Role of plant growth-promoting Ochrobactrum sp. MC22 on triclocarban degradation and toxicity mitigation to legume plants

Accepted Manuscript Title: Role of plant growth-promoting Ochrobactrum sp. MC22 on triclocarban degradation and toxicity mitigation to legume plants Authors: Merry Krisdawati Sipahutar, Alisa S. Vangnai PII: DOI: Reference:

S0304-3894(17)30032-8 http://dx.doi.org/doi:10.1016/j.jhazmat.2017.01.020 HAZMAT 18317

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

11-10-2016 10-1-2017 11-1-2017

Please cite this article as: Merry Krisdawati Sipahutar, Alisa S.Vangnai, Role of plant growth-promoting Ochrobactrum sp.MC22 on triclocarban degradation and toxicity mitigation to legume plants, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2017.01.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Role of plant growth-promoting Ochrobactrum sp. MC22 on triclocarban degradation and toxicity mitigation to legume plants

Running title: Biodegradation of triclocarban by Ochrobactrum sp. MC22

Merry Krisdawati Sipahutara,b, and Alisa S. Vangnaib,c*

a

Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok

10330, Thailand b

Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330,

Thailand c

Center of Excellence on Hazardous Substance Management (HSM), Chulalongkorn

University, Bangkok 10330, Thailand

*Corresponding author

Correspondence: Alisa S. Vangnai Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Tel: +662 218 5430; +662 218 5418; e-mail: [email protected]

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Highlights 

Ochrobactrum sp. MC22 is a plant-growth promoting, triclocarban-degrading bacterium.



MC22 has a versatile TCC degradation under both aerobic and anaerobic conditions.



Biodegradation kinetic study of TCC reveals the pathway and the rate-limiting step.



MC22 can completely degrade and detoxify TCC and all chloroaniline metabolites.



Role of MC22 on TCC toxicity mitigation to legume plants was clearly demonstrated.

Abstract Triclocarban (TCC) is an emerging and persistent pollutant once released into environment. In this study, TCC-degrading Ochrobactrum sp. MC22, was isolated and characterized. This is the first report on plant-growth promoting bacterium with versatile capability of TCC degradation under aerobic and anaerobic conditions. The aerobic degradation of TCC occurred completely of which the kinetic analysis revealed a non-self-inhibitive substrate effect, and broad-concentration-range degradation efficiency (ranging from 0.16-30 mg L-1). Anaerobic TCC degradation was feasible, but was significantly enhanced up to 40-50% when ferric, or acetate was provided as electron donor, or acceptor, respectively. TCC biodegradation under both conditions was proposed to initially occur through hydrolysis leading to transient accumulation of chloroanilines, which could be completely metabolized and detoxified. With concern on TCC adverse effect to plants, role of MC22 on toxicity mitigation was investigated using two legume plants: Vigna radiata and Glycine max (L.) Merr. Upon TCC exposure, damage of both plant structures, especially root system was observed, but was substantially mitigated by MC22 bioaugmentation. This study not only provides thorough TCC degradation characteristic and kinetics of MC22, but also suggests a

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potential role of this bacterial strain for a rhizoremediation in crop area with TCC contamination.

Abbreviation: Triclocarban (TCC); Plant-growth promoting bacteria (PGPB); 3,4-dichloroaniline (34DCA); 4-chloroaniline (4CA)

Keywords: Triclocarban; Ochrobactrum sp. MC22; Aerobic and anaerobic degradation; Plant-growth promoting bacteria; Toxicity mitigation

