Biodegradation α-endosulfan and α-cypermethrin by Acinetobacter schindleri B7 isolated from the microflora of grasshopper (Poecilimon tauricola)

Extensive use of pesticides has led to the contamination of ecosystem. Therefore, it is important to isolate potential new pesticide-degrading bacteria. For the biodegradation of α-endosulfan and α-cypermethrin, a new bacterium was isolated from the body microflora of grasshopper (Poecilimon tauricola). Based on biochemical, morphological, and 16S rRNA sequence analysis, the isolated strain B7 was identified as Acinetobacter schindleri. This bacterial strain was screened for its α-cypermethrin and α-endosulfan degrading potential with minimal salt medium (MSM) and non-sulfur medium (NSM), respectively. When glucose was added to non-sulfur medium containing α-endosulfan (100 mg/L) and minimal salt medium containing α-cypermethrin (100 mg/L), both pesticide degradation and bacterial growth were increased. Acinetobacter schindleri B7 was able to degrade 67.31% of α-endosulfan and 68.4% of α-cypermethrin within 10 days. The degradation products of pesticides were determined by HPLC. As a result, A. schindleri, a Gram-negative bacterium, can inevitably be used in the biological treatment of environments exposed to pesticides.


Introduction
Pesticides are used against pests to obtain more products in agriculture and their use is increasing day by day. These chemicals, which are toxic to the environment, are widely used in agriculture to protect stored products (grain or seed) and to remove harmful insects and weeds (Jiang et al. 2019;Gao et al. 2020). According to their chemical structure, pesticides can be grouped as carbamates, organochlorinates, organophosphorus and synthetic pyrethroids. Endosulfan is an insecticide in the cyclodine group of organochlorinated pesticides (Ozdal et al. 2016a, b). The use of endosulfan is banned in many countries due to its high toxicity. However, studies have shown that endosulfan continues to be used in an uncontrolled manner (Gao et al. 2020). Endosulfan and its degradation products have been detected in atmosphere, soil, food products, groundwater and surface waters. It can remain intact in nature for a long time and threaten the environment and public health. It has immunosuppressive, neurotoxicity, hepatotoxicity, respiratory toxicity, reproductive toxicity, and mutagenic effects in mammals (Sebastian and Raghavan 2017;Ahmad 2020;Nazir et al. 2021). Cypermethrin is a commercially available synthetic pyrethroid and generally used to control insects and pests in many environments. Synthetic pyrethroids usually have less toxicity for organisms than organophosphate and organochlorines. However, synthetic pyrethroids have negative effects on the environment and human health due to their high rate of use (Gur et al. 2014;Aguila-Torres et al. 2020).
Degradation of the pesticides in nature can occur under biotic or abiotic conditions. Pesticides are biodegradable in nature due to the activities of microorganisms. Endosulfan can be used by microorganisms (bacteria and fungi) as a source of carbon and/or sulfur (Mudhoo et al. 2019;Rodriguez-Peña et al. 2020). Also, α-cypermethrin can be used as the sole carbon source in the energy metabolism of microorganisms (Gur et al. 2014;Ramya and Vasudevan 2020).
Insects are one of the richest living groups in terms of species diversity. Insects are also rich in microbial diversity. The symbiotic interactions between microorganisms and insects can be in very different ways (defense against pathogens, produce enzymes that aid digestion, decomposition of harmful compounds) (Paniagua Voirol et al. 2018). Insect microflora forms a suitable habitat for microorganisms that produce some important enzymes. It is known that insects host antibiotic-and pesticide-resistant microorganisms (Pai et al. 2005;Ozdal et al. 2016a, b). Microorganisms can adapt to new environments by acquiring different characteristics with horizontal gene transfer, conjugative plasmids and simple mutations (Itoh et al. 2018; Ramakrishnan et al. 2019). In particular, insect intestines provide a suitable environment for gene transfer that provides resistance (such as antibiotic and pesticide resistance) gains between bacteria. Bacterial colonization in the gut of insect depends on pH, oxygen and nutrient availability (Saati-Santamaría et al. 2021). According to this information, it is possible to isolate pesticide-resistant microorganisms from insect intestines.
The aim of this study is to investigate the biodegradation of α-endosulfan and α-cypermethrin using A. schindleri B7 bacterial strain isolated from insect flora and determination of the product(s) resulted from biodegradation. In addition, the effects of glucose on bacterial growth, biodegradation of pesticides and degradation products were also investigated.

