Microbial approaches for remediation of pollutants: Innovations,future outlook,and challenges

Introduction

Earth’s ecosystem is being contaminated continuously by different pollutants nowadays. Diverse groups of pollutants are found in different places. Ecological restoration has come out as one of the most notable issues in environmental biotechnology. Ecosystem recovery is made with various paradigms and driven by complex processes considering multiple biological levels.^1,2 Long ago, nobody would have thought to find microbiologists possess an important role in the field of ecological restoration. In contrast, now the scenario has been changed because it is not possible to find a single ecosystem on earth where microorganisms are not present and have not been identified as key players for various functions. Microorganisms are found to be key players and vital components of all biogeochemical cycles (C, S, N, P, and metals, etc.). ³ Recent findings have integrated the physiological, biochemical, ecological, genomic, and metagenomic bases of microorganisms with various aspects, viz. anaerobic oxidation of methane, photosynthesis, uptake of phosphorus, sulfur, and nitrogen cycles in the process of bioremediation.^2,4 Advancement in environmental biotechnology allows us to understand the mechanistic characterization of relatively easy, simple, and less complicated biological systems to naturally occurring complex microbial communities that can dwell soils, air, and waters. There are various pollution causing agents (fertilizers, garbage, oil spills, toxic chemicals, and sewage disposals) that are being found around the globe. The global pollutions in the form of soil, water, air have occurred due to these pollutants.^5,6

The major diversity of microbial colony is notable to the functional role they execute in their natural habitat. They show response towards the changing environmental conditions (temperature, pH, terrestrial inputs, lights changing patterns, rise in sea level, and tropical storms, etc.) quickly. These behaviors make them suitable for potential bioremediation purposes. ⁷ However, microorganisms acclimatize to environmental conditions by creating a different pattern of gene expression, rate of growth, enzymatic activities, and physiological changes. Some of the microorganisms are found to have a unique strategy like the synthesis of biosurfactants, formation of biofilm, and production of bioactive compounds in the environment when they are being exposed to extreme environmental conditions like changes in the pressure, temperature, salinity, or micronutrients depletion. In the molecular biology perspectives, the microorganism which is used for bioremediation processes must possess resistant genotype towards the pollutant to be degraded by them.^8,9

Culture-dependent methodologies are widely used by researchers for studying microbial processes. The majority of microbiological studies for various applications have involved selective enrichment methodology to observe the certain activity of interest exhibited by microorganisms. This is found that only 1% of microorganisms can be cultured in existing laboratory conditions by using the culture-dependent technique. This is a limitation of this methodology. Additionally, the information may be lost during the investigation of an environmental sample due to dependencies upon specific dynamics of nutrients in the ecosystem. In some cases, the synergistic or antagonistic associations may affect the complex network of metabolic interdependencies.^10,11

With the advancement of molecular biotechnology, researchers had access to the activities and features of the various uncultured microbial world. ¹² In the year 1998, “metagenomics” came to the picture which involved the assessment of genomes from the microbial community directly in the environmental sample without preparing to culture them.^{13– 15} There are some remarkable achievements in microbial community analysis that also emerged in the 21st century. These include fluorescence in situ hybridization, denaturing gradient gel electrophoresis, restriction fragment length polymorphism (RFLP), polymerase chain reaction, temperature gradient gel electrophoresis, cloning and sequencing of rRNA gene, terminal RFLP (T-RFLP), and microarray.^15,16 Gradually, the orientation and insights to study the microbial communities in the pollutant sites have included the advanced “omics” and “next-generation sequencing” approaches like metatranscriptomics, metaproteomics, metabolomics, etc.

The main objective of this review is to provide insights and in-depth knowledge of a microbial community with special reference to their potential to biodegrade the pollutants and their functional activities in the polluted environments by adopting various omics approaches and microbial electrochemical technologies.

Organic pollutants in environment

Organic chemicals are highly soluble in lipids and least soluble in the water. These properties allow them to accumulate in the fatty tissue of the living system. Some of the pollutants are persistent, called persistent organic pollutant (POP). The POP can disperse and move to very long distances without any barrier in the atmosphere and deposited, where they have never been used. These produce an adverse effect on both human health and the associated environment.^17,18