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1. Introduction Biosolid generated from municipal and industrial treated sludge is rich in nutrients and organic matters, and thus has been commonly applied to agricultural land to improve soil properties and fertility [1]. However, a major concern regarding the use of biosolid-amended soil is the presence of incompletely-removed hazardous organic contaminants [2]. Among several toxic elements, triclocarban (TCC), which is a high-production-volume synthetic antimicrobial agent widely used in pharmaceutical and personal care products, has been extensively detected in the concentration range of g kg-1 level [3] to mg kg-1 level [4, 5] in biosolid-amended soil and sediments [6], and up to 51 mg kg-1 in biosolid [7, 8]. There are reports on adverse effects to human and other living organisms from endocrine-disrupting and biocidal activities of TCC [9], as well as toxicity of chlorinated-hydrocarbon products formed and accumulated from natural degradation of TCC [10], such as chloroanilines [11] and chlorocarbanilides [12]. In addition, it was recently reported that TCC contaminated in poorly-treated biosolid could be toxic to some plant species causing damage to seed emergence and growth of crop plants [13]. Concerned by the risk posed by TCC-containing biosolid and soil, several TCC removal techniques have been used. So far it has been shown that municipal sludge treatment through aerobic and anaerobic digestions could only remove TCC by 15-68% within 30 days [14]. Moreover, although microbial degradation is considered the main mechanism of TCC transformation [10], until now biodegradation behavior of microbial community in wastewater, soil, or sludge was reported to only partially degrade TCC [15]. For instance, it was reported that agricultural soil microbes could only aerobically degrade 58-67% of TCC at high initial concentrations (1.07-2 mg kg-1), and 4760% of TCC at low initial concentrations (0.05-0.2 mg kg-1) within 70-100 days [16, 17], while TCC degradation under anaerobic conditions was hardly detected [17]. Up to now, the

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only reported pure bacterial strain able to degrade TCC is Sphingomonas sp. YL-LM2C with an ability to degrade TCC up to 35% of the initial concentration of 4 mg kg-1 within 5 days and partially degrade the chloroaniline intermediates [11]. As a consequence, it is apparent that the bacteria with efficient TCC degradation ability is required for effective applications of bioremediation and rhizoremediation where pollutant degradation may be presented within the plant root zone in agricultural land [18]. Accordingly, this study aimed for the isolation and in-depth characterization of plantgrowth promoting bacterium with versatile biodegradation capability of TCC. Ochrobactrum sp. MC22 was successfully isolated, and its TCC biodegradation capacity was tested under aerobic and anaerobic conditions. The current work also focused on positive role of MC22 on cytogenotoxicity reduction assessed by Allium cepa root-tip cell test as well as on the mitigation of TCC harmfulness to plant physiological health tested with the selected legume plants, i.e. mung bean (Vigna radiata) and soybean (Glycine max (L.) Merr.). To our knowledge, this work is the first report on capability of a plant-growth promoting bacterium (PGPB) for TCC degradation under aerobic and anaerobic conditions, for TCC toxicity mitigation towards legume plants, and thus suggests a potential role of this bacterium for a rhizoremediation in crop area with TCC contamination.

2. Materials and methods 2.1 Chemicals, culture medium and bacterial cultivation conditions. All chemicals including TCC (99% purity; Sigma-Aldrich, MO, USA) were analytical grade. TCC stock solution was dissolved in acetone, and then diluted to the indicated concentration. The cultivation medium was a carbon-free mineral-salt medium (MSM) containing per liter of deionized water (g L-1) NaH2PO42H2O 0.66, Na2HPO4 5.8, KH2PO4 3, NaCl 0.5, MgSO4 0.25, and NH4Cl 2 (pH 7.0  0.1). For solid medium, 15.0 g L-1 of agar was

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added. Unless indicated otherwise, bacterial cultivation was conducted in a 250-mL Erlenmeyer flask using an inoculum of 4% (v/v) in a 100-mL MSM medium supplemented with TCC at 30C under a shaking condition on a rotary shaker at 150 rpm.

2.2 Isolation, identification of TCC-degrading bacterium, and characterization of bacterial plant growth promoting traits Soil samples were collected from agricultural areas and fruit fields for bacterial isolation. Five grams of soil sample were suspended in 100-mL MSM medium and enriched with 9.40 mg L-1 TCC. Then, the suspension was spread onto TCC-containing MSM agar. The bacterial colonies appeared on the plate were then purified. Then, growth of each isolates in TCCcontaining liquid medium at 9.40 mg L-1 was tested. The isolate MC22 grew with the highest specific growth rate [19] and thus was selected for further investigation. The identification of the selected strain was conducted on the basis of colony morphology, Gram-staining, and 16S rRNA sequence analysis. The following plant growth promoting traits of the selected strain were examined under various TCC concentrations (0, 9.40, and 15.67 mg L-1) using the methods previously described: phosphate solubilization, production of indole-3-acetic acid (IAA) and extracellular polymeric substance (EPS) [20], organic acid production [21], siderophore production [20, 22], and ammonia production [23].

