Introduction

Chloroacetanilide is an important class of systemic selective herbicides used worldwide for controlling both pre-emergent and early post-emergent weeds in several crops such as corn, rice and soybean. (Dwivedi et al. 2012). These herbicides inhibit the synthesis of proteins, lipids, alcohols, flavonoids, lignin, etc. (Seok et al. 2012). Butachlor, a chloroacetanilide class of herbicide, is one of the most extensively recommended class of chloroacetanilide class of herbicide for controlling pre-emergent broadleaf weeds, annual grasses and submerged macrophytes in freshwater water bodies. In addition to the lipid biosynthesis, this compound also disturbs several other important metabolic pathways and redox homoeostasis negatively (Götz and Böger 2004). It is one of the most abundantly used herbicides in Asia, Africa and South America. It has been reported that yearly butachlor consumption in Asia alone is more than 4.5 × 107 kg (Ateeq et al. 2002). Extensive usage of butachlor resulted in deleterious effects on the environment. Higher concentration of butachlor residues, as well as its intermediate degradation products, has been identified in various soils as well as ground and surface waters (Kim et al. 2013). Several studies pertaining to the pernicious effect of the herbicide in the environment have been undertaken (Wang et al. 2007; Fang et al. 2009; Abigail et al. 2015). Research suggests that butachlor is known to induce apoptosis in mammalian cells and exerts genotoxic effects on amphibians (Geng et al. 2005). It is also reported to exhibit toxicity to earthworms and impact microbial community structures and enzymatic activities (Muthukaruppan et al. 2004). Various in vitro studies have established the mutagenic property of butachlor towards aquatic organisms such as freshwater fish, Tilapia zillii (Nwani et al. 2013) and Salmonella typhimurium (Hsu et al. 2005) leading to the impairment of the water environment. Moreover, some studies also reported probable carcinogenicity and oxidative DNA damage in human cells due to butachlor (Dwivedi et al. 2012).

Owing to the persistent nature of the butachlor in the agricultural soil and the threats to the environment, great interest and concernment have been invoked about the behaviour and efficient remediation of the herbicide and its metabolites in the ecosystem (Debnath et al. 2002; Yu et al. 2003; Fang et al. 2009). Several studies have reported that butachlor could be removed from the environment by both biotic and abiotic processes (Pal et al. 2006). However, the chemical hydrolysis and photo-oxidation of the herbicide are relatively insignificant to its removal since these are time-consuming procedures and they involve other issues such as high cost, secondary effluent problems. Hence, microbial transformation is one of the most prodigious route for determining the fate of butachlor and its dissipation from the ecosystem (Zhang et al. 2011; Rajasankar et al. 2013). In the recent years, microbial degradation has received recognition as an effective, economical and dependable alternative for remediation of various pesticide contaminations. Earlier, a few microorganisms have been reported that have the capability to use butachlor as the sole carbon and energy source (Torra-Reventos et al. 2004; Dwivedi et al. 2010). A couple of studies on the metabolic pathway and enzyme studies have also been carried out (Zhang et al. 2011; Liu et al. 2012; Gao et al. 2015). However, previously isolated microorganisms are not competent enough regarding degradation potential in most of these studies. Nevertheless, it is essential to isolate new microbes with higher butachlor removal efficiency.

Environmental parameters play an exceptionally significant factor in determining the biodegradation efficiency of any microorganism. Their degradation and cell growth potential are highly affected by both environmental and nutritional parameters as in temperature, pH, nitrogen and carbon sources (Zhou et al. 2011; Kong et al. 2014). However, as per the available literature, no study regarding the nutritional and environmental parameters affecting butachlor biodegradation exists. Hence, it is highly essential to sketch an appropriate model design for enhancing the butachlor removal efficiency by the microbe. Optimized factors improve the biodegradation efficiency and reduces the process time and cost significantly. Considering the several parameters that vary simultaneously, statistical models like response surface methodology (RSM) and Plackett–Burman designs (PBD) can optimize the concerning parameters simultaneously to dismiss the drawbacks of one factor at a time technique (Singh et al. 2017). Although several studies regarding statistical modelling and optimization of biodegradation of many toxic pollutants exist in the literature, application of mathematical optimization for the biodegradation of butachlor is yet to be studied. RSM, endorsed by a software, is a sensible approach to estimate the relationship of an array of controlled and influencing parameters. Hence, it is highly essential to draft a strategy to enhance degradation of butachlor by the isolated microbial strain.

In the present study, a butachlor-catabolizing bacterium was isolated from the agricultural soil. Statistical optimization techniques such as Plackett–Burman design and RSM were being engaged in defining the significant factors affecting butachlor biodegradation and identifying the optimum levels of those variables for maximizing the butachlor degradation by the isolated strain.

