Biodegradation and Subsequent Toxicity Reduction of Co-contaminants Tribenuron Methyl and Metsulfuron Methyl by a Bacterial Consortium B2R.

AllyMax is a widely used herbicide formulation in wheat-rice cropping areas of the world. The residues of its active ingredients, tribenuron methyl (TBM) and metsulfuron methyl (MET), persist in soil and water as co-contaminants, and cause serious threats to nontarget organisms. This study was performed to assess the potential of a bacterial consortium for the degradation and detoxification of TBM and MET individually and as co-contaminants. A bacterial consortium (B2R), comprising Bacillus cereus SU-1, Bacillus velezensis OS-2, and Rhodococcus rhodochrous AQ1, capable of degrading TBM and MET in liquid cultures was developed. Biodegradation of TBM and MET was optimized using the Taguchi design of experiment. Optimum degradation of both TBM and MET was obtained at pH 7 and 37 °C. Regarding media composition, optimum degradation of TBM and MET was obtained in minimal salt medium (MSM) supplemented with glucose, and MSM without glucose, respectively. The consortium simultaneously degraded TBM and MET (94.8 and 80.4%, respectively) in cultures containing the formulation AllyMax, where TBM and MET existed as co-contaminants at 2.5 mg/L each. Mass spectrometry analysis confirmed that during biodegradation, TBM and MET were metabolized into simpler compounds. Onion (Allium cepa) root inhibition and Comet assays revealed that the bacterial consortium B2R detoxified TBM and MET separately and as co-contaminants. The consortium B2R can potentially be used for the remediation of soil and water co-contaminated with TBM and MET.

Introduction

Sulfonylurea (SU) herbicides were introduced for the eradication of weeds due to their low-dose application and crop selectivity.[1] These SU herbicides are known to inhibit acetolactate synthase, an enzyme responsible for the synthesis of branched-chain amino acids. This inhibition causes starvation of essential amino acids, which leads to the cessation of plant growth, and prevents mitosis and cell elongation.[2,3] The use of SU herbicides leads to their residual persistence in soil for a longer time, which affects seed germination and growth of the successive crops,[3−5] and adversely affects microbial fauna.[4,6] Residues of the SU herbicides have also been detected in surface and ground water, which were ascribed to their chemical properties, i.e., solubility in water, low partition coefficient Kow, moderate to high mobility, and slow degradation in the environment.[7]

AllyMax 28.6% SG (14.3% of each of tribenuron methyl, TBM, and metsulfuron methyl, MET), manufactured by DuPont de Nemours, France, is one of the most used herbicides in wheat, barley, oats, and other cereal crops.[8] Both TBM and MET are effective against a large number of annual dicotyledonous weeds,[9] but together in AllyMax they provide a higher level of control to a wider range of weeds. Potentially, the two different molecules (TBM and MET) impart synergistic effects on different species of weeds.

As a result of the widespread use of AllyMax, the residues of its active ingredients, TBM and MET, exist in the soil and water as co-contaminants. Many studies have reported the negative impacts of AllyMax active ingredients, i.e., TBM and MET, on the environment.[10] Recently, TBM was reported to assert detrimental effects on liver tissues of zebrafish, a nontarget organism.[11] Moreover, the persistence of TBM in soil was observed after 126 days of application.[12] MET residues have long been reported to contaminate surface and ground waters and induce unintended side effects on nontarget organisms.[13−15]

Microbial degradation is an appropriate solution to remediate the soil and water contaminated with herbicides.[16,17] In this regard, microbial degradation of TBM is well documented.[18−21] Similarly, there are studies on biodegradation of MET by the bacteria isolated from soil and other metrics.[22−24] However, there are no studies regarding biodegradation of MET and TBM as co-contaminants.

Effective biodegradation depends on different factors, i.e., inoculum size, herbicide concentration, pH, and temperature.[25−27] As a time-saving strategy, the use of statistically designed experimental approaches that offer optimized conditions for direct as well as interactive effects of variable factors at the same time is recommended. The Taguchi design of experiment (DOE) is a comprehensive approach that uses signal-to-noise (S/N) ratio for the analysis of different variables and is helpful for the evaluation of an optimal combination of factors for enhanced biodegradation of the contaminants.[28] Biodegradation of xenobiotics supposedly renders the nontoxic molecules; however, it can lead to the formation of more toxic metabolites as compared to the parent compounds itself.[29] Thus, while investigating the biodegradation of MET and TBM, there is a need to identify the metabolites generated during biodegradation and investigate the associated toxicity reduction.

The present study aimed to develop bacterial strains/consortia for degradation of a mixture of MET and TBM as these are frequently used together, e.g., in the formulation AllyMax. Initially, various factors were optimized for degradation of TBM and MET separately, and optimized conditions were further exploited for their degradation as co-contaminants. Mass spectrophotometry (MS) was used to identify the metabolites produced during their biodegradation. Onion (Allium cepa) root growth inhibition test coupled with comet assay was performed to examine the toxicity of the individual herbicides and their mixture after treatment. This is a pioneer report for simultaneous biodegradation of TBM and MET as co-contaminants.