2.3 Triclocarban biodegradation under aerobic and anaerobic conditions and analysis of TCC biodegradation kinetic and biodegradation pathways A time course TCC biodegradation was examined in MSM medium supplemented with 9.40 mg L-1 TCC under aerobic condition. A sample was collected at time interval to measure cell growth (OD600), then centrifuged to obtain cell-free medium, each of which was used for the analysis of TCC residual concentration by high performance liquid chromatography

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(HPLC) and degradation metabolites (as described below). Abiotic test system was carried out as a control. All experiments were performed at least in triplicates. Effect of co-substrate either as additional carbon or nitrogen at 1 g L -1 on triclocarban degradation was also investigated. TCC biodegradation kinetics and TCC-dependent growth kinetics were examined using various initial TCC concentrations ranging from 0.16-30 mg L-1. Microbial growth kinetics and the parameters were analyzed by fitting with a non-linear regression Haldane substrate inhibition model (Equation 1):

 = (𝑚𝑎𝑥 𝑆)/(𝑆 + (𝑆2/𝐾𝑖) + 𝐾𝑠)

(Eq. 1)

Where S is substrate concentration; max is a maximum specific growth rate; KS is a substrate affinity constant; and Ki is an inhibition constant [24]. Biodegradation data were normalized with cell protein concentration representing cell biomass [25] and fitted with a non-linear method of Monod-type kinetic paradigm (Equation 2) where the parameters were determined. 𝑞 = 𝑞𝑚𝑎𝑥𝑆/(𝐾𝑠 + 𝑆)

(Eq. 2)

where q is a specific substrate utilization or removal rate; qmax is the maximum volumetric degradation rate; and Ks is a substrate half-saturation constant [26]. Regression analysis was achieved with the data analysis tool pack of Microsoft Excel® and the model equations and kinetic parameters were solved using GraphPad Prism 5 software. (GraphPad Prism 5.00, CA, USA). All experiments were performed at least in triplicates. TCC biodegradation under anaerobic conditions was investigated in MSM medium containing 9.40 mg L-1 TCC. The influence of electron acceptor was determined under nitrate-, sulfate-, or iron-reducing conditions as previously described [27]. The influence of electron donor on TCC degradation was then performed by supplying succinic acid, or glucose, or acetate at 1g L-1. The test was conducted in comparison to abiotic control and the control without supplement. The test was carried out in a gas-tight serum bottle flushed with

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nitrogen gas, and resazurin (1 mg L-1) was used as a redox indicator. During the incubation at 30C under a shaking condition at 150 rpm, a sample was withdrawn at 2-day interval to measure residual concentration of TCC and degradation metabolites.

2.4 Analytical analysis and toxicity assessment of TCC and TCC degradative intermediates The residual concentration of TCC and transiently accumulated degradative intermediates was analyzed by a reverse phase HPLC and compared with standard compounds with known concentration as previously described [28]. The retention time of TCC under this analytical condition was at 6.421  0.050 min. The intermediates formed were collected and analyzed using LC-MS [29]. To demonstrate TCC detoxification, TCC degradative intermediates were collected during aerobic-, and anaerobic biodegradation test at 3-day and 14-day, respectively, and then subjected to cytotoxicity assessment using Allium cepa chromosome aberration test where mitotic index (MI) and aberration index (AI) were calculated from the total of 1,050 cells [28].

2.5 Analysis of growth and physiological characteristics of legume plants exposed to TCC with and without bioaugmentation of TCC-degrading bacterium Seeds of Vigna radiata and Glycine max (L.) Merr. were sterilized [30], pre-germinated for 3 days, and then aseptically transferred into a jar containing a half-strength Hoagland’s solution agar. Three experimental sets of at least six-seedlings were grown under the following conditions: (i) normal growth condition; (ii) TCC exposure (9.40 mg L -1); and (iii) TCC exposure with bacterial suspension (2.9  0.3 x 108 CFU mL-1). The cultivation was done in a growth chamber at constant humidity (80%) and temperature (25 ºC) with a cycle of 14-h of light and 10-h of darkness [31]. The plant biomass, plant- and root length were

