamination becomes a global environmental issue. The petroleum compounds have not only adverse impact on plant growth and development but also on human and ecological health. Therefore, their removal becomes a necessity in the current scenario for a healthy environment. Bioremediation is one of the most reliable, environmentally friendly techniques for the removal of hazardous compounds using biological sources like plants and microbes or the metabolites obtained from them. The microbial communities … play great role in the degradation of petroleum contaminants from the environment effectively. In this chapter, we have highlighted the microbial remediation of petroleum-contaminated soil for environmental clean-up. The chapter also discussed the different enzymes involved in bioremediation, mechanisms of petroleum compounds degradation, and factors influencing the bioremediation processes. This chapter may provide different clues for new research approaches for microbial-assisted methods of remediation.

#### **2. Different sources of petroleum contamination**

Petroleum comprises a number of aliphatic, branched, and aromatic hydrocarbons [14, 15] and several other organic compounds including some organometallic constituents [16]. The contamination of soil by petroleum hydrocarbons includes various sources such as the activities like industrial and municipal runoffs, effluent release, offshore and onshore petroleum industry activities as well as accidental petroleum spills (**Figure 1**). Most of them are toxic to human beings, animals, and vegetation [17–20]. The anthropogenic activities also lead to the release of petroleum hydrocarbons from oil and gas exploration and production units, tank leakages and overflow, petrochemical industry effluent discharge, accidental spills during loading and discharging, bunkering, oil tanker incident, transportation and storage, fugitive emissions, ballasting, and de-ballasting, burst in old underground

**Figure 1.** *Different sources of petroleum contamination.*

*Bioremediation of Petroleum-Contaminated Soil DOI: http://dx.doi.org/10.5772/intechopen.100220*

pipelines, war and political crisis, sabotage, and natural disasters. Such type of incidents has posed adverse impacts on terrestrial and marine biodiversity. The petroleum contamination directly or indirectly affects the wellbeings of all kinds of life inhabiting in the affected environment by altering population dynamics thereby interrupting the natural interaction among organisms at various trophic levels consequently misbalancing the natural community structure within the ecosystem ([17–20]; Belousova et al. 2001; Bejarano and Michel, 2010). In long term, this pollution affects the environment. Due to the adverse impact of these chemicals on human health and the environment, they are classified as priority environmental pollutants by the U.S. Environmental Protection Agency [21]. Wood fires or volcano eruptions account for the natural sources of petroleum contamination whereas major anthropogenic sources include industrial combustion processes, refining processes: coking (coal), cracking (petroleum products e.g., tar, waxes, oils) and fireplaces, tobacco smoke is categorized under indoor sources. Releasing hydrocarbon pollutants through spillages and leakage from underground tanks, steamers, unplugging of oil wells, or abandoned oil refinery sites causes contamination of surface soil, groundwater and ocean as well [20, 22–24].

#### **3. Effect of petroleum contamination on the environment and human health**

Contamination of soil by petroleum hydrocarbons can affect physicochemical properties of soil such as texture, compaction, structural status, penetration resistance, saturated hydraulic conductivity, mineral and heavy metal concentration (Hreniuc et al., 2015). The toxicity of petroleum compounds is of worldwide environmental concern and has an adverse effect on the environment and human health. The polyaromatic hydrocarbons (PAHs) are natural constituents of fossil fuels, coal, and petroleum comprising 0.2% and 7% PAHs [25]. The PAHs adsorb to dust or soot particles and enter into the atmosphere and are transported to far distances. Naturally, in a cyclic process, PAHs undergo a cycle of entry, deposition, and percolation. PAHs enter the environment via rain/fog thereafter get deposited on soil and plants and ultimately percolate in surface waters [26]. Dust produced by anthropogenic activities namely coal mining, automobile exhaust, transportation, and drilling of oil, stockpiles, and tailings is the major unnatural sources of PAHs that contaminate the various spheres of the atmosphere [27].

PAHs are present in all the spheres of the environment mainly evaporated into the atmosphere. Primarily when adsorbed on dust particles, PAHs undergo photolysis in the existence of sunlight. Upon oxidation, the complex structure of the compound can be broken in days or weeks [28]. The PAH compounds are hydrophobic in nature, immiscible in water; however miscible in other hydrophobic matter. PAHs can get easily adsorbed on dust particles as well as a precipitate on sediments of aquatic bodies. Therefore, these pollutants can easily mix with other hydrophobic matter and pollute the aquatic systems. Terrestrial and water system microbes possess the adaptability to degrade and mineralize PAHs over a longer or shorter time duration [29].

Under the influence of UV light, PAH metabolites produced are usually more toxic. PAHs in soil is unlikely to employ toxicity influence on terrestrial invertebrates [30]. In plants absorption of PAHs by roots from soils and thereafter translocated to other parts. Mobility of the absorbed contaminants is usually influenced by the dose, solubility, along other physicochemical properties of soil. The plant response against PAHs differs; certain plant species consist of components that may wear off a toxic consequence of PAHs; whereas, some plants have the ability

to synthesize PAHs and perform as growth hormones [29, 31]. PAHs are persistent organic compounds and have a longer half-life which accounts for the PAH bioaccumulation in terrestrial invertebrates (shellfish expected to consist much higher concentration of PAH than in the environment) observed. Nonetheless, the metabolism of PAHs is effective to preclude biomagnifications [32–35]. Organisms are adversely affected because of tumors, reproduction, growth development, and immunity. In mammals route to PAH absorption is by inhalation, dermal contact, and ingestion [31, 36, 37].

#### **4. Strategies used for removal petroleum contamination from soil**

The soil contamination by petroleum has drawn increasing attention to develop and implement innovative techniques to remove petroleum compounds from the soil in the past decades. Different strategies are being used for the removal of petroleum contamination from the soil that includes traditional physical and chemical remediation, which are less efficient. Bioremediation is one of the most reliable and efficient techniques used for the restoration of petroleum-contaminated soil in an eco-friendly way.

#### **4.1 Bioremediation of petroleum-contaminated soil**

Bioremediation is a process that naturally or artificially takes advantage of living organisms or their products to remove the pollutants of the contaminated environment [38]. Although it is time-consuming, due to their eco-friendly approach and very low cost, efficient and sustainable for restoring the contaminated soil in the context of sustainability, are extensively noticeable at present [39]. For this purpose, living organisms having the potential to grow under contaminated soil are usually used. The number of studies have revealed that selecting petroleumtolerant plants for bioremediation in cases of soil petroleum pollution is a feasible and sustainable technology. Many plants like ryegrass, alfalfa, *Mirabilis jalapa* are capable to grow in petroleum stress conditions [40–42].

The microorganisms that are utilized in petroleum pollutants removal can be bacteria, fungi, or yeasts. These microbes are the essential component in soil that play a crucial role in the remediation of petroleum contamination [43, 44]. Some of them have a high capacity to degrade contaminants and are widely used for environmental clean-up [45]. In the bioremediation of petroleum-contaminated soils, the most widely used organisms are bacteria which have high frequency, rapid growth, and a broad spectrum of degradation of petroleum products [46]. The development of microbial biotechnology is beneficial for screening and identifying microorganisms from petroleum-contaminated soils [47]. Many microorganisms have been isolated and exploited for the bioremediation of petroleum hydrocarbons. A number of bacteria having the capacity to degrade petroleum hydrocarbons have been identified [48]. Furthermore, some microorganisms were crucial for petroleum hydrocarbons since the abundance of these microorganisms was dominant increased after petroleum contamination [49]. Different indigenous bacteria have different degrading enzymes, the blend of several efficient bacteria was employed to remediate the contaminated soils. The combined activity of indigenous bacterial consortium and exogenous bacteria can efficiently enhance the degradation of petroleum [50].

Biostimulation is a leading strategy of bioremediation to decontaminate petroleum polluted soil. It includes regulating various environmental conditions such as temperature, moisture, pH, redox potential, aeration, mineral nutrition, etc. to

#### *Bioremediation of Petroleum-Contaminated Soil DOI: http://dx.doi.org/10.5772/intechopen.100220*

increase the growth and the metabolic activity of microorganisms. Consequently, various hydrocarbons can be tolerated and utilized as a carbon source to fulfill its growth requirements [51]. Bioaugmentation is another strategy of bioremediation, which refers to the inoculation of exogenous microorganisms into the contaminated soils to degrade the target contaminants [52]. The inoculated microorganism can be one strain or a consortium of microbial strains with diverse functional degradation capacities [53]. Bioaugmentation was considered to be more effective for the degradation of the light fraction of petroleum hydrocarbons [54]. Besides this, the microbial electrochemical system was considered as an emerging technique for bioremediation, which integrates microbial and electrochemical processes to convert the pollutants to less-toxic or value-added products [55]. With numerous integral benefits, the microbial electrochemical system was frequently applied in the remediation of petroleum contaminants in soil. The microbial electrochemical system can be utilized for different contaminants owing to the oxidation and reduction transformation involved in remediation processes [56].

In the last decay, the role of fungi in bioremediation has been increasingly recognized [57, 58], in which mainly saprotrophic and basidiomycetes, groups of fungi are highlighted to degrade or to transform toxic compounds [59, 60]. Mycoremediation is the bioremediation technique that employs fungi in the removal of toxic compounds; it could be carried out in the presence of both filamentous fungi [61] and macrofungi such as mushrooms [62, 63]. Both classes possess enzymes for the degradation of a large variety of pollutants [64, 65]. Fungi are noted for their colonizing abilities. They can colonize and adapt in diversified heterogeneous environments including complex soil matrices at extreme environmental conditions. Furthermore, they can decompose the organic matter and easily colonize both biotic and abiotic surfaces [66, 67]. Filamentous fungi have shown some different characteristics that make them more preferable for soil bioremediation than yeasts and bacteria [66, 68]. The most important is the type of growth i.e., multicellular mycelial growth, suitable to soil colonization and translocation of nutrients and water, the production of many bioactive compounds and extracellular enzymes, and the unique capability to co-metabolize many environmental chemicals [69].

#### *4.1.1 Mycoremediation of petroleum-contaminated soils*

Mycoremediation offers an alternative environmentally friendly technique for remediation of contamination in environmental matrices [70–73]. Different species of fungi have been used for the remediation of petroleum-contaminated soils. These include microfungi such as *Arbuscular mycorrhiza* and yeast [74–76] as well as *Penicillium* and *Aspergillus* species [77, 78]. Mycoremediation with macrofungi (mushrooms) is also identified [79, 80]. Abioye et al. [76] reported crude oil degradation by yeast *Saccharomyces cerevisiae*. It was inoculated in a sterile mineral salt media containing 1 g of crude oil under control conditions at 30°C for 28 days. Obire et al. [81] recognized micro-fungal communities were actively involved in the remediation potentials of cow & poultry compost on petroleum polluted soil sites. Isolated yeasts and molds from cow dung comprised of *Alternaria* sp., *Aspergillus* sp., *Cephalosporium* sp., *Cladosporium* sp., *Geotrichum* sp., *Moniliasp., Mucor* sp., *Penicillium* sp., *Rhizopus* sp., *Sporotrichum* sp., *Thamnidum* sp., *Candida* sp., *Rhodotorula* sp., and *Torulopsis* sp., *Saccharomyces* spp (Yeast) has also been revealed to transform polycyclic aromatic hydrocarbons (PAHs) into eco-friendly products [82].

One significant class of fungi, which demonstrate mycoremediation of petroleum-contaminated soils are the ligninolytic mushrooms such as white rot fungi [83–85]. Lebo et al. [86] and Fetzer [87], identified that white rot fungi are able to degrade recalcitrant organic pollutants, the fact is mushroomed naturally feed on and degrade lignin, a substance with the similar monomeric unit to organic contaminants. Stamets [73], validated up to 99% degradation of naturally diesel-contaminated soils at 20,000 parts per million concentration of PAHs after 8 weeks using the white rot mushroom i.e., *P. ostreatus.* Kristanti et al. [88], reported that up to 93% degradation of crude oil in the soil can be obtained using the white rot mushroom *Polyporus* sp. *S133* pre-grown on the wood meal. It has been established that the litter decomposing mushroom i.e., *Stropharia coronilla,* can metabolize PAHs such as benzo[a]pyrene at 200 μM and this activity could be increased up to 12 times in presence of supplementary Mn2+ as electron acceptor. Mohammadi-Sichani et al. [89], reported that the litter decomposing mushroom *A. bisporus* can yield a higher ability to degrade total petroleum hydrocarbons in soils than white rot mushroom such as *Pleurotus ostreatus* and *Ganoderma lucidum.*

The most suitable fungal genera used for remediation of hydrocarbon contaminated soil are the basidiomycetes group [90]. The saprotrophic basidiomycetes, utilize dead organic substances as a carbon source, consist of the wood-degrading fungal groups. Overall, white-rot fungi are reflected for a prominent role in the biodegradation of petrochemicals [91]. These fungi can degrade efficiently both lignin and cellulose biopolymers till the complete mineralization [92], by producing an extracellular enzymatic complex, which comprehends lignin peroxidases (LiPs), manganese-dependent peroxidases (MnPs), versatile peroxidases (VPs), laccases, H2O2-generating oxidases and dehydrogenases, produced during the idiophase, usually under nitrogen depletion. The most common example of white rot fungi, that are capable to degrade pollutants, include *Phanerochaete chrysosporium*, *Pleurotus ostreatus*, *Trametes versicolor*, *Bjerkandera adusta*, *Lentinula edodes*, *Irpex lacteus*, *Agaricus bisporus*, *Pleurotus tuber-regium* and *Pleurotus pulmonarius* [93, 94]. Among these fungi, *Phanerochaete chrysosporium* has been the most investigated for its ability to degrade toxic or insoluble compounds to CO2 and H2O, more efficiently than other fungi.

