Microbial bioremediation techniques and practices (some case studies, commercial products)

Being economic and ecologically sustainable, biorernediation with microbes is always regarded as the most important option for pesticide decontamination. However, various environmental factors may limit its application, particularly at the field level. The bioremediation technique at the lab scale may not succeed or fail at the field scale. The reason may be the difficulties in simulating the controlled laboratory condition to the field level. Several biotic and abiotic factors which control the microbes may also control the outcome of biorernediation experiment at field level. Another important factor is the bioavailability of pesticides, particularly persistent pesticides like DDT towards the microbes. Therefore, it is important to determine ways of increasing pesticide bioavailability. Table 3 lists some success stories where microbial biorernediation of pesticides was validated at the field level or mesocosnr level. Strong et al. (2000) succeeded in 97% degradation of atrazine at the filed level by using recombinant E. coli overexpressing the atrazine chlorohydrolase gene derived from Pseudomonas sp. ADP. The addition of phosphate as a biostirnulant increases the biodegr adation of atrazine in soil plots. Sagarkar et al. (2013) carried out a mesocosnr study (100 Kg soil) for biodegradation of atrazine herbicides and almost 90% atrazine degradation was observed with an atrazine degrading consortium comprising of 3 novel bacterial strains. John et al. (2018) used Klebsiella sp. isolated from pesticide-contaminated agricultural soil for in situ biorernediation of msecticide chlorpyrifos and demonstrated that the microbes can degrade the toxic clilorpyrifos into non-toxic products which increased the growth of soil microorganisms and dehydrogenase activity. Several field level studies for biorernediation of DDT contaminated site resulted in 68-95% removal of DDT.

Table 3. Microbial biorernediation of pesticides at fieldinesocosui level.

So, for on-site bioremediation, a systematic understanding of the surrounding nature and level of contamination of pesticides is important. Attention should be given in case of mixed pesticides contamination, since the degradation of one pesticide may stimulate or hinder the activity of microbes associated with the degradation of other pesticides. Prior knowledge is useful in choosing the best bioremediation strategy for field applications.

Biotechnological intervention in microbial remediation

Biotechnology is considered a prospective resource of safe, inexpensive, and effective methods for the bioremediation of contaminated sites including pesticides. Even though the use of microorganisms for bioremediation has gamed some success, the use of biotechnological tools may help hi gaining momentum. The genetic engineering approach may help in generating microbes with the best mix of biochemical pathways for remediation of contaminated sites. The recent development in the evolution of pesticide degradation pathways, along with the organization of catabolic genes involved, enabled the researcher to develop genetically engineered microbes with enhanced decontamination potential. Advancement of recombinant DNA technology gives a more clear understanding of the degradation process. Catabolic genes involved in the degradation pathway have been identified for several pesticides. By manipulating these degradation genes, attempts are made to generate hybrid pathways by developing microbial strains with enhanced degradation capabilities.

Genetically modified microorganism (GMO) for pesticide bioremediation

The use of microbes and their enzymes for the degradation of pesticides is considered as an eco-friendly and sustainable approach as they are self-sustainable and low-cost. However, the biodegradation potential of native microorganisms for different pesticides with diverse chemistry is very much limited. To overcome these constraints, designing transgenic microbes through genetic engineering approaches is a highly important step for enhancing the biodegradation of pesticides. Due to theh adaptation to wider environmental conditions, genetically modified organisms (GMOs) offer better potential for faster degradation of pesticides and thus environmental remediation. Thus, this branch of biotechnology helps in the remediation of pesticide pollution by converting them into a non-toxic or low toxic form. Microbes have been continuously and consciously introduced into the environment for a specific reason. However, current knowledge of biotechnology helps in developing a new strain with thrilling capabilities. Here, through modification/alteration of genetic material, i.e., DNA, one particular organism is modified to get the necessary character and is usually called as genetically modified. Several terminologies are associated with this technology and among them, the most used are “gene technology,” or “recombinant DNA technology” (RDT), or “genetic engineering,” and the modified organism is known as “genetically modified,” “genetically engineered,” or “transgenic.”

