Isolation and characterization of synthetic pyrethroids-degrading bacterial strains from agricultural soil

Abstract Pyrethroid pesticides are commonly used for pest control in agriculture setup, veterinary and home garden. They are now posing increased risks to non-targeted organisms associated to human beings due to their considerable use. The present work deals with the isolation of bacteria with tolerance to high concentrations of bifenthrin and cypermethrin from contaminated soil. Enrichment culture technique (bifenthrin concentration = 50-800 mg/L) was used for bacterial isolation. Bacteria that showed growth on minimal media with bifenthrin were also sub-cultured on minimal media with cypermethrin. Bacteria showing luxurious growth on both the pyrethroid, were screened out based on their morphological, biochemical parameters and by API 20NE Kit. Phylogenetic studies revealed that, one bacterial isolate (MG04) belonging to Acinetobacter lwoffii and other five bacterial isolates (MG06, MG05, MG01, MG03 and MG02) cluster with Pseudomonas aeruginosa, Pseudomonas putida respectively. Isolated members of genera Pseudomonas and Acinetobacter could be used for further detailed degradation studies by using FTIR, HPLC-MS or GC-MS analysis.

Isolation and characterization of synthetic pyrethroidsdegrading bacterial strains from agricultural soil tolerance potential to higher concentrations of bifenthrin and cypermethrin using different optimizing conditions.

Chemicals and media
Synthetic pyrethroids (SPs) bifenthrin and cypermethrin were purchased from Sigma-Aldrich (USA).All the chemicals were of analytical grade and 98% pure.Stock solutions of both the pyrethroids (1000 mg/L) were prepared in methanol and filtered by membrane filtration (pore size 0.22 µm).Mineral salt medium (MSM) used for enrichment and degradation studies was composed of the following components (in g/L): 1.0 NH 4 NO 3 , 1.0 NaCl, 1.5 K 2 HPO 4 , 0.5 KH 2 PO 4 , 0.005 FeSO 4. 7H 2 O and 0.2 MgSO 4 .7H 2 O, and final pH was adjusted to 7.0.For solid media, 15 g/L agar was added to mixture of all the media constituents.

Isolation and enrichment of SPs degrading bacteria
A conventional enrichment technique was performed for isolation of pyrethroids degrading bacteria.Enrichment was carried out in MSM supplemented with bifenthrin (initial concentration 50 mg/L and final concentration 800 mg/L) as an additional carbon source.The soil samples at 4-6 cm depth were collected from the agricultural fields of NARC Islamabad, having five years' history of pyrethroid usage.One-gram soil was transferred to 50 mL media in a flask and incubated for 5 days at 30 °C temperature and 180 rpm in a shaker incubator.Five (5 mL) of the incubated culture was sub-cultured into 50 mL fresh MSM medium for another 5 days.The process was further repeated seven times with media containing bifenthrin at different concentrations (200,300,400,500,600,700 and 800 mg/L).After enrichment, the culture was serially diluted and spread on MSM agar plates (supplemented with 50 mg/L bifenthrin) for isolating individual colonies.Bacterial isolates were then cultured on MSM media supplemented with 50 mg/L cypermethrin.Distinct bacterial colonies were checked for tolerance to various pyrethroids concentrations (50 to 800 mg/mL) and those possessing high degree of tolerance were selected for further analysis.

Phenotypic characterization of bacteria
Colony morphology and biochemical analysis of the bacterial isolates was done through their physical appearance on culture plates, gram-staining, capsule staining, spore staining and motility test (Ali et al., 2022;Holt et al., 1994).Biochemical analysis involved tests such as gelatin liquefaction, starch hydrolysis, triple sugar iron agar test, nitrate reduction, catalase test, oxidase test, methyl red, Voges Proskauer, citrate utilization and fermentation of sugars.Further API (Analytical Profile Index) 20NE, BIOMERIEUX, France was used to confirm the biochemical characterization of isolated bacterial strains.

