Characteristics of two terbutylazine-degrading bacteria and the construction of a live bacterial agent for effective degradation of terbutylazine in soil

: T wo kinds of bacteria, named TDJ-7 and TDJ-9, were isolated from the soil, which could degrade terbutylazine effectively. TDJ-7 and TDJ-9 were identifi ed as Bacillus pumilus and B acillus subtilis . T he degradation effi ciency of 1 0mg/L of terbutylazine by TDJ-7 could reach 95% within 6 days, and the strain TDJ-9 could reach 98% under the same conditions. Both strain TDJ-7 and strain TDJ-7 could also effectively degrade simazine, metribuzin, atrazine and ametryn. In addition, s train TDJ-7 and TDJ-9 had been successfully developed into a live bacterial agent that could be used to degrade terbutylazine residue. These results suggest that strain TDJ-7 and TDJ-9 can be used for the bioremediation of terbutylazine or other s-triazine herbicides contamination.


INTRODUCTION
T erbutylazine (CAS: 5915-41-3.)was widely used for weed control in crops (Brown 2018).Longterm use of terbutylazine resulted in its high residue levels in water and soil (Bottoni et al. 2013).Terbuthylazine and its major Intermediate metabolites were highly mobile and present considerable leachability in soil (European Food Safety Authority 2011, Viegas et al. 2019, Lewis et al. 2016).T erbutylazine was also considered to be an endocrine disruptor and carcinogen (Velisek et al. 2016).I t has many negative effects on crop photosynthesis, environmental microbes and public health (Andrea et al. 2018, Tang et al. 2019, Elsheekh et al. 1994).T herefore, the rapid elimination of terbuthylazine p ollution was considered to be essential to the safety of the environment.
T he use of terbutylazine-degrading strains was an ideal way for the remediation of terbutylazine pollution because it was cost-effective and environmentally friendly (Vaishampayan et al. 2007, Zhu et al. 2019b, Li et al. 2020).R ecently, a b ioremediation tool consisting of live cells of A. aurescens TC1 has been successfully used to repair the terbuthylazine-contaminated soil (Viegas et al. 2019, Silva et al. 2015).This method required prior acquisition of pure strains or microbial consortium with high degradation performance ( Zhu et al. 2019c, Sagarkar et al. 2014, Lu et al. 2020, Kou et al. 2020).In addition, many factors such as temperature, pH and oxygen content usually affect the biodegradation of terbuthylazine (Mueller et al. 2010, Getenga et al. 2009, Zhao et al. 2019, Hou et al. 2019).Therefore, in addition to screening high-efficiency terbutylazinedegrading strains, attention should also be paid to the effects of various environmental factors on the biodegradation of terbutylazine (Pinto et al. 2012, Zhu et al. 2018, Ding et al. 2019, Caracciolo et al. 2010).Cienc (2022) 94(1) e20200658 2 | 10 In this study, two high-efficiency terbutylazine-degrading strains have been isolated from soil.They could degrade many other s-triazine herbicides in addition to theirs excellent terbutylazine degradation ability.These two strains have now been developed into a microbial agent that could be used for bioremediation of terbutylazine pollution.

Chemicals and media
Terbutylazine (99.2%) was purchased from Fisher Company.All other chemicals used were of analytical grade or chromatographic grade.

Determination of terbutylazine
The various samples were pretreated and extracted for the determinations of terbutylazine according to the method described in the literature (Zhu et al. 2020).The extracts were analyzed by LC-MS/MS on 1260HPLC-6430Triple quadrupole mass spectrometer (Agilent™, USA).Terbutylazine was separated on a C18 chromatographic column (Agilent™, 3.0×100 mm, 1.8-Micron) with a flow rate of 0.3 ml/min.0.1% formic acid-water solution and acetonitrile were used for gradient elution.MS/MS analysis was performed in ESI positive mode and transitions were measured in MRM.The parameters of terbutylazine for MS/MS analysis: (precursor ion: 230, product ion: 174/132, fragmenter: 124, CE: 15/25).

Isolation of terbutylazine degrading strains
Soil samples were taken from the forest land of Nantong, China, where terbutylazine has been used for about ten years.Add 6.0g of soil sample to 100ml of isolation medium, mix well, and then shake culture at 30 o C. The content of terbutylazine in medium was determined every 24 hours, the assay method refer to previous studies (Cabras et al. 1989, Bichon et al. 2006, Mercadante et al. 2012).Transfer 5 ml of culture solution with 3-day degradation efficiency >70% to an enriched medium containing 10 mg/L terbutylazine, and continuously subculture for more than 5 times.After the degradation ability was retested to ensure that the 3-day degradation efficiency was more than 70%, the above-mentioned enriched culture solution was inoculated in an inorganic salt culture plate containing terbutylazine, and inverted culture was carried out (30 °C).Select the colony with vigorous growth, and draw lines repeatedly on the culture plate to obtain a pure culture.The strains were identified by 16SrRNA analysis, and build a phylogenetic tree (Wang et al. 2012, Min et al. 2019, Fan et al. 2020).