Generally, POPs present in the environment are categorized into two main groups: (a) intentionally released POPs and (b) unintentionally released POPs. The intentionally released POPs are the essential products that are synthesized by chemical reactions, for example, the organochlorine compound that includes a chlorine atom in the reaction. These compounds are highly lipid soluble. Some groups of POP are also found to be neurotoxic in nature. Based on their application, these can be further classified into two types: organochlorine pesticides and industrial chemicals. Organochlorine pesticides include hexachlorobenzene, dichlorodiphenyltrichloroethane (DDT), mirex, heptachlor, chlordane, endrin, dieldrin, and aldrin, etc. Industrial chemicals mainly include polychlorinated biphenyls (PCBs).^19,20 These chemicals are not used frequently because of their harmful impact on the environment. Some researchers had thoroughly investigated the applications of various groups of pesticides in different counties like the United States, China, Japan, Canada, and Australia. The frequently used insecticides were found to be carbofuran, chlorpyrifos, dichlorvos, and dimethoate. The fungicide groups, viz. thiophanate-methyl, mancozeb, carbendazim, and chlorthalonil were found to be applied more. Similarly, glyphosate, paraquat, 2,4-D, and acetochlor were frequently used as herbicides.^21,22

Unintentionally released POPs are the by-product of chlorine compounds that are produced during the combustion process. The pollutants like furans, dioxin, and polycyclic aromatic hydrocarbons (PAHs) are coming under this category. The Environmental Protection Agency has listed a group of a total of 16 PAHs which are hazardous and also found to be carcinogenic. ²⁰ The PAHs can be produced through a natural process like rangeland fires, oil seeps that exudates from trees. The anthropogenic activities like burning of fossil fuels, petroleum spills, cooking, agricultural fires, etc., are also responsible for the generation of PAHs.^20,22 These organic pollutants are considered as very harmful as they can be absorbed via the skin, respiratory epithelium, cornea, and gut through the ingestion of contaminated food. It also affects many organs, viz. skeletal muscles, gastrointestinal tract, secretory glands, nervous system, and respiratory system, etc., upon exposure.^20,23,24 The organic pollutants can be treated broadly by various methods, viz. physical method or chemical method (photo decomposition, partitioning, leaching, volatilization, etc.) and biological method (microbiological action and plant action). Apart from these, exploiting microorganisms for remediation of waste sites is a promising alternative to other methodologies. Depending upon types of pollutants, contaminated site, and application types, microbial treatments have been broadly classified as bioaugmentation, biostimulation, biosparging, and bioventing. The various approaches to the degradation of organic pollutants have been highlighted in Figure 1.

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Figure 1.

Degradation techniques for organic pollutants.

Effect of pollutants on human and environmental health

The POPs are found everywhere and everyone’s body in the present scenario. Surprisingly, embryos and fetuses are also having these POPs. Their availability is found in all age groups of people. Generally, these are noticed more concentrated in elderly persons. ²⁵ Serious and major health problems like cancer, obesity, neurological problems, reproductive issues, diabetes, and hormonal disruption, etc., occurred due to exposure of POPs in the living body. The negative effect of groups of POPs was investigated altogether. This is known as a synergistic approach.^18,26 It was also proved that the toxicity of POPs mixtures always found to be more than the laboratory experiment.^27,28 The various health problems and environmental problems associated with these POPs have been illustrated in Figure 2.

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Figure 2.

Health and environmental problems associated with POPs.

Organic pollutants degrading microorganisms

The bioremediation technique has been used for a long time, but modern developments are different in terms of sophisticated new technologies and systematic approaches. Research is focused mainly in the area of development and discovery of potential biodegrading organisms. Microorganisms, plants, and some lower eukaryotes have shown potential biodegradation activity for organic pollutants. The following section focuses on recent research (2013–2019) on biodegradation strategies using different species of microorganisms.

The selection of potential strains of bacteria and yeasts that are used in the combinations during biodegradation studies is shown to be very effective. Using these concepts, recently some researchers have used a different proportion of bacteria and yeast as a microbial consortium to determine their potential for the biodegradation of diesel oil. In this study, a combination of Aeromonas hydrophila MR4 and Yarrowia lipolytica EH 56 could degrade 87% of the diesel. Similarly, a combination of Xanthomonas maltophila MRP7 and Candida maltosa EH15 could degrade 90% diesel in seven days. The addition of rhamnolipids to these microbial combinations during the degradation process could reduce the removal time for diesel oil from the contaminated water. ⁵⁸

Oily sludge or waste generated is found to be highly stable and forms complex emulsions due to the presence of additives with surfactant properties. Hence, it is very essential to separate this residual oil from the aqueous phase implementing demulsification activity. In this context, the surfactants produced by yeasts (examples: Candida lipolytica, Candida guilliermondii, and Candida sphaerica), bacteria (examples: Bacillus sp., Pseudomonas aeruginosa, and Pseudomonas cepacia) were tested for the demulsification capacity. In this study, bacterial surfactants could recover about 65% of the seawater emulsified with motor oil, whereas surfactant synthesized by the yeasts could recover 35–40% only. ⁵⁹ Rahma et al. have investigated four consortia of microorganisms which can degrade up to 92.0% of total petroleum hydrocarbons in the two months of incubation time. Functional analyses of degrading genes were found to be alkB, ndoB, xylA, cat23, and nidA1. ⁶⁰ Some researchers had investigated the screening of mutant strain of Pseudomonas putida through gamma-ray irradiation for efficient degradation of crude oil. The degradation rate was 46.3% higher as compared to the parent strain. ⁶¹