Wasilkowski et al. (2012) affirmed that integration of conventional microbiology, biochemistry, ecology along with genetic engineering led to effective measures for in situ bioremediation. A wide variety of genetically modified microorganisms has shown great potential for effective and faster degradation of pesticides into non-toxic metabolites (Rayu et al. 2017). A large number of native microorganisms have been genetically modified for faster biodegradation of pesticides. For remediation of soil contaminated with multiple pesticides, Cao et al. (2013) constructed a genetically engineered microorganism by fusing the organophosphorus hydrolase with INPNC (ice nucleation protein) of Pseudomonas syringae onto the cell surface of Sphingobium japonicum UT26, an HCH- degrader.

Several genes have been isolated and identified with an ability of faster degradation of pesticides (Table 4), and can be suitably used for the construction of genetically engineered microbes. Encoding of atrazine chlorohydrolase by gene atzA resulted in faster degradation of atrazine (Neumann et al.

2004). Atrazine bioremediation in field-scale was accomplished by using a killed and stabilized whole-cell suspension of recombinant Escherichia coli engineered to atrazine chlorohyrolase, where

Table 4. List of genes from different species involved in pesticide degradation.

dechlorination of atrazine resulted in the formation of non-toxic and non-phytotoxic metabolites (Strong et al. 2000). For degradation of liexachlorocyclohexane (HCH) and methyl paratliion simultaneously, Lu et al. (2008) constructed genetically engineered Sphingonionas sp. BHC-A-mpd by overexpressing methyl paratliion hydrolase gene (mpd) to Sphingomonas sp. BHC-A, a highly efficient HCH-degrader. Gu et al. (2006) constructed an engineered strain P putida KT2440-DOP for degradation of organophosphoms pesticides along with some aromatic hydrocarbons. Yang et al. (2012) developed a genetically modified organism by overexpressing organochlorines (OCs) and organophospliates (OPs)-degradation gene linA and mpd, respectively, in E. coli for degradation of OCs and OPs simultaneously. An innovative approach for increasing the biodegradation of herbicides 2,4-D by native microorganism was achieved by the natural conjugative transfer of catabolic plasmids from E. coli to the indigenous soil bacteria (Top et al. 1998).

Other than the overexpression gene related to pesticide degradation, the fusion of protoplast is emerging as a promising technology for constructing multifunctional genetic strains where the gene for specific functionalities is transferred from one species to another and thus getting the benefit of both parents (Dillon et al. 2008). In the field of bioremediation of pesticides, this is a novel and reformative technique. By protoplast fusion of Rhodococcus sp. BX2 and Acinetobacter sp. LYC-1, Feng et al. (2013) constructed a functional strain FI for faster degradation ofbensulfuron-metlryl and butachlor simultaneously. For simultaneous degradation of neorricotinoid pesticides chlorotlralouil and acetanriprid, Wang et al. (2016) also developed a functional strain, AC, through a protoplast fusion of Pseudomonas sp. CTN-4 and Pigmentiphaga sp. strain AAP-1. With the advancement of recombinant DNA technologies, “suicidal GEMs” (S-GEMs) technology has also been emerged for remediation of contaminated sites (Patti et al. 2005, Kumar et al. 2013) which is safe and more efficient. However, survival and biodegradation ability of GMO in the field depends on the several biotic and abiotic factors in the environment. Factors like high clay and pH and moisture content increase the survivability of GMO, whereas factors like prolonged dry periods, presence of competing microorganisms, lytic bacteriophage, and protozoan predation will negatively affect the introduced microbes. Although a considerable number of GMOs have been constructed throughout the world for biorernediation of pesticides, their successful field-scale application is rare. Furthermore, constrain in upscaling the laboratory experiments, low bioavailability of pesticides to the induced microbes, and legislative problems related to the legal use of GMOs have prohibited wide-scale application.