Mostly the biodegradation studies at the laboratory & pilot scale are covered micro-fungi, but in last year, much attention has been given to mushrooms which are frequently present in soil and also easily cultivated [95]. Bioremediation by macro-fungi basidiomycetes is advantageous because, simultaneous remediation process, and soil enrichment with organic matter, nutrients and result in enhanced plant growth. Macro-fungi are potent degraders due to secretion of the similar intracellular enzymes (LiP, MnP, and laccase) labeled for the lignin-degrading fungi thus are attention-grabbing in the field of bioremediation. Altogether, they grow to a great extent and produce high biomass, when cultivated on carbon sources, like stubble or sawdust [79]. The mushroom biomass can be a protein source or can contain biologically active compounds such as phenols with antioxidant activity [64, 96]. Furthermore, mushroom biomass can be applied in biosorption treatment thanks to its ability to accumulate ions and xenobiotics from contaminated soils [97].

Different types of petroleum compounds such as phenanthrene, naphthalene, anthracene, pyrene, benzo[a]pyrene, fluoranthene, acenaphthene, etc. are earlier reported to be degraded by fungal biomass (**Table 1**). Phenanthrene can be degraded by *Pleurotus ostreatus* [98], *Phanerochaete chrysosporium* [99–101], *Phanerochaete sordida* [100, 102], *Ganoderma lucidum* [103], *Trametes versicolor* [104]. Naphthalene is degraded by *Penicillium oxalicum* [105], *Penicillium* sp. [106], *Penicillium fastigiata,* and *Penicillium digitatum* [107]. Govarthanana et al. [106] also reported degradation of acenaphthene and benzo[a]pyrene by


#### *Bioremediation of Petroleum-Contaminated Soil DOI: http://dx.doi.org/10.5772/intechopen.100220*

#### **Table 1.**

*Fungi involved in the degradation of different petroleum compounds.*

*Penicillium* sp. Similarly, fungus *Phanerochaete sordida* is reported to *degrade* fluoranthene [100, 102]. The petroleum compound like anthracene is reported to be degraded by various fungal species such as *Aspergillus fumigatus* [108], *Stropharia coronilla* [109], *Pleurotus ostreatus* [100]. The earlier study revealed the *Fusarium solani* and *Mucor* sp. can degrade benzo[a] pyrene [110, 111], while pyrene is degraded by *Trichoderma harzianum* [112], *Penicillium janthinellum* [113] and *Penicillium* sp. M 1 [114].

#### **5. Mechanism of petroleum hydrocarbon degradation**

Fungal genera catabolize hydrocarbons to intermediates analogous to those formed by a mammalian enzymatic system via Cytochrome P450 [115]. Whereas, several mechanisms have been proposed includes both direct and indirect oxidation of the organic molecule by the fungal enzymes namely Lignin peroxidase, Manganese Peroxidase, versatile peroxidase, and Lacasses (**Figure 2**) [116–118]. The promising combination of multi enzymatic mechanisms could play a key role in the degradation process [119]. Radical mediated reaction initiated by manganese peroxidase involves indirect oxidation of benzene rings with hydroxyl group may be led to spontaneous ring opening. Produce derivatives like muconic acid and carbon dioxide by decarboxylation of carboxyl groups [120, 121].

**Figure 2.** *Fungal enzymatic degradation mechanism of PAH compounds.*

#### **5.1 Enzymes involved in biodegradation of petroleum contamination**

Most of the enzymes are extracellular and allow to attack and then degrade large molecules into smaller units which can enter the cells for further transformations [122]. Extracellular laccases start ring cleavage in the biodegradation of aromatic compounds [61]. Laccase are multicopper oxidases with low substrate specificity & could act on o- and p-phenols, phenylenediamines via four-electron transfer from the target organic substrate to molecular oxygen [123]. Fungal peroxidases generate oxidants that initiate substrate oxidation in the extracellular environment [61]. They belong to the class II peroxidases [124] & catalyze the oxidation of various organic substrates by utilizing peroxide (H2O2) as an electron acceptor. Lignin peroxidase (LiPs), Manganese peroxidase (MnPs), and Versatile or non-specific peroxidase (VPs) are the leading fungal high redox class II peroxidase enzymes as reported earlier. These intracellular enzymes are convoluted in the biodegradation process of the complex lignocellulosic structure and, subsequently, could biotransform various organic compounds into inorganic substrates [125]. Some reported fungal genera could also secrete the dye-decolorizing peroxidase (DyPs), which have the ability to oxidize and hydrolyze phenolic and non-phenolic compounds [126]. Heme-thiolate peroxidase (HTPs) allocates peroxide-oxygen, from H2O2/R-COOH to organic substrate; in this group chloroperoxidases (CPOs) and aromatic peroxygenase (APOs) are involved. APOs can mainly be active on heterogeneous substrates via aromatic preoxygenation, epoxidation, or hydroxylation of aliphatic organic compounds [124].

Biodegradation by intracellular enzymatic pathway includes a class of cytochrome P450 monooxygenase and glutathione transferase, mainly possessed by lignocellulosic and plant litter fungi [125]. These enzymes show a functional role in the primary and secondary metabolism of fungi. Cytochrome P450 monooxidase, heme-thiolate containing oxidoreductase, could act on numerous organic substrates in stereo- and region selective manner, requiring molecular oxygen for the reaction initiation. These enzymes are triggered via reduced heme iron and single molecular oxygen to a substrate. Hydroxylation, epoxidation, sulfoxidation, and dealkylation intermediate reactions can occur and entail NADPH as electron donor [127]. Glutathione transferase enzyme is positioned in different cellular partitions and catalyze the nucleophilic attack of carbon, nitrogen or sulphur atom in non-polar compounds by reducing

*Bioremediation of Petroleum-Contaminated Soil DOI: http://dx.doi.org/10.5772/intechopen.100220*


**Table 2.**

*Major enzymes involved in petroleum compounds degradation.*

glutathione. These enzymes consist a wide range of substrate specificity and involved in the detoxification of several endogenous or exogenous toxic metabolites [128]. Major enzymes involved in petroleum compounds degradation are summarized in **Table 2**.

#### **6. Factors influencing bioremediation of petroleum contaminants**

Bioremediation is a technique to remove the contamination in a cost-effective and environment-friendly way. The remediation of contaminants depends on various factors like availability of contaminants for microbes, temperature, pH, relative humidity etc.

#### **6.1 Availability of contaminants**

Solubility and bioavailability are important factors in the bioremediation of petroleum-contaminated soils. Boopathy and Manning [129], stated that the rate of contaminant conversion during bioremediation depends on their uptake and metabolism rate and the rate of contact with the cells of the organism. Mannig et al. [130] reported that activities that result in the homogenous spread of contaminants in soils can drastically stimulate their biodegradation. Singh and Agarwal [131], demonstrated that the bioavailability of organic contaminants decreases with time. According to Boopathy and Manning [129], some physicochemical progressions such as sorption, desorption, diffusion, & dissolution stimulate contaminants bioavailability. So, these elements must be measured during planning for bioremediation of petroleum-contaminated soil. The use of surface-active agents (surfactants) could aid to combat the contaminants bioavailability issue during the remediation process [129].

#### **6.2 Temperature**

Temperature plays a critical role in bioremediation processes [132]. It has been reported that the rate of degradation of organic contaminants is comparably higher at higher temperatures [133]. Higher rates of degradation of organic contaminants are also reported in tropical soils compared to soils from temperate regions [134, 135]. Dimond and Owen [136], stated that temperature affects the half-life of organic contaminants which increases with lowering temperatures. Hong et al. [137], demonstrated that a temperature range of 20–40°C was optimal for degradation of the contaminant fenitrothion. Siddique et al. [132] further demonstrated that the highest degradation of Hexachlorocyclohexane in water and a soil slurry was achieved at an incubation temperature of 30°C.

#### **6.3 pH**

The soil environment is contaminated with different types of organic compounds that are causing an adverse effect on the soil microbial diversity. The pH of the soil is highly mutable and ranges from 2.5 to 11 which may significantly affect the biodegradability of hydrocarbons. The aptness of a pH range in any bioremediation process is site-specific, & swayed by the complex relation among the organism, contaminant, and soil properties. The pH range may also affect the solubility and availability of contaminants in soil. The organic contaminant present in the soil is degraded at high pH because of the increased solubility [138]. The study carried out by Owen et al. [135], demonstrated faster degradation of organic contaminants in alkaline soil pH compared to in acidic soil. The report also suggested that at low soil pH of 4.5 to 4.8, degradation of organic contaminants is inhibited [139]. Nash et al. [140], reported the effects of pH on the stability of DDT and observed maximum degradation in both moist and dry soils were obtained at pH values above 7. In another study, Hong et al. [137] reported bioremediation of fenitrothion-contaminated soil using *Burkholderia* sp. *FDS-1* with an optimal degradation at a slightly alkaline pH of 7.5. Thus, pH is one of the factors that should be considered in the bioremediation of petroleum-contaminated soils.

#### **6.4 Relative humidity**

Relative humidity is an important parameter in the removal of contaminants from the soil. In bioremediation of contaminated soils, generally more than 60% relative humidity is maintained [141, 142]. Several studies have been reported a different range of relative humidity. The utilized relative humidity for bioremediation of contaminated soil was 70% [142] and 60% [141], while it was between 60 and 70% [143]. The relative humidity values up to 85–95% have also been reported for remediation of contaminated soil [116]. The growth of mushrooms and their fruiting is also reported at a relative humidity of 70–80% [144].

#### **7. Conclusion**

Bioremediation could serve as a sustainable alternative for complex pollutant clean-up. Though this technique has been explored and studied for years. But still, it is not been maximized for practical solutions and field-scale application for the treatment of petroleum-contaminated soil. Hence, it is necessary to carry out an assessment of microbes for bioremediation of petroleum-contaminated soil site and its evolvement, limitations, and perspectives in the field. Present literature provides

#### *Bioremediation of Petroleum-Contaminated Soil DOI: http://dx.doi.org/10.5772/intechopen.100220*

an understanding of bioremediation for petroleum-contaminated soils, in which different types of fungi, mechanisms of the technique are highlighted. The findings offer mycoremediation is capable of providing reliable options for the treatment of petroleum-contaminated soils. This is because fungi can provide cheaper and safer means for the simultaneous degradation of organic contaminants for environmental clean-up.

