Biodegradation of the fungicide carbendazim by bacteria from Coriandrum sativum L . rhizosphere

The biocidal agrochemicals commonly used in agriculture can remain in the soil, affecting the environmental conditions and causing serious risks to health. Knowing that soil microorganisms, especially those from the rhizosphere, can degrade environmental xenobiotics, it was evaluated the potential of bacteria isolated from Coriandrum sativum L. rhizosphere to biodegrade carbendazim (MBC), a fungicide extensively used by agriculturists from rural farming communities in Manaus, Amazonas. Cultures carried out in medium containing carbendazim as a sole carbon source enabled the isolation of 80 bacteria, in the established conditions. Assays to determine degradation potential allowed the selection of the two elite isolates identified as Stenotrophomonas sp. and Ochrobactrum sp. Quantitative assays with each strain individually or in consortium, were carried out using minimal salt medium added with carbendazim (250 μg mL) and incubated at 30°C, under agitation (125 rpm) for 21 days. Samples used in the biodegradation test were HPLC analyzed for final fungicide quantitation. The Stenotrophomonas sp. strain was more efficient (68.9%) to degrade carbendazim and showed no toxicity in tests with Artemia salina.

The increased demand for food and the introduction of non-native cultures and therefore more susceptible to pests, imposed to the agriculturists in Amazon a more intensive use of pesticides (Römbke, Waichman, & Garcia, 2008), mainly insecticides, herbicides and fungicides (Ecobichon, 2001).
Usually, sandy soils present low water holding capacity and low organic matter content (Oliveira, 2010).According to Santos et al. (2017) the soil at Nova Esperança, a rural farming community located within the limit of Manaus, AM, Brazil, where the Coriandrum sativum (coriander) rhizosphere soil samples were collected, presents a sandy texture and at 10-20 cm depth is considered fertile.Moreover, this soil exhibit slow alkalinity, with pH values within the range of 7.5 and 7.6, low ratios of potassium, magnesium and boron, and absence of aluminum.
However, C. sativum rhizosphere soil samples were collected from a cultivation area used for growing vegetables, where the replacement of organic matter is continuous, providing sufficient nutrient to supply the needs of the plants and to maintain a microbial community in the rhizosphere.Multi-residue analysis of the soil samples investigated in this study lead to the detection of 0.189 μg kg -1 of MBC fungicide.
So, the aim of this paper was to characterize the bacterial rhizosphere community of C. sativum from cultivated areas in Nova Esperança (Manaus, AM, Brazil), and evaluate the biodegradation of carbendazim (MBC) by the isolated bacteria.

Selective isolation of bacteria
Portions (6.6 g) from 5 samples of soil were homogenized totalizing 33 g of a composite soil sample, which was inoculated in a 2 L Erlenmeyer flask containing 500 mL of minimal salts medium -MMS (K 2 HPO 4 (2.78 g L -1 ), (NH 4 ) 2SO 4 (1.0 g L -1 ), MgCl 2 .6H 2 O (0.2 g L -1 ), NaCl (0.1 g L -1 ), FeCl 3 .6H 2 O (0.02 g L -1 ), CaCl 2 (0.01 g L -1 ) -to 1 L) and carbendazim -MBC (250 μg mL -1 ).A negative control was prepared containing only MMS (500 mL) and carbendazim (250 μg mL -1 ), without the soil sample.The flasks were incubated at 28°C under agitation (125 rpm) for seven days for rigorous selection process.After that period, aliquots of 100 mL were removed from each flask and inoculated again in 400 mL of MMS and MBC for more seven days, under the same conditions.This procedure was repeated for two more times.After the last incubation period, aliquots of 1 mL from each flask were submitted to serial dilution (10 -1 , 10 -2 , 10 -3 , 10 -4 ), using 0.85% saline solution.Aliquots (100 μL) from each dilution were plated on Petri dishes containing MMS agar + MBC (250 μg mL -1 ).The experiments were carried out in triplicate.With the aid of a Drigalski handle, the cells were uniformly distributed on the plates which were incubated in BOD at 28°C for ten days to isolate bacteria and fungi.The bacteria which grew were transferred to test tubes containing 5 mL Luria Bertani -LB agar (Himedia) culture medium.

Inoculum preparation for biodegradation assays
The bacterial inoculum was prepared from stock cultures LB kept at -20°C, which were transferred to plates containing LB and incubated at 30°C for 24 hours.With the aid of a platinum handle, an aliquot containing approximately 10 6 CFU mL -1 bacterial suspension was transferred to 15 mL Falcon type tubes containing 3 mL of LB broth and were incubated at 30°C and under agitation (250 rpm) for 4 hours.The resultant culture was centrifuged (1792 rcf) for 10 minutes.The supernatant was discarded and the cell pellet suspended with MMS.Bacterial biomass was diluted in MMS to obtain O.D. equal to -1 absorbance at 610 nm, which corresponds to a population of 10 9 CFU mL -1 .In the biodegradation tests were used aliquots (1 mL) of bacterial inoculum suspension.In the consortium of selected bacteria, were utilized aliquots (500 μL) of each bacterial isolate.