Bacterial inocula
Terbutylazine-degrading strains, Bacillus pumilus TDJ-7 and Bacillus subtilis TDJ-9 have been isolated from soil.The strains were inoculated into an enriched medium containing 10 mg/L terbutylazine for shake culture at 30°C (100 rpm).After 24 hours of culture, collect bacterial cells by centrifugation and rinse with sterile water (Zhu et al. 2019a, Xu et al. 2020, Luo et al. 2021).The bacterial cells were resuspended in normal saline as inoculum of the subsequent experiments, and the concentration was approximately 2×10 6 cells/ml.

Degradation of terbutylazine by strain TDJ-7 and TDJ-9
Inoculate 5% strain TDJ-7 inoculum into the isolation medium containing 10mg/L terbutylazine, and incubate at 30°C (100rpm).The amount of terbutylazine residue was measured every 24 hours.In addition, the strain TDJ-9 was studied synchronously according to the same experimental process.

Tolerance of strain TDJ-7 and TDJ-9 on terbutylazine
The content of terbutylazine in isolation medium was adjusted respectively to 5, 10, 20, 50 and 100 mg/L by applying terbutylazine wettable powder.Then, strain TDJ-7 was inoculated for shake culture, and OD 600 was determined periodically for evaluating the tolerance of strains on terbutylazine.The strain TDJ-9 was studied synchronously according to the same experimental process.
Degradation of atrazine, metribuzin, simazine and ametryn by strain TDJ-7 and strain TDJ-9 Four kinds of media containing different herbicides were prepared by replacing terbutylazine in the isolation medium with atrazine, metribuzin, simazine and ametryn respectively, the final concentration of herbicide was 20 mg/L.5% strain TDJ-7 inoculum was inoculated in the above prepared medium containing 20mg/L atrazine for shake culture at 30°C (100rpm).The amount of atrazine residue was determined periodically.Testing for other 3 s-triazine herbicides were carried out simultaneously in the same manner.
The degradation of 4 s-triazine herbicides by strain TDJ-9 was tested synchronously as described above.

Development of the microbial agent for bioremediation of terbutylazine pollution
2.5% strain TDJ-7 inoculum and 2.5% TDJ-9 inoculum were simultaneously inoculated into enrichment medium, incubated for 6 hours at 30°C (100rpm).Finally, this microbial consortium was stored at -4°C for subsequent studies.
Isolation medium containing 10mg/L terbutylazine was divided into 3 groups.I.In the first group, 3% (v/v) of the above microbial consortium were added, and Populus alba rhizosphere soil was also added (add 2g soil per 100ml medium).II.Add only 3% microbial consortium.III.Control group.Finally, the above 3 groups were cultured at 30°C, and the content of terbutylazine was determined periodically.
Preparation for microbial agent containing strain TDJ-7 and TDJ-9 (Zhu et al. 2019a): TDJ-7 inoculum, TDJ-9 inoculum and sterile nutrient broth were mixed (1:1:2, v/v), the mixed solution was cultured at 30°C for 12 hours.Mixture of fresh Populus alba rhizosphere soil and rice bran flour (1:9, w/w) was used as carrier, and each 0.4 kg carrier was mixed with 100 ml pre-made mixed solution.The mixture was incubated at 30°C for 12 hours, and agitated every 4 hours.Finally, store them in a sterile jar at -4°C.
Degradation of terbutylazine by new microbial agent: Add terbutylazine to fresh soil (the parameters of soil were shown in Table I) to a concentration of 10 mg/kg, and put the soil in 0.3 m x 0.3 m plastic boxes making 0.1 m depth, to ensure that the weight of each box of soil was the same.Follow the steps below to add the microbial agent to the soil: Mix 1.5 g of the microbial agent with 100 ml of sterile water and sprinkle it all on a box of soil.Only 100 ml of sterile water was sprinkled into the control group.Then all of them were incubated at 30 °C for more than 7 days and turned every 6 hours.The content of terbutylazine in soil was determined periodically.Cienc (2022) 94(1) e20200658 4 | 10

Degradation of terbutylazine by strain TDJ-7 and TDJ-9
The results in Fig. 2 indicated that both strain TDJ-7 and strain TDJ-9 could rapidly degrade terbutylazine.The 6-days degradation efficiency of terbutylazine (10mg/L) by TDJ-7 was approximately 95%, while the strain TDJ-9 could reach 98% in 6 days.It could also be seen from Fig. 2 that the strain TDJ-9 degraded terbutylazine faster than TDJ-7.In addition, the experimental results showed that the combined application of TDJ-7 and TDJ-9 has achieved higher degradation rate, and the degradation efficiency of terbutylazine (10mg/L) could reach 99% in 6 days.Therefore, the combined application of two terbutylazine-degrading bacteria was considered to be a more effective way for degrading terbutylazine.

