Biodegradation of 4-nitroaniline by plant-growth promoting Acinetobacter sp. AVLB2 and toxicological analysis of its biodegradation metabolites

Journal of Hazardous Materials 302 (2016) 426–436

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Biodegradation of 4-nitroaniline by plant-growth promoting Acinetobacter sp. AVLB2 and toxicological analysis of its biodegradation metabolites Sivagnanam Silambarasan a , Alisa S. Vangnai a,b,∗ a b

Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Center of Excellence on Hazardous Substance Management (HSM), Chulalongkorn University, Bangkok 10330, Thailand

h i g h l i g h t s • • • • •

Acinetobacter sp. AVLB2 is a PGPB able to degrade high concentration of 4-NA. Growth and degradation kinetics for 4-NA removal by AVLB2 were studied. A novel biodegradation pathway for 4-nitroaniline has been proposed. Toxicological studies revealed non-toxic nature of 4-NA biodegraded metabolites. Acinetobacter sp. AVLB2 could maintain PGP traits under 4-NA stress.

a r t i c l e

i n f o

Article history: Received 23 August 2015 Received in revised form 2 October 2015 Accepted 6 October 2015 Keywords: Acinetobacter sp. AVLB2 4-Nitroaniline Biodegradation pathway Kinetic analysis Plant growth promotion

a b s t r a c t 4-nitroaniline (4-NA) is one of the major priority pollutants generated from industrial productions and pesticide transformation; however very limited biodegradation details have been reported. This work is the first to report 4-NA biodegradation kinetics and toxicity reduction using a newly isolated plant-growth promoting bacterium, Acinetobacter sp. AVLB2. The 4-NA-dependent growth kinetics parameters: max , Ks and Ki , were determined to be 0.039 h−1 , 6.623 mg L−1 and 25.57 mg L−1 , respectively using Haldane inhibition model, while the maximum biodegradation rate (Vmax ) of 4-NA was at 0.541 mg L−1 h−1 and 0.551 mg L−1 h−1 , following Michaelis–Menten and Hanes–Woolf models, respectively. Biodegradation pathway of 4-NA by Acinetobacter sp. AVLB2 was proposed, and successfully led to the reduction of 4-NA toxicity according to the following toxicity assessments: microbial toxicity using Escherichia coli DH5˛, phytotoxicity with Vigna radiata and Crotalaria juncea, and cytogenotoxicity with Allium cepa root-tip cells. In addition, Acinetobacter sp. AVLB2 possess important plant-growth promoting traits, both in the presence and absence of 4-NA. This study has provided a new insight into 4-NA biodegradation ability and concurrent plant-growth promoting activities of Acinetobacter sp. AVLB2, which may indicate its potential role for rhizoremediation, while sustaining crop production even under 4-NA stressed environment. © 2015 Elsevier B.V. All rights reserved.

1. Introduction 4-Nitroaniline (4-NA) is an aromatic amine extensively used as an intermediate in the production of several industrial and highvolume chemicals including pesticides [1,2], and thus it has been found as a contaminant in agricultural biosolid generated from industrial wastewater sludge [3]. 4-NA is a ubiquitous contaminant in environment up to 100 mg L−1 [4] especially in agricultural

∗ Corresponding author at: Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Fax: +662 218 5418. E-mail address: [email protected] (A.S. Vangnai). http://dx.doi.org/10.1016/j.jhazmat.2015.10.010 0304-3894/© 2015 Elsevier B.V. All rights reserved.

soil from the application of such biosolid as a part of fertilizer [3] and from pesticide natural transformation [1,5]. It has been enlisted as one of the major priority pollutants and subjected for treatment [2] due to its chemical stability and persistence [6–8] as well as toxicological effects to living organisms and human health even at low concentrations [9,10]. The chemical treatment of 4-NA using advanced oxidation process or photocatalytic degradation has been used [2,11–13], but they are costly and usually cause secondary pollution. Alternatively, microbial degradation is known as an environmental-friendly, economical and efficient technology to decontaminate the sites polluted with toxic compounds. However, since 4-NA is classified as a non- or hardly