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determined after 7 days of cultivation. Anatomical analysis of plant roots was conducted by cross-sectioning the primary root at a distance of 1.2-1.5 cm from the apex to prepare 2-mmthick semi-thin sections, which were then stained with Safranin O for tissue differentiation [32]. The sections were mounted on slides, observed under a light microscope (Olympus BX51) coupled with a digital imaging system (Olympus DP70), and analyzed for total root area, vascular bundle area, and area of xylem element using RootScan v2.0 image analysis software [33]. The observation of TCC-degrading bacterium colonization on the plant root surface was also carried out using a scanning electron microscope (JEOL Model JSM5410LV, Tokyo, Japan) at 15 kV [34].

2.6 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 Dennett’s multiple comparison test using Graphpad Prism, v5.03 (CA, USA).

3. Results and discussion 3.1 Isolation and characterization of TCC-degrading bacterium strain MC22 The bacterial isolate MC22 enriched from guava field soil was isolated on a selective medium agar where TCC was provided as a sole carbon source. It is a Gram-negative, nonspore forming, motile and short rod-shaped bacterium. The 16S rRNA analysis indicated that it is a member of the genus Ochrobactrum (KT808621). Taxonomical analysis revealed that MC22 has a phylogenetic relationship to the preceding pollutant-degrading, soil-inhabiting Ochrobactrum (Fig. S1), e.g. a hexadecane-degrading Ochrobactrum sp. P1(2013) (KF987808),

an

industrial

site-inhabiting

Ochrobactrum

pituitosum

CCUG50899

(NR_115043), and a soil-inhabiting Ochrobactrum pseudogrignonense (JX266314). As a

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result, the isolate MC22 was identified as triclocarban-degrading Ochrobactrum sp. MC22 (referred to as MC22 hereafter).

3.1.1 TCC biodegradation kinetics, the proposed degradation pathway, and toxicity assessment of the degradative metabolites under aerobic conditions It has been stated that an effective treatment approach for TCC mainly relies on microbial activity [15]; however, several reported microorganisms either as mixed or pure cultures so far exhibited only limited capability in degrading TCC either under aerobic or anaerobic conditions [15-17]. Since TCC has been detected in various environmental compartments with different oxygen levels [3], it is worthwhile to explore TCC degradation capability of MC22 under both conditions. Under aerobic condition, TCC-dependent growth profile of MC22 was initially determined simultaneously with time-course TCC degradation in MSM medium with TCC at 9.40 mg L-1. MC22 could utilize TCC as a sole carbon and energy source to grow exponentially without lag phase reflecting a rapid adaptation of MC22 to TCC (Fig. 1A). Under the test condition, MC22 could degrade TCC up to 78  4.9% within 6 days of incubation with a specific degradation rate of 0.0015  0.0001 mg L-1 [mg protein] -1. The supplementation of additional carbon or nitrogen source did not significantly improve TCC biodegradation, while glucose markedly repressed cell degradation activity (Fig. 1B). Up to present, the only information of TCC degradation by a pure bacterial culture available for comparison is from a report of a triclosan-degrading Sphingomonas sp. strain YL-JM2C [11]. Strain YL-JM2C was able to degrade up to 35% of 4-mg L-1 TCC in a yeast extractcontaining medium even after the prolonged incubation time up to 5 days [11]. Accordingly, it is clearly shown that Ochrobactrum sp. MC22 has higher capability for TCC degradation compared to the previously reported microorganism.