Materials and methods

Chemicals and medium

Butachlor (purity = 90.5%) from Insecticides India, Ltd., was used in this study. The rest of the chemicals and reagents were obtained from Merck (India) of the highest analytical reagent grade. The composition of the mineral salts medium (MSM) used in this study is as follows: (NH4)2SO4 (1.0 g/L), NaCl (1.0 g/L), K2HPO4 (1.5 g/L), KH2PO4 (0.5 g/L) and MgSO4·7H2O (0.2 g/L) (pH 7.0) (Liu et al. 2012).

Isolation and identification of butachlor-catabolizing microorganism

The soil samples were obtained from an agricultural field in Odisha, India (20.266°N, 86.166°E) having a history of butachlor application for many years (DAFP ODISHA 2008). The enrichment culture for the isolation of butachlor-catabolizing microorganism was carried through as reported by Mohanty and Jena (2017). About five grams of the contaminated soil sample was added to 100 mL MSM comprising butachlor (100 mg/L), and the setup was incubated for five days at 35 °C, 180 RPM in an orbital shaker incubator. Then, five millilitre of the enrichment culture suspension was aseptically transferred into fresh flask containing MSM and further incubated for five more days. The concentration of butachlor was determined after each transfer to confirm its degradation. After sixth transfer, the enrichment culture was subjected to serial dilution and was transferred to MSM agar plates comprising 100 mg/L of butachlor. The pure culture obtained was evaluated for their ability to degrade butachlor. Eventually, the bacterial strains showing maximum butachlor tolerance and highest degradation potential were chosen for further studies.

For the identification of the microbial strains, 16 s rRNA gene sequences were obtained and were homologized using BLAST algorithm with the archived 16 s rDNA sequences already submitted at GeneBank, NCBI. A phylogenetic tree is developed as presented in Fig. 1 using MEGA 6.0.5 software (Thompson et al. 1997). The partial 16 s rDNA gene sequences obtained were submitted to GeneBank, NCBI.

Fig. 1
figure 1

Phylogenetic tree based on the 16S rRNA gene sequences of strain AS1 and related species. The GenBank accession number for each microorganism used in the analysis is shown along with the species name

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Analytical method

The butachlor residue in the medium was estimated by HPLC analysis of the culture medium in regular interval of time. The HPLC analysis was carried out with 5-µm, C-18 column (Agilent Technologies, USA). The mixture of methanol and water (both HPLC Grade) in a ratio of 70: 30 was asserted as the gradient mobile phase. While the flow rate was held at 1 ml/min, butachlor was quantified at wavelength of 225 nm (Dwivedi et al. 2010) (Liu et al. 2012). Each reading was performed in triplicates.

Design of experiments

Plackett–Burman design

In the present investigation, a 16-run Plackett–Burman design (including four centre points) was employed for seven variables (along with two dummy variables). The factors were been evaluated both for higher level denoted by + 1 and lower level denoted by − 1 while the centre value was denoted as level zero. The boundary value as well as the value of the centre point of variable has been defined as per the results obtained in preliminary studies (data not shown here). The parameters considered for the study, their corresponding − 1 and + 1 values, the design and the corresponding response (butachlor degradation) values have been enlisted in Table 1. The effect of each parameter on butachlor degradation has been calculated using the following equation

$$Y = M_{0} + \varSigma M_{i} X_{i}$$
(1)

where Y denotes the response (percentage butachlor removal), M0: model intercept; Mi: linear factor coefficient; Xi: Participant variable (Dayana Priyadharshini and Bakthavatsalam 2016). The factors having p value less than 5% in the regression analysis were considered to have a crucial effect on butachlor biodegradation and were studied further by RSM.

Table 1 Plackett–Burman design of factors with butachlor degradation (percentage) as a response
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Response surface methodology

Utilizing the Plackett–Burman analysis, three influential factors (pH of the medium, growth temperature and biomass concentration) imperative for biodegradation of butachlor were shortlisted. To optimize the significant factors for the biodegradation of butachlor, a 23 full factorial central composite design (CCD), each at five levels (− α, − 1, 0, + 1, + α) with six replicates at the centre points and eight axial points, was employed for improving butachlor degradation by the isolated strain. An altogether of 20 experiments was carried out, and the particulars of the experimental design have been enlisted in Table 2. A second-order polynomial equation was engaged for analysing the experimental data obtained and calculates the relationship between the studied parameters as shown by Eq. (2):