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biodegradable pollutant [8], very limited information is currently available for the microbial degradation of 4-NA. To date, there are only two 4-NA-degrading bacteria reported, i.e., a refinerysoil enriched Stenotrophomonas maltophilia HPC 135 [14] and an industrial-effluent isolated Rhodococcus sp. FK48 [1]. As a practical and sustainable remediation of pollutants in agricultural area, the use of pollutant-degrading, plant-growth promoting rhizospheric bacteria (PGPB) is advantageous not only to mitigate soil toxicity, but also to simultaneously promote plant activities and growth [15]. Nevertheless, since previous studies had demonstrated adverse effects of soil pollutants on PGPB growth and their role in plant-growth promoting activities [16–18], it is important to employ such bacteria whose ability is actively remained even in the presence of toxic pollutant. In this study, Acinetobacter sp. AVLB2 was isolated as 4-NA-degrading, plant-growth promoting bacterium from rhizosphere soil of a legume crop (long bean; Vigna unguiculata). The 4-NA degradation kinetics study was conducted, and further analysis showed that it could efficiently degrade 4-NA at high concentrations, while maintained in vitro PGPB traits. The degradation pathway of 4-NA in Acinetobacter sp. AVLB2 was then proposed. This study provides significant details of 4-NA biodegradation not only in term of scientific point of view, but also in term of toxicological standpoint as it is the first to elucidate PGPB performance for 4-NA toxicity reduction. In addition, it illustrated the feasibility and potential application of 4-NA-degrading PGPB for rhizoremediation of sites contaminated with 4-NA.

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Sequence divergences were quantified by Kimura’s two-parameter distance model. 2.4. Analysis of 4-NA-dependent bacterial growth and 4-NA biodegradation kinetics Effect of various initial concentrations of 4-NA (0, 5, 10, 15, 20, 25 and 50 mg L−1 ) on cell growth and biodegradation kinetics was investigated. The overnight-grown cells (3%, v/v) were inoculated to 4-NA-containing MSM supplemented with 4 mM succinate, and incubated at 30 ± 2 ◦ C under shaking conditions (120 rpm). Un-inoculated media with the same concentrations of 4-NA were carried out as abiotic controls. At time interval, a sample was collected to measure cell growth as cell optical density at 600 nm using a spectrophotometer, then centrifuged at 10,000 rpm for 10 min to obtain cell-free medium, each of which was used for the analysis of residual concentration of 4-NA by high performance liquid chromatography (HPLC) (as described in Supplementary material) and degradation metabolites (as described below). All experiments were performed in triplicates. 2.5. Analysis of 4-NA degradation metabolites

2. Materials and methods

The cell-free medium sample collected at 120-h during the timecourse biodegradation test with 25 mg L−1 of 4-NA was extracted with an equal volume of ethyl acetate, and subjected for analysis using a gas chromatography-mass spectrometry (GC–MS) as described in Supplementary material.

2.1. Chemicals and media

2.6. Toxicological studies

All chemicals and reagents including 4-NA (98% purity; Merck, MA, USA) were analytical grade. The cultivation media included carbon-free minimal salt medium (MSM) (pH 6.8 ± 0.2) consisting of (g L−1 ): Na2 HPO4 5.8, KH2 PO4 3, NaCl 0.5, NH4 Cl 1, MgSO4 0.25, and Luria-Bertani (LB) medium (pH 7.0). For solid medium, 15.0 g of agar was added per liter.

To evaluate the biodegradation efficiency, the toxicity level of the degradation metabolites was analyzed in comparison to that of the parental compound (4-NA) using microbial toxicity, phytotoxicity and cytogenotoxicity studies. The degradation metabolites collected at 120-h during the time-course biodegradation test with 25 and 50 mg L−1 of 4-NA was extracted by ethyl acetate, dried, redissolved to the initial volume in distilled water, and subjected to toxicity tests. The microbial toxicity test was carried out using Escherichia coli DH5˛ according to Wen et al. [20] method with minor modifications. In brief, the impact of degraded metabolites or 4-NA on cell growth (OD550 ) in LB broth was determined. The growth inhibition was estimated using the inhibition index (I) defined as: I = (Ao −A)/Ao × 100%, where Ao and A are cell turbidity in the absence or presence of 4-NA or its degraded metabolites, respectively, determined after 24-h of incubation at 37 ± 2 ◦ C at 120 rpm in a slanted position. Phytotoxicity test was carried out using seeds of legume plants: mung bean (Vigna radiata) and sunn hemp (Crotalaria juncea). In each test, five surface-sterilized seeds were placed on the Petri dish lined with filter paper where 2-ml of the test solution (4-NA or its degraded metabolites) or deionized water (a control) was added. The seed-containing plates were then placed in an environmental chamber with controlled temperature (28 ◦ C) and humidity (90%), in the dark for 72 h. At the end of the exposure period, toxicity level was assessed in terms of inhibition of seed germination and their sprouting length in comparison to those of the control in which distilled water represented non-toxicity effect to the seeds with 100% seed germination and the maximum sprouting length. Cytogenotoxicity was assessed using Allium cepa chromosome aberration test [21]. Briefly, small healthy A. cepa bulbs of which outer scales and dried roots were removed were kept in clean water for 72 h in the dark for root development. The roots with approximately 15.0 mm in length were then exposed to 4-NA or its degraded metabolites for 3 h whereas those treated with