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Furthermore, the kinetic study was conducted to determine TCC-dependent growth kinetic and a growth-linked TCC biodegradation kinetic because not only it provides insight dynamic performance of pollutant-degrading cells, but the kinetic parameter values are also important for pollutant biodegradation process [26]. Accordingly, TCC-dependent growth of MC22 was examined at various TCC concentrations. The result revealed that MC22 cells could survive under all TCC concentrations tested. However, the increase of TCC concentration adversely affected specific cell growth rate indicating substrate inhibition [24]. The growth kinetic curve constructed in the relationship between specific growth rate and TCC concentration fitted well with a non-linear regression Haldane inhibition model (with R2 of 0.9895) (Fig. 1C) where the following apparent growth kinetic parameters were derived, i.e µmax = 0.019 ± 0.001 h-1, Ks, = 1.87 ± 0.28 mg L-1, and Ki = 16.67 ± 2.28 mg L-1. These Ks and Ki values describe key cellular information that will be beneficial for subsequent application regarding the affinity concentration of TCC, and the maximum TCC concentration at which MC22 can tolerate the shock load, respectively. Further analysis in term of biodegradation capability showed that MC22 exhibited a remarkable capability to completely degrade TCC at the initial concentrations of 1.57, 3.13, and 6.27 mg L-1 after 1.5, 2, and 3 days of inoculation, respectively (data not shown), while the degradation was prolonged at higher TCC concentrations ( 9.40 mg L-1). The biodegradation kinetic curve fitted well with Monod-type kinetic model for a single-substrate biodegradation process (with R2 of 0.977) (Fig. 1D) in which the following apparent parameters were depicted: the maximum specific TCC degradation rate (qmax) = 0.0036 ± 0.0002 mg h-1 [mg cell protein]-1; and the TCC concentration at half qmax (Ks) = 10.2 ± 1.2 mg L-1. Further study was then conducted including identification of the corresponding degradative intermediate(s) to gain the biodegradation pathway information. During aerobic

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degradation of TCC, the two transiently accumulated intermediates were detected having a HPLC retention time at 3.928 and 3.444 min (Fig. 1A), and were then identified by LC-MS to be 3,4-dichloroaniline (34DCA), and 4-chloroaniline (4CA), respectively (Fig. S2A), in line with previous observations involving characterizations of a wastewater bacterial consortium [15], and Sphingomonas sp. strain YL-JM2C [11]. However, in this study, further examination was comprehensively performed to prove that MC22 was able to degrade 34DCA and 4CA, and that the detoxification of the entire biodegradation system was occurred. MC22 was capable of degrading 34DCA and 4CA (Fig. S2E), but with a 2.6-3 times slower rate than that of TCC, having a qmax at 0.0014 ± 0.00008, and 0.0012 ± 0.00002 mg h-1 [mg cell protein] -1, respectively. This result may suggest that the oxidation of TCCdegradative intermediates may be rate-limiting step(s) in this degradation pathway. As a final assessment for toxicity threat assurance, the Allium cepa root-tip chromosomal aberration assay was conducted since it is validated as an efficient standard test for toxicity monitoring of environmental substances [35]. In comparison to the untreated cell control, root-tip cells exposed to TCC at 9.40 and 15.67 mg L-1 showed sign of chromosome damages (Fig. 2; B1-B5, C1-C5) with decreasing mitotic index (MI), and noticeably increasing chromosome aberration index (AI) in a dose-dependent manner (Table 1). When the similar test was performed with the corresponding intermediate(s) collected at day 3 of the aerobic degradation where approximately 70% of TCC was transformed, it was shown that less chromosome abnormalities were observed (Fig. 2; D1-D5, E1-E5) having increased values of MI and decreased values of AI. As MC22 exhibited TCC degradation and detoxification capability, these results demonstrated that the potential application of MC22 for biological treatment of TCC.

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3.1.2 Exploring MC22 capability for TCC biodegradation, and toxicity assessment of the degradative metabolites under anaerobic conditions Report on TCC biodegradation under anaerobic condition is very limited. The only two reports so far by either wastewater- or soil-microbial consortium under anaerobic condition indicated that TCC is highly resistant to biodegradation (i.e. about 20% TCC degraded from the initial 1 mg L-1 TCC over 2.5 month period) [15, 17]. Since it is precedent that Ochrobactrum sp. is known as a facultative anaerobe capable of surviving in the absence of oxygen [36], it was intriguing to examine if MC22 is capable of TCC degradation under anaerobic condition. In this study, the test was carried out in a defined condition with respect to electron donor and acceptor to have a better understanding of supporting factor for TCC biodegradation activity of MC22. When tested with various electron acceptors over 2-week incubation period, TCC dissimilation by MC22 was occurred up to 40% under iron (III)reducing condition (Fig. 3A). In general, anaerobic biodegradation activity can be enhanced by providing more cellular energy [37]; therefore, the effect of various exogenous electron donors on TCC dissimilation stimulation was evaluated. Among all organic compounds each supplied as an electron donor, acetate served as the most effective donor promoting TCC dissimilation up to 50% (Fig. 3B). Under this condition, three transient intermediates, i.e. 34DCA, 4CA, and aniline were consecutively detected at 2, 4, and 8 days of incubation, respectively (Fig. 3B). The toxicity assessment of the intermediates at day-14 using Allium cepa root tip cells indicated that anaerobic degradation by MC22 could only detoxify TCC to a certain level (Fig. 2; F1-F5, G1-G5 and Table 1) because TCC dissimilation under anaerobic condition occurred at much slower rate. So far, two biodegradation pathways of TCC have been proposed from mixed culture studies. While the reductive dechlorination of TCC was reported to be key dissimilation mechanism of TCC under either aerobic and anaerobic conditions by the river sediment