Results

TBM and MET Degrading Bacterial Strains

Rhizospheric and endophytic bacterial isolates, OS-2 and SU-1, identified as Bacillus velezensis and Bacillus cereus, respectively, were found efficient for the degradation of TBM and MET. One previously reported bacterial strain, Rhodococcus rhodochrous AQ1,[28] also degraded both TBM and MET efficiently.

A consortium B2R comprising these three strains showed significantly higher potential to degrade both TBM and MET (Figure ) as compared to individual strains. At 10 mg/L initial concentrations in MSM, 92% TBM and 89% MET were degraded by the consortium B2R. However, 71.5, 81.8, and 70.6% TBM, and 78, 81, and 72.6% MET degradation occurred with individual strains AQ1, SU-1, and OS-2, respectively, after 2 weeks of incubation.

Figure 1

Biodegradation of metsulfuron methyl (MET) and tribenuron methyl (TBM) at 10 mg/L initial concentration in MSM by the strains R. rhodochrous AQ1, B. cereus SU-1, and B. velezensis OS-2 separately and their consortium B2R. Each data point is the average of three replicates. Error bars represent the standard deviations of the means, and the letters describe the significant difference between the treatments.

Optimization of Culture Conditions for Biodegradation of TBM and MET

By applying Taguchi DOE, in a factorial experimental design L9 orthogonal array, four variable factors, i.e., pH, temperature, culture composition, and initial herbicide concentration at three different levels of each, were examined (Table ). Among the set of experimental runs, maximum degradation (99.4%) of TBM in terms of higher S/N ratio (39.8) and percent degradation was found in L9, where the levels of factors were pH (8.0), temperature (35 °C), medium (MSM-G), and 5 mg/L initial TBM concentration (Table ). Experiment No. 1 at pH, 6.0, temp, 25 °C, media, MSM, and PC, 5 mg/L resulted in minimum degradation (53.1%) of TBM with 34.6 S/N ratio. On the other hand, the highest degradation (93% and 39.4 S/N ratio) of MET was achieved in experiment No. 6 at pH, 7.0, temperature, 35 °C, MSM, and MET at 10 mg/L initial concentration.

Table 1

Factors and Their Levels (L1–L3) Selected to Optimize the Biodegradation of Tribenuron Methyl (TBM) and Metsulfuron Methyl (MET) by the Consortium B2R by Using the Taguchi Design of Experiment

Sr., no.	Factor	Level 1 (L1)	Level 2 (L2)	Level 3 (L3)
1	pH	6	7	8
2	Temp	25 °C	30 °C	35 °C
3	Media	MSM	MSM-G	MSM-Y
4	PC (herbicide concentration)	5 mg/L	10 mg/L	15 mg/L

Table 2

Experimental Runs Consisting of Combinations of the Selected Factors at Three Levels (L1–L3) Generated by the Taguchi Design of Experiment Designed, L9 Orthogonal Array Schemea

	Factors with their levels				Response (% TBM biodegraded)					Response (% MET biodegraded)
Exp. no.	pH	Temp	Media	PC	R1	R2	R3	Average	S/N ratio	R1	R2	R3	Average	S/N ratio
1	L1	L1	L1	L1	55.5	51.0	52.9	53.12	34.6	75.0	71.5	72.5	72.8	37.0
2	L1	L2	L2	L2	68.3	62.5	63.4	64.7	36.2	80.4	78.5	79.0	79.0	38.0
3	L1	L3	L3	L3	72.1	73.6	76.3	74.0	37.3	69.0	72.0	74.0	71.6	37.1
4	L2	L1	L2	L3	95.6	93.5	91.9	93.6	39.4	61.5	64.5	62.7	62.8	35.9
5	L2	L2	L3	L1	76.3	72.5	73.3	74.0	37.3	81.3	84.0	80.1	81.8	38.2
6	L2	L3	L1	L2	95.7	96.1	93.4	95.0	39.5	91.0	93.2	95.0	93.0	39.4
7	L3	L1	L3	L2	65.6	67.3	65.4	66.1	36.4	71.5	68.3	69.4	69.7	36.8
8	L3	L2	L1	L3	88.6	90.2	87.4	88.7	38.9	75.2	76.0	73.2	74.7	37.4
9	L3	L3	L2	L1	98.2	100	100	99.4	39.8	83.6	84	81.5	82.9	38.4

Biodegradation of tribenuron methyl (TBM) and metsulfuron methyl (MET) by the consortium B2R is presented as response.

Biodegradation of TBM and MET was found to depend upon all of the selected variables (Figure S1). Temperature was the major contributing factor for degradation of TBM. Conversely, herbicide concentration showed the highest impact on degradation of MET. Increase in pH of the media from L1 (pH 6) to L3 (pH 8) and that of temperature from L1 (25 °C) to L3 (35 °C) increased the degradation efficiency of B2R, as indicated by the high S/N ratios (Figure S2). Degradation of TBM was highest at L3 of pesticide concentration (15 mg/L), whereas that of MET was highest at L1 (5 mg/L).