Growth kinetics
The growth kinetics of all bacterial isolates grown on LB agar were estimated by inoculating into 50 mL MSM concerns and put people at risk of various diseases and conditions such as damage to endocrine, reproductive and immune system and increased chances of cancer (ATSDR, 2003;Zhang et al., 2010).Pyrethroids also cause environmental issues like air, soil and water pollution (Cycoń et al., 2006;Decourtye et al., 2005;Wendt-Rasch et al., 2003).SPs are enormously toxic to aquatic life, even only 10 ng/L concentration is enough to eradicate the entire invertebrate population of a river or lake (Pearce and Warford, 2012).The SPs pollution occurs through treated livestock in dipping tanks and/or directly from agricultural soil run off (Armstrong and Phillips, 1998).In Pakistan, increased than standard (European Union Standard for Drinking Water) pyrethroid residues have found in Rawal and Simly Dam Lakes, Islamabad (Iram et al., 2009).One of local newspaper also reported high fish mortality in these lakes that is probably due to excessive use of SPs in agricultural fields of National Agriculture Research Council (NARC) which are adjacent to these lakes.
Nowadays photolysis, hydrolysis and biodegradation are the commonly used approaches for disposal of SPs in agricultural lands (Fan et al., 2012).Biodegradation is the most suitable method for the disposal of pesticides residues and is accomplished by microbial forging activities.However, rate of biodegradation largely depends on type of microbial community composition and diversity, type of soil and pesticide and climatic conditions as well.Important to note is that microbial degradation of SPs is also highly environmental friendly, effective, cheap and safe practice to clean up these environmental contaminants (Singh and Walker, 2006).Unfortunately, biodegradation approach is still not in practices to detoxify these environmental pollutants in Pakistan.Thus, it is necessary to develop rapid and effective methods to remove these toxic SPs constituents that may reduce the environmental and public health risks associated with pyrethroid usage.Biodegradation is a major practical and applicable approach for the detoxification of SPs in various conditions such as either in soil or in aquatic environment.
To date, many researches have done on isolation and evaluation of pyrethroid degrading bacteria and fungi (Cycoń and Piotrowska-Seget, 2016;Fan et al., 2012;Zhang et al., 2010).However, previous studies on biodegradation of SPs used microbes that could only use carbon as sole energy source (Grant et al., 2002), and these conditions may not be suitable for biodegradation in open environment.
Also, bifenthrin a type-I pyrethroid (Hintzen et al., 2009) which is classified as toxicity class-II moderately hazardous compound (WHO, 2009) is the most persistent and obstinate pyrethroid to microbial degradation (Wang et al., 2009).Only few studies have reported bifenthrin degrading microbes (Chen et al., 2012).Similarly, cypermethrin -a type-II pyrethroid has become one of the dominant insecticides among retail sales to consumers (Weston et al., 2009) and regarded as a possible human carcinogen by the Environmental Protection Agency (EPA) USA (Zhang et al., 2010).So, there is a strong need to develop effective strategies for the removal of SPs residues due to their high toxic effects on mankind and environment.The present study was aimed to isolate and identify the bacterial strain from agricultural soil with degradation and Brazilian Journal of Biology, 2023, vol.83, e271790 3/7 Pyrethroids degrading bacteria isolated from soil media supplemented with cypermethrin and bifenthrin each at 800 mg/L concentration.During incubation at 37 °C, the growth curve of strains was measured at 620 nm at different time intervals (4, 20, 40, 60, 80 and 96 hours).The medium without SPs was considered negative control and best strains were selected on the base of growth log phase.

Temperature and pH optimization
For pH optimization, different aliquots of 50 mL MSM supplemented with SPs were set up at different pH (4.5, 5.0, 6.0, 7.0, 8.,0, 9.0 and 10.0) and inoculated with the isolated bacteria.The growth curve of 40 h at 37 °C incubated cultures was estimated at 620 nm using spectrophotometer (BioRad, USA).Negative control was left blank and optimum pH was determined by plotting absorbance values and pH.