$${\text{Response}} = M_{0} + \sum\limits_{i = 1}^{n} {M_{i } X_{i} } + \sum\limits_{i = 1}^{n} {M_{ii } X_{i}^{2} } + \sum\limits_{i = 1}^{n} {\sum\limits_{j = 1}^{n} {M_{ij } X_{i} X_{j} } }$$
(2)

wherein response denotes the predicted percentage of butachlor removal; X symbolizes the input variables influencing the response; M0 is the constant (intercept coefficient); Mi represents the ith linear coefficient; Mii intends to be ith quadratic coefficient, and Mij stands for the ijth interaction coefficient (Zhao et al. 2017). For establishing the interaction among the variables, surface plots (three-dimensional) of the predicted responses were constructed. The statistical software Minitab (Version 17.1) has been used for designing the experiment and further determination of the regression coefficients by analysis of variance (ANOVA) and the coefficient of determination (R2).

Table 2 Effect of factors and statistical analysis of factors using Plackett–Burman Design
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Results and discussion

Isolation and characterization of butachlor-catabolizing strain

From the preliminary screening of 12 bacterial isolates obtained from the enrichment culture, three bacterial strains designated AS1, AS2, and AS5 were initially selected for the study taking into account their high butachlor tolerance potential. Among the isolates, the bacterial strain-AS1 was capable of tolerating butachlor concentration as high as 1000 mg/L and was able to degrade up to 100 mg/L of butachlor within 48 h. Hence, the strain was selected for further investigation. The strain-AS1 is a Gram-negative, motile, rod-shaped, non-spore forming bacterium. The microbial strain has been positive for starch hydrolysis, urease, Voges–Proskauer and nitrate reduction test while negative for oxidase and lactose hydrolysis. The 16S rDNA sequence obtained has been submitted in the GenBank repository with Accession No. KT427634. The multiple sequence alignment of the obtained sequence and the sequences available in GenBank archives revealed the highest degree of similarity with the members of the genus Serratia and forming a subclade with S. ureilytica KJ722485 (100% homology) (Fig. 1). Thus, because of the aspects stated above, the strain-AS1 was identified as S. ureilytica.

Optimization of biodegradation of butachlor by Serratia ureilytica

The seven parameters contemplated in this study for their influence on biodegradation of butachlor by the microbial strain were statistically analysed using PBD. Table 1 enlists the design of experiments and their corresponding butachlor degradation percentage. The wide variation in responses from 32.93 to 75.69, in the 16 trials, emphasizes the dependency of butachlor removal efficiency of the microbe on various process parameters. Table 3 exhibited that parameters such as pH of the medium, growth temperature and inoculum size have a substantial impact on the biodegradation of butachlor and were hence incorporated in the subsequent optimization study. However, the rest of the parameters had no significant effect on biodegradation of butachlor. The factors are having a confidence level more than 95% were considered as important for their incorporation in the subsequent studies of optimization. The model equation for the percentage butachlor biodegradation (Y):

$$Y = 34.8255 + 0.762556X_{1} - 1.80056X_{2} - 0.380500X_{3} + 21.9867X_{4} + 24.8222X_{5} - 6.5733X_{6} - 6.9200X_{7}$$
(3)
Table 3 ANOVA for PBD (Plackett–Burman Design) model
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The correlation coefficient (R2) of the value 0.984 suggested that up to 98.4% variabilities in the butachlor degradation could be calculated. The statistical analysis of the experimental data using F-test is presented in Table 4.

Table 4 Experimental design and results of CCD
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The CCD was put to use for determining the interactions among the significant parameters viz. Growth temperature (X1), pH of the medium (X2) and biomass concentration (X3) for further optimization studies. Table 2 showcases the experimental design matrix and the corresponding results obtained. The following second-order polynomial equation has been deduced after the application of multiple regression analysis for the analysis of the experimental data:

$$Y = 75.146 + 5.806X_{1} - 1.611X_{2} + 8.802X_{3} - 7.329X_{1}^{2} - 11.803X_{2}^{2} - 4.520X_{3}^{2}$$
(4)

where Y denotes the percentage butachlor degradation. Table 5 presents the ANOVA of the proposed model for the percentage butachlor degradation. The significance of the model was suggested by its “F value” which was found to be 127.79. The linear as well as the quadratic terms were significant for the estimation of butachlor biodegradation efficiency. Both the experimental and the predicted response are in good agreement with each other as suggested by the R2 value of 0.9833. The “Lack of Fit” of 0.067 suggested it to be non-significant compared to the pure error which means only 6.7% probability of occurrence of “Lack of Fit” due to noise. The established model is sufficiently competent to predict the biodegradation of butachlor within the ranges of the dedicated variables.