### **Author details**

Raman Kumar Ravi1 \*, Shalini Gupta<sup>2</sup> and Reeta Verma3

1 Department of Environmental Science, Uttaranchal College of Science and Technology, Dehradun, Uttarakhand, India

2 Environment Studies, Delhi College of Arts and Commerce, University of Delhi, Delhi, India

3 School of Environment and Sustainable Development, Central University of Gujarat, Gandhinagar, India

\*Address all correspondence to: smartramanravi@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[137] Hong Q, Zhang Z, Hong Y, Li S. A microcosm study on bioremediation of fenitrothion-contaminated soil using *Burkholderia* sp. FDS-1. International Biodeterioration and Biodegradation. 2007;**59**(1):55-61

[138] Xu B, Jianying G, Yongxi Z, Haibo L. Behaviour of DDT in Chinese tropical soils. Journal of Environmental Science and Health. 1994;**B29**:37-46

[139] Andrea MM, Tomita RY, Luchini LC, Musumeci MR. Laboratory studies on volatilization and mineralization of 14c-p, p'-DDT in soil, release of bound residues and dissipation from solid surfaces. Journal of Environmental Science and Health Part B. 1994;**29**(1):133-139

[140] Nash RG, Harris WG, Lewis CC. Soil pH and metallic amendment effects on DDT conversion to DDE. Journal of Environmental Quality. 1973;**2**:390-394

[141] Migliore L, Fiori M, Spadoni A, Galli E. Biodegradation of oxytetracycline by *Pleurotus ostreatus* mycelium: A mycoremediation technique. Journal of Hazardous Materials. 2012;**215**:227-232

[142] Xiao X, Chen H, Si C, Wu L. Influence of biosurfactant-producing strain Bacillus subtilis BS1 on the mycoremediation of soils contaminated with phenanthrene. International Biodeterioration and Biodegradation. 2012;**75**:36-42

[143] Singh M, Srivastava PK, Verma PC, Kharwar RN, Singh N, Tripathi RD. Soil fungi for mycoremediation of arsenic pollution in agriculture soils. Journal of Applied Microbiology. 2015;**119**(5): 1278-1290

*Crude Oil - New Technologies and Recent Approaches*

[144] Seidu A, Quainoo AK, Addae G, Stenchly K. Mycoremediation of diesel contaminated soil with oyster mushroom (*Pleurotus ostreatus*) using maize (*Zea mays*) as the test crop. UDS International Journal of Development. 2015;**2**(2):1-8

#### **Chapter 5**

## Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment

*Chioma Bertha Ehis-Eriakha, Stephen Eromosele Akemu, Simon Obgaji Otumala and Chinyere Augusta Ajuzieogu*

#### **Abstract**

Globally, the environment is facing a very challenging situation with constant influx of crude oil and its derivatives due to rapid urbanization and industrialization. The release of this essential energy source has caused tremendous consequences on land, water, groundwater, air and biodiversity. Crude oil is a very complex and variable mixture of thousands of individual compounds that can be degraded with microbes with corresponding enzymatic systems harboring the genes. With advances in biotechnology, bioremediation has become one of the most rapidly developing fields of environmental restoration, utilizing microorganisms to reduce the concentration and toxicity of various chemical pollutants, such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters, nitroaromatic compounds and industrial solvents. Different remediation methods have been introduced and applied with varied degrees of success in terms of reduction in contamination concentration without considering ecotoxicity and restoration of biodiversity. Researchers have now developed methods that consider ecotoxicology, environmental sustainability and ecorestoration in remediation of crude oil impacted sites and they are categorized as biotechnological tools such as bioremediation. The approach involves a natural process of microorganisms with inherent genetic capabilities completely mineralizing/degrading contaminants into innocuous substances. Progressive advances in bioremediation such as the use of genetically engineered microbes have become an improved system for empowering microbes to degrade very complex recalcitrant substances through the modification of rate-limiting steps in the metabolic pathway of hydrocarbon degrading microbes to yield increase in mineralization rates or the development of completely new metabolic pathways incorporated into the bacterial strains for the degradation of highly persistent compounds. Other areas discussed in this chapter include the biosurfactant-enhanced bioremediation, microbial and plant bioremediation (phytoremediation), their mechanism of action and the environmental factors influencing the processes.

**Keywords:** environment, bioremediation, phytoremediation, genetic engineered microorganisms, crude oil

#### **1. Introduction**

One of the major environmental problems facing industrialized nations in recent times is hydrocarbon contamination resulting from oil and gas exploration and exploitation activities. As the demand for liquid petroleum increases, the release of this essential energy source into the environment becomes inevitable and has caused devastating consequences to marine/coastal waters, shorelines and land as well. Human activities such as accidental release of petroleum products, uncontrolled landfills, sabotage, leaking of underground storage or improper storage of crude oil are of particular concern in the environment. Hydrocarbon components have been known to belong to the family of carcinogens and neurotoxic organic pollutants which constitutes a major health challenge globally. Oil spill on land, penetrates to a depth of about 10–30 cm and sometimes beyond, results in the loss of soil fertility and also initiates environmental degradation [1]. This consequently alters the physicochemical properties of the soil making it impossible for the soil to produce at its optimal capacity.

The application of biotechnology today as a tool for environmental clean-up has been widely studied. Biotechnological tools in eco-restoration of crude oil impacted sites involves the use of biological agents to decontaminate/detoxify, mineralize, transform or degrade toxic/harmful substances into innocuous forms. The process known as bioremediation is genetically-driven, whereby microbes with inherent enzymes harboring catabolic genes utilize these xenobiotics as a source of carbon and energy thereby decontaminating the environment. The biological agents in bioremediation; microbes (microbial bioremediation), plants (phytoremediation) or plant-microbe interaction and their mode of operation will be extensively discussed in this chapter. Nigeria and some other nations in Africa have experienced devastating consequences of pollution in all environmental compartments which till date is still a major challenge [2].

With the advances of biotechnology, bioremediation has become one of the most rapidly developing fields of environmental restoration, utilizing microorganisms to reduce the concentration and toxicity of various chemical pollutants, such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters, nitroaromatic compounds, industrial solvents and the very recalcitrant substances [3]. This has been made possible through a very important, emerging and next generation approach, called genetic engineering which involves the modification of the genetic structure of an organism to increase/enhance their activity. This approach is one potential key to a very successful, and swift bioremediation, whereby the catabolic potentials of an organism is enhanced by the introduction of hydrocarbon catabolic genes into the microbe. This paper highlights the various biotechnological tools that can be practically adopted especially in Nigeria and Africa at large to encourage environmental sustainability and eco-restoration of crude oil polluted environments.

#### **2. Crude oil pollution and environmental consequences**

#### **2.1 Crude oil as an environmental pollutant**

The intensification and rapid increase of manufacturing industries and the intensive use of fuels has led to an increased release of a wide range of xenobiotic compounds to the environment. Overtime, continuous loading of excess hazardous waste and xenobiotic compounds into the water bodies and soil has led to the destruction of soil structure, component and biodiversity, scarcity of clean water

#### *Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*

thereby limiting crop production [4, 5]. One of the major types of pollution that have caused so much harm/damage to the ecosystem generally is crude oil pollution. Crude oil contains so many toxic compounds such as hydrocarbons which can be easily converted to activated metabolites or free radicals during their oxidation [6]. The high toxicity of crude oil is usually attributed to its low molecular weight hydrophobic petroleum hydrocarbons. Other larger constituents of crude oil include alkyl PAHs with three or more rings which are less soluble in water [7]. In the past, several incidents have occurred which caused devastating damage to the ecosystem and have revealed the importance of preventing the escape of effluents into the environment, one of such incidents is the Exxon Valdez oil spill [8]. The Exxon valdez, a cargo ship carrying crude oil was grounded on the 24th march, 1989 along the Bligh Reef in Alaska, northeastern Prince William Sound. This resulted in the release of about 20% of the entire cargo (about 36,000 metric tons) [9]. Another significant oil spill that occurred in the Gulf of Mexico in 2010 is the BP Deepwater Horizon spill. Approximately 4 million barrels of crude oil spilled from the Macondo Wellhead (MW) making it the largest accidental marine oil spill in history. The biological impacts of the oil spill were severe, including in the deep sea, a habitat typically characterized by high biodiversity and generally economic and ecological impact [10].

In Nigeria, crude oil and gas production contributes to 25% of the nation's gross domestic product (GDP) and about 90% of the foreign exchange. The exploration and production of crude oil has caused devasting impact to all environmental compartments within the country, especially in the Niger Delta Region [11, 12]. A constant reoccurring phenomenon is the leaks from oil tankers and petrol leakage into the soil and these slicks formed contribute to reduction of dissolved oxygen and co-marine environment which causes oil slick. Polycyclic aromatic hydrocarbons (PAHs) which are one of the major components of crude oil have been found in water ways as a result of pollution caused by the effluents from petrochemical industries. Some of the major activities that cause petroleum hydrocarbon pollution of the environment are oil well drilling production operations, refining, storage, transportation, marketing in the upstream and downstream industry, anthropogenic sources [13]. Some of the causes of oil pollution may also occur in form of spillages due to corrosion of pipelines, oil well blowout, vandalization of pipelines or accidental discharges.

#### **2.2 Environmental consequences of crude oil pollution**

Crude oil pollution has been reported to cause devastating environmental consequences. Its effects range from the destruction of the soil structure and biodiversity, to limitations in plant growths which may further affect the farmer's source of income, and health hazards in man. It has also been reported that plants that grow in oil polluted soils show signs of chlorosis on their leaves and are also retarded due to the water deficiency. These have led to a complete halt in some farming activities like fishing or even death in some cases when contaminated water or food crop is consumed [14, 15].

There are countless literatures on the study of the causes and effects of petroleum hydrocarbon contamination on human health, soil, plant growth and the environment in general [16–18]. Ojimba [19] conducted a research to determine the effects of crude oil pollution on crop production. He analyzed data from 17 out of the 23 Local governments in Rivers state, Nigeria. His results showed there has been a significant reduction in the size of available farmlands due to crude oil pollution, this further reduced the physical farm products by 1.09016 tons. His results also indicated that 78% of farm lands had less than 80% efficiency due to

crude oil contamination. The study concluded that crude oil pollution on farmlands and crops has negative effects on the output of crops. Abii and Nwosu [20] reported that Crude oil pollution causes reduction in the fertility of the soil such that the major essential nutrients necessary for the plants to grow are almost completely lost. Other effects of crude oil pollution on plants may range necrosis, chlorosis, yield reduction, bleaching, spotting of leaves, malformations to mesophyll cells and epidermal layers [21].

Al-Qahtani [16] investigated the effects of sludge from oil refineries on soil properties and the rate of plant growth. He carried out the experiment by applying the refinery sludge in a plant *Vinca rosea (Catharonthus roseus).* The results showed that with increase in the application of the sludge, the soil chemical composition showed a reduction in dry matter yield and decrease in plant yield significantly. There was also increase in soil salinity with the application of oil refinery sludge. With the continuous introduction of the sludge, there was a significant decrease in the essential mineral elements of plants such as phosphprus and nitrogen compared to the control treatment. Ibemesim [22] conducted an experiment on the tolerance of sour grass (*Paspalum conjugatum Bergins*) in a crude oil polluted system. In their results, the crude oil polluted soil did not have any significant effect on the major growth parameters. Their result showed that polycyclic aromatic hydrocarbons (PAHs) was able to modify the absorption, uptake and availability of sodium (Na+) in the plant.

Sun et al. [18] conducted an experiment to study ability of the eggs and larvae of a marine medaka (*Oryzias melastigma*) to survive in crude oil polluted environment. The experiment was carried out by treating the eggs and larvae with three different treatments. The first treatment was with CO2, the second was with a water-soluble fraction of crude oil which was prepared using crude oil and sea water in a volumetric ratio of 1:100 respectively and the third was mixed with a CO2/water soluble fraction of crude oil mixture. The combined treatment (CO2 and water soluble fraction) had no detectable effect on the size or survival rate, however there were significant anomalies of the tissues in treatments with the water-soluble fractions of crude oil. They concluded that crude oil pollution has the ability to perform as a contributory factor to natural mortality. Agbogidi et al., [23] carried out a study to examine the environmental and socio-economic impacts of oil exploration in two oil producing communities in Delta state, Nigeria. The study showed that crude oil spillage due to oil and gas industry activities (exploration and production) caused damages to arable soils and water bodies which have led to a reduction in crop yield and hence the income capacity of the farmers. The results also showed a heightened deforestation and increased health hazards due to the crude oil activities in the communities.

Obire and Anyanwu [24] also conducted an experiment to investigate the Fungal population at different concentrations of crude oil pollution in a soil sample. Their analysis showed high significant difference between the control and the oil treated soils, the total fungal counts of petroleum-utilizing fungi were relatively higher. Some of the fungi species isolated from the soil were *Candida, Alternaria, Mucor, Rhodotorula, Penicillium, Saccharomyces, Trichoderma, Rhizopus,* etc. they concluded that high concentration of crude oil has a significant adverse effect on the fungal population and diversity. These are effects of crude oil pollution in the ecosystem. They further recommended that this harmful effect justifies the need for bioremediation.

#### **3. Biotechnological tools in eco recovery of crude oil polluted soil**

Biotechnology is defined as the set of scientific techniques that makes use of biological systems or living organisms to make, modify or improve products which

#### *Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*

may be products mays be plants or animals [25]. It has also been defined as a process which involves developing organisms for specific purposes and it includes the use cell fusion, recombinant DNA and other novel bioprocess technologies [26]. Biotechnological tools in eco-recovery of crude oil polluted sites are those biotechnological processes that involves the use of bio-products and also microbes for production of environmentally friendly products, reduces pollution and its effect, and all general restoration and maintenance of the environment to its pristine (natural) state for the benefit of man and the environment [2]. Biotechnological tools in eco recovery are also concerned with prevention of processes capable of causing an unsustainable environment for man and eco-components. There are no known number of bio tools used in prevention or restoration of a polluted environment, however the most successfully applied, eco-friendly and cost effective tool in environmental decontamination is bioremediation. The different types of bioremediation (biosurfactant-enhanced bioremediation, microbial bioremediation, plant bioremediation, genetically modified microorganisms in bioremediation), mechanism of action and factors influencing the process will be discussed in this chapter.