Analysis of variance was carried out to quantitatively determine the significance of each factor for biodegradation of TBM and MET, as indicated by the F-ratio (Table ). Temperature had the highest influence on TBM degradation, followed by media composition. In case of MET, temperature and concentration showed the most significant effect of degradation.

Table 3

Analysis of Variance Revealing the Percent Contribution of Each Variable Factor, i.e., pH, Temperature (Temp), Media Composition, and Herbicide Concentration (PC), to the Biodegradation of Tribenuron Methyl and Metsulfuron Methyl by the Consortium B2R

			Tribenuron methyl					Metsulfuron methyl
Sr., no.	Factors	DOF (f)	Sum of squares (S)	Variance (V)	F-ratio (F)	Pure sum (S’)	Percent P (%)	Sum of squares (S)	Variance (V)	F-ratio (F)	Pure sum (S’)	Percent P (%)
1	pH	2	317.51	158.75	45.84	310.58	9.68	58.14	29.07	8.41	51.23	2.65
2	Temp	2	1581.24	790.62	228.29	1574.31	49.07	726.58	363.23	105.15	719.68	37.24
3	Media	2	1204.29	602.28	173.86	1197.36	37.32	336.213	168.11	48.65	329.30	17.04
4	PC	2	42.66	21.33	6.16	35.74	1.11	749.58	374.79	108.48	742.67	38.43
	other factor	18	62.34	3.46			2.81	62.188	3.45			4.65
	total	26	3208				100	1932.7				100

The sets of optimum conditions, their contributions, and estimated degradation of TBM and MET under these conditions are shown in Table . For TBM and MET, 98.21 and 93.45% degradation can be attained by employing optimized levels of pH, temperature, media composition, and concentration.

Table 4

Taguchi DOE Output Presenting Optimal Levels of Variable Factors for Enhanced Biodegradation of Tribenuron Methyl and Metsulfuron Methyl by the Consortium B2R

		Tribenuron methyl			Metsulfuron methyl
Sr., no.	Factors	Level description	Level	Contribution	Level description	Level	Contribution
1	pH	7	L2	08.77	7	L2	2.02
2	Temp	35	L3	10.88	35	L3	5.42
3	Media	MSM-G	L2	07.33	MSM	L1	4.97
4	PC	15	L3	06.77	5	L1	3.92
Total contribution from all factors		33.77			16.33
Current grand average of performance		78.44			77.13
Expected biodegradation at optimum conditions		98.21			93.45

An experiment was conducted to validate the fitness of the Taguchi optimization method, using predicted optimized conditions for all of the four factors for both herbicides individually. The observed TBM and MET degradation was 97.21 and 94.5%, respectively, which was similar to the predicted values at 95% level of confidence (data not shown).

Biodegradation of TBM and MET as Co-contaminants

The biodegradation rates and other kinetic parameters were determined for simultaneous degradation of TBM and MET as co-contaminants. Degradation was assessed in cultures containing AllyMax, having 2.5 mg/L each of TBM and MET, and compared to those having 2.5 and 5.0 mg/L TBM and MET separately. Culture conditions optimized using TOE, an orthogonal array where substantial degradation of both the herbicides occurred, i.e., pH (7.0), temperature (35 °C), and medium composition, MSM, were used. In all cases, the consortium efficiently degraded TBM and MET under the conditions tested.

The biodegradation of TBM and MET followed first-order kinetics, as depicted by plotting a graph between ln Ct/Co and time (Figure ). TBM (2.5 mg/L) was degraded at the highest rate with a K (day–1) of 0.968, followed by 0.772 at 5 mg/L and 0.575 when both the herbicides were present as co-contaminants (Table ). MET was also degraded at the highest rate at 2.5 mg/L; however, the rate of degradation was lower than TBM under the same set of conditions. In cultures where TBM was present as co-contaminant, the lowest degradation rate (0.337) was observed.

Figure 2

Semi-logarithmic plot of Ct/C presenting biodegradation of TBM (red circle solid) and MET (red diamond solid) at 2.5 mg/L each, TBM (blue circle solid) and MET (blue diamond solid) at 5 mg/L each, and TBM (●) and MET (⧫) where AllyMax was added to achieve 2.5 mg/L of each herbicide. The consortium B2R was inoculated in MSM cultures. Dotted lines show the experimental data, whereas solid lines depict the first-order fit.