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microbes leading to the formation of two lesser chlorinated congeners, i.e. 3,4’dichlorocarbanilide, and 4,4’-dichlorocarbanilide [12], the hydrolysis of TCC was reported to be a primary biodegradation pathway by mixed cultures obtained from wastewater and activated sludge leading to the formation and accumulation of 34DCA and 4CA [15, 38]. In this study, 34DCA and 4CA were found to be key intermediates from both aerobic- and anaerobic TCC degradations; therefore it is most likely that TCC undergoes oxygenindependent hydrolysis as an initial dechlorination step in biodegradation pathway to form its chloroaniline components, which later on could be completely degraded in MC22.

3.2 Influence of MC22 on growth and physiological characteristics of legume plants in the presence and absence of triclocarban Prior to apply TCC-degrading bacteria to cleanup agricultural soil contaminated with TCC in the process called rhizoremediation, it was essential to examine that such bacterial cells would not cause detrimental effect to plant, or even better to promote plant growth. As a consequence, plant-growth promoting activities of MC22 were initially determined, and then its influences on plant growth and physiological characteristics with and without TCC were investigated.

3.2.1 Plant-growth promoting traits of TCC-degrading Ochrobactrum sp. MC22 By using in vitro test methods to assess PGPB traits, MC22 could be classified as PGPB exhibiting all of the following three key activities, and maintaining them even under TCC stress: (i) synthesis of useful compounds for plants; (ii) facilitating plant nutrient uptake; and (iii) alleviation of plant environmental stress [39] (Table 2). MC22 could support plant growth by synthesizing macronutrient ammonia as well as phytohormone auxin, IAA, to accelerate plant growth. The two main detected organic acids, i.e. gluconic acid and succinic

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acid, released from MC22 potentially play a major role for phosphate solubilization in order to facilitate plant phosphate uptake, while significant production of SA-, and DHBA-type siderophores by MC22 could provide direct benefit for iron sequestration for bacterial cells and for plant. EPS produced by PGPB is not only required for plant stress protection, but is also important for root colonization process of bacteria [40]. In this case, although cell exposure of TCC slightly reduced EPS production level (Table 2), further investigation showed that MC22 could form and maintain colonization well on the legume-plant roots tested under TCC stress condition (Fig. 5), in which EPS may play a part to facilitate this colonization. The overall in vitro test result confirmed stably-maintained PGPB activities of MC22 in the presence and absence of TCC.

3.2.2 Role of MC22 on TCC toxicity mitigation of legume plants To fundamentally establish potential application of MC22 as a TCC-degrading culture for rhizoremediation, it is important to substantiate role of MC22 on TCC toxicity mitigation to plant, and how it influences plant growth and health. The selected plants for this investigation were mung bean (Vigna radiata), and soybean (Glycine max (L.) Merr.), which belong to the plant family Leguminosae. Legumes have been considered one of the promising plants for pollutant remediation not only because it has deep-reaching root system and high biomass rate [41], but also has symbiotic relationship with both rhizobial [41] and non-rhizobial bacterial species [42]. Although nitrogen fixation ability of MC22 has not yet been studied, the ability of other Ochrobactrum strains to form nitrogen-fixing nodules on plant roots has been documented [43, 44]. Upon exposure to TCC at 9.40 mg L-1, one-week grown mung bean and soybean plants showed signs of damages (Fig. 4) with slow growth rate leading to 30-50% lower plant weight (Fig. 4A, D), 25-30% lower whole-plant length (Fig. 4B, E), and unhealthy roots with