Before now, remediation of contaminated/polluted environments have been carried out using conventional methods such as to cap and contain the contaminated areas of a site or digging up contaminated site and removing it to a landfill. These methods have however had some drawbacks. The first method is however just a temporary solution as the contaminants may still linger on the site and may further require monitoring and maintenance in the future, this leads to increased cost. In the landfill method, the contaminated soil is excavated moved to a different site and the excavation and transporting of the contaminants may pose a serious environmental risk, it might also prove expensive to find new sites for the disposal of the contaminated soil [27]. These drawbacks have led to the search for a better approach which would include transforming the pollutants to a harmless substance or a complete destruction of the pollutants if possible [28].

The use of biotechnology which entails the application of genetic modifications to improve the ratio of work done and reduce cost associated with remediation and eco restoration process have become a major factor for the increased exploitation of biological systems in waste reduction and eco restoration. Due to the urgency in the need for an effective and efficient biotechnological process and the need for a process that completely destroys the pollutants, researchers have come up with a technique for rehabilitating either contaminated sites or sites that have been degraded due to anthropogenic activities and the mismanagement of the eco system. This process is called bioremediation and it involves the application of living microorganisms to degrade environmental pollutants or to prevent pollution. The different strategies/tools used in bioremediation of oil spills include bio-stimulation, bio-augmentation, use of genetically engineered microbes, nutrient application, seeding with competent or adapted hydrocarbonoclastic bacteria or their consortium. Some of these Environmental biotechnological tools for the clean-up of crude oil contaminated sites are highlighted here.

#### **3.1 Bioremediation**

Bioremediation has been defined as the process of removing toxic waste from the environment using biological agents. According to Kumar et al. [27]. It was defined as the most effective tool to manage waste polluted environment and recovery of contaminated soil. Bioremediation have been carried out both in situ and ex situ in several sites around the world with very successful outcomes. This method is considered a non-invasive, cost effective and sometimes logistically favorable clean-up technology which attempts to accelerate the naturally occurring biodegradation

of contaminants through the optimization of the prevailing conditions [29, 30]. Bioremediation alongside natural attenuation have provided solution for emerging contaminant problems using actions such as biological carbon sequestration, landfill stabilization, endocrine disrupters and mixed waste biotreatment. Plants and microorganisms play roles in the remediation of contaminated environment; thus, the purpose of bioremediation is to reduce the potential toxicity of chemical contaminants in the environment via degradation, transformation, and immobilization of the undesirable compounds through the introduction of biosystems such as higher organisms like plants (phytoremediation), microbes, and animals. Some of the microbes involved in bioremediation process may include aerobes, anaerobic bacteria, fungi and other microbes with degradative potentials. Several microorganisms including *Mycobacteria, Pseudomonas*, *Bulkhoderia*, *Enterobacter, Acinetobacter, Alcaligenes*, *Bacillus*, *Proteus* various *Corynebacteria* and some yeasts have been confirmed to degrade or utilize oil as a source of food [12, 31, 32]. Papadaki and Mantzouridou [33] reported that microorganisms involved in bioremediation are capable of converting or degrading contaminants such as crude oil that can be used as energy source. Microorganisms can also adapt to the stresses caused by pollutants in the environment due to some characteristics such as metabolic potentials that are inherited during natural selection or resistance to the pollutants and this contributes to the recovery and soil restoration process [34]. In a report by Adetutu et al. [35] microbes were able to remediate up to 46.6% of the oil in a contaminated soil in 320 days.

Several bioremediation strategies have been explored for treating different sites but most have been designed for land oil spill control. These strategies attempts to increase the efficiency of natural attenuation process and they include: landfarming, composting, use of bioreactors, bioventing/biosparging, pump and treat, bioslurping, biostimulation, and bioaugmentation [36]. A description of the in-situ and ex-situ bioremediation techniques is presented in **Table 1.**

#### *3.1.1 Biosurfactant-enehanced bioremediation*

Many microorganisms involved in bioremediation produce potent surfaceactive compounds that can emulsify oil in water called biosurfactants and unlike chemical surfactants, the microbial emulsifier is biodegradable and non-toxic thereby facilitating the removal of hydrocarbon pollutants especially in the marine environment [43]. Biosurfactants can improve hydrocarbon bioremediation by two methods; the first incorporates the increment of substrate bioavailability for microorganisms, while the other method includes interaction with the cell surface which builds up the hydrophobicity of the surface allowing hydrophobic substrates to relate more effectively with bacterial cells [44]. By bringing down surface and interfacial tension, biosurfactants causes an increment to the surface areas of insoluble compounds prompting expanded portability and bioavailability of hydrocarbons. In outcome, biosurfactants upgrade biodegradation and removal of hydrocarbons. Biosurfactants are known to increase biodegaradation of highly hydrophobic compounds such as aromatics, alkanes, resins, cycloalkanes [45] by increasing bioavailability of the hydrophobic compound through facilitated transport of the pollutants from the solid phase (such as communication between surfactants and hydrocarbons, communication between contaminants and single biosurfactant molecules), improvement on the apparent solubility of the contaminants (improve the apparent solubility of the hydrophobic organic compound), and emulsification of non-aqueous phase liquid contaminants (in this process biosurfactants can lower the surface tension between non-aqueous and aqueous phases, this then leads to an increase in improving mass transport, the contact area, and mobilization liquid-phase contaminants).


*Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*

> **Table 1.**

*The most applicable in-situ and ex-situ bioremediation techniques for hydrocarbon removal.*

Biosurfactants may be secreted outside the cells (extra-cellular) or located inside the cells (intracellular) [46]. Based on myriads of documented reports available on bacterial bio-surfactants, it has been established that the spectrum of activity depends on the chemical composition of the pollutant. A strain of *Pseudomonas aeruginosa* was reported by Patel et al. [47] to produce the rhamnolipid type of biosurfactant which was mono as well as di-rhamnolipid. Rhamnolipid and its producing microorganism has been implicated in the specific degradation of hexadecance which clearly shows that there is a strong relationship between the type of surfactant and the type of hydrocarbon/oil that gets degraded. In another related study, a group of bacteria producing glycolipids and sophorolipids significantly degraded polycyclic aromatic hydrocarbons. Chakrabarti, [48] reported that in the presence of glycolipids Surface active glycolipids when introduced in to the hydrocarbon polluted environments have improved the biodegradation of 2,4-DCPIP.

Bacteria produce biosurfactants in the form of biofilm which interacts with an interface and alters the surface properties such as wettability and other properties. Biosurfactant producing bacteria have been to be isolated from different environmental compartments including the marine environment. A marine bacterium, *Pseudomonas aeruginosa* was isolated from sea water polluted with oil. This organism successfully degraded nonadecane, heptadecane, hexadecane and octadecane, after 28 days of incubation. This same bacterium has also effectively degraded other components of hydrocarbons such as pristane, tetradecane and 2-methylnaphthalene [49, 50]. The degradation ability of this bacterium has been proven to be due to the production of a bio-surfactant. In another experiment, two biosurfactantproducing strains; *Pseudomonas* ML2 and *Acinetobacter haemolyticus* were inoculated into a hydrocarbon contaminated soil to monitor and study the biodegradation potentials. After two months incubation period, a drastic reduction in the hydrocarbon concentration (11–71%) and (39–71%) was observed by *Pseudomonas* ML2 and *Acinetobacter haemolyticus*, respectively. These results suggests the remarkable hydrocarbon degradation ability of cell free biosurfactant produced by bacteria. Several biosurfactants have been produced by various microbes which include: rhamnolipids (*P. aeruginosa*), liposan (*C. lipolytica*), surfaction (*B. subtilis*), emulsan (*A. calcoaceticus*), sophorolipids (*T. bombicola*), carbohydrate-protein-lipid (*Microbacterium sp*.) viscosin (*P. fluorescens*) and serrawettin (*S. marcescens*) [50–53].

#### *3.1.2 Mechanism of action of bioremediation*

Microbial-assisted bioremediation explores the potentials of naturally occurring hydrocarbon degrading microbes (oleophilic microbes) or plants in the detoxification/degradation/mineralization of hazardous substances to human health and the environment. These microbes can either be native to the contaminated area or could be introduced from a similar site into the contaminated soil, a process called bioaugmentation [54]. Bioremediation occurs most frequently by the action of microbialmediated degradation. This process is often achieved by the action of consortia of organisms and for bioremediation to be effective, there must be complete mineralization of the hydrocarbon which occurs through a series of enzymes harboring catabolic genes to produce harmless products such as CO2 and H2O [55].

Biodegradation of petroleum hydrocarbons is a complex process that depends on the nature and on the amount of the hydrocarbon present. Petroleum hydrocarbons are divided into four broad categories: Saturates (branched, unbranched and cyclic alkanes), aromatics-ringed hydrocarbon molecules such as monocyclic aromatic hydrocarbons (MAHs) and polycyclic aromatic hydrocarbons (PAHs), resins (Polar oil-surface structures dissolved in saturates and aromatics) and asphaltenes (dark-brown amorphous solids colloidally dispersed in saturates and aromatics).

#### *Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*

These various categories respond differently to biodegradation as a result of their chemical structures and molecular weight. For example, PAHs, asphaltenes and resins are considered highly recalcitrant because of their high molecular weight [56].

Microbial degradation is a major route and ultimate natural mechanism by which one can clean up petroleum hydrocarbon pollutants from soil environment [57]. Typically, an individual microorganism will biodegrade a limited number of hydrocarbons whereas a microbial consortium can biodegrade an impressive array of hydrocarbons collectively [58, 59]. Onuoha et al. [60] reported that Nigerian soil especially in the Niger Delta region, may harbor a significant population of hydrocarbon degraders as a result of the increased multifarious activities of the oil industry within the region. The result of the investigation revealed that an appreciable number of bacterial isolates showed different degrees of degradation in mineral salt medium using spent oil as sole source of carbon. In a similar study, Chikere and Ekwuabu [61] conducted an investigation in Bodo community, Ogoniland, Nigeria to characterize the active culturable indigenous hydrocarbon utilizing microbial population. A significant population of hydrocarbon utilizing bacteria and fungi corresponding to the long-term impact of crude oil in the study area was observed. The hydrocarbon degrading microbes have an inherent genetic capacity to assimilate hydrocarbons and/or its products [62]. The process is therefore regarded as a complex biological oxidation process involving mostly aerobic organisms which may be enhanced by supplementation with fixed nitrogen, phosphate and other rate-limiting nutrients. Microorganisms have enzyme systems that can degrade and utilize different hydrocarbons as source of carbon and energy [63, 64]. The driving force for petroleum biodegradation is the ability of microbes to utilize hydrocarbons, to satisfy cell growth and energy.

Biodegradation may occur spontaneously and the process is called natural attenuation. In most cases however, this might take a longer time and this could be as a result of inability of the natural circumstances of the contaminated site to favor the natural attenuation process [65]. Also, it may be due to inadequate number or diversity of microorganisms with specific enzyme system required to break down the contaminant and lack of favorable environmental conditions to support the process. Such situations can be improved by supplying one or more of the missing/ inadequate microbes, developing oil eating bugs through genetic engineering/ recombination, introducing rate-limiting nutrients or enhancing environmental factors to favor the active degraders. It was reported that extra nutrients were added to accelerate the breakdown of oil spill caused by the super tanker Exxon Valdez on the Alaskan shoreline [66]. Since numerous types of pollutants are to be encountered in a contaminated site, diverse species of microorganisms are likely to be required for effective mediation [67].

#### **3.2 Microbial remediation**

Microbial bioremediation strictly involves the use of microbes or their derivates (Enzymes, biomass) to degrade or transform xenobiotics for the detoxification of crude oil polluted environments. Microorganisms are ubiquitous, therefore pollutants in the different environments come in contact with these oleophilic microbes. Specifically, the hydrocarbon degrading microorganisms (bacteria, fungi, algae) are able to breakdown these pollutants because of their inherent genetic capabilities to mineralize these hydrocarbons through metabolic pathways. Microbial bioremediation technology in the long run promotes the growth of specific microflora or the microbial consortia, indigenous to the contaminated sites that are able to perform the desired activities. In the process, microorganisms use the contaminants as source of energy or nutrient. The microbial consortia can perform this

role optimally by either adding terminal electron acceptor or promoting microbial growth by adding nutrients [27]. In oil contaminated sites as it relates to this review, oil spills can be broken down using multiple techniques which includes the microbes feeding on the crude oil or addition of fertilizers/nutrients to the contaminated site to accelerate the decomposition of crude oil by the microorganism present in the soil or by introducing hydrocarbon degrading bacteria from exogenous sources to augment the indigenous population. As regards to crude oil contaminated environment, bioremediation process exploits the catabolic ability of microorganism to feed on oil. Research frontiers globally have described various application of microorganisms in the bioremediation of oil pollution under controlled conditions, field scale and in different environmental conditions, with very encouraging results [13, 15, 55]. The natural existence of a large diversity of microbial species expands the variety of chemical pollutants that are degraded or detoxified [68].