Table 5

Kinetic Parameters for Biodegradation of Tribenuron Methyl (TBM) and Metsulfuron Methyl (MET) and Active Ingredients in AllyMax Using Consortium B2R

Treatmentsa	Concentration mgL-1	Regression equation	K (day–1)	T1/2 (days)	R2	Degradation (%)
Biodegradation of TBM
TBM control	2.5	Ct = 2.40 × 10–0.027	0.027	110.0	0.81	13.6
TBM control	5	Ct = 4.90 × 10–0.024	0.024	124.0	0.83	16.0
(TBM + MET) control	2.5 + 2.5	Ct = 2.50 × 10–0.028	0.028	135.0	0.78	18.2
TBM + B2R	2.5	Ct = 2.36 × 10–0.968	0.968	4.5	0.99	99.2
TBM + B2R	5	Ct = 4.80 × 10–0.722	0.722	6.2	0.99	97.0
(TBM + MET) + B2R	2.5 + 2.5	Ct = 2.20 × 10–0.575	0.575	7.3	0.99	94.8
Biodegradation of MET
MET control	2.5	Ct = 2.50 × 10–0.025	0.025	148.0	0.82	13.6
MET control	5	Ct = 5.00 × 10–0.018	0.018	189.0	0.84	10.8
(TBM + MET) control	2.5 + 2.5	Ct = 2.50 × 10–0.022	0.022	169.0	0.74	12.0
MET + B2R	2.5	Ct = 2.45 × 10–0.887	0.887	5.2	0.98	98.6
MET + B2R	5	Ct = 4.90 × 10–0.402	0.402	10.6	0.98	87.0
(TBM + MET) + B2R	2.5 + 2.5	Ct = 2.40 × 10–0.337	0.337	13.2	0.98	80.4

TBM + MET indicates that AllyMax was added to the medium and both herbicides’ active ingredients were present as co-contaminants.

In cultures containing 2.5 and 5.0 mg/L TBM separately and 2.5 mg/L TBM and MET each, 99.2, 97.0, and 94.8% degradation was achieved after 15 days of incubation. At 2.5 mg/L, 98.6% MET was degraded, followed by 87.0% at 5.0 mg/L and 80.4% when both TBM and MET were present as co-contaminants. Although the presence of TBM as co-contaminant had some effect on degradation of MET as indicated by degradation and half-lives, substantial degradation of TBM and MET was observed as co-contaminants.

Identification of TBM and MET Metabolites Produced during Biodegradation

In high-performance liquid chromatography (HPLC) chromatograms of TBM and MET culture extracts, peaks were observed at 2.0 and 3.44 min retention times, respectively, which were attributed to their metabolites. With increasing incubation period, a decrease in these peak areas was observed, indicating that the TBM and MET metabolites were further degraded (Figures S3 and S4).

For confirmation, these extracts were subjected to ESI-MS/MS in both positive ion and negative ion mode. In the full MS of TBM and MET, various ion peaks, corresponding to their respective metabolites, were detected. For the confirmation of these metabolites, their further tandem mass spectrometry was conducted. The ESI-MS2 of the ion peak at m/z 155.1 generated daughter ion peaks of m/z 114.0 that resulted from the fragmentation of the parent ion peak at m/z 155.1 at positions C-2, N-3 and C-4, N-5, respectively. The m/z 98.1 was fragmented at N-1, C-1 and N-3, C-4 positions. Similarly, the daughter ion peak at m/z 85.0 and m/z 71.1 were due to the cleavage at N-1, C-2 and C-4, N-5 positions along with a base peak at m/z 57.1 (Figure A).

Figure 3

ESI-MS2 of metabolites produced during biodegradation of tribenuron methyl and metsulfuron methyl using consortium B2R. (A) ESI-MS2 of m/z 155.1@CID 3.5 in positive ionization mode, (B) ESI-MS2 of m/z 182.0@CID 4.0 in negative ionization mode, and (C) ESI-MS2 of m/z 272@CID 3.5 in negative ionization mode.

The ion peak at m/z 182.0 was fragmented into m/z 164.0 due to possible water loss. The base ion peak was generated at m/z 155.0 due to cleavage at the carbonyl position. Similarly, the peak at m/z 138.1 was generated during its cleavage at S–N and the bond between carbonyl carbon and the benzene ring. Few other fragmentations, e.g., m/z 129.8 and m/z 106.0, were also observed, which suggest further rearrangements during its ESI-MS2 (Figure B). During the tandem mass spectrometry, another metabolite with m/z 213 was also detected (data not shown).

During MS/MS analysis of MET, a metabolite with m/z 272.2 was detected in negative ionization mode at the 7th day of incubation. The MS2 of this ion peak generated daughter ion peaks of m/z 241.2 due to methoxy loss and m/z 150.0 due to cleavage at the aryl and sulfonyl linkage (Figure C). This metabolite is potentially a phenyl ring hydroxylation product of MET after the cleavage of the sulfonylurea bridge. The other two identified metabolites (m/z 155.1 and 182.0) produced during biodegradation of MET were similar to the one produced during biodegradation of TBM. Based on these results, the proposed biodegradation pathways for TBM and MET using consortium B2R are presented in Figures and 5, respectively.

Figure 4

Proposed biodegradation pathway of tribenuron methyl by consortium B2R.