Table 5 ANOVA for CCD model
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The regression model was graphically represented by surface plots (three-dimensional) and corresponding contour plots (two-dimensional) (Figs. 2, 3, 4) which were used to contemplate the effect of each parameter individually as well as their mutual interaction among themselves, on biodegradation of butachlor. The plots are based on Eq. (4), where holding one variable at its optimal level, the effect of the other two variables was studied by varying them within the experimental boundaries, the pattern of which indicates the significance of the mutual interaction among the independent variables. As demonstrated in the figures, a clear peak as response surface for butachlor degradation meant that the optimum points are well within the design boundaries.

Fig. 2
figure 2

Surface and contour plot showing interactions between temperature (X1) and pH (X2) on butachlor biodegradation by the isolated S. ureilytica strain AS1 while inoculum size (X3) at zero level

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Fig. 3
figure 3

Surface and contour plot showing interactions between pH (X2) and inoculum size (X3) on butachlor biodegradation by the isolated S. ureilytica strain AS1 while temperature (X1) at zero level

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Fig. 4
figure 4

Surface and contour plot showing interactions between temperature (X1) and inoculum size (X3) on butachlor biodegradation by the isolated S. ureilytica strain AS1 while pH (X2) at zero level

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Figure 2 depicts the effect of growth temperature (X1) versus pH of the medium (X2) on butachlor biodegradation keeping the initial inoculum size (X3) constant at level zero. The response surface plot indicates that change in the growth temperature as well as the in surrounding pH has a significant impact on the degradation efficiency of the microbial strain. The butachlor removal efficiency increases with increasing temperature, but after a certain point, the biodegradation efficiency declines on further increase in the temperature. A similar pattern was observed for the change in the surrounding pH. The uniformly elongated diagonal pattern of the contour plots suggested the significance of the interaction between temperature and pH on butachlor biodegradation. Similar results were reported during the biodegradation of profenofos by a novel bacterial consortium (Jabeen et al. 2015).

The influence of pH of the medium (X2) and the inoculum size of the microbial strain (X3) on butachlor biodegradation keeping the temperature constant has been depicted in Fig. 3. The figure suggests that the optimum butachlor degradation can be attained by keeping the surrounding pH value within 7–8 and increasing the concentration of the microbial inoculum for the study. The elongated two-dimensional contour plot of butachlor degradation versus pH, inoculum size suggests that the mutual interaction among the parameters is significant to the butachlor biodegradation by the isolated strain. Similar results were suggested in the previous study of biodegradation of phenol by the Chlorella pyrenoidosa where the concentration of the biomass in the medium plays a major role in the biodegradation of phenol (Dayana Priyadharshini and Bakthavatsalam 2016).

The plot representing the significance of growth temperature (X1) and the inoculum size (X3) on biodegradation of butachlor while keeping the pH of the medium (X2) at zero level has been depicted in Fig. 4. As presented in the figure, with an increase in the concentration of the biomass and the temperature, the butachlor biodegradation increases. However, the degradation efficiency decreases with increasing the initial biomass concentration as well as the incubation temperature further beyond the optimal conditions. The elongated running two-dimensional contour plot suggests that at a particular pH, the growth temperature and the initial biomass concentration are interdependent on each other and this relationship is vital for the biodegradation of butachlor by the microbial strain. This is in agreement with the earlier works where inoculum size portrays a major role in the biodegradation of the organic contaminant (Zhou et al. 2011).

To validate the obtained statistical model and propose a better understanding of the biodegradation of butachlor, triplicate experiments were performed at optimum levels of the independent variables. The predicted butachlor removal efficiency under the optimal conditions was 79.42% against the actual experimental removal efficiency of 81.08%. Since the predicted and the actual measured values were found to be very close, the validity of the predicted RSM model was thus substantiated.

Biodegradation of butachlor by the strain AS1

Figure 5 demonstrates the biodegradation of butachlor by the isolated S. ureilytica strain AS1 under the optimized condition. The figure exhibits the gradual decrease in the butachlor concentration as a function of time. HPLC analysis quantifies the concentration of the butachlor at a particular time point. Significant reduction in the major butachlor peak at the retention time of 14 min along with the subsequent emergence of numerous secondary peaks of unknown metabolites has been observed at different time points (data not presented). The microbial S. ureilytica strain AS1 displayed complete butachlor degradation of 500 mg/L of butachlor within 10 days at optimized conditions. Increasing the concentration of the butachlor in the medium resulted in the inhibition of the degradation efficiency of the bacterial strain. A prolonged lag phase observed in MSM comprising more than 500 mg/L is attributed to the adaptation phase, possibly owing to the fact that the microorganism takes time to process signal transduction and subsequently induce a metabolic pathway for biodegradation. Similar results have been observed in previous studies reporting prolonged lag phase in similar intensified conditions (Dwivedi et al. 2010; Mohanty 2012).