So many microorganisms have been reported with hydrocarbon remediation potentials which are *Bacillus* spp. (degradation of hydrocarbons and phenoxy acetates) [15], *Pseudomonas* spp. (degradation of benzene, anthracene, and PCBs) [69, 70] also *Azotobacter* species (degradation of benzene and cycloparaffin) [70] and so many other microbes as previously discussed. White rot fungi have also been reported to have greater access to poor bio-available substrates, since they secrete extracellular enzymes involved in the oxidation of complex organic and inorganic matters [13]. For example, direct application and incubation of fungal laccase in hydrocarbon contaminated soils for 14 days led to the reduction of the PAHs such as benzo(a)pyrene and anthracene by about 80% [71, 72].

Microbial bioremediation technique has some advantages over other clean-up methods such as: public acceptance, a naturally occurring process, low cost technology, it can be done in situ and ex situ, instead of contaminants being transferred from one form to another or one medium to another, complete destruction of target organic pollutants is possible to produce non-toxic substance and it can lead to eco-restoration of the polluted medium [40, 68]. Some oleophilic microbes and their hydrocarbon specificity are presented in **Table 2.**

#### *3.2.1 Factors that influence microbial bioremediation*

There are a few factors that contribute to the success of microbial bioremediation, some of these factors may include the growth and survival of microbial populations, the ability of these organisms to come into contact with the substances that need to be degraded into less toxic compounds, cation exchange capacity, relevant nutrient availability, acidity (soil pH), aeration or oxygen (electron acceptor level), water solubility, temperature, enzyme activity, hydraulic properties, [31] water content, site condition, microbial communities, sufficiency of the numbers of microorganisms and the habitability of the microbial environment for the microbes to thrive [89]. Sometimes the environment might be too toxic for the microorganisms to survive, in this case, the microbes should be engineered to be able to survive the high toxicity. Also, bioremediation works best in soils that are relatively sandy because sandy soils allow mobility and greater likelihood of contact between the microbes and the contaminant [90]. Therefore, for any bioremediation process to be successful, the environmental factors that play major roles in the process must first be understood.

The process of bioremediation may not always result in the complete mineralization of organic compounds, some of the organic compounds are transformed naturally to other metabolites and the toxicity and persistence of these new metabolites are mostly unknown [91]. Compliance analysis requires examination of the contaminated site in the light of the governing regulation and the action plan.

*Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*


#### **Table 2.**

*Oleophilic microorganism and hydrocarbon specificity.*

For a successful bioremediation, the site must first be examined and characterized and this is a very challenging and difficult aspect of a bioremediation efforts. Some factors that influence microbial degradation of hydrocarbons in the environment is present are presented in **Table 3.**

#### **3.3 Plant bioremediation (phytoremediation)**

This is one of the biotechnological approach/tools in which plants are used in the clean-up of contaminated environments. It is an emerging technology and it promises a cost friendly, less-intrusive and effective clean up and restoration of crude oil contaminated soils [65]. It can also simply be defined as a process of using plants and plant-associated microorganisms such as Arburscular Mycorrhizal fungi (AMF) or plant growth promoting rhizobacteria (PGPR) to clean up contaminated soils. It is an inexpensive, non-invasive alternative for other remediation methods such as the chemical/engineering-based methods [31]. Green plants are solar-driven, and are an effective filtering system endowed with fouling and degradative abilities [92]. It has been reported that salt marsh plants such as *Spartina alterniflora, Sagittaria lancifolia, Spartina patens* and *Juncus roemeriannus* are able to take up hydrocarbons from oil-contaminated sediment [93, 94]. Godheja et al. [95] reported that *Dioscorea* sp. have been reported to be able to metabolize petroleum hydrocarbons such as n-hexane and also Enzymes such as peroxidases and cytochrome P450 found in the plant *Dioscorea composita* was involved in the biotransformation of hydrocarbon.

In an experiment by Olusola and Anslem [96]. A plant (*Amaranthus hybridus*) was cultivated in a nursery and then transplanted into experimental pots containing crude oil contaminated soils. A white rot fungus (*Pleurotus pulmonarius*) and a mycorrhizal fungus (*Glomus mosseae*) were introduced into some of the different pots to study the ability and the degree of bioremediation of crude oil contaminated soils by these fungal species. The results showed that plants which were grown in the crude oil polluted pots without any of the fungal species died within two weeks while the pots with the soil samples which were inoculated with the fungus survived. The contaminated soil sample inoculated with *Glomus mosseae* showed the best result in terms of plant growth. They concluded that biological treatments are the best methods for cleaning or remediating contaminated soils. They also suggested that


#### **Table 3.**

*Factors that influence microbial degradation of petroleum hydrocarbon in the environment.*

#### *Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*

certain plants which have associations with microorganisms such as the AMF species *Glomus mosseae* could play roles in the clean-up of crude oil contaminated soils.

Plants and plant-associated microorganisms are both involved in phytoremediation process. The plants used must first be tolerant to the pollutants, encourage the growth of rhizospheric microorganisms and in turn these microorganisms can secrete oil degrading enzymes and thereby generate energy in a process called rhizodegradation. However, there is a major setback with this process in that plants tend to compete with the hydrocarbon degrading microorganisms for the available nutrients like fixed nitrogen and phosphorus.

#### *3.3.1 Mechanisms of action of phytoremediation*

Phytoremediation offers potential for restoring large areas of contaminated environments requires certain mechanisms for a successful remediation process. Plants are able to remove pollutants through processes such as biodegradation, phytovolatilization, accumulation, and metabolic transformation. Several factors determine the most effective phytoremediation mechanism to adopt, such as the bioavailability of the contaminant, type of contaminant, soil properties and other environmental factors that support plant growth and activities. There are several routes through which plants decontaminate polluted sites, however, the primary channel for plant uptake of contaminants is through the root systems (rhizosphere) which harbors the essential components required for decontaminating toxic substances. The rhizosphere of plants has a large surface area responsible for the absorption and accumulation of essential nutrients and water required for growth. A large diversity of microorganisms are usually found in this region because of the exudates and enzymes released which stimulates the activities of microorganisms capable of degrading hydrocarbons present in the soil, direct biochemical transformation of petroleum hydrocarbons, and have also shown resistance to crude oil toxicity [97]. Rhizospheric interactions between host plants and the microorganisms that are resident in the rhizosphere are critical to the phytoremediation process. Host plants enrich the rhizosphere by releasing root exudates that help in recruiting the beneficial pollutant degrading bacteria and other microorganisms to the rhizosphere. In a report, a plant growth promoting rhizobacteria *Pseudomonas putida KT2440* was recruited due to the production of 2,4-dihydroxy-7-methoxy-1,4-bezoxazin-3-one, a root exudate produced in Maize seedlings [98].

There are several other phytoremediation mechanisms which include; phytoextraction, phytostabilization, phytofiltration, phytodegradation, phytovolatilization rhizodegradation and phytostimulation (**Figure 1**). *Phytoextraction/ phytoaccumulation* is a process of absorption or translocation of contaminants from the roots to other parts of the plants. Rhizofiltration is a process which involves the roots removing contaminants from water bodies, thus causing the water to be filtered. This mechanism is closely related to phytoextraction but it strictly applies to the aquatic environment. Phytostabilization involves binding the contaminants to the roots of plants which leads to the immobilization of the contaminants and thus reducing leaching of the contaminants from soil [98]. Phytodegradation involves the secretion of exudates by the roots to break down contaminants which are then removed via transpiration and uptake. Phytostimulation involves the enhancement of the microbial activity in the rhizosphere to facilitate the breakdown of organic contaminants. Phytovolatilization is a process that involves the removal of contaminants from the soil by volatilizing them into thin air [99]. These various methods have proven to be effective for petroleum hydrocarbon degradation.

**Figure 1.** *Mechanism of phytoremediation of crude oil.*

#### **3.4 Genetically modified microorganism for enhanced eco-recovery**

Generally speaking, microbial degradation of xenobiotics involves the utilization of microbes with specific enzyme systems responsible for the degradation, mineralization, transformation or detoxification of pollutants [100]. Nevertheless, under certain growth conditions, composition, type and concentration of the pollutant, effective degradation is not expected even with the availability of microbes with degradation potentials. Compounds like Polychlorinated biphenyls (PCBs), synthetic group of chlorinated aromatic hydrocarbons and other Organic compounds, due to their complex organic structure, is posing persistent and deleterious threats to the ecology and human health even for decades [101–107]. Therefore, it has become imperative to design and develop alternative hydrocarbon degradation arrangement with specific degradation genes to the available pollutants in the environment by cultivating microbes with engineered catalytic capabilities [108].

Genetically Engineered Microorganisms (GEMs) can be obtained by recombinant DNA technology/genetic engineering of microbes or by natural exchange of genes by bacteria in the environment through horizontal gene transfer of plasmid-borne genes. The application of GEMs in bioremediation of xenobiotics have shown great potentials in soil [103], groundwater [102] and other environmental components exhibiting improved mineralization abilities over a broad-range of contaminants.

The use of GEMs represent a research frontier with wide application which extends to phytoremediation. Jain and Bajpai [108] reported a number of applications available in enhancing the degradative performance of oleophilic microbes using genetic engineering approaches. A very significant example is the genetic modification of rate-limiting steps in the metabolic pathway of hydrocarbon degrading microbes to yield increase in mineralization rates or the development of completely new metabolic pathways incorporated into the bacterial strains for the degradation of highly persistent compounds.

The first GEM, *Pseudomonas fluorescence* HK44GEM was designed to perform diverse functions in petroleum hydrocarbon degradation. The wild type,

#### *Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*

*Pseudomonas fluorescence* strain was cultivated from a PAH contaminated soil. Naphthalene catabolic compound (Vector PUTK21), a transposon-based bioluminescence producing lux gene fused with promoter naphthalene catabolic gene were introduced into the *P. fluorescence* to form *P. fluorescence* HK44GEM. Upon trial in the presence of naphthalene or its intermediate (Salicylate) enhanced catabolic gene expression, naphthalene degradation and concomitant bioluminescent response was observed. The GEM was capable of sensing and responding to environmental pollutants through an early detectable signal such as bioluminescence. The bioluminescence signaling in strain HK44GEM also served as a reporter for naphthalene bioavailability and biodegradation.

Additionally, since oil is a mixture of various hydrocarbons (n-alkanes, aromatic hydrocarbons, polycyclic aromatic hydrocarbons), the construction of engineered bacteria capable of degrading various petroleum hydrocarbons by genetic engineering technology is a development direction to control crude oil pollution. The degradation of some petroleum components by microorganisms is controlled by an extrachromosomal plasmid; therefore, superbugs (product of genetic engineering: oil eating bug) can be constructed by introducing plasmids with capabilities for degrading different components in a single cell.

A recombinant *Acinetobacter baumannii* S30 pJES was constructed by inserting the lux gene into the chromosome of the *A. baumannii* S30, a strain with the biodegradation efficiency for total petroleum hydrocarbon (TPH) of crude oil. Thus, the persistence of strain *A. Baumanni* S30 PJES was observed and confirmed at the bioremediation site after the genetic engineering process site [109]. Also, a recombinant strain M145-AH constructed by overexpressing alkane monooxygenase (encoded by alkB gene) in a non-alkane-degrading actinomycete *Streptomyces coelicolor* M145 wasto exhibit a high ability observed to degrade n-hexadecane [110].

Genetically modified microorganisms such as bacteria including *E. coli* and *Pseudomonas*, fungi including *Aspergilus niger* and *Rhizopus arrhizus* and also algae, e.g., *Chlorella vulgaris* and *Anabaena variabilis* and others microbes, have been engaged in degradation of various compounds such as toluene, oil spills, naphthalene, camphor, hexane, octane, xylene, halobenzoates and others. Engineered microbes are more potent than the natural strains when it comes to degradation due to their higher degradative capacities. Advantageously, this engineered microorganism can quickly adopt pollutants as their substrates [111–114].

#### **4. Future prospects**

Microbe-assisted contaminant reduction and in-depth analysis of the organisms' metabolisms have over time accelerated the overall bioremediation process. However, in the next decade, molecular manipulations and the decryption of the cellular mechanisms using an integrated OMIC tool approach will play major roles in bioremediation processes [115].