2.2. Enrichment and screening of 4-NA-degrading bacterial strains Long-bean crop soil with history of pesticide use was used as a source for bacterial enrichment and isolation. One gram of the soil sample was enriched with 25 mg L−1 of 4-NA in MSM at 30 ± 2 ◦ C on a rotary shaker at 120 rpm for one week. Then, 1% (v/v) of the suspension was transferred to fresh 4-NA-containing MSM, and incubated under the same conditions. The process was repeated three times. Bacterial isolates were purified on 4-NA containing MSM agar. The isolates were then screened for 4-NA tolerance by a minimum inhibitory concentration (MIC) method using various 4-NA concentrations (25–50 mg L−1 ) provided on MSM-agar plates. The fastest growing bacterial strain with higher tolerance of 4-NA within 24-h incubation was selected for further degradation study. 2.3. Identification and characterization of 4-NA degrading bacteria The identification and characterization of the selected 4-NA degrading bacterium was conducted on the basis of colony morphology, Gram-staining and 16S rRNA gene sequence analysis. Genomic DNA was extracted using a standard protocol [19]. The analysis of 16S rRNA gene fragment amplified from 27F and 1492R primers was thoroughly conducted by Macrogen (Seoul, Korea). The phylogenetic tree was inferred by MEGA 6.0 software using neighbor-joining method with a bootstrap value of 1000 replicates.

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Fig. 1. 16S rRNA sequence-based phylogenetic position of AVLB2. The evolutionary distance was inferred using the neighbor-joining method in comparison to sequences available in the GenBank database. Numerical values represents bootstrap percentile from 1000 replicates. The scale bar shows the branch length corresponding to 0.005 nucleotide substitutions per position.

distilled water were used as negative controls. The root tips exposed to different treatments were cut, placed on a glass slide, fixed in Carnoy’s solution for 90 min, hydrolyzed in 1N HCl at 60 ◦ C for 10 min, washed, and stained with 1% aceto-orcein for 5 min. After destaining with water, they were observed under a light microscope at 1,000 × magnification for cell division and cytogenetical abnormalities. The mitotic index (MI) and aberration index (AI) were then calculated from the total of 1250 cells [21] as follows:

Mitotic index (MI, %)=

Aberration index (AI, %)=

number of the dividing cells × 100 1, 250

number of the chromosomal aberrations cells × 100 1, 250

2.7. Bioassays of plant growth promoting activities with and without 4-NA stress The following plant growth promoting traits of the selected strain were qualitatively and quantitatively examined with and without addition of 4-NA at 25 or 50 mg L−1 using the methods previously described: phosphate solubilization [16] and the corresponding organic acid production by HPLC analysis [17]; an indole-3-acetic acid (IAA) production [22,23]; qualitative test on siderophore production using Chrome-azurol S (CAS) blue medium

[24] and quantitative analysis [25]; and the production of exopolysaccharide [25]. All experiments were conducted in triplicates. 2.8. Statistical analysis The data were statistically analyzed and significant differences among the treatment means were calculated at P ≤ 0.05 by one-way ANOVA with Dunnett’s multiple comparison test using Graphpad Prism, v5.03 (CA, USA). 3. Results and discussion 3.1. Enrichment, isolation, screening and characterization of 4-NA degrading bacteria After one-week enrichment of the rhizosphere soil of V. unguiculata with 4-NA, bacterial colonies with two clearly distinguished morphologies were obtained on 4-NA-selective plates. Further studies on growth of the bacterial isolates in 4-NA-containing liquid medium differentiated their growth ability and rate. Among all, the bacterial isolate AVLB2 (referred to as AVLB2 hereafter) was specifically chosen based on its relatively highest growth efficiency, tolerance to 4-NA and its PGP activities as described later on. The 4-NA tolerance test results showed that while most isolates could survive in the presence of 4-NA with MIC value below or at 25 mg L−1 , AVLB2 had higher 4-NA tolerance with MIC value up to

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50 mg L−1 . Therefore, AVLB2 was selected for further investigation. AVLB2 is an aerobic, Gram-negative, non-spore forming and short rod-shaped bacterium, oxidase negative, catalase positive. The colonies grown on LB agar are circular, convex with entire margins and non-pigmented. The analysis of a 1460-bp 16S rRNA sequence revealed 99% similarity to that of Acinetobacter sp., and thus the strain was identified as Acinetobacter sp. AVLB2. The nucleotide sequence of AVLB2 has been deposited in the GenBank database under the accession number of KT304816. Phylogenetic position of Acinetobacter sp., AVLB2 in relation to the species of this genus reported in the GenBank database is as illustrated (Fig. 1). Among all, AVLB2 is mostly phylogenetically related to a diesel-degrading Acinetobacter oleovorans DR1 (CP002080). There are many reports available on the degradation of environmental pollutants by Acinetobacter sp. [6,26,27], but this current study is the first to report Acinetobacter sp. as an individual, 4-NA-degrading bacterium with efficient plant-growth promotion ability.