Identification of endosulfan degrading microorganisms
Species growing on NSM agar (containing α-endosulfan) and MSM agar (containing α-cypermethrin) were defined by the morphology and biochemical methods (Gram staining, nitrate reduction, catalase and oxidase tests, and starch hydrolysis). Isolated bacteria were also identified according to fatty acid methyl ester (FAME) profiles using the MIS software package (Ozdal et al. 2016a, b). Besides, the isolate that degraded both pesticides was determined by sequencing the 16S rRNA gene region (Gur et al. 2014).

Preparation of uniform bacterial inoculum
Isolates were grown in TSB at 30 °C, 150 rpm overnight as pre-culture. Bacteria grown in broth medium were centrifuged for 5 min at 5000 rpm, washed several times with 0.9% SFS and adjusted to optical density (OD 600 ) of 0.5.

Biodegradation of α-endosulfan and α-cypermethrin by Acinetobacter schindleri B7
α-endosulfan and α-cypermethrin (100 mg/L) were added into 100 mL of NSM and MSM in 250 mL conical flasks, and thereafter 1 mL of inoculum (OD 600 0.5) was inoculated (Ozdal et al. 2017a, b, c). The flasks were shaken on a reciprocal shaker at 150 rpm at 30 °C for 10 days and at a pH of 8.0. The effects of glucose (1 g/L) on bacterial growth, biodegradation of pesticides and degradation products were also investigated.

Analytical methods
HPLC was used for the determination of pesticides (α-cypermethrin and α-endosulfan) and their biodegradation products. After 10 days, the samples were extracted with ethyl acetate (1:1, v/v). Upper phases were dried with sodium sulfate anhydrous, and concentrated with a rotary evaporator. The biodegradation samples of α-endosulfan were analyzed by HPLC with ODS C18 Hypersil Column (250 × 4.6 mm, 5 µm) equipped with UV-VIS detector at 214 nm. A mixture containing acetonitrile/water (70:30, v/v) was used as the mobile phase (Ozdal et al. 2017a, b, c). The biodegradation samples of α-cypermethrin were analyzed by HPLC using a SUPELCOSIL C18 DB Column (250 × 4.6 mm, 5 µm) with an acetonitrile/water (85:15) mobile phase. The solutes were detected using a UV-Vis detector at 235 nm (Gur et al. 2014). The retention times of pesticides and degradation products are given in the supplementary file Figs. S1-S2. To determine bacterial growth, the culture broths were centrifuged (8000 rpm for 5 min at 4 ℃), and the bacterial biomass was washed with SFS two times. Bacterial cell growth was determined by measuring the optical density (OD) of culture broth using a UV-Vis spectrophotomer at 600 nm.

Statistical analysis
All experiments were conducted in triplicates. The variance analysis was carried out according to the one-way ANOVA test using SPSS 13.0 for Microsoft Windows, and the averages were compared with the Duncan test at a confidence level of 0.05.