A thermotolerant strain of Gordonia sp. strain 1D was successfully screened from the contaminated soils of the oil refinery. It was found to utilize crude oil and diesel fuel at temperatures up to 50°C. Successively the responsible gene (gyrB) for the degradation was also isolated. Recently, the stable isotope probing technique was used to identify active 13C-labeled phenanthrene degraders. They have found dominant fungal and bacterial species capable of degrading various petroleum-based compounds. ⁶² A recombinant strain of Pseudomonas aeruginosa DAB was constructed that can able to degrade the crude oil through the production of rhamnolipid. ⁶³ Immobilized cells of Bacillus sp. E3 with algae materials (Enteromorpha and kelp residues) have studied to investigate the oil degradation rates, which were found to be 65% higher after 21 days. ⁶⁴ Shchemelinina et al. had isolated potential of fungal strains (Cadophora malorum, Mucor circinelloides, Trichoderma viride, and Mycelia sterilia) having the ability to degrade oil sludge. ⁶⁵ In one study, bacterial species have been isolated from the oil-contaminated sites of Khurais oil field, Dhahran, Saudi Arabia. Three bacterial strains were selected depending upon their capacity to grow in the presence of hydrocarbons. They were identified as Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus cereus. ⁶⁶ A petroleum oil-degrading microorganism Pseudomonas aeruginosa NCIM 5514 was isolated from the crude oil polluted site of Ankleshwar, Gujarat, India. ⁶⁷

The degrading potential of microorganisms for various pollutants, viz. hydrocarbons, pesticides, and furans has been reported earlier for many compounds including PCB, PAH, DDT, etc. Various PAH-degrading strains were screened from the mangrove sediments. The microbial strains like Microbacterium sp. BPW, Novosphingobium sp. PCY, Alcaligenes sp. SSK1B, Ralstonia sp. BPH, and Achromobacter sp. SSK4 are capable of degrading various PAHs. ⁶⁸ The latest information concerning with organic pollutants and key degraders has been summarized in Table 1.

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Table 1.

Biodegradation of selected organic pollutants by microbial strains and their specific activities.