Figure 5

Proposed biodegradation pathway of metsulfuron methyl by consortium B2R

Toxicity Reduction of the B2R-Treated TBM and MET

A. cepa bulbs were allowed to grow in water containing TBM, MET, their mixture, and the formulation AllyMax. Roots of onion bulbs started to germinate and grow after two days of incubation in control, while it was delayed in the water having the herbicides. A statistically significant and differential response of the same concentrations of TBM, MET, their mixture, and the formulation AllyMax towards root inhibition was observed (Table ). After biodegradation of TBM, MET, their mixture, and AllyMax with the consortium B2R, root inhibitions (%) in onion cells were decreased.

Table 6

Onion Root Growth Inhibition (%) of TBM, MET, Their Mixture, and the Formulation AllyMax (Added to Achieve Equivalent Concentrations of TBM and MET) after Treatment with the Consortium B2Ra

		Root length (cm) ± SD		% Inhibition over control
Herbicides	Concentration mg/L	Untreated	B2R treated	Untreated	B2R treated
Negative control (water)	NIL	6.30 ± 0.32a	NA	NA	NA
TBM	0.5	0.63 ± 0.03e	4.40 ± 0.12b	90.0	30.0
MET	0.5	0.35 ± 0.01f	3.27 ± 0.09c	95.0	49.4
TBM + MET (mixture)	0.25 + 0.25	0.24 ± 0.02f	2.07 ± 0.11d	96.2	67.3
TBM + MET (AllyMax)	0.25 + 0.25	0.21 ± 0.03f	1.95 ± 0.06d	96.7	69.2

NA = not applicable (in case of −ve control, i.e., water B2R treatment was not required).

In Comet analysis, three parameters, tail length, tail DNA (% DNA), and olive tail movement (OTM), were measured to assess the DNA damage in onion roots (Table ). In contrast to negative control, significantly high DNA damage was observed in onion roots exposed to TBM and MET. However, the mixture of TBM and MET at 0.25 mg/L each as co-contaminants and AllyMax showed significantly higher DNA damage than that of TBM and MET separately. In consortium B2R degradates of the cultures with the above-mentioned concentrations of herbicide active ingredients, there was significant reduction in DNA damage. It showed that the toxicity induced by each herbicide and their formulation was decreased by treating with the bacterial consortium B2R.

Table 7

Scored DNA Damage (±SD) in A. cepa Root Cells Exposed to Herbicides TBM, MET, Combination of Both and the Formulation AllyMax, and BR2 Degradates of Cultures Containing the Respective Herbicides as Determined by Comet Assaya

Treatments	Tail DNA%	Tail length	Tail moment	Tail DNA%	Tail length	Tail moment
	Un-inoculated control			B2R inoculated degradates
Control	0.43 ± 0.18a	3.5 ± 0.88a	0.6 ± 0.2a	-	-	-
TBM	12 ± 1.21b	9.8 ± 1.3b	2.9 ± 0.5b	2.5 ± 0.5b	5.16 ± 0.9b	0.43 ± 0.2a
MET	17 ± 2.1c	17.9 ± 1.6c	3.5 ± 0.24b	6.5 ± 1.6c	8.53 ± 2.1bc	1.32 ± 0.4b
TBM + MET (mixture)	26 ± 2.8d	35.4 ± 1.9d	6.6 ± 0.4c	5.7 ± 1.3c	16.3 ± 1.8c	1.21 ± 0.7b
TBM + MET AllyMax	25 ± 2.3d	37 ± 4.4d	7.1 ± 2.3c	8.5 ± 2.0c	14.33 ± 0.2c	1.8 ± 0.3b

The control represents onion roots growing in water. All values present the mean of 5 replicates ±SD.

Discussion

TBM and MET are frequently used together in various formulations, e.g., AllyMax for broad-spectrum weeds control in wheat, barley, and other grain crops. As co-contaminants, these are more toxic and persistent; therefore, their residues have an augmented impact on subsequent crops and ecosystems. Exogenous bacteria specialized for degradation of TBM and MET have been investigated as an effective strategy to remove their remnants in the environment. The majority of the earlier studies primarily focused on degradation of a single herbicide. In routine agricultural practice, both TBM and MET are components of formulations that are effective against a variety of weeds. This results in a combination of TBM and MET contamination in the environment. Therefore, in this research, simultaneous degradation of TBM and MET as co-contaminants was addressed.

In this study, a consortium B2R was developed that successfully degraded TBM and MET separately and together as co-contaminants. The consortium B2R comprised three bacterial strains, B. velezensis OS-2, B. cereus SU-1, and R. rhodochrous AQ1. The genus Rhodococcus has already been reported for biodegradation of pesticides, e.g., cyhalothrin,[30] p-nitrophenol[31] metribuzin,[28] metamitron,[32] and atrazine.[33] Similarly, Bacillus spp. are well known to degrade a number of pesticides, including atrazine, malathion and parathion,[34] imidacloprid,[35] and fipronil[36] among many others.