3.2. Degradation of 4-NA by Acinetobacter sp. AVLB2 and growth studies The initial test conducted to determine whether AVLB2 could utilize 4-NA as a sole carbon source showed that cells could utilize 4-NA for growth with 4-NA degradation rate of approximately 0.2 ± 0.05 mg L−1 h−1 OD600 −1 . Nevertheless, the biodegradation rate could be substantially enhanced by addition of succinate at 4 mM as a co-substrate as it evidently increased the average 4-NA degradation rate by 82 ± 3% (up to 1.2 ± 0.05 mg L−1 h−1 OD600 −1 ). Accordingly, the following tests were thoroughly conducted with succinate addition. This result agreed with the previous reports on positive effect of co-substrate on biodegradation of aniline derivatives where citrate, succinate, or yeast extract could accelerate chloroaniline degradation rate in Pseudomonas putida CA16 [28], and Delftia tsuruhatensis H1 [29]. Then, the growth of AVLB2 supplemented with various initial concentrations of 4-NA and its ability to degrade 4-NA were tested (Fig. 2a–f). In all cases, a short lag phase of growth and of 4-NA degradation (up to 3-h lag period) were observed, which indicated rapid bacterial acclimatization of 4-NA. Compared to Rhodococcus sp. FK48’s ability to degrade 4-NA at a similar concentration range (20–25 mg L−1 ), where at least 24-h acclimatization period was required [1], this characteristic of AVLB2 reflects its efficient ability not only to cope with high toxicity and to degrade 4-NA at high concentration, but also suggest its prompt induction and response to 4-NA when detoxification process is required. Further monitoring showed that AVLB2 could completely degrade 4-NA to undetectable level within 24–48 h when low 4-NA concentration was initially provided (5–15 mg L−1 ) (Fig. 2a–c), while it was prolonged up to 144 h with increase 4NA concentration (Fig. 2e and f). In abiotic control, without AVLB2, there was no change in 4-NA concentration indicating that autolysis, photo-oxidation and other abiotic effects did not take part in this degradation during the test period. A complete 4-NA biodegradation even at high concentration within a relatively shorter period by AVLB2 was shown to be relatively superior feature to those previously reported. Up to present, 4-NA degradation performance has been reported by only two pure bacterial cultures and a mixed bacterial culture. A report on S. maltophilia HPC 135 showed that it could degrade 4-NA as high as 138 mg L−1 in the presence of acetate, but the efficiency was only up to 70% after 48 h with no further degradation [14], while Rhodococcus sp. FK48, or a mixed culture acclimated from municipal wastewater biosludge could completely degrade 4-NA, but with the initial concentration as low as 20 mg L−1 [1], and 13 mg L−1 [6], respectively.

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Table 1 Biodegradation kinetic parameters of 4-nitroaniline by Acinetobacter sp. AVLB2. Kinetic parameters

−1

Ks (mg L ) Vmax (mg L−1 h−1 ) Vmax /Ks (h−1 ) R2

Apparent kinetic parameter values obtained from the two kinetics models Michaelis–Menten model

Hanes–Wolf model

2.438 0.541 0.222 0.972

2.944 0.551 0.187 0.991

3.3. Influence of initial 4-NA concentration on bacterial specific growth rate and biodegradation kinetics of 4-NA Bacterial growth kinetics and biodegradation kinetics of the target pollutant are fundamental information to understand bacterial characteristic and degradation performance; therefore growth kinetic profile of AVLB2, and 4-NA degradation kinetics were determined. Growth kinetic profile of AVLB2 was determined in terms of specific growth rate (, h−1 ) with various initial 4-NA concentrations (S) using a non-linear regression of Haldane inhibition model (Eq. (1)) because it commonly involves microbial growth when toxic substrate with potential inhibitory effect is considered. Then, the maximum specific growth rate (max ), a half-saturation growth constant (Ks ) and inhibition constant (Ki ) were determined from the graph plotted (Fig. 3). 

= S+

S

 max S2 Ki

(1)

+ Ks

The experimental values fitted well with the calculated values with an R2 value of 0.9394. The max of AVLB2 was determined to be 0.039 h−1 . The estimated Ks value was at 6.623 mg L−1 , which indicates that AVLB2 has high preference towards this toxic pollutant. The growth kinetic profile revealed that there is a partial inhibition of cell growth in the presence of high concentration of 4-NA as the Ki value was estimated to be 25.57 mg L−1 . Nevertheless, even with partial inhibition, the bacterial specific growth rate remained 75% of the maximum value. This result strengthens the fact that this strain could tolerate high concentration of 4-NA while maintaining satisfactory specific growth rate. Further analysis on 4-NA biodegradation kinetics was conducted at different 4-NA concentrations by fitting experimental data with a non-linear method of Michaelis–Menten paradigm of microbial kinetics (Eq. (2)) [30], and a linear regression method of Hanes–Woolf plot models (Eq. (3)) as follow: ds S = −Vmax S + Ks dt S