Organic pollutants	Microbial strains	Important findings with respect to the microbial strains	Performance outcome/pollutant removal efficiency	References
Crude oil and diesel fuel	Gordonia amicalis	hydrocarbon-oxidizing thermotolerant strain can be used in remediation of oil-contaminated soils in hot climates.	The degree of crude oil degradation by Gordonia sp.1D at 45°C was 38% in liquid medium and 40% in soil.	111
Petroleum oil	Corynebacterium species	GC–MS analysis confirmed that the biosurfactant was 13-docosenamide (z). Biosurfactant also induces the growth of petroleum degrading bacteria, i.e. Corynebacterium species.	Maximum growth was found at 96 h with biosurfactant (13-docosenamide (z)) and 144 h without biosurfactant.	112
Crude oil	Pseudomonas aeruginosa DAB	Production of rhamnolipids (17.3 g/L) and crude oil was utilized as a sole carbon source. Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB.	After 14 days, nearly all of the contents in crude oil were decreased, and the maximum removal rate of 1% crude oil was 95.7% which compared to 70% by the wild-type strain.	63
Crude oil	Mycobacterium AGS10	The strain of Mycobacterium AGS10 was identified as acidophilic. Strain AGS10 is an efficient degrader of both light and heavy, high-viscosity oil at рН 2.5.	After 28 days of incubation at 30°C and pH 2.5, the degradation of n- and iso-alkanes was 99 and 44%, respectively, for the light, low-viscosity oil of the Nizhnevartovsk deposit, 58 and 32%, respectively, for the medium-density oil of the Moscow oil-processing plant, and 80 and 16%, and 99 and 69%, respectively, for the heavy, viscous oils of the Cheremukhovskoe and Usinkoye oil fields.	113
Petroleum hydrocarbon	Pseudomonas spp.	Biodegradation of total petroleum hydrocarbon (TPH) with syringafactin Emulsification. The activity of the produced biosurfactants was stable over a pH range of 6–12, at high salinity (10%), and after heating at 80°C.	The strains could only use diesel oil as sole carbon source. At 28 days of biodegradation test, a removal of around 70% of total petroleum hydrocarbon (TPH) was achieved in bioreactors.	114
Marine oil spill	Bacillus E3	Immobilized Bacillus E3 with Enteromorpha residue and kelp residue Compared with the Enteromorpha residue, the kelp residue demonstrated a better oil absorption ability.	Immobilized Bacillus E3 degraded more than 70% of the total alkane and PAHs. Free Bacillus E3 degraded 63% of the total alkane and 66% of total PAHs, respectively.	64
Petroleum hydrocarbon	Pseudomonas aeruginosa SNP0614	P. aeruginosa SNP0614 and the mixed oil-degrading bacteria enhanced 30% degradation rate.	Emulsification activity (E24) by the surfactant produced by the specific strain for crude oil was found to be 90%.	115
Petroleum hydrocarbon	Pseudomonas ZS1	Degradation of petroleum by Pseudomonas sp. ZS1 was prevented by the presence of Alcaligenes sp. CT10.	Degradation rate of crude oil by Pseudomonas sp. ZS1 reaches as high as 97.6% in 36 days. However, when Alcaligenes sp. CT10 is present, the degradation rate reduces by 4.3-fold (degradation rate of 22.5% vs. 97.6%).	116
Petroleum hydrocarbon	Pseudomonas putida 300-B	Pseudomonas putida 300-B was a mutant strain screened by gamma irradiation having strong positive correlation with the gene abundance and expression.	Degradation was 46.3% higher than the parent strain.	61
4-bromophenol	Ochrobactrum HI1	Ochrobactrum sp. HI1 was isolated from soils in the vicinity of a bromophenol production plant, which can degrade 4-bromophenol in aerobic degradation path way.	4-bromophenol and its isotopes could be degraded successfully. Isotopic effect for normal carbon was also observed (εC = −1.11 ± 0.09‰).	117
2,4-dichlorophenoxy acetic acid	Thauera DKT	This strain of Thauera DKT could degrade 2,4D to 2,4-dichlorophenol (2,4DCP) and then dechlorinated to form the intermediates products like 2-chlorophenol (2CP), 4-chlorophenol (4CP), and phenol before complete degradation through anaerobic path way.	The maximum degradation rate was found to be 0.017 ± 0.002 mM/day.	118
Chlorothalonil	Stenotrophomonas acidaminiphila BJ1	This strain was found to be a probiotic in nature, which could successfully colonized the soil by competing with the native biota. Moreover, the bacterial inoculation stimulated some members of indigenous soil microbial communities.	Rate of degradation of chlorothalonil was faster and its half-life decreased from 9.0 to 4.9 days.	119
Propiconazole	Pseudomonas aeruginosa PS-4	The metabolic intermediates like 1,2,4-triazole, 2,4-dichlorobenzoic acid, and 1-chlorobenzene were produced during the degradation. Induction of monooxygenase activity and the CYP450 gene was observed, which enhanced the degradation process.	Eight microgram per liter of propiconazole out of 10 μg/L could be utilized by this strain in the culture media as a sole carbon source under optimized conditions.	120