Generally, the bioremediation systems that use microbes are strongly affected by several factors such as temperature, pH, carbon sources, substrate concentration, nutrients etc. Such factors that can influence degradation of TBM and MET by the consortium B2R were optimized using Taguchi DOE, which helped to reduce the time and energy required for the design and execution of the experiments and analysis of the results.[28] The method is very helpful to investigate the significant interactions between different factors at the same time. In this investigation, increase in temperature from 25 to 35 °C significantly increased the biodegradation of TBM and MET separately. This is mainly because lower temperatures can impart a negative effect on the growth of bacteria, which concurrently affect the metabolic and enzymatic activities of microbes to reduce the degradation rate.[37]

Media pH is reported as an important factor in the degradation of sulfonylurea herbicides. The pH can directly affect cell growth and metabolic activities, thereby affecting their degradation capacity.[37] In this study, consortium B2R was able to degrade both herbicides over the range of pH (acidic to basic), showing the bacterial efficiency to degrade the TBM and MET in diverse pH environments.

In this study, both TBM and MET were degraded in all of the media used, i.e., MSM, MSM-G, and MSM-Y. Degradation of TBM was enhanced in the presence of glucose. Similar findings have been previously reported, and it was speculated that tribenuron methyl would be co-metabolically degraded in the presence of the easily available carbon source.[10,19] The authors also hypothesized that glucose was converted into organic acids, which lowered the pH of media and might have assisted in degradation of the TBM. In the present study, a buffered medium was used, and only a slight difference in media pH was noted throughout the experiment. The higher degradation of TBM in the presence of glucose observed in the present studies can be attributed to the enhanced bacterial growth and activity in the presence of an easily available carbon source. Significantly higher MET degradation was observed in minimal medium without any added carbon/nutrient source. Taguchi DOE was found to be a good approach for the optimization of variable factors on the basis of the comprehensive results obtained. It is also worth noting that, despite the fact that both herbicides belong to the same chemical group, their optimized conditions in terms of biodegradation by the same bacterial consortium were different.

In the current investigation, biodegradation of TBM and MET as co-contaminants was carried out by applying the optimum levels of media pH and incubation temperature as suggested by Taguchi DOE whereas lowest levels of concentration were employed. Degradation of TBM and MET followed first-order kinetics. For both compounds, a high degradation rate and low half-lives were observed at the lowest concentration tested, e.g., 2.5 mg/L followed by that at 5.0 mg/L. Comparatively low degradation was observed when AllyMax was used to obtain 2.5 mg/L each of TBM and MET, indicating that the presence of one compound affected the degradation rate of the other and vice versa. After 15 days of incubation, 94.8 and 80.4% TBM and MET were degraded, respectively, when these were present as co-contaminants, indicative of the capability of B2R for the degradation of TBM and MET separately and as co-contaminants when their formulations are used. In independent studies, Yang and Yu reported microbial degradation of TBM and MET in soil separately.[20,21] Some studies on degradation of TBM by bacteria, e.g., Bacillus sp., Serratia sp., and Pseudomonas sp.[18,19] are available, whereas studies regarding bacterial degradation of MET are scarce. Lu[23] and Huang[24] reported MET biodegradation by Ancylobacter sp. and Methylopilla sp. in liquid cultures, respectively, and described the biochemical pathway and enzyme involved in degradation. However, there are no studies regarding biodegradation of MET and TBM when these co-exist, e.g., when the herbicide formulation AllyMax is used for weed control.

In this study, the mass spectral data revealed that metabolites were produced by cleavage of the sulfonylurea bridge of both TBM and MET. Metsulfuron methyl was metabolized into the hydroxylation product after cleavage of the sulfonylurea bridge. This product is produced by incorporation of the hydroxyl ion into the aromatic ring through the enzymatic action of cytochrome P450, as suggested earlier.[38] Both herbicides were mineralized into saccharin and 2-amino-4-methoxy-6-methyl-1,3,5-triazine. The disappearance of peaks corresponding to these metabolites on the HPLC chromatograms after a longer incubation indicated that these metabolites were further degraded.

Sometimes, metabolites produced as result of bacterial degradation of pesticides are more toxic as compared to the parent compounds.[39] However, as observed in the present study, after degradation of TBM and MET, separately and as co-contaminants, by the consortium B2R, phyto- and genotoxicity were drastically reduced. This indicates the potential of consortium B2R for remediation of TBM and MET separately and as co-contaminants.

To the best of our knowledge, this is the first report demonstrating the ability of a bacterial consortium to degrade TBM and MET simultaneously. Subsequent studies are necessary to evaluate the ability of this consortium to remediate the water and soil environments contaminated by these herbicides.