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45-50% shorter root length (Fig. 4C, F), compared to those under normal growth conditions. Bioaugmentation of MC22 to the final cell concentration at 2.9  109 CFU ml-1 could clearly mitigate TCC toxicity to both plants of which overall plant structure became healthier with significantly improved plant morphological parameters including increased biomass, longer plant length, and longer root length in comparison to those under TCC exposure alone (Fig.4A-F). From overall plant structure observation, mung bean plant seemed to suffer from TCC toxicity more than soybean plant. However, it was obvious that both plant root systems were significantly damaged as both of them had fewer numbers of lateral roots compared to those of the control. Therefore, effect of TCC on the primary-root anatomical changes of both legume plants upon TCC exposure was subsequently analyzed on transverse root sections. TCC exposure to both plants caused a thinner root with significant decrease of total cross section root area (Fig. 5A, D), smaller areas of vascular bundle (Fig. 5B, E), and xylem vessel (Fig. 5C, F). Although plant responsive mechanisms to toxic pollutant generally differ among plant species, type and concentration level of the pollutant, this result is in agreement with some reports elaborating that one of the plant defensive mechanisms is to minimize the pollutant uptake system by developing a narrower root diameter, smaller vascular bundles, and less number of xylem strands [45, 46]. Upon MC22 bioaugmentation, normal root anatomy of both plants was observed (Fig. 5). This result clearly reflected TCC detoxification role of MC22, which in turn supported plant health recovery from TCC toxicity damage. Further investigation confirmed TCC detoxification by revealing that under the symbiotic condition with mung bean and soybean growth, MC22 could colonize both plant root surfaces with high density in the taproot zone (Fig. 6) and facilitated TCC biodegradation in halfstrength Hoagland’s medium up to the total degradation of 60  2% and 65  2% of the initial TCC concentration after 7 days of incubation with the degradation rate of 0.75  0.08 and

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0.85  0.12 mg L-1 TCC degraded day-1, respectively, while TCC level was insignificantly changed in abiotic control to which MC22 was not provided (data not shown).

4. Conclusions Ochrobactrum sp. MC22 was isolated and identified as plant-growth promoting bacterium with broad and versatile capability of TCC degradation under aerobic and anaerobic conditions. The degradation kinetics analysis revealed a non-self-inhibitive substrate effect, and broad-concentration range degradation efficiency from low to high concentration of TCC, which may suggest a feasible role of this bacterium to treat lowconcentration TCC remained in agricultural soil amended with treated-biosolid, and pretreat high-concentration TCC in biosolid. The in-depth details of degradation pathway and key reaction were proposed through metabolite identification and kinetic analysis. The potential application of MC22 in rhizoremediation of TCC-contaminated crop areas was demonstrated by the fact that MC22 possesses plant-growth promoting traits, which are steadily maintained under pollutant stress, can colonize on plant root to promote symbiotic relationship with plant, and simultaneously, is able to detoxify TCC to reduce adverse effect to plants.

Acknowledgements This research was funded by the office of higher education commission (OHEC) and the S&T postgraduate education and research development office (PERDO) through the research program in hazardous substance management in agricultural industry, Center of Excellence on

Hazardous

Substance

Management

(HSM).

Additional

supports

from

Rachadaphiseksomphot Endowment Fund, Chulalongkorn University through Faculty of Science (Sci-Super III), and from Thailand Research Fund (IRG 5780008) were acknowledged.