Thiabendazole	Sphingomonas B13	The proposed pathways for degradation of thiabendazole resulted in the formation of thiazole-4-carboxamidine.	TBZ degradation was observed as of the first enrichment cycle with approximately 90% of the fungicide degradation occurring in three days.	121
Atrazine	Anthracophyllum discolor	The Anthracophyllum discolor was immobilized with various substrate, and the activity of the degradation rate was checked.	The half-life (t1/2) for degradation of atrazine decreased from 14 to 6 days.	122
Chloroanilines	Geobacter KT5	The obtained isolate was found to utilize a wide range of trichloroanilines (TCAs), dichloroanilines (DCAs), monochloroanilines (MACs), and aniline as sources of carbon and energy. It also used Fe(III) as a terminal electron acceptor under anaerobic conditions.	The rate of utilization for trichloroaniline was found to be 2.48 ± 0.32 µM day−1.	123
2,4,5-trichlorophenoxyacetic acid	Cellulosimicrobium NPZ-121	The identified intermediates of 2,4,5-T conversion by strain NPZ-121 included the products of sequential dehalogenation of the substrate molecule: 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chlorophenoxyacetic acid (4-CPAA), and phenoxyacetic acid (PAA).	The concentration of 2,4,5-trichlorophenoxyacetic acid was decreased by 36%, 43.6%, and 75% after three, five, and six days of incubation, respectively.	124
Pendimethalin	Paracoccus P13	An alkane metabolite, 1,3-dinitro-2-(pentan-3-ylamino)butane1,4-diol, was identified as a product of P13-mediated pendimethalin degradation by UHPLC–MS/MS. This finding suggested that there may be a new degradation mechanism of pendimethalin in strain P13 that differs from those that are commonly observed.	During the time of two days, 100 mg/L pendimethalin was degraded. During the time of five days, 200 mg/L pendimethalin was degraded.	125
2,4-dichlorophenoxyacetic acid	Achromobacter LZ35	Strain LZ35 harbored the conserved 2,4-D/alpha-ketoglutarate dioxygenase (96%) and 2,4-dichlorophenol hydroxylase (99%) and catabolized 2,4-D via the intermediate 2,4-dichlorophenol.	The strain of LZ35 could remediate 75% of 100 mg and 90% of 50 mg 2,4 dichlorophenoxyacetic acid upon inoculation with 7.89 ×106 CFU g−1 of contaminated soil.	126
Beta-cypermethrin (Beta-CP)	Brevibacillus parabrevis BCP-09	This strain was isolated from the activated sludge samples in pyrethroid-manufacturing, wastewater-treatment system in Chengdu, China.	Beta-cypermethrin was degraded 75.87% within three days subjected to specific optimal conditions.	127
Glyphosate	Comamonas odontotermitis P2	Presence of GOX (glyphosate oxidoreductase) and phnJ (C-P lyase) genes in C. odontotermitis are responsible for the degradation of glyphosate through GOX and C-P lyase metabolic pathways.	Degradation of glyphosate (1.5 g/L) within 104 h resulting in a maximum degradation of 90%.	128
Acetamiprid	Fusarium CS-3	This strain can degrade acetamiprid over a wide range of pH (5.0–8.0) and temperature (20–42°C).	Degraded 50 mg/L of acetamiprid is equal to 99.6% in 96 h.	129
Organophosphorus	Aspergillus niger NCIM 563	The degradation of organophosphorus compounds was mediated by the phytase production.	Aspergillus niger NCIM 563 could degrade 91% degradation of organophosphorus.	130
Chlorpropham	Bacillus licheniformis NKC-1	The activity of enzymes synthesized by these strains (CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase) was observed, and these are found to be responsible for the degradation of Chlorpropham completely.	The complete degradation of chlorpropham was achieved after 144 h.	131
2-chloro-4-nitro phenol (2C4NP)	Cupriavidus CNP-8	Complete biodegradation of 2C4NP occurred via a hydroxyquinol pathway	The strain CNP‑8 can degrade 2C4NP as high as 3.2 mM completely under optimum condition in the period of 24 h.	132
Di(2-ethylhexyl) phthalate	Gordonia alkanivorans YC-RL2	The strain was found to be halotolerance and could tolerate up to 0–5% NaCl. It also showed an outstanding performance in a wide range of pH (6.0–11.0).	Degradation rates were all above 94%. Gordonia alkanivorans YC-RL2 could withstand high concentrations of Di(2-ethylhexyl) phthalate (up to 800 mg/L).	133
Methyl parathion	Burkholderia zhejiangensis CEIB S4-3	Genes coding for enzymes in the benzoquinone pathway and the benzenotriol pathway were found, which could completely degrade methyl parathion.	B. zhejiangensis CEIB S4-3 was able to completely degrade Methyl parathion using it as the only source of carbon.	134
Malachite green	Klebsiella aerogenes S27	Klebsiella aerogenes S27 was an endophyte having novel oxidoreductase activity; thus, facilitating the study of the plant–endophyte symbiosis in the bioremediation processes.	Malachite green (100 mg/L) was decolorized completely in 8 h at 30°C and at pH 7.0.	135