v

=

1 Ks [S] + Vmax Vmax

(2) (3)

where Vmax is the maximum biodegradation rate obtained from a slope value of a linear plot as 1/V max , and Ks is a half saturation concentration determined with intact bacterial cells as described previously and obtained from an x-axis intercept of a linear plot as—Ks [31,32]. The degradation dynamics of 4-NA by AVLB2 exhibited good compliance with both Michaelis–Menten model (Fig. 4a) and Hanes–Woolf model (Fig. 4b), and the apparent kinetic parameters: Ks , Vmax and Vmax /Ks , deduced from both plots are presented in Table 1. Instead of considering Ks and Vmax values individually, the ratio of Vmax /Ks known as the specific substrate affinity could be considered as a useful index for the whole-cell reaction efficiency [31] in which nutrient (in this case, 4-NA) uptake efficiency by bacteria is taken into account in addition to substrate assimilation ability [33]. The higher value means that bacteria can

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Fig. 2. Biodegradation of 4-NA by Acinetobacter sp. AVLB2 and bacterial growth in MSM supplemented with various 4-NA concentrations: (a) 5 mg L−1 , (b) 10 mg L−1 , (c) 15 mg L−1 , (d) 20 mg L−1 , (e) 25 mg L−1 and (f) 50 mg L−1 . During incubation period, the following parameters were monitored: residual 4-NA (%) ( ); 4-NA-dependent ); abiotic control with 4-NA ( ), and cell turbidity in the absence of 4-NA (OD600 ) ( ). The data are mean ± standard deviation cell growth (OD600 ) ( from triplicate treatments.

well uptake and assimilate the target pollutant. In our study, the values of Vmax /Ks were estimated to be 0.222 and 0.187 by each model, respectively (Table 1). Since this is the first to report on 4-NA biodegradation kinetic parameters and there is no data for comparison, the reported value of other pollutants was used for comparison purpose. As a result, the values of Vmax /Ks for 4-NA degradation by AVLB2 was found to be in a similar range to those reported with

the high biodegradation efficiency towards chlorpyrifos [32,33] and trichloroethylene [31]. 3.4. Biodegradation pathway for 4-NA by Acinetobacter sp. AVLB2 GC–MS analysis of the metabolites produced during 4-NA degradation by AVLB2 revealed four peaks with retention time

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oxygenation(s), which is similar to the degradation pathway proposed for 4-aminophenol in Burkholderia sp. AK-5 [39]. Accordingly, the possible 4-NA biodegradation pathway adopted by AVLB2 was proposed as shown in Fig. 5.

3.5. Toxicity assessment of the entire 4-NA degradative products

Fig. 3. 4-Nitroaniline-dependent specific growth kinetics of Acinetobacter sp. AVLB2 fitted with Haldane model.

of 4.210, 6.416, 14.544, and 16.024 min (Supplementary Fig. 1a). The fragmentation pattern and molecular ion analysis of the 4.210-min-, and the 16.024-min metabolites indicated their m/z values to be 91.0 [(M-2H)− , where M = 93], and 125.9 [(M-0.21H)− , where M = 126.11], which corresponded to aniline (Supplementary Fig. 1b), and 1,2,4-benzenetriol (1,2,4-BT) (Supplementary Fig. 1c), respectively, while the identification of the other two was unsuccessful. 1,2,4-BT is a transient metabolite often found in biodegradation pathway of a number of nitroaromatic compounds [34–36] including 3-nitrophenol, 4-nitrophenol [37], 4-NA [1] as well as aniline derivatives including 4-chloroaniline [38] and 4-aminophenol [39] before it is further transformed to maleylacetate and eventually enters tricarboxylic acid (TCA) cycle. Interestingly, in this study, aniline was also identified as one of the degradation intermediates suggesting nitrite removal as the first reaction occurred. This reaction appears to be similar to the degradation reaction of 2-chloro-4-nitrophenol by Burkholderia sp. RKJ800 where nitrite removal was initially catalyzed by monooxygenase [40]. The detection of 1,2,4-BT suggested that the following reactions via aniline may include deamination and