Recently, a key area of modern-day scientific advancement in the removal of pollutants from the environment (either in soil or groundwater) is the nanoparticles empowered remediation. Green nanoremediation as a nature-based technology offers numerous promises for the cleanup and restoration of polluted soils such as crude oil polluted soils with reference to the efficiency of the process, energy consumed and the global need for eco-friendly processes [116]. Wang et al. [117] reported the use of silica nanoparticles capped with lipid bilayers of *Pseuodomonas aeruginosa* as method of cleaning up of PAH (benzo[a]pyrene) from contaminated soil surface.

Some of the all-round benefits of the use of green nanotechnology as a biotechnological tool for remediation of crude oil polluted sites may include the rapid removal of pollutants, reduced usage of hazardous substances and the cost effectiveness. Nanobioremediation might contribute immensely to the sustainability of the environment because of these benefits when compared to other methods of remediation. The copulation of biological entities with nanomaterials have furthermore demonstrated enhanced effectiveness in the degradation of contaminants in soil and water. This can be seen as a future possibility in facing environmental challenges. Dave and Das [118] reported that nanoparticles can potentially bind with xenobiotic compounds and can either transform them into less harmful byproducts or completely degrade them, this process can help in the clean-up of contaminated environment. The requirement for any ideal bioremediation process relies on the use of an environmentally friendly and efficient approach. These above-described technologies are complete for the effective bioremediation process. Also, as part of Nanotechnological tools in bioremediation, nanobiosurfactants provides unique properties which makes them potentially strong candidates for ecofriendly nanobioremediation in the future [119].

Some inorganic sensors have also been developed and applied in nanotechnology [120] to trace and identify contaminants/pollutants in the environment which will inform the most suitable/appropriate biotechnological tool to be applied for clean-up process. In another study, it was reported that the use of oxygen-sensitive proteins to develop oxygen biosensors is an emerging field which can be adopted for the preparation of nanomaterials that are able to respond to oxygen levels and other specific components of pollutants [121]. Ryu et al. [122] extensively reviewed the field of Transmembrane proteins which were incorporated into membranes coupled to several trandusctors and observed that this approach can be successfully applied in pesticide detection, monitoring of gases, microarray etc. Based on this, [123] recommended that in the future, hydrocarbon catabolic enzymes may also be incorporated to monitor more complex pollutants such as Polycyclic aromatic hydrocarbons (PAHs). These concepts, explored with proteins will open a wide area of sensing and detoxification opportunities in bioremediation. Techniques such as biofilm formation and whole-cell immobilization for the removal and recovery of soils containing pollutants such as heavy metals and PAHs have also gained attention [124].

#### **5. Conclusion**

The various biotechnological tools in ecorestoration of crude oil polluted environment outlined in this review has been confirmed to be eco-friendly and effective in the mineralization of the pollutants. Biosurfactant-producing microbes contribute significantly to enhancing microbial bioremediation by increasing bioavailability. Microorganisms produce a wide range of surfactants with hydrocarbon specificity. Microbial bioremediation and phytoremediation have both yielded positive results in environmental studies under favorable conditions and growth conditions, respectively. The genetic engineered microbes in bioremediation favor the degradation of recalcitrant hydrocarbons and increase the rate of degradation. Although this method is still under investigation based on environmental and ecological risk. This review has highlighted known eco-friendly approaches of bioremediation of polluted sites using several biotechnological tools.

*Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment DOI: http://dx.doi.org/10.5772/intechopen.98808*

### **Author details**

Chioma Bertha Ehis-Eriakha1 \*, Stephen Eromosele Akemu1 , Simon Obgaji Otumala1 and Chinyere Augusta Ajuzieogu2

1 Department of Microbiology, Edo State University Uzairue, Edo State, Nigeria

2 Department of Microbiology, Renaissance University, Enugu State, Nigeria

\*Address all correspondence to: bertha\_chioma@yahoo.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Actinomycetes as An Environmental Scrubber

*Sutaria Devanshi, Kamlesh R. Shah, Sudipti Arora and Sonika Saxena*

#### **Abstract**

Biotechnological tools engaged in the bioremediation process are in reality, sophisticated and dynamic in character. For specialized reasons, a broad variety of such devices are employed to produce a safe and balanced environment free of all types of toxins and so make life simpler for humans on planet Earth. Actinomycetes is one of these extremely important and functionally helpful groups. They can be used for a variety of bioremediation objectives, including biotransformation, biodegradation, and many more. Actinomycetes are one of the most varied groups of filamentous bacteria, capable of prospering in a variety of ecological settings because to their bioactive capabilities. They're famous for their metabolic diversity, which includes the synthesis of commercially useful primary and secondary metabolites. They produce a range of enzymes capable of totally destroying all of the constituents. They are well-known for their ability to produce bioactive secondary metabolites. Members of various genera of Actinomycetes show promise for application in the bioconversion of underutilized urban and agricultural waste into high-value chemical compounds. The most potential source is a wide range of important enzymes, some of which are synthesized on an industrial scale, but there are many more that have yet to be discovered. Bioremediation methods, which use naturally existing microbes to clear residues and contaminated regions of dangerous organic chemicals, are improving all the time. In the realm of biotechnological science, the potential of actinomycetes for bioremediation and the synthesis of secondary metabolites has opened up intriguing prospects for a sustainable environment.

**Keywords:** Actinomycete, Bioremediation, Biotransformation, Biodegradation, Sustainable environment, Bioactive compounds

#### **1. Introduction**

The word 'actinomycete' comes from the Greek words 'atkis' (ray) and'mykes' (fungus), both of which contain bacterial and fungal features. Actinomycetes are gram-positive bacteria that generate spores and are found in nature aerobically. They are one of the most important taxonomic units among the 18 major lineages currently recognized within the domain bacteria. The Actinobacteria class is an important part of the microbial population in soils. Their metabolic variety and unique growth features make them ideal bioremediation agents [1–12]. Actinobacteria are a worldwide collection of microorganisms that may be found in

a variety of natural habitats [13]. They are Gram-positive bacteria with a high guanine plus cytosine (G1C) content base in DNA (5575 mol%), however new species have been discovered that do not follow this norm. This group is extremely diverse, as it contains a wide range of microorganisms that differ chemically, morphologically, and physiologically [14]. This morphological variety is demonstrated by a constant shift from basal and bacilliform cells to hyphae that break and branch, generating aerial mycelium with lengthy chains of spores. Actiospores are generated as a result of nutrient deficiency and may withstand protracted desiccation [15]. This capacity to sporulate is critical for their survival in the wild. Temperatures of 2530C and neutral pH are ideal for growth in most cases, however several species have been separated from harsh settings. Most of these bacteria are aerobic, although some may also be microaerophilic or anaerobic. They are heterotrophic, which means they can use both simple and complicated carbon sources [16]. The creation of a significant number of biotechnologically significant metabolites (antibiotics, enzymes, enzyme inhibitors, immunomodulators, and so on) demonstrates physiological variety [14]. Additionally, due to the formation of a metabolite called geosmin, they require a specific odor of damp soil as part of their unique traits.

#### **1.1 Occurrence and habitat**

Actinomycetes are the foremost abundant life style saprophytes that form thread-like filaments within the soil. They grow as hyphae like fungi liable for the characteristically "earthy" smell of freshly turned healthy soil. The actinomycetes exist in various habits in nature and represent a ubiquitous group of microbes cosmopolitan in natural ecosystems around the world. Actinomycetes are widely distributed in soil and ocean. There are many reports for isolation of actinomycetes from terrestrial soils [17, 18], marine ecosystem [19, 20], mangrove ecosystem [21, 22], composts, vermicomposts [23]. Environmental factors influence the type and population of actinomycetes in soil. They are found both on mesophilic (25-300C) and thermophilic (400C) environments. The pH is also a major environmental factor determining the distribution and activity of actinomycete. Most of the actinomycetes grow at optimum pH around 7. Vasavada et al. [24] showed that pH, salinity, use of media and carbon and nitrogen sources affect the growth and antibiotic production by actinomycetes. Many mesophilic actinomycetes are active in compost in initial stages of decomposition. However the capacity for self-heating during decomposition provides ideal conditions for thermophilic actinomycetes. Actinomycetes diversity also can be influenced by the range of plant species grown thereon particular soil. Since different plants produce different chemical metabolites, so as to survive the microbes (actinomycetes during this case) got to adapt to the environment [25]. As soil is the best source of Actinomycetes, much research has been focused on the soil ecology. They may be found in a variety of soils, both cultivated and uncultivated, fertile and infertile, in diverse parts of the world. pH is a major environmental element that determines the distribution and activity of soil Actinomycetes. Neutrophiles are found in less numbers in acidic soils with pH < 5.0, but acidophilic streptomycetes are plentiful. Many active mesophilic Actinomycetes may be found in compost. The research have recently been conducted in aquatic settings such as fresh and marine waters. Many researchers saw Actinomycetes as part of the native microflora of marine ecosystems, whereas others saw them as wash-in elements that persist as spores in marine and littoral sediments. Salt tolerance has been demonstrated in creatures from maritime settings. Actinomycetes have been studied for their presence, survival, and activity in a variety of severe settings. Streptomycetes, both acidophilic and aciduric, are common in acidic soils. Actinomycetes have been found in hot springs, marine sediments, and crater lakes, among other harsh habitats

(soda lakes). Lonar Lake, a crater produced by meteorite impact, provides a unique opportunity. Because of the high sodium carbonate concentration, the lake water is salty and alkaline (pH 9.5 to 10.0).

#### **1.2 Structure**

The development of usually branched threads or rods distinguishes actinomycetes. In most cases, the hyphae are non-septate. The sporulating mycelium may be branching or non-branching, straight or spiral. Spherical, cylindrical, or oval spores are found. They resemble fungus morphologically, which might be related to the fact that their cell wall composition is similar to that of gramme positive bacteria. They have been separated from ordinary bacteria because of their filamentous form and cultural traits.

#### **1.3 Actinobacteria: growth and reproduction**

This diversified group has a lot of morphological differentiation, including septate and nonseptate multicellular strands and a filamentous-type structure. Strains often form compact colonies on solid culture medium, consisting of mycelium, a mass of hyphae pertaining to the microbe, and distinguishing into aerial and substrate mycelium. Actinobacteria have a modest growth rate in general. After 24 hours of incubation, a branching mycelium forms on the surface of a solid medium, which may be examined under a microscope; colonies form after 34 days, but mature aerial mycelium with actinospores occurs after 714 days as shown in **Figure 1**. Some strains that develop slowly may take up to a month to incubate. The development and stability of the substrate and aerial mycelium can be influenced by the culture medium composition. Colonies of Actinobacteria can be elevated or laid flat. Their texture ranges from incredibly soft to exceedingly hard and pasty. White, yellow, orange, pink, red, purple, blue, green, brown, and black are among the colors available. They might have smooth, grooved, wrinkled, granular,

**Figure 1.** *Life cycle of Actinomycetes.*

or flaky surfaces. Their appearance is frequently totally compact, or a mixture of both, with diverse developing zones in concentric rings of radial orientationThe colony size ranges from one millimeter to a few centimeters in diameter, depending on the species, age, and cultivation circumstances. Actinobacteria may grow on liquid medium as well, but only under certain circumstances. To achieve uniform growth, liquid cultures require agitation and aeration, as well as suspension in culture media. Actinobacteria are a common microbial community found in soil, with an average of 5631010 CFU/g of soil. Actinobacteria are found in the soil as latent spores that produce their mycelia only when specific environmental circumstances are ideal, such as nutrition availability, humidity, temperature, or physiological interactions with other microbes. These organisms are investigated for biotechnological applications, particularly in bioremediation of harmful chemicals, because to their metabolic variety and relationship with the environment. The interaction of Actinobacteria with accumulated pollutants in the environment, such as oil, rubber, plastics, pesticides, and heavy metals, has been investigated for more than 20 years [6, 10, 24]. The life cycle of the actinomycetes is shown **Figure 1**. Actinomycetes are mostly mycelioid and Gram-positive. They begin as unicellular creatures, but eventually evolve into branching filaments or hyphae, which multiply rapidly by generating new branches, forming the mycelium. As seen in **Figure 1**, this type of mycelium is known as "substratum or primary mycelium." After a period of development, hyphae of a different sort emerge from the mycelium substratum and begin to grow in the air. Aerial hyphae and aerial or secondary mycelium are the terms used to describe these hyphae. Sheath is an additional cell wall layer seen on aerial hyphae.