The algae play a vital role in the biomonitoring of organic pollutants in the aquatic ecosystems. In some cases, the addition of the bacterial culture as activated sludge to the microalgal consortium (Chlorella vulgaris) significantly speed-up the biodegradation of hydrocarbons in oil-polluted site. Recently, an algal–bacterial co-cultivation method had been implemented for the remediation of nitrogen rich wastewater along with the co-production of lipids. The ratio of 1:0.75 for bacteria and microalgae was found to be optimum for the highest lipid yield of 130 mg/L. ⁶⁹ A similar type of research was also performed for the remediation of seafood wastewater effluent to investigate the relationship between lipid production and bacterial growth by an initial microalgae Chlorella vulgaris density culture. The lipid content was approximately two times higher compared to that of the control. ⁷⁰ In order to remediate the wastewater generating from a fertilizer manufacturing industry, Chlorella vulgaris was grown onto a polyurethane foam support material in the form of a fluidized bed bioreactor. In this study, along with the bioremediation the authors had also reported for co-production of biodiesel in the medium. ⁷¹

Microbial treatment in the polluted site

Culture-based approaches

Culture-dependent techniques are generally executed to study the diversity of microorganisms present in the ecosystem. ⁶⁷ These techniques have used to provide notable findings of the interactions of microorganisms with petroleum hydrocarbons oil-contaminated fields. ⁷² In this method, isolation and cultivation of microorganisms have been performed from the pollutants site using basic microbiology techniques like enumeration in solid media, most probable number (MPN)-type liquid media, and culturing in specialized Biolog plates.^73,74 The characterization of microbial communities relies upon various limiting factors for microbial activities in the ecosystem of the oil reservoir that describe the ecosystem’s community.^75,76 The isolation media for the microorganisms are formulated to be rich in carbon and nitrogen sources as compared to prevailing environmental conditions. ⁷⁷ The potential oil-degrading strains are obtained a lesser number in the low-temperature oil reservoirs ecosystem as compared to high-temperature petroleum oil reservoirs.^78,79 In order to isolate and enumerate the oil-degrading microorganism from the petroleum oil-contaminated soils, keeping proper control along with the test samples is compulsory.^72,73 Along with hydrocarbon metabolizers on the agar plate, some non-hydrocarbon metabolizing microorganisms were also isolated on the agar plate in a previous study. ⁸⁰ Silica gel was used by some researchers as a solidifying agent to minimize the problem associated with trace carbon in the agar plate. The MPN has been used previously in order to investigate the metabolism of microorganisms in petroleum-based hydrocarbon polluted sites by studying the formation of an emulsion. Here, the isolates of microorganisms are not required. Generally, MPN assay is mostly used for studying microorganisms in anaerobic environments.^73,81 To achieve more insights of petroleum hydrocarbon degradation mechanisms of petroleum pollutants degradation, a detailed molecular level understanding is required to be, which will help to correlate the gene expression to these activities.^82,83 Presence of different strains of facultative anaerobes, sulfate reducers, and oil-degrading methylotrophs in the petroleum oil-degrading environment was characterized by implementing the MPN methodology. The substrate utilization pattern for the growth of the microorganisms isolated from the petroleum hydrocarbon polluted site can be studied with the technique of biolog system.^84,85 The functional and metabolic diversity of microbial communities can be characterized in continuous-flow cultures by using this biolog system. Implementing this metabolic diversity of microorganisms with respect to hexadecane utilization was also studied by considering its substrate utilization patterns for the same. ⁸⁴ Various methodologies for microbial community analysis in the petroleum hydrocarbon polluted site have been illustrated in Figure 3.

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Figure 3.

Methodologies for microbial community analysis in the polluted site.

Omics-based approaches

The onset of next generation sequencing has evolved the “omics” approaches in order to study the functional aspects of microbial remediation of organic pollutants. The “omics”-based approaches are otherwise called as “cultivation-independent technique.” Steps involved in “omics” approach for the selection and characterization of microbial communities present in petroleum oil reservoir samples are schematically represented in Figure 4.

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Figure 4.

Work-flow of “Omics” approaches with special reference to biodegradation strategy for environmental pollutants.

The major drawbacks in DNA-based techniques are found that they do not provide information on the gene expression as it occurs under in situ conditions. ⁸⁶ Hence, the approach for “omics” has come into the picture. The “omics” can be classified into various categories based upon the implemented methods like metagenomics, metatranscriptomics, metaproteomics, and metabolomics. Recently, functional diversity of bacteria from Vagator and Morjim beaches of north Goa, India was analyzed by using a metagenomic approach that is associated with tarballs. The proteobacteria species were found to be a dominant group in both Vagator and Morjim tarballs. ⁸⁷ Metagenomics is used to monitor microbial communities, providing information to the functional gene composition of the microbial communities in the polluted sites. ⁸⁸ Recent studies have highlighted the composition of the microbiome in the presence of PAH changes. The groups of alpha-proteobacteria and actinobacteria have dominated in the hydrocarbon contaminated soil. Further in-depth studies revealed that the community of proteobacteria was found to be more in the hydrocarbon contaminated sites. ⁸⁹ Analysis of transcription (mRNA) in accordance with gene expression is dealt with metatranscriptomics. Various microbial communities from the polluted sites have been characterized with the help of metatranscriptomics techniques for ecosystem services like agriculture, ^{90 – 92} bioremediation,^{93– 95} and carbon cycling. ⁹⁶ The results obtained from the nucleic acids and protein profiles have shown a good correlation for the analysis of functional communities. ⁹⁷ The uses of metaproteins to evaluate the functioning of the ecosystem in the polluted site have drawn the interest because it carries both phylogenetic and functional information. ⁹² Some of the reported remarkable findings of “omics” application in the bioremediation processes are summarized in Table 2.

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Table 2.

Findings of “Omics” applications in bioremediation process.