The most important goal for pollutant biodegradation process towards safe environment is to eliminate or minimize the overall toxicity caused by the parental toxic pollutant or toxic metabolites formed during the degradation; therefore toxicity assessment is strictly required as a final test step for risk assurance. This present work employed the following three toxicity assessment techniques to assure 4-NA toxicity reduction achieved by AVLB2 biodegradation ability. Microbial toxicity test using E. coli DH5˛ clearly showed that 4NA are highly hazardous to E. coli cells as the inhibition index was at 81.9 ± 3.8%, and 86.1 ± 3.4 % in the presence of 25 and 50 mg L−1 of 4-NA, respectively (Fig. 6a). Nevertheless, the degraded metabolites became less toxic to cells as the inhibition index was minimized to 18.9 ± 3.4% and 34.4 ± 3.0%, respectively. Phytotoxicity study was essentially conducted because of the safety concern of 4-NA reportedly contaminated in agricultural area and its potential impact on plant growth. Accordingly, legume seeds of V. radiata (Fig. 6b) and C. juncea (Fig. 6c) were used for the toxicity assessment of 4-NA at 25 and 50 mg L−1 as well as their corresponding degraded metabolites collected at 120-h of degradation period. The results were then compared to the normal growth of seeds in water at 72-h of incubation as positive controls with 100% germination and the maximum sprouting length of 5.36 ± 0.3 cm, 5.86 ± 0.6 cm for V. radiata and C. juncea seeds, respectively. 4-NA at both concentrations caused noticeable adverse effect to both seed types as the germination percentage was up to 60% reduction, and it slowed down or inhibited the root elongation by >50% as shown by more than half shorter sprouting length compared to that of the controls (Fig. 6b and c). Biodegradation treatment of 4-NA by AVLB2 evidently reduced toxicity effect to the test plants as the

Fig. 4. 4-Nitroaniline biodegradation kinetics analysis of Acinetobacter sp. AVLB2 using: (a) Michaelis-Menten paradigm for microbial kinetics, and (b) Hanes-Wolf model.

Fig. 5. A proposed aerobic biodegradation pathway for 4-Nitroaniline in Acinetobacter sp. AVLB2. Underlined compounds are the experimentally detected metabolites. Numbers in a bracket represent the proposed reactions including (1) nitrite removal, (2) deamination and oxygenation, which may occur simultaneously or sequentially (dashed arrows), and (3) aromatic ring cleavage, respectively.

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Fig. 6. Toxicity assessment of 4-nitroaniline at 25 and 50 mg L−1 and its degraded metabolites: (a) microbial toxicity test using E. coli DH5˛ expressed as inhibition index (%); (b) phytotoxicity test with V. radiata expressed as sprouting length; (c) phytotoxicity test with C. juncea expressed as sprouting length. The data are mean ± standard deviation from triplicate treatments.

germination percentage of all test seeds treated with the degradative metabolites were up to 100%. The root elongation ability was nearly fully recovered when the seeds were treated with the degradative metabolites of 4-NA at 25 mg L−1 since 4-NA at this initial concentration was completely degraded at a 120-h (Fig. 2e). In the test starting with the metabolites of 4-NA at 50 mg L−1 , the negative impact on root elongation was mitigated with less extents (Fig. 6b and c) because 4-NA was partially remained at a 120-h (Fig. 2f). This result strengthened that 4-NA even at low concentration has substantial phytotoxicity, and the biological treatment with AVLB2 could achieve the significant reduction in phytotoxicity associated with 4-NA. Cytogenotoxicity test using A. cepa root tip cells is routinely used as one of the key eco-toxicity assays and required as an in vivo evaluation of cytotoxic and genotoxic potential of chemicals and/or environmental samples [41,42]. In this study, this in vivo onion test was employed and determined as the mitotic index (MI) and chromosome aberration index (AI) values [41,42] to evaluate the harmful effect of 4-NA and the corresponding degraded metabolites. The MI value represents a cell proliferation biomarker, which measures the proportion of cells in the mitotic phase of cell cycle. Thus, the decrease of MI value of A. cepa meristematic cells

may infer as cell death caused by cytotoxic agent. On the other hand, chromosomal aberration may be induced by genotoxic agent, which disrupts the normal chromosome number or chromosome structure by DNA break, inhibition of DNA synthesis and replication of altered DNA. Therefore, the increase of AI number indicates increase of genotoxic and chromosomal damages. As agreed with the above results, this in vivo test showed that 4-NA has cytotoxicity effect at the two concentrations tested, 25 and 50 mg L−1 in which the MI value of the root tip cells was reduced from that of the normal condition control by 73% and 82%, respectively (Table 2). Biological treatment of 4-NA by AVLB2 resulted in nearly elimination of cytotoxicity as the MI values of the metabolites were statistically in the similar range of that of the non-toxic control. In addition, the chromosomal structures were observed in the meristematic root cells of A. cepa treated with 4-NA, the degraded metabolites and distilled water (as a negative control) (Fig. 7). Meristematic root tip cells treated with distilled water showed a normal physiology in the mitotic stages (Fig. 7a–d) with negligible aberrations of 0.05 ± 0.04%. The chromosome abnormalities were clearly observed in the root tip cells exposed to 4-NA at 25 and 50 mg L−1 , and thus increased the AI value by 26 and 31 folds. On the other hand, when tested with the metabolites after 4-NA