Selection of microorganisms and carbendazim biodegradation assays
Degradation tests were carried out with the two finest strains selected after preliminary tests for evaluation of bacterial growth measured by optical density after 72 hours of culture.Concurrently, the degradation was quantified by high-performance liquid chromatography -HPLC.The biodegradation experiments with each isolate or with a consortium of both bacteria were performed in triplicate.Aliquots (1 mL) of the bacterial suspension (D.O. 1) were inoculated in 125 mL Erlenmeyer flask containing MMS (50 mL) and 250 mg L -1 MBC (97% purity, Sigma-Aldrich) diluted in dimethylsulfoxide (DMSO) and incubated at 30°C under agitation (125 rpm) for 21 days.The positive control group (LB and bacteria) was used to monitor the bacterium viability.

Extraction of carbendazim
The extraction of MBC was performed using ethyl acetate.To each Erlenmeyer flask, 50 mL of ethyl acetate was added.The material was transferred to a 125 mL separatory funnel and subjected to vigorous agitation for 1 minute.The organic phase was collected and the aqueous phase was extracted twice more, by the addition of 50 mL of ethyl acetate.The end of the extraction was monitored by thin layer chromatography (TLC).In the collected organic phase, anhydrous sodium sulfate was added and after filtration the samples were subjected to vacuum rotary evaporator at 40°C until reduction of the volume of the sample to approximately 10 mL.The sample was transferred to 10 mL volumetric flask containing 10 μg mL -1 of an internal standard solution.The volume of 1 μL of the sample was injected into HPLC.

Biodegradation data analysis
The internal standard and surrogate solutions were added with phenanthrene-d10 (phe-d10) and naphthalene d8 (nap-d8) with a purity ≥ 98% (Sigma-Aldrich, 98%), solubilized in HPLC grade hexane.The internal standard (nap-d8) was inserted in the samples diluted in a volumetric flask before injected into HPLC.The surrogate (phe-d10) was inserted in Erlenmeyer flasks before starting the extraction process.The use of these patterns is needed to recognize possible losses during the extraction process.The chromatographic area of the analyte was corrected by extraction recovery rate, which was obtained by the formula: [(CAs/CAis] x 100, where CAis corresponds to the chromatographic area of the internal standard and the CAs for the chromatographic area of the surrogate.The MBC concentration was obtained by a calibration curve using different MBC concentrations: 100, 200, 300, 500, 700, 900 and 1200 μg mL -1 .The percentage of MBC degradation was calculated by the formula: [(CC -CE)/CC] x 100, where CC is the MBC concentration in the control group and CE the remaining concentration of MBC from experiment.Finally, it was calculated the average and the standard deviation of the biodegradation rate in order to obtain greater accuracy of MBC concentration.

Toxicity test
Following the biodegradation assays, a toxicity test was carried out with Artemia salina in triplicates for both strains and the consortium as described by Atayde, Carneiro, Martins, and Palheta (2011).Determination of lethal concentration (LC 50 ) values was performed as described by Harwig & Scott (1971) in triplicate only for extracts that showed toxicity.