“Omics” approaches	Specific findings	Performance outcomes	References
Metagenomics	Biodegradation efficiency of the native microbiota and PAH-degrading microbial consortium was analyzed comparing various effects (e.g. disappearance, displacement, permanence, etc.).	PAH biodegradation in soil using the microbes found to be 84% after 30 days.	136
	Identification of groups of dioxygenases and its functional targets for degradation of aromatic compounds were executed from the native bacterial community.	Fourteen distinct groups of genes from the Gram positive bacteria of the genera Rhodococcus, Mycobacterium, Nocardioides, Terrabacter, and Bacillus were characterized.	137
	Biodegradation efficiency of anthracene in polluted soils was studied in which it was found that spiked soil was having most of the Actinobacteria, and unspiked soil was having most of the Acidobacteria.	Incubating the soil and contaminating it with anthracene changed the bacterial community structure, but spiking the soil with acetone had no effect on the bacterial community structure.	138
	Uncultured bacterial species were having the potential to remove most of the pollutants as per analyzed sequences on polluted sites.	Over-expression of Ring-hydroxylating dioxygenases (RHDs) was studied with E. coli, which could degrade PAH.	139
Metaproteomics	The relative abundance of proteobacteria was increased in the petroleum pollution site, whereas for Rhizobiales it was decreased.	Addition of compost (0.55% of the metaproteome) promoted the removal PAHs and alkanes up to 88% after 50 days.	134
	Degradation of the aromatic compounds by Pseudomonas sp. K82, Acinetobacter strains, and responsible enzymes were identified.	Degradation of low molecular-weight aromatic-hydrocarbon and associated gene responsible for that was identified which is located in plasmid pLA1.	140
	Degradation pathway for Naphthalene and responsible groups of bacteria for the same from complex microbial communities were identified.	Biodegradation capabilities for growth-supporting aromatics, which results in bio-stimulated communities being extremely fit to naphthalene utilization completely.	94
	Information on the diverse dioxygenase genes for the catabolism of PAHs has been partially provided in a few bacteria, Pseudomonas rhodesiae KK1.	Degradation of pyrene and fluoranthene was achieved completely.	141
Metatranscriptomics	The metatranscriptome was analyzed from the soil and has a long history of usage of pesticides and chemical fertilizers used for agriculture purpose. Pesticide degraders like Pseudomonas, Serratia, Bacillus, Sphingobium, Achromobacter, Micrococcus, and Streptomyces were characterized. Similarly, heavy metals detoxifier like Desulfobacterium, Desulfobulbus, Arthrobacter, Acinetobacter, Ralstonia, Thiobacillus, and Rhodospirillumwere characterized.	Transcripts like catechol 1, 2-dioxygenase, benzoate 1, 2-, Intradiol ring-cleavage dioxygenase, gentisate 1,2-dioxygenase, etc., demonstrated high abundance which helps to degrade the aromatic compounds. Furthermore, analysis of various metabolisms revealed that bacteria in this ecosystem are dependent on organosulfonated compounds for their growth and development.	142
	The microbial communities are found in the orders: Rhodospirillales, Burkholderiales, Actinomycetales, Alteromonadales, Solirubrobacterales, and Caulobacterales.	The rhizosphere found in the polluted soils is enriched in transcripts related to PAH degradation.	143,144
	Degradation of atrazine can be mediated by a strong link between the relative abundance of mRNA by quantitative predictive capabilities of metatranscriptomics.	Undefined microbial community with varying fractions of an Escherichia coli strain can catalyze a specific transformation reaction for the herbicide atrazine.	145
	More abundance of transcripts of genes in the phenanthrene polluted sites related to dioxygenase, detoxification, and the stress response is due to the presence of native soil microbial communities to phenanthrene.	A community responses in soil to contamination with the PAH phenanthrene using a metatranscriptomic approach were evaluated.	146

Microbial electrochemical technologies: a way forward

Microbial electrochemical technology (MET) is an emerging area applied in the field of interdisciplinary subjects with biological intervention that has been used in the last decade. Broadly, it is applied in the field of microbial fuel cell (MFC) technology, bioelectrochemical treatment (BET) system, microbial electrolysis cell (MEC), and microbial electrosynthesis system (MES). MFC technology is used to generate electricity whereas BET system is used to remediate the complex and recalcitrant pollutants present in the wastewaters. Other techniques like MEC and MES are applied for the production of hydrogen and different chemicals, respectively. ^{6 ,98– 100}

The researchers have introduced microbial remediation cells for the removal of pollutants, viz. perchlorate, uranium, and chlorinated solvents in cathode through the reduction. ^{1 01– 103} Other applications of MET are the microbial electron mediating cells (MERCs) that are useful to maximize the biodegradation of herbicides isoproturon ¹⁰² and atrazine ¹⁰³ in the polluted sites. Recently, Yang et al. had investigated the removal of perchlorate from the wastewater by using MET. ¹⁰⁴ Dibenzothiophene (DBT) removal was enhanced by more than three-fold compared to the natural attenuation in a MERCs. ¹⁰⁵ Recent applications of MET for pollutants removals have been summarized in Table 3.

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Table 3.

Application of microbial electrochemical technologies with respect to pollutants removal, responsible microorganisms, and key findings.