Genomic DNA isolation
Isolation of genomic DNA was done by CTAB (Cetrimonium bromide) method with some modifications (Wilson, 2001).Bacterial isolates were inoculated on nutrient agar plates and incubated for at 37 °C.Loops (n=3) of the fresh culture were added to 300 µL of TE buffer and vortexed at high speed.Five (5 µL) 20 mg/mL proteinase K and 30 µL of 10% SDS were added, and mixture was incubated at 37 °C for 1 hour in water bath, followed by the addition of 100 µL CTAB solution and 100 µL 5 M NaCl and incubation again at 65 °C for 30 minutes in water bath.After incubation, 500 µL mixture of phenol, chloroform and isoamyl alcohol at 25:24:01 ratio was applied, and solution was subjected to centrifuge for 20 minutes at 12000 rpm.The supernatant was collected in sterile Eppendorf tubes and pellets were discarded.For DNA extraction, 500 µL isopropanol and 200 µL 3 M sodium acetate was added to the supernatant and kept at room temperature for 30 minutes followed by overnight incubation at freezing temperature at 4 °C.The DNA pellet was recovered by centrifuging an overnight freeze supernatant at 10,000 rpm and 4 °C for 15 minutes.Supernatant was discarded and pellets were washed with 200 µL 70% ethanol by centrifugation at 10,000 rpm for 5 minutes.Ethanol was removed by using blotting paper and DNA pellets were dissolved in 50 µL TE buffer for amplification of DNA.
The thermal profile used for amplification reaction was initial denaturation at 95 °C for 5 minutes, 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 90 seconds and a final extension for 10 minutes.The hold was set at 4 °C and PCR product was visualized by 1% (w/v) electrophoresis using UV-Transilluminator.Amplified products were purified using the PCR purification kit (Thermo Scientific) and sent for sequencing.
Upon retrieval of nucleotide sequences, they were confirmed through blasting at NCBI and EzBioCloud databases and MEGA-X software to construct phylogenetic tree.The nucleotide sequences were submitted in NCBI GenBank database under the accession numbers OK148706 to OK148710 and MG957215.