Fig. 5
figure 5

Degradation profile of the isolate S. ureilytica strain AS1 at different initial concentration of butachlor

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Discussion

Biological treatment methods have been proved to be promising alternatives to remediate contaminated environments. In this study, twelve bacterial strains were isolated from soil samples obtained from an agricultural field with a history of application of the herbicide. Using the selective enrichment technique, an efficient butachlor-catabolizing strain AS 1 has been isolated. The microbial strains were assessed for their butachlor tolerance ability within the range of 100–300 mg/L of butachlor. The prime focus of the study was to isolate and identify the major bio-remediating microbial agents and assess their butachlor remediation capability in the optimized conditions. Based on the comparative analysis of the phenotypic attributes of the isolated strain AS1 and its phylogenetic analysis revealed to form a subclade with similarity with S. ureilytica sp. nov. KJ722485 (100% similarity) (Bhadra et al. 2005). Strain AS 1 showed higher tolerance and better degradation characteristics from previously reported butachlor degraders. This study might be the basis for screening, production and utilization of microbial strains as bioremediation agents for commercial purposes in the future. The microorganism Serratia has earlier been reported to utilize few hydrocarbons for the production of the dyes and remediate some heavy metals (Pakala et al. 2007; Rahman et al. 2009; Venkateswar Reddy et al. 2015). However, to the very best of our understanding, this is the first of its kind report pertaining to the exploration of the herbicide remediation aspect of the strain S. ureilytica. Even though, a few microorganisms have been reported for the biodegradation of butachlor, thus far, no precise and methodological study on butachlor biodegradation has been carried out. This study provides the first evidence that the strain AS1 has the potential in the efficient degradation of butachlor which is extensively used in the agriculture of various crops and is of global concern (Abigail et al. 2015).

The novelty of the present investigation is the isolation of a high tolerating butachlor-catabolizing microorganism and mathematical optimization of butachlor degradation by the same. Application of the statistical design of experiment for screening and optimizing various experimental parameters renders a prompt recognition of the significant factors and interaction among them. To the best of our knowledge, this is the first study to report on the application of statistical design of experiments and mathematical modelling for optimization of the process of butachlor biodegradation. Application of PBD elucidated the facts that the parameters such as pH of the medium, growth temperature and initial biomass concentration has a significant impact on the biodegradation of butachlor and was hence incorporated in the subsequent optimization study. RSM elaborated the influence of the above-mentioned parameters in a more obvious way and reported the interactive effect of different variables on butachlor biodegradation.

Even though a few microorganisms having the capability to degrade butachlor have been reported earlier, there is no report of complete degradation by any microorganism (Dwivedi et al. 2010; Kim et al. 2013). Another distinguished characteristic of this particular microorganism is its ability to withstand high concentration of butachlor of 1000 mg/L. In contrast to the previous studies pertaining to the biodegradation of butachlor or other chloroacetanilide herbicides which evaluated the biodegradation potential of the microorganism only up to 100 mg/L, this bacterial strain is capable of complete biodegradation up to 500 mg/L of butachlor within 240 h. The microorganism S. ureilytica strain AS1 is able to utilize the butachlor exclusively as the source of carbon and energy. However, on increasing the concentration of the herbicide, inhibition in terms of both growth and degradation potential can be observed. Serratia ureilytica strain AS1 proves to be a promising candidate as an accomplished butachlor degrading strain and may be utilized for the treatment of effluents with high butachlor concentration.

Conclusion

The present study is the first of its kind that presents an account of the application of statistical designs for optimizing the biodegradation of chloroacetanilide class of herbicide butachlor by the newly isolated S. ureilytica strain. The purpose of the study was to identify the significant parameters that affect the degradation efficiency of the microbial strain and to achieve the maximum output. Among the parameters evaluated, incubation temperature, medium pH and the initial microbial biomass were found to be the important ones that influence the biodegradation of butachlor by the microbial strain the most. Statistical methods were proven to be an effective and competent tool for elucidation of optimization of various process parameters for maximizing butachlor degradation by the isolated strain and the effect of mutual interaction among them. The bacterial S. ureilytica strain AS1 was established to be a highly efficient and promising candidate for the biodegradation of butachlor. The results obtained in this study laid the groundwork for the employment of this isolated microbial strains for the remediation of highly concentrated butachlor contaminated water, soil and sediments.