#### **2. Classification of actinomycetes**

The "Bergey's Manual of Systematic Bacteriology - 2nd edition" for Actinobacteria classification has five volumes, which contain internationally recognized names and descriptions of bacteria. Classification of Actinobacteria has been rearranged. In Volume 5, the phylum Actinobacteria is split into six classes, namely Actinobacteria, Acidimicrobiia, Coriobacteriia, Nitriliruptoria, Rubrobacteria, and Thermoleophilia. The class Actinobacteria is further divided into 16 orders that are Actinomycetales, Actinopolysporales, Bifidobacteriales, Catenulisporales, Corynebacteriales, Frankiales, Glycomycetales, Jiangellales, Kineosporiales, Micrococcales, Micromonosporales, Propionibacteriales, Pseudonocardiales, Streptomycetales, Streptosporangiales, and Incertae sedis. In the order of abundance in soils, the common genera of Actinobacteria are Streptomyces (nearly 70%), Nocardia, and Micromonospora, although Actinoplanes, Micromonospora, and Streptosporangium also are generally found.


**Table 1.** *Classification of actinomycetes.* At present, the molecular identification is predicated on 16S rDNA sequences, which is most vital for Actinobacteria (**Table 1**).

#### **3. Actinomycetes—a biofactory of novel enzymes**

Actinomycetes is a genus of bacteria belonging to the Actinobacteria class. They're all gram-positive bacteria. Actinomycetes species are facultatively anaerobes (with the exception of A.meyeri and A. israelii, which are both obligate anaerobes), and they thrive in anaerobic environments. Individual bacteria of Actinomyces species can generate endospores, and colonies of Actinomyces develop fungus-like branching networks of hyphae. The appearance of these colonies led to the false belief that the creature was a fungus, and the name Actinomyces, which means "ray fungus," was given to it (from Greek actis, ray, beam and myles, fungus). Actinomycetes species may be found in soil as well as in animal microbiota, including the human microbiome. They are well-known for their importance in soil ecology; they generate a variety of enzymes that aid in the decomposition of organic plant material, lignin, and chitin. As a result, their presence is critical in the composting process. Humans and cattle have commensal flora on their skin, mouth flora, gut flora, and vaginal flora. They're also renowned for causing infections in humans and cattle, mainly by gaining entrance to the inside of the body through wounds. People with immunodeficiency are more susceptible to opportunistic infections, as they are to other opportunistic illnesses. They are comparable to Nocardia in all of the qualities listed above, as well as in their branching filament production. Actinomycetes species, like many other anaerobes, are finicky and difficult to cultivate and isolate. Although clinical laboratories culture and isolate them, a negative result does not rule out infection since unwillingness to grow in vitro might be the cause.

#### **4. Role of actinomycetes as environmental cleaner**

Actinomycetes work as an environmental scrubber for the cleaning of the contaminant place and release potential enzymes for the same. The widely used application of Actinomyectes is shown in **Figure 2**.

#### **4.1 Bioremediation**

Actinomycetes are well-known for their bioremediation abilities. Antibiotics and chemical complexes are effectively consumed by actinomycetes. Pesticides and chemical complexes at large dosages can be degraded by them. Petroleum hydrocarbons are widely employed as chemical components and fuel in our daily lives. Petroleum has become one of the most prevalent pollutants of large soil surfaces as a result of increased use, and is now regarded a serious environmental hazard. In the environment, hydrocarbons degrade in a variety of ways. Bioremediation is one of the methods for removing them from the environment.

The utilization of soil organisms to breakdown contaminants into innocuous chemicals is known as bioremediation. Pesticides and other xenobiotics in the environment are successfully disintegrated and bioremediated by the actinomycetes. Actinomycetes play an essential role in the environmental destiny of hazardous metals, altering transitions between soluble and insoluble phases through a variety of physico-chemical and biological processes, and producing considerable quantities of biosurfactants. These mechanisms are key components of natural

#### **Figure 2.**

*Implications of actinomycetes in different domains.*

biogeochemical cycles for metals and metalloids, and some of them might be used to remediate polluted materials. Actinomycetes' role in bioremediation and stress-related behavior has been well investigated. Numerous Actinomycetes strains from composts are presently being studied to see if they can breakdown certain petroleum hydrocarbons and decolorize several synthetic dyes, which might lead to bioremediation applications. Actinomycetes have a number of characteristics that make them ideal candidates for bioremediation of organically polluted soils. They are capable of degrading complex polymers and play a vital role in the recycling of organic carbon. According to certain research, Streptomyces flora may play a critical role in hydrocarbon breakdown. Many strains produce cellulose- and hemicellulose cellulose degrading enzymes as well as extracellular peroxidase, which may solubilize lignin and destroy lignin-related compounds. Actinomycetes are the leading category of degraders in some polluted locations [26]. Actinomycetes have the capacity to survive in an oily environment. As a result, these bacteria can be used in Bioremediation to remove oil contaminants.

#### **4.2 Actinomycetes to subtract environmental oil pollutants**

A dark sticky naturally occurring liquid (petroleum) that is called as Crude oil, a complex mixture of compounds having varying molecular weight and containing 30% polyaromatic hydrocarbons (PAHs). PAH compounds such as naphthalene, acenaphthene, fluorene, phenenthrene, fluoranthene, pyrene, acenaphthylene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k] fluoranthene, benzo[a]pyrene, dibenz[a, h]anthracene, benzo[g, h, i]perylene, anthracene, and indeno[1,2,3cd] pyrene) are pollutants nominated by the United States Environmental Protection Agency as priority PAHs. The most widespread organic pollutants and potentially health hazards are targeted for measurement in environmental samples. On the other hand it is also found in environmental components like cereals, oils, fats, vegetables, cooked that are carcinogenic, mutagenic, and teratogenic. So removal of PAHs is an issue of big interest [27]. Crude oil containing various PAHs, focuses on metabolic pathways for its degradation and microbial degraders. In aerobic or anaerobic conditions, bacterial and fungal strains are able to target the specific PAH through effective and eco-friendly

#### *Actinomycetes as An Environmental Scrubber DOI: http://dx.doi.org/10.5772/intechopen.99187*

bioremediation approach currently. Additionally, a new approach will be needed to design for dearomatization of crude oil to shoot a solution in numerous PAH inhabitants.

The nocardioform actinomycetes of the genera *Mycobacterium*, *Rhodococcus* and *Gordona* are the soil microflora able to mineralize PAH. These novel actinomycetes *Sphingomonas paucimobilis* BA2, *Gordona sp*. BP9, *Mycobacterium sp.* VF1 were able to grow on anthracene, pyrene or fluoranthene as the sole carbon source and mineralizing PAH with up to four rings [28]. Actinomycetes were potent to metabolize phenanthrene present at roadside soil polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH polluted soils from industrial sites [26]. *Rhodococcus and Gordonia* were potentially selected actinomycetes to remediation of polycyclic aromatic hydrocarbons in liquid culture and spiked soil. Biosurfactant or degrade phenanthrene when cultured on medium contains glucose, hexadecane and rapeseed oil at 300 c. Gordonia sp. APB and G. rubripertincta produced emulsion from rapeseed oil whereas Rhodococcus sp. DSM44126 ability to degrade phenanthrene as sole source of carbon and anthracene [29].

A novel anthracene degrading actinomycete was isolated from a hydrocarbon contaminated soil at mechanical engineering workshop. Haloalkalitolerant actinomycetes, Kocuria rosea, Kocuria palustris, Microbacterium testaceum, and Nocardia farcinica were used in investigation of the correct fluorescence method to check the PAHs biodegrading capacity of actinobacteria. In the fluorescence method, excitation and emission fluorescence were used in study of the PAHs biodegrading to determine the residual anthracene concentration [27].

*Rhodococcus opacus 412* and *R. rhodnii,* firstly adapted to phenanthrene and antracene containing solid mineral medium that accelerated metabolism of polyaromatic hydrocarbons. It provides microbial growth on phenanthrene as the sole source of carbon and energy in liquid medium, phenanthrene was utilized by these strains. Additionally, first *Rhodococcus opacus 412* was grown to anthracene on solid developed variant strains that transform anthracene in liquid medium to anthraquinone and 6, 7-benzocoumarin [30].

A glass bead system was developed for growth of PAH-degrading actinomycetes in liquid culture. Here, R*. wratislaviensis* was able to degrade phenanthrene and anthracene whereas an actinomycete, *Mycobacterium LP1* with a high capacity to degrade phenanthrene and pyrene. Strains were isolated from an agricultural soil that screen for biosurfactant activity and phenanthrene degradation in the presence of different co-substrates in liquid cultures and in soil. Mycobacterium LP1, favoring biological degradation of low-molecular-weight PAH at the first time of inoculation, and in second step addition of rapeseed oil, which promoted the abiotic transformation, and probably the solubilization, of the high-molecular-weight PAH [29].

Biodesulfurization was a selective and cost-effective method for subordinating the sulfur content of petroleum products. DBT, used as a model polyaromatic sulfur heterocycle for microbial isolation and characterization to check capability of transforming organosulfur compounds found in an assortment of fossil fuels. However, Biotransformations were occurred through metabolic degradative pathways or growing with it as a sole sulfur source and biocatalytic desulfurization for the selective removal of polyaromatic sulfur heterocycle. *Rhodococcus erythropolis* I-19 was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) from hydrodesulfurized middle-distillate petroleum (MD 1850) with the aid of multiple copies of key dsz genes present in cell [31].

The sulfur oxides released after incineration of fossil fuels, the main environmental problem, cause air pollution and are the key for acid rain. Organosulfur compounds, including dibenzothiophene (DBT) are metabolized by microbes.

*Rhodococcus erythropolis* IGTS8 have gene clusters of the dsz, three genes, dszA, dszB, and dszC. Genetic analysis of the dsz Promoter and its associated regulatory regions of Rhodococcus erythropolis IGTS8 were done. In genetic investigation, dsz gene clusters are involved in conversion of dibenzothiophene (DBT) to 2-hydroxybiphenyl and sulfite. *Rhodococcus* can use DBT as the sole source sulfur [32].

*Rhodococcus* sp. strain SY1, dibenzothiophene (DBT)-desulfurizing bacterium utilized dimethyl sulfide (DMS), dimethyl sulfoxide (DMSO), and several alkylsulfonates as sole sulfur sources. Strain SY1 were able to degrade DMS in the oxidative pathway *via* DMSO, DMSO2 (dimethyl sulfone), and methanesulfonate to methane and sulfate, reducing a part of DMSO back to DMS. Sulfate produced can reduce enzymatic expression by the addition of BaCl2 enhanced the degradation rate of DBT about 14%. Spent motorcycle lubricating oils degradation was explored using microbiological standard procedures. Actinomycetes such as Nocardia sp., Gordonia sp., Micromonospora sp. and Rhodococcus sp. were able to degrade 1.035% to 7.53% of the spent lubricating oil [33].

An application of biosurfactants while one needs to clean up oil. Arthrofactin, a novel biosurfactant was produced by Arthrobacter species strain MIS38. Arthrofactin is one of the most effective lipopeptide biosurfactants and it effectively removes oil [34].

#### **4.3 Actinomycetes in pesticide degradation**

India, an agricultural country, has lost 30% of agricultural produce to pests. As a result insecticides, fungicides, pesticides and herbicides have rising demand for protection of crops. Monocrotophos (MCP) is organophosphorus pesticide and hazardous and extensively utilized in India to protect economically important crops. Biomineralization of Monocrotophos, by soil bacteria *Arthrobacter atrocyaneus* MCM B-425 and *Bacillus megaterium* MCM B-423 were capable to degrade MCP (concentration of 1000 mg l−1) to the extent of 93% and 83%, respectively. MCP is degraded by metabolic pathway involving the enzymes phosphatase and esterase to carbon dioxide, ammonia and phosphates through formation of one unknown compound – Metabolite I, valeric or acetic acid and methylamine, as intermediate metabolites. Two cultures will be used for bioremediation of waste water treatment and MCP contaminated soil [35].

A pollutant 4-chlorophenol (4-CP), toxic and recalcitrant compound which is formed from chlorination of waste water, in pulp mills, from breakdown of herbicides such as 2,4-dichlorophenoxyacetic acid and from anaerobic degradation of more highly chlorinated phenols, such as pentachlorophenol and 2,4,6-trichlorophenol. In Bioremediation, certain microbial strains are able to degrade 4-CP. A novel strains Arthrobacter chlorophenolicus sp. capable of degrading up to 350 p.p.m. (2.7 mM) high concentration of 4-CP (Westerberg et al., 2000). Arthrohacter ureafaciens CPR706 degrades 4-chlorophenol through the hydroquinone pathway [36].