Results
Soil samples collected from pyrethroid containing agricultural fields were processed for isolation, identification and characterization of SPs-degrading bacteria.Six SPs-degrading bacteria were isolated through conventional enrichment culture technique using MSM supplemented with bifenthrin and cypermethrin pyrethroids as sole carbon source.All the bacterial strains showed growth even at higher concentrations on SPs supplemented MSM, indicating the capability of strains to utilize the bifenthrin and cypermethrin as C source.The growth kinetics of all six strains were varied from each other, however MG-04 showed highest growth on 800 mg/L bifenthrin and cypermethrin as compared to other strains (Figures 1a and b).
The morphological and biochemical characteristics of the isolated organisms are summarized in the (Table 1).All the isolates were gram-negative, non-spore forming, rod shaped bacteria.MG-04 was non-motile and positive for capsule staining, whereas the rest of the isolates were non-capsulated motile bacteria.Three strains MG-03, MG-05, and MG-06 were biochemical identical.They appeared positive for starch hydrolysis, nitrate reduction, catalase, oxidase, urease, indole production, methyl-red, citrate utilization and dextrose fermentation and negative for gelatin liquefaction, Voges-Proskauer and fermentation of lactose, maltose and sucrose.Based on presumptive identification, MG-01, MG-02, MG-03, MG-05 and MG-06 strains were identified as members of genus Pseudomonas, however MG-04 was an Acinetobacter sp.API 20NE further confirmed this identification.
From the results of growth kinetic studies, bacterial strain Acinetobacter sp.MG-04 was selected best candidate for the biodegradation of SPs and determined its temperature and pH optimization.The results of temperature and pH optimization of Acinetobacter sp.MG-04 in MSM containing 800 mg/mL bifenthrin and cypermethrin are summarized in Figures 2a and 2b respectively.Acinetobacter sp.MG-04 indicated highest growth at 40 °C in both MSM supplemented with bifenthrin and cypermethrin after 40 hours of incubation (Figure 2a).The log phase of MG-04 growth declined after the 40 °C, and highest bacterial growth decline was observed at 55 °C.Likewise, optimum pH for strain MG-04 was determined 7 and 8 after 40 hours' incubation at 40 °C (Figure 2b).The minimum bacterial growth was noticed at 4 pH, however, after 8 pH a drastic decline in bacterial growth against bifenthrin and cypermethrin was recorded after 40 hours of continuous incubation.
The NCBI blast of 16S rRNA gene sequence of the best selected strain MG-04 indicated its 98% homology with Acinetobacter lwoffii strains.The phylogenetic analysis of this strain indicated its similarity with previously identified Acinetobacter strains isolated from different agricultural soils worldwide.Therefore, MG-04 was submitted to GenBank database as Acinetobacter lwoffii MG-04 [MG957215] (Figure 3).
The remaining five bacterial isolates (MG-02, MG-06, MG05, MG01 and MG03) of the present study were   Pyrethroids degrading bacteria isolated from soil identified as phylogenetically identical to bacteria of the genus Pseudomonas.The MG-06 (98%), MG-05 (100%), MG-01 (99%) and MG-03 (99.20%) made clusters with the Pseudomonas aeruginosa spp., while the bacterial isolate MG-02 showed 98% resemblance with the Pseudomonas putida.The results were also supported by high bootstrap value along with high similarities with their respective top hit sequences mentioned above (Figure 4).

Discussion
The constant increase in human population, the use of different types of pesticides and fertilizers input gained acceptance in agricultural practices to enhance crops yield (Sabir et al., 2021).Though the literature confirmed that these fertilizers and pesticides enhance crop yield, but studies have also documented unfavorable effects of these fertilizers on soil microorganisms which made these living species adoptable/resistant to surrounding environment (Martelli and Giacomini, 2018;Ryall et al., 2012) similar to antibiotic resistance development in bacteria (Shakeela et al., 2020).Different bacterial and fungal strains have been found resistant to many pesticides (Bhatt et al., 2019), for example, Candida spp., Aspergillus spp., Pseudomonas spp., and Bacillus spp., have been reported as pesticides (including pyrethroids) degrading bacteria (Bhatt et al., 2019;Paul and Mandal, 2019;Singh and Walker, 2006).It has also confirmed that bacteria degrade pyrethroids and use them as carbon source for their growth (Jin et al., 2014).
The current study isolated many bacterial strains from agricultural lands applied with pyrethroid pesticides.Jin et al. (2014) concluded same results from their research.Our study reported that Acinetobacter spp., hydrolyzed pyrethroids and used them as nutrient source for their growth.This is confirmed from previous studies which successfully isolated pyrethroids degrading bacteria (Cycoń and Piotrowska-Seget, 2016;Grant et al., 2002), and their enzymes from soil habitat (Fan et al., 2012).
As mentioned above, the present study isolated Acinetobacter lwoffii and Pseudomonas spp., from pyrethroids containing agricultural soil.One of the previous research studies also isolated Acinetobacter spp., from pyrethroids applied soil (Jin et al., 2014), while Gong et al. (2018) isolated Pseudomonas species from the same environment.Acinetobacter lwoffii and Pseudomonas spp., of the present research have found to have high levels of resistance to bifenthrin and cypermethrin.Acinetobacter species are ubiquitous in nature and known to be involved in removal of pesticides such as organophosphate, carbamates and diclofop-methyl herbicides (Bhatt et al., 2021).They are well recognized for remediation of heavy metals (Abdel-El-Haleem, 2003).Previous studies have also shown their role in decontamination of diesel, phenol and crude oil (Jung et al., 2010), but there are very few reports of pyrethroid degradation by members of this genera (Akbar et al., 2015;Jin et al., 2014).Jin et al. (2014) isolated Acinetobacter spp.strain JN8 from soil by enrichment culture technique that could utilize a broad range of only type-II pyrethroids as carbon source for cell growth.Akbar et al. (2015) isolated Acinetobacter calcoaceticus MCm5 which degraded both types of pyrethroids.Other previous studies isolated and identified different species of Acinetobacter from agricultural soil responsible for degrading pyrethroids and other pesticides (Singh et al., 2004;Singh and Walker, 2006; Chen et al.,  The optimal tree with the sum of branch length = 0.02 is shown.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.Most of the species belonging to Acinetobacter genera were used in the tree, whereas Bacillus tequilensis KCTC 13622 was used as out group.2012).There is no report of Acinetobacter lwoffii having high resistance to pyrethroids earlier.So, this specie is successfully isolated for the first time in the current research effort.
Pseudomonas spp.are an effective bioremediating agents of environmental pollutants such as oil, phenol, azo dyes, organophosphates, organochlorines, carbamates and pyrethroids (Cycoń and Piotrowska-Seget, 2016;Wasi et al., 2013).A previous study (Song et al., 2015) isolated Pseudomonas aeruginosa JQ-41 with potential to degrade both bifenthrin and cypermethrin.The newly isolated members of both the genera could have more potential application for treatment of waste from pyrethroid polluted environments after detailed degradation studies and optimization of conditions for degradation.By the use of HPLC, GCMS or other more precise techniques, metabolites produced by bacterial degradation of bifenthrin and cypermethrin could also be identified that will be helpful in designing degradation pathways for pyrethroids by these bacteria.Additionally, bacterial enzymes responsible for degradation of pyrethroids could be purified and applied in similar bioremediation studies in the future.