Isolation and molecular analysis of bacteria
Coriandrum sativum rhizosphere soil inoculated in selective medium containing carbendazim as a sole carbon source enabled the isolation of 80 bacteria.It was possible to identify 60 strains and 52 different possible species.Table 1 shows the 20 most common bacteria identified by rDNA sequencing.The predominant genera were Bacillus sp., Ochrobactrum sp., Enterobacter sp. and Stenotrophomonas sp.Among the identified isolates, the genus Bacillus was the most representative, with 28% frequency.From an area adjacent to the area investigated in this study, which had been treated with the fungicide Mancozeb, Rebière (2015) isolated 23 bacteria from rhizosphere soil of Allium fistulosum L. (Welsh onion) crops of which 81.81% were identified as Bacillus sp.Among 23 bacteria isolated from rhizosphere soil collected in China, Hu, Zhao, Liu, Song, and You (2013) identified the genus Ochrobactrum which is able to grow in MMS medium added with the insecticide Imidacloprid (50 μg mL -1 ).
After 72 hours of culture in medium containing carbendazim as sole carbon source, two bacterial isolates were selected (Stenotrophomonas sp. and Ochrobactrum sp.) for qualitative and quantitative biodegradation tests.The two selected genera had their sequence annotation submitted to the GenBank-NCBI and were deposited as Stenotrophomonas sp.(accession number KX376306) and Ochrobactrum sp.(accession number KX376307).
The genus Stenotrophomonas sp. is found in harsh environments such as Antarctica (Vazquez, Ruberto, & Mac Cormack, 2005) and can produce bioactive substances, such as laccase enzyme (Galai, Limam, & Marzouki, 2009).Some bacterial strains can degrade PAHs (Zafra, Absalón, Cuevas, & Cortés-Espinosa, 2014), as well as the fungicide Chlorothalonil (Zhang et al., 2014).Stenotrophomonas sp. and Ochrobactrum sp. can degrade Chlorpyrifos (Talwar, Mulla, & Ninnekar, 2014;Deng et al., 2015.), but there is no report on the efficiency of Stenotrophomonas sp. and Ochrobactrum sp. to degrade carbendazim.Therefore, it is important further studies to validate the potential of those microorganisms for degrading carbendazim in Amazonian soils.The growth of the bacterial isolates, cultured alone or in consortium, for 21 days in culture medium containing carbendazim, was determined by optical density and chromatographic methods.
The Stenotrophomonas sp. and the consortium of both Stenotrophomonas sp. and Ochrobactrum sp. when cultured at a concentration of 250 μg mL -1 carbendazim showed the best growth rates (Figure 1).Zhang et al. (2014) evaluated the ability of Stenotrophomonas sp.strain to degrade the fungicide chlorothalonil cultured in minimal salt medium added with chlorothalonil (20 μg mL -1 ) for 7 days.Stenotrophomonas sp.optical density ranged 0.17.
The obtained results indicate that both Stenotrophomonas sp. and Ochrobactrum sp. can degrade carbendazim without supplementary sources of carbon.Fang et al. (2010), Zhang et al. (2013), andSalunkhe et al. (2014) showed that bacterial strains used in biodegradation tests can be isolated using carbendazim as the only source of carbon.According to Fang et al. (2010), such characteristics are required when isolating microorganisms to be used for bioremediation purposes.Several bacteria efficient in degrading MBC have been cited in the literature, such as Pseudomonas sp.(Xiao et al., 2013), Bacillus subtilis TL-171 (Salunkhe et al., 2014), and Brevibacillus borstelensis (Arya & Sharma, 2015).Salunkhe et al. (2014) reported that four strains of B. subtilis individually degraded from 75.7 to 95.2% MBC in a concentration of 10 μg mL -1 after 15 days of incubation.The concentration of MBC used to validate the degradation potential by the bacterial isolates Stenotrophomonas sp. and Ochrobactrum sp. was 25% higher than that used by Salunkhe et al. (2014).
The lowest degradation percentage showed by the consortium of both bacterial isolates may have occurred due to competition for nutrients caused by the co-cultivation of isolates of different genera.However, Arya & Sharma (2015) investigating the degradation ability of bacteria alone or in consortium found a significant reduction of MBC caused by bacteria in consortium.Luo et al. (2009) isolated three marine bacterial lineages: Ochrobactrum sp., Stenotrophomonas sp., and Pseudomonas sp. to degrade benzopyrene an aromatic polycyclic hydrocarbon of high molecular weight.Faster degradation was observed when the isolates were cocultured in consortium.
The obtained results indicate that the isolate Stenotrophomonas sp.cultured alone shows greater potential to degrade carbendazim if compared to Ochrobactrum sp.MBC degradation potential, no matter if cultured alone or in consortium with Stenotrophomonas sp.

Toxicity tests using Artemia salina
Values obtained in toxicity tests carried out with A. salina (Table 2) ranged 0-0.7% using Stenotrophomonas sp.extract and the consortium of both isolates.Harwig & Scott (1971) established criteria for the extract toxicity classification using only the number of microcrustaceans and considered as non-toxic (NT), mortality less than 0.9%.However, Ochrobactrum sp.extracts showed slight toxicity (LT).From the result with the Ochrobactrum sp., culture extracts of this lineage were tested in A. salina at concentrations of 50, 25, 13, 7 and 3% to determine the lethal concentration (LC50), median lethal dose values indicated that extracts from this isolate are not toxic in all tested concentrations.

Conclusion
The Stenotrophomonas sp.strain was able to degrade 68.9% of carbendazim in a mineral culture medium containing 250 μg mL -1 of the fungicide.In

Figure 1 .
Figure 1.Optical density of culture medium containing Ochrobactrum sp. or Stenotrophomonas sp.bacterial isolates from Coriandrum sativum L. rhizosphere cultured for 21 days at 30°C under agitation (125 rpm) separately or in consortium.Error bars represent standard errors of the means.Carbendazim biodegradation analyzed by HPLC HPLC analysis of extracts from Stenotrophomonas sp.cultures indicated the higher potential (68.9%) of this bacterial isolate to degrade carbendazim, if compared to the isolate Ochrobactrum sp.cultured separately or in consortium with Stenotrophomonas sp.(Figure 2).

Figure 2 .
Figure 2. Biodegradation of carbendazim in 21 days by bacteria isolated from Coriandrum sativum L. rhizosphere.Error bars represent standard errors of the means.

Table 1 .
The twenty major bacterial isolates able to grow in selective medium containing carbendazim as sole carbon source.

Table 2 .
Toxicity tests using Artemia salina