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  • Jonathan Jinghan Xiao & Katherine E. Graham

Novel Cypermethrin Bioremediation using Genetically Engineered Bacillus. Subtilis SCK6


Pyrethroids are a major commercial insecticide used for their high effectiveness against a broad spectrum of pests. Cypermethrin, a synthetic pyrethroid, is highly toxic to aquatic life and some vertebrates. While chemical treatment of pesticide contamination exists, a biological approach has better application potential and sustainability. The designed bioremediation system was intended to optimize its removal efficiency of cypermethrin through genetic engineering and statistical modeling.

Bacillus subtilis SCK6 with its non-pathogenic trait and high transformation efficiency was selected. The overexpression plasmid of cypermethrin metabolizing enzymes was constructed on Benchling. Box-Behnken design of the Response Surface Methodology was then used to optimize and analyze factor interaction for the transformant's biodegradation efficacy.

With Pxyl-XyIR inducible-operon for esterase, in which only the treatment of xylose activate the SCK6* to hydrolyze pesticide residuals. The RSM model generated by the Box-Behnken design demonstrated comprehensive analysis and optimization of the data with multiple interacting factors. The B. subtilis SCK6* can be incorporated with novel xylose-containing biofertilizers for real-world application, and corresponding analytical models can help adjust the bioremediation system for optimized degradation efficacy.

The Pesticide Problem:

With humanity on the rise, natural resources are continuously strained to support increasing consumption. More people results in higher demand for essentials like food and water (Bish, 2020). One method of increasing agricultural production is the utilization of pesticides, any substance used to control, repel, or kill unwanted organisms known as pests (Why We Use Pesticides, 2022). Conventional chemical pesticides are most commonly integrated into pest management systems due to their effectiveness and low price. Without pesticides, U.S. food production would not only drastically decrease causing prices to skyrocket, but also weakening the overall American economy: as lower employment, price instability, reduced international food aid.

Pesticides are a foundational part of modern agriculture, yet their lingering side effects of pesticides pose the danger of contaminating their surroundings due to their long stability and high transmissibility via air and waterways (Saleh et al., 2020). When a pesticide is applied, it does not immediately break down to remain effective; the half-life of a pesticide determines its environmental significance as the introduction of chemicals has a high risk of disrupting the natural ecosystem. Pesticide persistence can lead to bioaccumulation resulting in toxic levels of chemicals in water, plants, and animals (Hanson et al., 2015).

Current methods to minimize the chemical damage include federal regulations and various remediation systems such as activated sludge and wastewater treatment. The Environmental Protection Agency (EPA) is a federal organization that registers and regulates all pesticide usage in the United States. Every company must present scientific research on the pesticide’s toxicity, its risks to humans, and environmental effects to the EPA before its approval (Delaplane, 1996). While this does ensure national pesticide regulation, chemicals are still introduced into the environment.

A prominent issue is the accumulation of pesticides in water systems from runoff. This is damaging as it spreads the toxic chemicals far from their point of application, increasing their impact radius. Standard removal practices are either physical, chemical or biological. Physical remediation is based on the adsorption process, a common large scale water purification method. Chemical remediation is the conversion of pollutants into harmless compounds through various chemical reactions, often coupled with physical processes. While not as effective as its counterparts, biological remediation is both low cost and more environmentally friendly (Andrunik & Bajda, 2021).

Pyrethroid Agricultural Runoff:

Pyrethroids were made to increase the effectiveness of natural occurring pyrethrins by improving their longevity (NCBI, 2023). Cypermethrin (CYP), a type of synthetic pyrethroid, is found in a variety of pest management products designed for crops, livestock, trees, and households (Hołyńska-Iwan & Szewczyk-Golec, 2020). CYP is highly toxic for small aquatic organisms and several vertebrates, as it has been proven to kill them after affecting their central nervous system (NCBI, 2023). Aquatic insects in particular are crucial due to their variety of roles in their ecosystem including water purification and acting as a food source (Suter & Cormier, 2015). CYP poses a significant risk due to the low concentration proven to cause damage, making even short exposures detrimental. Human trials have proved low doses of CYP have minimal effect as it is excreted rapidly. However, the US EPA classified cypermethrin as a possible human carcinogen (group C) due to evidence linking it to cancer in mice test groups (NCBI, 2023).

Once cypermethrin has contaminated the environment, its only removal is through water treatment to reverse pesticide damage. Most pesticide water remediation requires specific treatment before reaching standard domestic wastewater management due to their variability in ideal conditions (Saleh et al., 2020). As a result, the remediated water often still contains toxic levels of unaddressed pesticides. A published California study of pyrethroid water treatment found that most residential, storm, and agricultural runoff contained pyrethroids concentrations that exceeded acutely toxic thresholds. After pyrethroids passed through secondary treatment systems at municipal wastewater treatment facilities, effluents still contained high pyrethroid concentrations (Weston & Lydy, 2010). Microorganisms are a promising alternative to standard practices due to their price and sustainability, but are restricted by uncertainties such as inconsistent performance and application difficulties (Sarker et al., 2021). If biological treatments of cypermethrin are improved, then the efficacy and sustainability of the remediation increases.