Triazine rings are found in pesticides, plastic resins, dye s-Triazine herbicides are broadly used in modern agriculture, where they kill susceptible plants by coordinating to the quinone-binding protein in photosystem II, thereby inhibiting photosynthetic electron transfer. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-s-triazine) is broadly used herbicides in the United States for the control of broadleaf weeds in corn, sorghum, and sugarcane. *Arthrobacter aurescens* TC1, potential organism to metabolize substantial quantities of s-triazine compounds in the environment. A. aurescens TC1 were able to degrade 3,000 mg of atrazine per liter in liquid culture as the sole source of nitrogen, carbon, and energy [37].

#### *Actinomycetes as An Environmental Scrubber DOI: http://dx.doi.org/10.5772/intechopen.99187*

A novel *Streptomyces spp.* VITDDK3, halo tolerant Actinomycete was isolated and screened from Saltpan Soil. The Stain was considered potentially for production of biosurfactant, heavy metal resistance activity (to cadmium and lead) and also dyes decolourization activity. 98% of the azo dye and Reactive red 5B were potentially degraded by *Streptomyces spp.* VITDDK3. The new strain will be used further for large scale production of the lead compound [38]. *Rhodococcus chlorophenolicus* were degrade tetrachloro-para-hydroquinone to 1, 2, 4-trihydroxybenzene by metabolic process of microbial enzymes through reductive aromatic dechlorination process [39].

The chloroacetanilides including alachlor [2- chloro-2′,6′-diethyl-N- (methoxymethyl) acetanilide] are selective herbicides extensively used for preemergent weed control. The extremely toxic alachlor (144 mg l−1 concentration) was biologically degraded enzymatically by the metabolic pathway of the strains *Streptomyces sp*. LS166, LS177, and LS182. The *Streptomyces sp.* were degraded around 60–75% of the alachlor in 14 days [40]. Herbicides are greatly significant for agriculture. Herbicides may act as pollutants, damaging the soil, ground water and surface water. Actinomyces play an important role in the cycle of the elements in nature and in degradation of organic xenobiotic substances. *Streptomyces albus, Streptomyces aureus* and *Streptomyces chrysomallus* were Influence Xenobiotic Substances- Sulfonylurea herbicides, tribenuron-methyl and nicosulfuron, which were commonly used in farming [41].

#### **4.4 Actinomycetes involved in plastic degradation**

Plastics, corrosion resistant materials, strong, durable and inexpensive polymers are derived from petrochemicals and chemical processes to produce long chain polymers. At room temperatures the plastic polymers are not considered as toxic, but when heat is released from plastics have undesirable effects on the environment and human health. Plastics are accessible in a variety of forms such as nylon, polycarbonate, polyethyleneterephthalate, polyvinylidene chloride, Urea formaldehyde, polyamides, polyethylene, polypropylene, polystyrene, polytetraflouro ethylene, polyurethane and polyvinyl chloride. Naturally occurring microorganism involved in degradation of organic material- plastic and plastic waste. Styrene is possibly carcinogenic to humans that cause mammary gland tumors in animals [42]. Polystyrene waste accumulates in the environment posing an increasing ecological threat [43].

The plastics of poly (β-hydroxybutyrate)(PHB)-and poly (ε-caprolactone) (PCL) were degraded by aerobic microorganisms that persist in the natural environment. The plastic depolymerizing microorganisms are distributed over many kinds of material, including landfill leachate, compost, sewage sludge, forest soil, farm soil, paddy soil, weed field soil, roadside sand, and pond sediment [44]. Actinomycete strains *Streptomyces* genus and *Micromonospora* genus were isolated and screened for the capability to degrade poly (ethylene succinate) (PES), poly(εcaprolactone) (PCL) and poly(β-hydroxybutyrate) (PHB) from upstream and downstream regions of the Touchien River in Taiwan [45].

*Streptoverticillium kashmirense* AF1 was able to degrade a natural polymer; poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. Extracellular enzymes PHBV depolymerases secreted by *Streptoverticillium kashmirense* AF1 was purified and degrade PHBV film [46]. *Actinomadura*, *Microbispora, Streptomyces, Thermoactinomyces* and *Saccharomonospora* were thermophilic actinomycetes strains able to degrade poly (ethylene succinate) (PES), poly (ε-caprolactone) (PCL) and poly (β-hydroxybutyrate) (PHB). Thermophilic actinomycetes

*Microbispora rosea, Excellospora japonica* and *E. viridilutea* were able to degrade aliphatic polyester, poly (tetramethylene succinate) (100 mg PTMS film) [47].

Biofilms are the favored bacterial mode of living and survival, most microorganisms—which tend to attach to surfaces—to gain physical support, increase nutrient utilization. Polyethylene (PE), synthetic polymer, is highly inert and virtually non-biodegradable. *Rhodococcus ruber* (C208) has formed a dense biofilm on polyethylene (PE) surfaces when degradation of their analogous substrates within the biofilm [48]. A biofilm-producing strain of *Rhodococcus ruber* was degraded polyethylene by organization yields "mushroom-like" 3D structures on the full-grown biofilm [43]. Rhodococcus sp. strain RHA1, strong polychlorinated biphenyl (PCB) degrader has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). Linear plasmids of Rhodococcus sp. strain RHA1 having degradative genes such as bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4 [49].

Polylactic acid (PLA), biodegradable plastic has broadly applicable in food packaging with respect to environmental concern in solid-waste management. Novel polylactic acid-packaging degrading actinomycete, Streptomyces sp. KKU215 biomass productions were carried out in PLA-packaging as sole carbon source. The potent strain was used in biodegradation of PLA-packaging [50]. Amycolatopsis strains, poly(L- lactide) degrader stain has ability to assimilate degradation product like poly lactic acids [51]. *Amycolatopsis sp*. strain HT-6, a poly(tetramethylene succinate) (PTMS)-degrading actinomycete, was observed to degrade poly(tetramethylene carbonate) (PTMC). Actinomycetes strain degrades polycarbonate PTMC in a liquid culture with 150 mg of PTMC film, completely and fast degraded with a high yield of cell growth [51]. Polylactide (PLA)-degrading microorganisms are sparsely distributed in soil environments. An *Amycolatopsis* was potent in degradation of PLA film (100-mg film) added was degraded by the strain in liquid culture after 14 days of incubations [52].

#### **4.5 Actinomycetes as biological degrader of dyes**

Synthetic dyes, coloring agents are mostly used in textile industries and spawn a huge amount of wastewater during the process of dyeing. The release of colored effluents in rivers and lakes are the key reason for reduction of dissolved oxygen concentration creating anoxic condition and foremost to the acute toxic effects on the flora and fauna of the ecosystem. In addition to colored effluents in water bodies reduces the photosynthesis as it hampers dispersion of light in water. The color of textile wastewater deduction is a major environmental concern [53]. These synthetic dyes are not easily removed in waste water treatment plants [54].

Azo dyes, water-soluble reactive dyes constitute the most versatile class of synthetic dyes used in the textile, pharmaceutical, paper, food and cosmetic industry due to their ease in production and variety in color compared to natural dyes. Azo dyes are widely used in textile industries. When Azo dyes are left in water bodies without any treatment, they result in environmental pollution and in turn are toxic, carcinogenic and mutagenic. Azo dye Reactive Yellow Biodegradation was carried out by microorganisms isolated from the activated sludge. The isolated actinomycetes were acclimatized to different concentrations of dye from 0.005–0.200% (mg/100 ml). The consortium was developed by mixing five actinomycetes and the found degradation of dye depends on the concentration of dye in addition to the growth of the actinomycetes. Lignin peroxidase, laccase and tyrosinase enzymes were responsible for steady degradation activities. Biosorption of Reactive Yellow dye as occurred by using dead biomass of actinomycetes [55]. The *Streptomyces* spp., indigenous in environment was able to degrade azo blue and azo orange

#### *Actinomycetes as An Environmental Scrubber DOI: http://dx.doi.org/10.5772/intechopen.99187*

dyes in optimization of conditions through metabolic pathways with responsible enzymes [53].

*Thermobifida fusca* BCRC 19214, the thermophilic actinomycetes was produced in laccase. The laccase are diphenol oxidases, could oxidize dye intermediates, especially 2,6-dimethylphenylalanine and p-aminophenol [56]. *Streptomyces spp*. was used in the decolorization of monosulfonated mono azo dye derivatives of azobenzene. Additionally strain was enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. The Change in dye structure was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. Streptomyces spp. was also decolorized Orange I [57]. Sulfonated Azo Dyes ("4C-radiolabeled azo dyes) and sulfanilic acid were used to study dye substitution patterns and biodegradability-mineralization by a white-rot fungus and an actinomycete. Phanerochaete chrysosporium and Streptomyces chromofuscust mineralization of modified dyes anionic azo dyes, containing lignin-like substitution patterns considered among the xenobiotic compounds. These very specific structural changes in the azo dye molecules enhanced their biodegradability [58].

Reactive dye is the foremostly used one type of azo dye containing different reactive groups. Reactive dyes that derive from dyeing industries increase Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD), change the pH of water bodies and it causes serious problems in plant, animal and human beings. The presence of dyes in water is highly visible and affects their transparency and esthetic even though the concentration of the dyes is low [59]. Actinomycete strains were decolorized effluents containing different types of reactive dyes anthraquinone, phthalocyanine and azo dyes. The absorption of reactive dyes was done by the strains cells outcome in the decolorization of the effluents [54]. Actinomycete *Streptomyces krainskii* SUK – 5 was potent to degrade and decolorize textile azo dye- Reactive blue–59 in nutrient medium in shaking condition. Actinomycete were induced enzyme lignin peroxidase, and NADH-DCIP Reductase and MR reductase play key roles in degradation [60].

#### **4.6 Actinomycetes and heavy metal**

Heavy metals were successfully removed from wastewaters, and industrial wastes are still a key study area today. Streptomyces coelicolor's application in heavy metal removal via interactions is consistent with traditional heavy metal responses, resistance mechanisms, and secondary metabolite formation. Some physiological features of the salt sensitive cultivar Giza 122 of *Zea mays*, L. plants maintained for 10 weeks in the greenhouse were affected by Streptomyces sp. HM1 and heavy metal Cd (10, 20, 40, and 60 ppm). The presence of Streptomyces spp. HM1 in the soil increased the tested plant's heavy metal tolerance marginally. As a consequence, the maize test cultivar seems prospective for use in heavy metal polluted soils, even when actinomycetes are present [61]. Phytoremediation is a promising method that cleans toxins from the soil, water, and air using plants and their associated Plant Growth-Promoting microorganisms. Plant beneficial actinomycetes have been widely exploited as a heavy metal phytoremediation tool for cleaning up metal-polluted soils, and they play an important role in plant growth, metal/nutrient acquisition, metal detoxification, and reduction of biotic/abiotic stress. It is conceivable to boost microbial inoculants as an ecologically acceptable bio-tool for use in heavy metal phytoremediation in metal-polluted soils based on these positive plant-actinomycetes interactions [62]. Copper bioaccumulation was caused by the actinobacterium Amycolatopsis sp. AB0. Amycolatopsis sp. AB0, a copper-resistant

actinobacterium, was isolated from contaminated sediments and shown excellent copper specific biosorption capacity (25 mg g–1). The existence of copper P-type ATPase genes in Amycolotopsis was discovered for the first time [63].

#### **4.7 Actinomycetes in removal of groundwater pollutant**

*Pseudonocardia dioxanivorans sp.* nov., a novel actinomycete was isolated from industrial sludge contaminated with 1,4-dioxane that grows on 1,4-dioxane which is a probable human carcinogen. Novel strain was also growing on tetrahydrofuran, gasoline aromatics and several other toxic environmental contaminants [64].

### **5. Conclusion**

Pollution of the environment is becoming more of a global issue. This circumstance necessitates rapid technological adaptation. Biological remediation, which involves the use of live organisms or their products, is a viable option. Actinobacteria have proven to be an effective instrument for carrying out this procedure on various matrices, under various growth conditions, as pure cultures or consortia, or as enzyme and emulsifier providers. The next stage is to use this information to a larger scale in the field. Actinomycetes strains can be identified and used for solid waste biodegradation with success. The consortium of selected actinomycetes strains might be used to assess their economic viability in the biodegradation process. This broadens their applications in biotechnology and environmental research.

### **Author details**

Sutaria Devanshi1 , Kamlesh R. Shah<sup>2</sup> \*, Sudipti Arora1 and Sonika Saxena1

1 Dr. B. Lal Institute of Biotechnology, Jaipur, India

2 Department of Biotechnology, Pramukh Swami Science and H.D. Patel Arts College, Kadi, North Gujarat, India

\*Address all correspondence to: skrlipase@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Actinomycetes as An Environmental Scrubber DOI: http://dx.doi.org/10.5772/intechopen.99187*

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Section 3