Results
A new bacterial isolate capable of degrading both α-endosulfan and α-cypermethrin was isolated from body microflora of grasshopper. Biochemical and morphological tests showed that it was a Gram-negative, aerobic, oxidasenegative, catalase-positive, coccobacilli, non-pigmented, non-motile and non-spore-forming organism. Genetic analysis of bacteria capable of degrading both α-endosulfan and α-cypermethrin was performed. A 910 bp of 16S rRNA sequence of the strain was screened with BLAST (GenBank, NCBI). The nucleotide sequence was registered at GenBank with accession number KC453989. A phylogenetic tree was shown in Fig. 1. According to these results, this isolate was identified as A. schindleri strain B7.
It was determined that A. schindleri B7 utilized α-endosulfan as sole sulfur and carbon source, and α-cypermethrin as the sole carbon source. In different NSM (α-endosulfan + glucose, α-endosulfan, and glucose), bacterial growth was significantly changed. Further bacterial growth (OD 600 1.74) was determined when only glucose was added to NSM. Bacterial growth in the presence of α-endosulfan and glucose was 1.58 (OD600), and it decreased to 0.61 (OD600) in the medium absence of glucose (Fig. 2a). In different MSM (α-cypermethrin + glucose, α-cypermethrin, and glucose), bacterial growth was significantly changed. Maximum bacterial growth (OD 600 1.75) was achieved with the supplement of glucose to MSM. Bacterial growth in the presence of-cypermethrin and glucose was 1.57 (OD 600 ) and dropped to 0.59 (OD 600 ) when glucose was not added (Fig. 2b).
The percentage of biodegradation of α-endosulfan and α-cypermethrin was determined with HPLC on the tenth day. It was observed that the α-endosulfan biodegradation rate by A. schindleri B7 was different at the same incubation period when inoculated in NSM supplemented with or without glucose. As seen in Fig. 3a, the addition of glucose increased biodegradation of α-endosulfan. In the absence of Fig. 1 Phylogenetic tree depending on the 16S rRNA sequences of strain of A. schindleri B7 and related species glucose, the α-endosulfan biodegradation efficiency of A. schindleri B7 was 59.74% at the end of 10 days. The addition of 1 g/L of glucose increased α-endosulfan biodegradation efficiency to 67.31%, which corresponds to an increase of 12.72%. As shown in Fig. 3a, endosulfan diol, endosulfan ether and endosulfan lactone were defined as degradation products in the biodegradation of α-endosulfan by A. schindleri B7. Intermediate products ratios increased when glucose was added to NSM. Endosulfan diol was determined as the main metabolite in both media. Also, endosulfan sulfate, a toxic metabolite, was not formed in both media.
As seen in Fig. 3b, it was determined that the degradation rates of α-cypermethrin by A. schindleri B7 in glucose-added medium and glucose-free medium were 68.4% and 59.53%, respectively. The addition of glucose (1 g/L) increased the α-cypermethrin degradation efficiency to 68.4%, corresponding to an increase of 14.90%. As a result of α-cypermethrin biodegradation, in the glucose containing medium, more muconic acid, 3-phenoxybenzoic acid and 3-phenoxybenzaldehyde were formed compared to the medium without glucose. But, less phenol was formed in glucose containing medium.
When Figs. 2, 3 are analyzed together, presence of glucose enhanced the growth of the A. schindleri B7 and the biodegradation efficiency of α-endosulfan and α-cypermethrin. The increase in pesticides biodegradation  . 3 Biodegradation of α-endosulfan (100 mg/L) (a) and α-cypermethrin (100 mg/L) (b) in the presence and absence of glucose (1 g/L) and its metabolites at 10 days was determined to be related to the increase in bacterial density resulted from the use of glucose.