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Fig. 7. Cytogenotoxicity assessment of 4-nitroaniline and its degraded metabolites in meristematic cells of A. cepa. A. cepa roots were treated with the following test solutions: (a–d) distilled water; (e–l) 4-NA; (m–p) the 4-NA degraded metabolites. Cell cycle and chromosome aberration are shown as follow: (a) prophase, (b) metaphase, (c) anaphase, (d) telophase, (e) chromosome missegregation, (f) irregular prophase, (g) abnormal metaphase, (h) sticky metaphase, (i) chromosome breaks at anaphase, (j) irregular anaphase with chromosome bridges, (k) anaphase bridge, (l) apoptosis, (m) prophase, (n) metaphase, (o) anaphase, (p) telophase.

biodegradation, chromosomal damages were markedly reduced (Fig. 7m–p) and negligible aberrations were occurred ranging from 0.1 ± 0.04% to 0.34 ± 0.12%. These results confirmed that the biological treatment of 4-NA by AVLB2 is an effective treatment technique resulting in less/non-toxic nature of the 4-NA degraded metabolites.

3.6. Plant growth promoting traits of Acinetobacter sp. AVLB2 To cleanup 4-NA as an agricultural soil contaminant, and simultaneously reclaim soil fertility, it would be advantageous if 4-NA-degrading microbe exhibits plant-growth promoting (PGP) activities even in the presence of toxic chemical stress. Since AVLB2 had been proven as an effective 4-NA-biodegrading microbe, subsequent characterizations were conducted to determine whether it possesses PGP traits including P-solubilization, production of organic acids, indole acetic acid, siderophores and exo-polysaccharides, and whether these traits remain during 4-NA stress.

In the absence of 4-NA, AVLB2 showed positive P-solubilization activity forming a clear halo zone with a diameter of 11 mm around the spot of inoculation on Pikovskaya medium agar, and yielded the maximum amount of tri-calcium phosphate solubilization in liquid medium of 104.23 ± 2.57 ␮g mL −1 . Under stress of 4-NA dosage at 25 and 50 mg L−1 , the P-solubilization activity was remained at 58% and 46%, respectively (Table 3). In addition, organic acid secretion was also determined because it is one of the major bacterial mechanisms for mineral phosphate solubilization and for plant-microbe symbiotic interaction [17,43]. In the absence of 4NA, AVLB2 secreted a significant amount of organic acids causing pH drop during the solubilization of inorganic phosphate (Table 3). HPLC analysis revealed that gluconic acid, formic acid, citric acid and acetic acid were found to be predominant acids. In the presence of 4-NA, the organic acid synthesis and secretion by AVLB2 could be fairly maintained as the similar profile of organic acid synthesized from the strain, but with less extent, was clearly observed (data not shown). The IAA produced by rhizobacteria is known to promote root growth [44]. In the absence of 4-NA, AVLB2 produced a

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Table 2 Mitotic indexes and chromosomal aberration index examined in A. cepa root tip cells exposed to 4-nitroaniline, the degraded metabolites and distilled water (as a negative control). Treatment

Concentration of the compound (mg L−1 )

Number of dividing cells

Control (H2 O)

–

208.66 ± 1.52

25 50

55.33 ± 1.52 37 ± 2a

Extracted metabolites

25 50

186.66 ± 1.07a 152.33 ± 1.52a

CB

MC

0

0

ML 0

8.33 ± 0.57 6.33 ± 0.57

7.66 ± 0.57 0

1.33 ± 0.5 0

0 0

0 7.33 ± 1.15 0 2.66 ± 0.57

MA

AB

AL

0

0

0.66 ± 0.57

0 4±1 0 0

0 2 ± 1.73 0 0

Mitotic index (MI) %

Aberration index (AI) %

16.69 ± 0.12

0.05 ± 0.04

4.42 ± .12 2.96 ± 0.16a

1.28 ± 0.08a 1.57 ± 0.16a

a

0 0 0 1.66 ± 1.15

14.93 ± 0.08a 12.18 ± 0.12a

0.1 ± 0.04* 0.34 ± 0.12c

Each value represents the mean ± SD of three replicates per treatment. In the same column according to Dunnett’s multiple comparison test significant differences at P ≤ 0.05 levels over control are indicated by different letters. Values followed by the asterisk (*) denote non-significant values over the control. Abbreviations: CB: chromosome breaks; MC: metaphase cluster; ML: metaphase lagging chromosome; MA: metaphase aberrations; AB: anaphase bridge; AL: anaphase lagging chromosome.