Targeted pollutants	Microorganisms responsible for electrochemical reaction	Key findings/applications	References
17α-ethynylestradiol	ShewanellaAeromonas	Removal of steroid hormone concentrations from the water	147
Sulfate	Desulfobulbus, Geobacter, and Desulfovibrio	Sulfate removal from the water	148
2,4,6-Trichlorophenol (TCP)	Methanosarcina, Methanoseata, Methanococcus, Methanobacterium, and Methanomicrobium	Remediation of recalcitrant pollutants along with the generation of energy in the form of electrical power	149
Petroleum hydrocarbon (PHC)	Gamma proteobacteria	Bioremediation of marine petroleum hydrocarbon pollutants	150
Methyl orange (MO)	Not mentioned	Dye wastewater treatment	151
Phenol	Not mentioned	Phenol removal from the industrial waste	152
Sulfide	Thioalkalivibrio, Thioalkalimicrobium, and Desulfurivibrio	Sulfate removal from the wastewater	153
Benzene, methyl-tert-butyl ether (MTBE) and ammonium	Not mentioned	Microbial electrochemical technology for remediation of Benzene and related aromatic pollutants	110
Copper	Actinobacteria and Acidobacteria	Treatment of wastewater along with recovery of copper by using single-chamber MFC along with simultaneous energy recovery	154
Sulfadiazine (SDZ)	Geobacter	Biodegradation efficiency of Sulfadiazine and antibiotic resistance genes (ARGs) development	155
p-nitrophenol	Corynebacterium, Comamonas, Chryseobacterium, and Rhodococcus	Removal of aromatic compounds	156
Petroleum and Polyaromatic hydrocarbon (PAH)	Not mentioned	Plant-microbial fuel cell (P-MFC) was fabricated for removal of PAH and other oil along with a generation of electricity	157
Cefazolin sodium (CFZS)	Acinetobacter, Stenotrophomonas, and Lysinibacillus	CFZS was degraded, and its concentration was found to be 1.2 to 6.8 mg L−1 h−1 along with the electricity production in MFC	158
p-Chloronitrobenzene (p-CNB)	Propionimicrobium, Desulfovibrio, Halanaerobium, and Desulfobacterales	Treatment of high-concentration p-CNB wastewater	159
	beta-Proteobacteria, nitrobacteria and denitrifying bacteria	Removal of nitrate during bioelectricity generation under slightly alkaline and neutral conditions	160
Polycyclic aromatic hydrocarbons (PAH)	Geobacter psychrophilus	Removal of PAH by using a benthic microbial electrochemical system (BMES)	161

Future outlook and challenges

The environment-friendly and low-cost bioremediation technology is considered as one of the novel approaches for the remediation of polluted area. Moreover, the degradation mechanisms and pathways, reaction model, degradation kinetics, the half-life of the degraded pollutants should be considered for the effective remediation process.^106,107 Risk of co-production of secondary contamination from the metabolites of degraded hydrocarbon and other pollutants into the ecosystem should be identified. The major impact of cultivating various groups of biodegrading microorganisms for different pollutants in the local ecological system should be assessed. ¹⁰⁸

This is a well-known fact that the rate of degradation for a complex mixture of pollutants by one species of microorganism is very time consuming. This can be time effective using the mixed local microorganisms present in the natural ecosystem. Due to the involvement of a special group of enzymes synthesized by the respective mixed local microorganisms, the rate of degradation is very fast. ¹⁰⁹

However, modern methodology with advanced tools like genetic engineering, metabolic engineering, bioelectronics, protein engineering, genomics, and proteomics, nano-biotechnology, and bioreactor technologies have been gaining momentum to improve bioremediation process in research and development. A new technique like “Biofilm systems” has also developed to remediate the recalcitrant pollutants from the wastewater. The application of “Biofilm system” for bioremediation purposes can be a future scope to work on this topic. ¹¹⁰

Application of nanotechnology for remediation of pollutants can be taken into consideration due to various advantages like (i) giving rise to sustainable products, (ii) remediation of hazardous substances, and (iii) development of biosensors to monitor the environmental pollution. Dioxin can be absorbed by carbon nanotube more easily than activated carbon.

Despite various advancements in the field of pollution monitoring system using microbial biosensors, certain limitations are found. These include (a) large variability in sensitivity, (b) response times, (c) detection thresholds, and (d) signal relaxation lengths. Future research can be addressed to overcome these limitations for the biosensors.

Conclusions

Microbial communities at the polluted sites are the major driving force behind the remediation of pollutants and other biological activities. The effective bioremediation strategies require consideration of physicochemical parameters and catabolic properties of degrading microbial communities. Most microorganisms are not readily cultivable and cannot be characterized easily, which is a big challenge for microbiologists. Biostimulation of indigenous microorganisms and bioaugmentation through introducing desired microorganisms can be used as an alternative tool to satisfy bioremediation goals. In the 21st-century, the emergence of specialized techniques for monitoring the genome, transcriptome, and proteome of microbial communities has broadened the way towards the successful and efficient bioremediation technique. However, due to the technological constraints and economic problems, the application of high-throughput genomic approaches is still at an early stage of development for the bioremediation.