Discussion
Insect intestines present specific environments for bacterial colonization, and bacteria in the intestine provide numerous useful services to their hosts. Interactions between insects and bacteria may be symbiotic or pathogenic. This symbiotic association has turned into a necessary evolutionary purpose for them to survive under different environmental conditions (Okay et al. 2013;Kurbanoglu et al. 2015;Jahnes and Sabree 2020;Ozdal and Algur 2020). Insect symbionts constitute a rich source of bioactive molecules (bio-surfactants, polysaccharides, and pigments) as well as detoxifying enzymes. It is inevitable that these molecules will contribute to the biodegradation of pesticides due to their protective properties against microorganisms (Das et al. 2014;Ozdal et al. 2017b;Ozdal, 2019).
In recent years, bacteria isolated from insect flora have been indicated to be a significant source to biodegradation of pesticides. Isolation of microorganisms that break down pesticides is made from different environments where pesticides are used (Gaonkar et al. 2019). Many studies have shown that pesticide-resistant insects' intestine provides a suitable environment for the isolation of potential microorganisms for biodegradation of pesticides (Ozdal et al. 2016a, b;Itoh et al. 2018;Ozdal and Algur 2020). Some symbiotic microorganisms mediate the detoxification of pesticides, providing pesticide resistance to their hosts (Cheng et al. 2017;Skaljac et al. 2018). Pesticide-degrading gut bacteria (Enterococcus casseliflavus, Enterococcus mundtii, Staphylococcus sciuri and Pseudomonas stutzeri) were isolated from the gut of fifth instars of Spodoptera frugiperda strains resistant to lambdacyhalothrin, deltamethrin, chlorpyrifos ethyl, spinosad and lufenuron, using insecticide-selective media (de Almeida et al. 2017).
It has been known that a carbon source (at low concentrations) other than the target chemical affects the rate of degradation of organic compounds. The presence of an easily degradable carbon source such as glucose at high concentration can cause catabolite repression and a decrease in transcription rate (Gur et al. 2014). Gaonkar et al. (2019) reported that Pseudomonas aeruginosa was able to degrade chlorpyrifos effectively in presence of glucose and the degradation rate of the isolate was enhanced. Of course, the presence of a carbon source in the growth medium significantly affects bacterial metabolism. Our study showed that pesticides were co-metabolized even in the presence of glucose. According to the results obtained from the current study, the addition of glucose accelerated the growth of the bacteria as well as the biodegradation process (Fig. 2). The increase in biodegradation of the pesticide was found to be due to the increase in cell density (Gur et al. 2014;John et al. 2016). This could potentially be because pesticide-degrading enzymes in A. schindleri are expressed even in the presence of glucose.
Endosulfan is generally biodegraded by oxidation and hydrolysis pathways. In the oxidation pathway, endosulfan sulfate is formed. In the hydrolysis pathway, endosulfan diol, endosulfan ether and endosulfan lactone, which are less toxic than endosulfan, are formed (Mudhoo et al. 2019). Microorganisms that produce endosulfan sulfate are not suitable for bioremediation applications because endosulfan sulfate is very toxic to organisms. Some microorganisms degrade endosulfan through oxidation by forming endosulfan sulfate. It is thought to be very valuable that bacterial species carrying the hydrolytic pathway for degradation of endosulfan. Many bacteria (Pseudomonas, Rhodococcus, Stenotrophomonas, Ochrobacterum and Alcaligenes) have been found to use the hydrolytic pathway for endosulfan degradation (Mudhoo et al. 2019). Ozdal et al. (2017a, b, c) reported the biodegradation of α-endosulfan by Stenotrophomonas maltophilia OG2 (isolated from the cockroaches, Blatta orientalis) with the degradation efficiency of 81.5% in 10 days. In the same study, α-endosulfan was converted to endosulfan diol, endosulfan ether and endosulfan lactone by bacterial isolate OG2. In light of this information, A. schindleri B7 breaks down the α-endosulfan by hydrolysis (non-oxidative, not forming endosulfan sulfate) which is less toxic pathway (Fig. 3a).
In conclusion, it was determined that pesticides-degrading new bacteria can be isolated from insects and used in biodegradation studies. This isolated bacterium can be investigated for the biodegradation of different pesticides. Also, the isolated bacterium has the potentials to be applied widely for environmental bioremediation. Based on our study, many insects living in pesticide contaminated environments should be investigated for the isolation of new pesticide-degrading microorganism(s).