Conclusions

The consortium B2R developed and characterized in this study is highly effective for simultaneous degradation of TEB and MET separately and as co-contaminants. The L9 orthogonal arrays in Taguchi DOE were found to be useful to determine the optimal levels of different factors to attain enhanced degradation. The optimized conditions enabled us to figure out a setup for simultaneous biodegradation of TBM and MET as co-contaminants (as in AllyMax), which was confirmed experimentally. Mass spectral analysis confirmed that TBM and MET were metabolized into simpler compounds, which were further degraded. A. cepa root inhibition and comet assay revealed that the phyto- and genotoxicity of individual herbicides and their formulation were reduced after biodegradation. It is postulated that the consortium efficiently degraded the herbicides TBM and MET, separately and as co-contaminants, and that degradation was associated with reduction of their toxicity. The consortium B2R will be used to investigate bioremediation of TBM and MET especially as co-contaminants in vegetated cropland soil, which will potentially be a step forward for sustainable agriculture.

Materials and Methods

Isolation and Selection of Bacterial Strains for Biodegradation of TBM and MET

Soil exposed to SU herbicides was collected from wheat fields in Sheikhupura (31° 42′ 04.0″ N, 73° 57′ 58.6″ E), District of the Punjab province, and spiked with TBM and MET (1 mg/kg soil, w/w). Wheat seeds were sown in this soil and the plants were allowed to grow for two months. For isolation of endophytic bacteria, the wheat roots were sterilized and crushed to achieve the root extract as demonstrated earlier.[28] The extract was serially diluted and spread on Luria broth (LB) agar plates. For the isolation of rhizospheric bacteria, rhizospheric soil (10 g) was added to 100 mL of MSM containing 5 mg/L each of TBM and MET, and enriched for two months as described earlier.[40] Pure isolates were obtained by dilution of the enriched culture and by spreading on LB agar plates containing the respective herbicides. Additionally, already isolated pesticides degrading bacterial strains were also evaluated for their ability to degrade TBM and MET. The bacterial isolates and already isolated strains were inoculated in MSM medium containing 10 mg/L MET and TBM separately to determine their ability to degrade these herbicides. Two isolates, OS-2 and SU-1, and a previously reported strain R. rhodochrous AQ1[28] degraded >70% of the added TBM and MET and were selected for further studies. The isolates OS-2 and SU-1 were identified by 16Sr RNA gene sequencing. The sequences obtained were used to identify the strains using NCBI BLAST.[40]

Development of Inocula for Biodegradation of TBM and MET

The strains AQ1, OS-2, and SU-1 were cultivated in LB medium at 35 °C and 100 rpm. After 24 h, the cells were centrifuged at 6000g for 5 min to get pellets of each bacterium. The cell biomass of each bacterium was suspended in saline (0.85% sodium chloride) individually to achieve 109 cells/mL as explained earlier.[41,42] All of the three bacterial strains were checked for compatibility with each other by the streak plate method.[43] The suspensions of compatible bacterial strains were mixed in 1:1:1 ratio to get a consortium B2R.[28] One percent of this consortium was used for degradation studies unless otherwise mentioned.

Biodegradation of TBM and MET by Individual Strains and the Consortium B2R

A shake flask study was conducted to compare the potential of individual strains and the consortium B2R to degrade TBM and MET. Each herbicide (10 mg/L) was added separately in 100 mL of MSM and inoculated (1 mL) with individual strains and the consortium B2R. The flasks were placed in a orbital shaker adjusted at 100 rpm and 35 °C. The un-inoculated media containing herbicides served as control. For herbicide residue analysis, samples were taken at three days interval up to two weeks.

Optimization of Culture Conditions for Maximum Biodegradation of TBM and MET

Four variables were selected to attain optimum culture conditions for degradation of TBM and MET using consortium B2R. A factorial experimental L9 orthogonal array scheme was designed by Taguchi DOE of Qualiteck-4 packages (Nutek Inc., MI). Four factors at three different levels, i.e., temperature (25, 30, 35 °C), pH (6, 7, 8), media composition [MSM, MSM and glucose (MSM-G), and MSM and yeast extract (MSM-Y)], and initial TBM and MET concentrations (5, 10, and 15 mg/L), were selected (Table ), and were arranged in the L9 orthogonal array (Table ). In the media MSM-G and MSM-Y, glucose and yeast extract were added in MSM@0.05%, respectively, to explore the co-metabolism effect on the biodegradation of TBM and MET. After two weeks of inoculation (1% consortium B2R), the residual concentration of TBM and MET in culture medium was quantified by the HPLC system. Qualitek-4 software was used to analyze the recorded data to determine the individual as well interactive effect of variables. The results thus obtained afforded the optimal conditions for removal of each herbicide on the basis of the signal-to-noise ratio (S/N ratio) with the selected option “bigger is better.” eq was used for calculation of the S/N ratio.In this equation, the response from the selected factor level combination in terms of percent biodegradation is denoted by “Y”, while “n” represents the number of responses in this combination. The experiment was conducted in triplicates.