Selection of the organism

Bacillus subtilis is an aerobic gram positive, non-pathogenic bacterium, easily engineered for heterogeneous protein expression (Su et al., 2020). The use of B. subtilis meets the criteria of biosafety, as it does not pose any ecological threats to local plants, animals and humans. Key enzyme laccase and esterase present in B. subtilis facilitate pyrethroid degradation. Therefore, Bacillus subtilis is selected as our model organism because of its high potential for agricultural biotechnology application and specific suitability for cypermethrin degradation. We plan to use recombinant B. subtilis strain SCK6 with engineered inducible super-competence for high efficiency transformation. SCK6 has an extra competence master regulator Comk under the control of the xylose-inducible promoter. The addition of xylose to 1% (w/v) to B. subtilis cultures in LB medium will allow for plasmid DNA transformation frequencies of up to 107 with multimeric plasmid preps or 104 with ligated plasmid DNA (Zhang & Zhang, 2010).

Selection of the Enzyme

As previously mentioned, laccase and esterase were identified as the key enzymes facilitating the biodegradation of cypermethrin in Bacillus subtilis via PCR gene amplification. Increase in esterase activity was elucidated both qualitatively and quantitatively in the presence of cypermethrin in B. subtilis Strain 1D (Gangola et al., 2018). Furthermore, the biodegraded products of cypermethrin from both esterase were proven to be environmentally safe. Bacterial esterase has been demonstrated as a powerful enzyme for hydrolyzing pyrethroid compounds (Bhatt et al., 2020). The protein coding sequence of Bacillus subtilis’ gene estB could be retrieved from NCBI (Eggert et al., 2000).

Construction of the Plasmid

The transformation plasmid pJet1.2 specR/estB was designed on the software Benchling (see Figure 1 for plasmid map) — full annotated sequence can be retrieved in the corresponding reference link (Xiao, 2023). The backbone of the plasmid is pJet1.2 specR cassette designed for spectinomycin resistance of Bacillus subtilis. It can be purchased from Addgene under Plasmid ID 117120 (Diebold-Durand et al., 2019). The goal is to overexpress the estB gene, thus a strong promoter of B. subtilis is necessary for high protein yield. An inducible operon system is included to control the esterase expression while maintaining the enhanced expression level when turned on. Pxyl-xylR is a xylose-inducible promoter from B. subtilis which maintains a high expression level of the downstream protein gene when activated (Zhang, Su, Duan, Liu, & Wu, 2017). Pxyl-xylR promoter sequence is retrieved from Synbiohub and integrated into the pJet 1.2 upstream to the estb gene The estB gene acquired from NCBI was then placed downstream to the P43 (Eggert et al., 2000). The gene fragment (marked as ORF) which contains the P43 promoter and estB will be synthesized and then ligated to the pJet1.2 specR backbone.

Figure 1

Protocol Design

To test the competency of the Bacillus subtilis, it undergoes transformation, testing, and analysis processes. The B. subtilis strain SCK6 will be plated on normal LB plate and plasmid cloning E. coli will be plated on LB+Ampicillin for selection. LB+Spectinomycin plate will also be poured for later transformation. A miniprep procedure is then carried out to extract our plasmid. The transformation protocol is based on previous research which designed the exact recombinant strain SCK6 with xylose-inducible super-competence to maximize the B. subtilis’s transformation efficiency (Zhang & Zhang, 2010; B. Subtilis Transformations, 2017). The M9 Minimal Salt and corresponding minimal salt medium (MSM) will be prepared to pH of 7. MSM were used for the cultivation and remediation efficacy testing of pesticide-degrading bacterial strains in related studies (Negi et al., 2014; Pankaj et al., 2016). The lack of carbon source in MSM medium will force the B. subtilis to major breakdown cypermethrin to acquire energy. The group of 15 samples generated from Box Behnken Design with different values under three critical factors—shaking speed, inoculum size and cypermethrin concentration will be tested for the optimized biodegradation at 20 ppm. Test group information is shown in Table 1. The bacteria will grow for 15 days at 33 °C. Samples will be withdrawn after incubation and residual pesticide with biodegradation percentage will be quantified by Gas Chromatography-Mass Spectrometry (GC-MS).

Table 1

Box-Behnken Design


With Pxyl-xylR inducible operon outputting high estB expression, the B. subtilis SCK6* can be incorporated with novel xylose-containing biofertilizers for real-world application with corresponding analytical models adjusting the bioremediation system for optimized degradation efficacy. Since Bacillus Subtilis fertilizers already include live strains to increase the intake, processing, and retention of nutrients via mutualism between microbes and plants, the addition of enhanced cypermethrin degradation capabilities (Bueno et al., 2022). By tackling the toxic pesticide problem at the source, leaching into waterways is minimized to optimize the ecological health of the waters around agricultural sites. An additional application of our modified B. subtilis strain is a candidate for activated sludge wastewater treatment due to its pre-existing usage in nitrate removal. Bio-augmented wastewater is a more sustainable and cost effective alternative to conventional chemical treatment (Rahimi et al., 2020). Control group testing has been completed to establish the initial rate of degradation before transforming the bacteria. Until necessary funding is acquired, following procedural steps cannot be completed. Leveraging genetic engineering, our proposed solution is a revolutionary design that is the first to materialize research connecting cypermethrin biodegradation to Bacillus subtilis.


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