Due to legal challenges and high expenses involved in transgenic research, this approach affects many companies and until now it is confined to academic and research institutes. Controversy related to the use and release of GMOs in the open environment restricts its use. Concerns have been expressed by several countries including India over the safety and ecological damage linked with the release of GMO. In India, release of GMOs for bioremediation in uncontrolled conditions is strictly prohibited. In the Indian scenario, for releasing GMOs, several step permission is required which starts from Institutional Bio-Safety Committee (IBSC) to Research Committee on Genetic Modification (RCGM) for field and other related tests and trials and GEAC (Genetic Engineering Approval Committee) approval is must for commercial releases. Since bioremediation with GMO is rarely practiced, the system as a consequence is not yet defined completely. Therefore, environmental and ecological concerns and regulatory constraints hamper the use of GMOs for field-scale bioremediation of pesticides.

Metagenomics and metaproteomics approach for evaluation of pesticide bioremediation

The indigenous microbial community establishes a complex ecological niche for remediation of the pesticide-contaminated site. But they are not readily cultured. Therefore, the analysis of the structural and functional composition of microbes is essential to determine their role during bioremediation of various pesticides. Concurrently, the knowledge of primary and secondary metabolites/protein will help in understanding the interaction between pesticide and microbe, how it works at the molecular level and response of organisms exposed to a pesticide-contaminated environment. The repeated or high dose of the application of pesticides may have negative effects on the ecosystem due to the formation of toxic metabolites which can disturb the endogenous metabolism as the microbes are exposed to changing environments. Furthermore, due to the persistent nature of pesticides, particularly organochlorine pesticides, there is a chance of partial mineralization by microbes which lead to toxic metabolites accumulation. Consequently, compared to the traditional method, there is a necessity of a more accurate method for pesticide bioremediatiou. Metagenornics and proteornetabolomics approach may create a system that can help to understand the site-specific microorganism during the active biorenrediation process. Onrics based approaches have great potential for microbial detection and community analysis. These ornics-based techniques have a great impact on the bioremediation potential of microbes for pesticide degradation.

The main prospect for the researchers is to find out the major mechanisms involved in biorenrediation processes for the degradation of pesticides. The integration of functional genomics, proteornics, transcriptonrics, and metabolornic data along with the biorenrediation process may help to get a clear-cut picture of the microbial biorenrediation process. The integration of all of these with high throughput techniques will enable a step forward in the researches on pesticide biorenrediation. Recently, in various studies, the nretagenonric approach has been used to investigate the potential biodegr adation pathways of persistent pesticides (Fang et al. 2014).

The metagenomic approach was used by Fang et al. (2014) to understand the abundance and diversity of biodegradation genes (BDGs) along with the potential degradation route of DDT, HCH, and atrazine in both freshwater and marine sediments by using 6 datasets. It was found that out of 69 genera identified, Plesiocystis, Anaerolitiea, Jannaschia, and Mycobacterium were found to be potential for pesticide biodegradation in all sediments. Dadhwal et al. (2009) proposed the biostimulation of indigenous HCH degrading microorganisms after analysing the diversity of culturable microorganisms for effective biorenrediation of severely HCH-contaminated site in India. Metagenomic analysis carried out by Sangwan et al. (2012) for HCH-contaminated soil samples from India confirms the horizontal transfer of HCH catabolism genes. The study also suggests developing an economically viable bioremediation technology for HCH contaminated sites. Upon degradation, insecticide chlorpyrifos produces a toxic metabolite 3,5,6-trichloro-2-pyridinol (TCP). Gene (tcp3A) encoding a TCP degrading enzyme was cloned from a nretagenonric library prepared from cow rumen (Math et al. 2010). TCP was used as a sole source of carbon by recombinant E. coli harboring the tcp3A gene. This was a breakthrough in the application of nretagenomics approaches for pesticide degradation. Besides, recently a combination of nretagenomics and metaproteomics approach has been used for remediation of emerging contaminants (acetaminophen and sulfonamides) and this approach can also be extended for bioi emediation of pesticides (Chang et al. 2018).