Biodegradation of TBM and MET as Co-contaminants in the Formulation AllyMax

The potential of bacterial consortium B2R was evaluated to degrade TBM and MET as co-contaminants in their formulation, AllyMax. The AllyMax, TBM, and MET were added to the MSM as follows: (i) AllyMax (2.5 mg/L TBM and MET each), (ii) TBM (2.5 mg/L), (iii) TBM (5 mg/L), (iv) MET (2.5 mg/L), and (v) MET (5 mg/L). The experiment was carried out in triplicates for two weeks. The representative samples were taken at three-day intervals for residual analysis of TBM and MET. The biodegradation rate (K, day–1) and half-life (T1/2, day) of each herbicide and as active ingredient in AllyMax was determined by plotting ln [Ct/C0] versus time by employing eqs and 3.In eq , Ct denotes the herbicide concentration in mg/L at a specific time interval “t” and C0 indicates the initial herbicide concentration (mg/L) at time “zero”.

Identification of Metabolites of TBM and MET

Metabolites of TBM and MET produced during biodegradation were determined by ESI-MS/MS (Model: LTQXL-Thermo Electron Corporation). The samples were extracted with methylene dichloride twice, dissolved in acetonitrile (LC–MS grade purity) and filtered through a poly(tetrafluoroethylene) (PTFE) membrane syringe filter (0.45 μm) before injecting them using a direct syringe pump to the mass spectrometer. The analysis of TBM and MET metabolites was done using mass spectrometry at a normal mass range from m/z 50 to 1000, and the mass spectra were recorded using electrospray ionization (ESI) probe in both positive and negative ion modes. The system was run on full scan as well as ion isolation mode to conduct selective ion monitoring (SIM). Further tandem mass spectrometry was conducted using various energies, ranging from 10 to 45 mV. The data obtained was processed using X-calibur software. The chemical structures (parent and daughter ion peaks) were drawn using ChemBioDraw Ultra 8.0 software. The compounds were identified by manually correlating their finger print fragmentation patterns with reference standards and published data.

HPLC Analysis of Residual TBM and MET

The extraction of the samples containing TBM, MET, and AllyMax was performed with 20 mL of dichloromethane (DCM). After vigorous shaking, the solvent mixture was allowed to settle in two layers. The organic layer was separated from the aqueous layer in 50 mL glass vials. The organic layer of DCM was evaporated overnight, and then herbicide residues were dissolved in HPLC-grade acetonitrile and filtered through 0.45 μm nylon disc filters.

The herbicide analysis was done using Ultimate dionex 3000 UHPLC equipped with a diode array detector and chromeleon software. The isocratic mobile phase (acetonitrile/water; 90:10) was programmed using a diode array detector ranging from 225 to 290 nm. The retention time for TBM and MET was calculated as 2.4 and 1.9 min at a flow rate of 1 mL/min, respectively, at 240 nm. The linear calibration curve was obtained by plotting the peak areas of 5, 10, 20, and 50 mg/L TBM and MET as a function of the concentration using the cobra run of Chromeleon software. TBM and MET concentrations were determined using a chromatography studio from the plot as a function of the peak area.

A. cepa Root and Comet Assays of the B2R-Treated Water

Equal-sized and healthy onion bulbs (135 ± 2 g) were purchased from the local market. The onion root growth inhibition assay was performed by following the procedure explained earlier.[44] TBM, MET, and AllyMax were subjected to degradation at 0.5 mg/L initial concentration. A. cepa tubers were exposed to the degradates and respective controls to investigate the reduction in toxicity. The experimental setup was placed at room temperature (23 ± 2 °C) for seven days. The average root length was determined by taking 5 roots from one bulb (25 roots from 5 bulbs) for each application.

The genotoxic potentials of the treatments were determined by Comet assay as described earlier.[45] The onion root tips were gently cut into the chilled isolation buffer (500 μL). The buffer medium contained Tris 4 mM, Triton X-100 (0.5%, w/v), and MgCl2–6H2O, pH 7.5. Nuclei were isolated from the root cells of A. cepa as previously described.[46] Nuclear suspensions (50 μL) were mixed with an equal volume of low-melting-point agarose LMPA (1.5%) and then were spread over the slides precoated with 1% normal-melting-point agarose NMPA. The slides were allowed to solidify for 5 min at 4 °C in an ice tray. The slides were placed in alkaline buffer (300 mM NaOH and 1 mM ethylenediaminetetraacetic acid, EDTA, pH > 13) for 20 min at 4 °C and electrophoresed at 25 V and 300 mA for 20 min at 4 °C. The slides were neutralized with 0.4 M Tris buffer (pH 7.5) three times and stained with ethidium bromide (20 μg/mL) for 5 min. Fifty comets per slide were scored using a fluorescence microscope equipped with a CCD camera.

Statistical Analysis

Microsoft Excel (2016) was used to calculate the means and standard deviations of the replicates. Statistically significant differences of the results among the variances were analyzed using SPSS 14.0 software. Statistical significance was measured at a level of p < 0.05.