Table 3 Plant-growth promoting activities of Acinetobacter sp. AVLB2 in the presence and absence of 4-nitroaniline. Treatments

Phosphate solubilized in the test liquid medium (␮g mL Control 4-NA (25 mg L−1 ) 4-NA (50 mg L−1 ) F value

IAA (␮g mL−1 )

Phosphate solubilization

104.23 ± 2.57 60.5 ± 1.11a 48.47 ± 3.01a 457.3

−1

)

−1

Final pH 4.42 ± 0.49 5.53 ± 0.29 5.68 ± 0.26 –

Siderophores SA (␮g mL

86.08 ± 3.76 63.25 ± 0.86a 49.66 ± 0.94a 193

)

48.44 ± 1.71 36.33 ± 1.2a 22.11 ± 2.87a 123.8

EPS −1

DHBA (␮g mL 9.83 ± 2.36 6 ± 1.32b 2.83 ± 0.57a 14.42

)

(␮g mL−1 ) 16.33 ± 1.52 18.66 ± 0.57* 22.33 ± 1.52b 16.47

Each value represents the mean ± SD of three replicates per treatment. In the same column according to Dunnett’s multiple comparison test significant differences at P ≤ 0.05 levels over control are indicated by different letters. Values followed by the asterisk (*) denote non-significant values over control. Abbreviations: IAA: indole acetic acid; SA: salicylic acid; DHBA: 2,3-Dihydroxy benzoic acid; EPS: exo-polysaccharides. The control was Acinetobacter sp. AVLB2 in the absence of 4-nitroaniline.

S. Silambarasan, A.S. Vangnai / Journal of Hazardous Materials 302 (2016) 426–436

4-NA

a

Chromosomal aberrations

S. Silambarasan, A.S. Vangnai / Journal of Hazardous Materials 302 (2016) 426–436

substantial amount of IAA at 86.08 ± 3.76 ␮g mL−1 (Table 3). Although IAA was progressively decreased with increasing dosage of 4-NA, its production activity was partially maintained (Table 3). Siderophores production was positively manifested in AVLB2 using SA and DHBA quantitative assays under a normal condition suggesting its ability to facilitate iron solubilization. Although this ability was reduced up to 25–70% in 25 and 50 mg L−1 of 4-NA amended medium, reasonable activity was remained under the toxic chemical stress (Table 3). EPS production was considered as an important PGP trait for cell protection from desiccation, phage attack, and phagocytosis [45]. AVLB2 was able to synthesize EPS naturally, and the synthesis was well maintained even in the presence of 4-NA (Table 3). These results indicated the innate capability of expressing multiple PGP traits of AVLB2 and demonstrated that it may serve well as an effective bioinoculant for 4-NA detoxification process in 4NA-contaminated agricultural area. 4. Conclusion Acinetobacter sp. AVLB2 was isolated and identified as an efficient 4-NA degrading, plant-growth promoting bacterium. It could not only utilize 4-NA as a sole carbon source, but its degradation capability towards 4-NA also occurred with high substrate affinity and high degradation rate, while PGP traits were well maintained even under toxic chemical stress. Biodegradation pathway of 4NA proposed in this study indicated a novel route with sequential nitrite removal and deamination reactions prior to oxygenation and ring cleavage. Toxicological studies proved that this biodegradation process achieved the ultimate goal for bioremediation in which less/non-toxic metabolites were generated. Therefore, Acinetobacter sp. AVLB2 could potentially be developed as a bio-inoculant to remediate 4-NA as well as for sustainable agronomic production programs in 4-NA contaminated area. Acknowledgments This research was funded by the Ratchadapisek Sompoch Endowment Fund (2014), Chulalongkorn University (CU-57-063CC). Ratchadaphisek Somphot Endowment Fund for Post-Doctoral Research through Graduate School, Chulalongkorn University, and The Thailand research fund (IRG 5780008) are also appreciated. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2015.10. 010. References [1] F. Khan, J. Pandey, S. Vikram, D. Pal, S.S. Cameotra, Aerobic degradation of 4-nitroaniline (4-NA) via novel degradation intermediates by Rhodococcus sp. strain FK48, J. Hazard. Mater. 254–255 (2013) 72–78. [2] J.H. Sun, S.P. Sun, M.H. Fan, H.Q. Guo, L.P. Qiao, R.X. Sun, A kinetic study on the degradation of p-nitroaniline by Fenton oxidation process, J. Hazard. Mater. 148 (2007) 172–177. [3] S.R. Smith, Organic contaminants in sewage sludge (biosolids) and their significance for agricultural recycling, Philos. Trans. A Math. Phys. Eng. Sci. 367 (2009) 4005–4041. [4] S.R. Smith, D. Riddell-Black, Sources and impacts of past, current and future contamination of soil, in: Research Project Final Report for Defra Project code SP 0547, Imperial College London, 2007, pp. 1–247. [5] S.B. Haderlein, K.W. Weissmahr, R.P. Schwarszenbach, Specific adsorption of nitroaromatic explosives and pesticides to clay minerals, Environ. Sci. Technol. 30 (1996) 612–622. [6] A. Khalid, M. Arshad, D.E. Crowley, Biodegradation potential of pure and mixed bacterial cultures for removal of 4-nitroaniline from textile dye wastewater, Water Res. 43 (2009) 1110–1116.

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