Tolerance of strain TDJ-7 and TDJ-9 on terbutylazine
In Fig. 3, when the concentration of terbutylazine was increased from 20mg/L to 100 mg/L, OD 600 of strain TDJ-7 did not change significantly (P>0.05).If the concentration of terbutylazine was as low as 10 mg/L or 5 mg/L, OD 600 would decrease.It might be due to the activation effect of terbutylazine on the growth of the strain TDJ-7, but the lower concentration of terbutylazine could not produce enough activation effect.In Fig. 4, there was no significant change in OD 600 of TDJ-9 when the concentration of terbutylazine was increased from 10 mg/L to 100 mg/L.Therefore, it was speculated that strain TDJ-7 and TDJ-9 were able to tolerate at least 100 mg/L of terbutylazine.Based on existing research results, it was considered that both strains had excellent degradation ability to terbutylazine, and could be used for the biodegradation of terbutylazine in a wide concentration range.Degradation of atrazine, metribuzin, simazine and ametryn by strain TDJ-7 and TDJ-9 According to the experimental results in Fig. 5 and Fig. 6, both strain TDJ-7 and strain TDJ-9 could effectively degrade simazine, metribuzin, atrazine and ametryn.The degradation efficiencies of 4 s-triazine herbicides by strain TDJ-7 were respectively 97.0%, 92.5%, 99.5% and 100% in 7d.The efficiency of strain TDJ-9 in degrading four s-triazine herbicides was higher than that of strain TDJ-7, and the 7-day degradation efficiencies were 98.5%, 96.0%, 100% and 100%, respectively.In addition, the above experimental results have also shown that the strain TDJ-9 degrade terbutylazine faster than the strain TDJ-7.Usually the s-triazine herbicides contained a common s-triazine structure.Some microorganisms or enzymes capable of cleaving s-triazine structure may have the ability to degrade a variety of s-triazine herbicides.It was speculated that both strains TDJ-7 and TDJ-9 may have the ability to cleave the s-triazine structure.Therefore, these two strains were also supposed to be used for bioremediation of some other s-triazine herbicides contamination, not just degradation of terbutylazine.

Degradation of terbutylazine by the microbial agent
The degradation efficiency of terbutylazine (10mg/L) by the microbial consortium consisting of TDJ-7 and TDJ-9 could reach about 96.0% in 5d (Fig. 7).If the microbial consortium and Populus alba rhizosphere soil were jointly applied to degrade terbutylazine, its degradation rate could be significantly improved, and the 5-day degradation efficiency could reach 99.0%.It was speculated that these should be caused by some special microorganisms, enzymes or trace organic compounds in Populus alba rhizosphere soil.Therefore, adding appropriate amount of Populus alba rhizosphere soil may be an effective way to improve the degradation effect in the preparation of microbial agents.
The degradation efficiency of 10 mg/kg of terbutylazine in soil could reach 96.0% by new microbial agent after 7 days of incubation (Fig. 7).In the control group (no microbial agent), the degradation efficiency of terbutylazine was 11.0% in 7d, which should be attributed to some natural microorganisms, soil enzymes and other physical or chemical factors.These results indicated that the development of new microbial agents has been successful and that their products could be used for bioremediation of terbutylazine contamination.

CONCLUSION
Two terbutylazine degrading strains were isolated from soil, and they were Bacillus pumilus TDJ-7 and Bacillus subtilis TDJ-9.The degradation efficiency of 10mg/L of terbutylazine by TDJ-7 could reach about 95% in 6d, and the strain TDJ-9 could reach 98% under the same conditions.The combination of the two strains could significantly accelerate the degradation of terbutylazine.Adding an appropriate amount of Populus alba rhizosphere soil during the microbial degradation of terbutylazine could also improve the degradation rate.In addition, both TDJ-7 and TDJ-9 could effectively degrade simazine, metribuzin, atrazine and ametryn.TDJ-7 and TDJ-9 have been successfully developed into a microbial agent.The new microbial agent could be used to degrade terbutylazine in soil, and have achieved many satisfactory results.According to the results of this study, we believed that it was an attractive choice to use the isolated terbutylazine-degrading bacteria to clean up contaminated sites.TDJ-7, TDJ-9 and their microbial agent could be used for the bioremediation of terbutylazine or other s-triazine herbicides contamination.

Figure
Figure 3.Effect of concentration of terbutylazine on the growth of TDJ-7.

Figure
Figure 2. The curve on degradation of terbutylazine by different microorganisms.

Figure
Figure 4. Effect of concentration of terbutylazine on the growth of TDJ-9.

Figure 5 .
Figure 5.The curve on degradation of 4 s-triazine herbicides by the strain TDJ-7.

Figure
Figure 6.The curve on degradation of 4 s-triazine herbicides by the strain TDJ-9.

Figure 7 .
Figure 7. ①Degradation of terbutylazine by microbial consortium and rhizosphere soil.②Degradation of terbutylazine in soil by microbial agent.

Table I .
Basic physic-chemical characteristics of fresh soil.