Conclusion
The widespread detrimental impacts of various pesticides on soil ecology and biodiversity due to recalcitrant nature compels the researcher to investigate the biodegradation potential of natural microbial flora of the soil to cope with this threat.The present study was aimed to detect soil microbial candidates with potential to degrade synthetic pyrethroids.Acinetobacter lwoffii and Pseudomonas spp.were found capable to degrade significant level of degradation of known concentration of synthetic pyrethroids, the optimization of degradation potential was achieved by testing on variable ranges of temperature and pH.

Figure 1 .
Figure 1.Growth kinetics of six bacterial strains by the degradation of 800 mg/mL (a) Bifenthrin and (b) Cypermethrin supplemented in Mineral Salt Medium (MSM).

Figure 2 .
Figure 2. The temperature (a) and pH (b) optimization for the biodegradation capability of bacterial strain Acinetobacter sp.MG-04 against the (SPs) Bifenthrin and Cypermethrin's supplemented in Mineral Salt Medium (MSM).

Figure 3 .
Figure 3.The evolutionary tree was inferred using the Kimura 2-parameter model with bootstrap value (n= 100) was used for computing evolutionary distances of Acinetobacter lwoffii MG-04.The optimal tree with the sum of branch length = 0.02 is shown.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.Most of the species belonging to Acinetobacter genera were used in the tree, whereas Bacillus tequilensis KCTC 13622 was used as out group.

Figure 4 .
Figure 4.The phylogenetic tree based on 16S rRNA gene of SPsdegrading Pseudomonas strain through Neighbor-Joining method.Kimura 2-parameter model with bootstrap value (n= 100) was used for computing evolutionary distances.Most of the species of Pseudomonas representing high resemblance with the studied bacterial isolates in different databases were used along with Acidiferrobacter spp.1013-28-CG34 (AY532575) as an out group in the phylogenetic analysis.All positions with <95% site coverage was eliminated.

Table 1 .
Physio-biochemical characteristics of the isolated bacterial strains from agricultural soil.