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[18] L.E. de-Bashan, J.P. Hernandez, Y. Bashan, The potential contribution of plant growthpromoting bacteria to reduce environmental degradation – A comprehensive evaluation, Appl. Soil Ecol. 61 (2012) 171-189. [19] J. Monod, The growth of bacterial cultures, Annu. Rev. Microbiol. (1949) 371-394. [20] M. Ahemad, M.S. Khan, Effect of fungicides on plant growth promoting activities of phosphate solubilizing Pseudomonas putida isolated from mustard (Brassica compestris) rhizosphere, Chemosphere 86 (2012) 945-950. [21] J.G. Busato, L.S. Lima, N.O. Aguiar, L.P. Canellas, F.L. Olivares, Changes in labile phosphorus forms during maturation of vermicompost enriched with phosphorussolubilizing and diazotrophic bacteria, Bioresour. Technol. 110 (2012) 390-395. [22] W. Chang, A. Akbari, J. Snelgrove, D. Frigon, S. Ghoshal, Biodegradation of petroleum hydrocarbons in contaminated clayey soils from a sub-arctic site: The role of aggregate size and microstructure, Chemosphere 91 (2013) 1620-1626. [23] D. Goswami, P. Dhandhukia, P. Patel, J.N. Thakker, Screening of PGPR from saline desert of Kutch: Growth promotion in Arachis hypogea by Bacillus licheniformis A2, Microbiol. Res. 169 (2014) 66-75. [24] M. Gharasoo, F. Centler, P. Van Cappellen, L.Y. Wick, M. Thullner, Kinetics of substrate biodegradation under the cumulative effects of bioavailability and selfInhibition, Environ. Sci. Technol. 49 (2015) 5529-5537. [25] A. Juhasz, R. Naidu, Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degradation of benzo(a)pyrene, Inter. Biodeterio. Biodeg. 45 (2005) 57-88. [26] K. Kovarova-Kovar, T. Egli, Growth kinetics of suspended microbial cells: from singlesubstrate-controlled growth to mixed-substrate kinetics, Microbiol. Mol. Biol. Rev. 62 (1998) 646-666.

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[27] J.H. de Best, P. Hunneman, H.J. Doddema, D.B. Janssen, W. Harder, Transformation of carbon tetrachloride in an anaerobic packed-bed reactor without addition of another electron donor, Biodegradation 10 (1999) 287-295. [28] S. Silambarasan, A.S. Vangnai, Biodegradation of 4-nitroaniline by plant-growth promoting Acinetobacter sp. AVLB2 and toxicological analysis of its biodegradation metabolites, J. Hazard. Mater. 302 (2016) 426-436. [29] N. Vinayavekhin, G. Mahipant, A.S. Vangnai, P. Sangvanich, Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress, Appl. Microbiol. Biotechnol. 99 (2015) 59715983. [30] T. Toyama, T. Furukawa, N. Maeda, D. Inoue, K. Sei, K. Mori, S. Kikuchi, M. Ike, Accelerated biodegradation of pyrene and benzo[a]pyrene in the Phragmites australis rhizosphere by bacteria-root exudate interactions, Water Res. 45 (2011) 1629-1638. [31] L. Yang, Y. Wang, J. Song, W. Zhao, X. He, J. Chen, M. Xiao, Promotion of plant growth and in situ degradation of phenol by an engineered Pseudomonas fluorescens strain in different contaminated environments, Soil Biol. Biochem. 43 (2011) 915-922. [32] H.P. Singh, D.R. Batish, R.K. Kohli, K. Arora, Arsenic-induced root growth inhibition in mung bean (Phaseolus aureus Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation, Plant Growth Regul. 53 (2007) 65-73. [33] A.L. Burton, M. Williams, J.P. Lynch, K.M. Brown, RootScan: Software for highthroughput analysis of root anatomical traits, Plant soil 357 (2012) 189-203. [34] S. Saiyood, A.S. Vangnai, P. Thiravetyan, D. Inthorn, Bisphenol A removal by the Dracaena plant and the role of plant-associating bacteria, J. Hazard. Mater. 178 (2010) 777-785.

22

[35] T.H. Ma, The international program on plant bioassays and the report of the follow-up study after the hands-on workshop in China, Mutat. Res. 426 (1999) 103-106. [36] G.H. Wang, C.Y. Cheng, M.H. Liu, T.Y. Chen, M.C. Hsieh, Y.C. Chung, Utility of Ochrobactrum anthropi YC152 in a microbial fuel cell as an early warning device for hexavalent chromium determination, Sensors (Basel) 16 (2016). [37] W.C. Evans, G. Fuchs, Anaerobic degradation of aromatic compounds, Annu. Rev. Microbiol. 42 (1988) 289-317. [38] W.E. Gledhill, Biodegradation of 3,4,4'-trichlorocarbanilide, TCC, in sewage and activated sludge, Water Res. 9 (1975) 649-654. [39] X. Zhuang, J. Chen, H. Shim, Z. Bai, New advances in plant growth-promoting rhizobacteria for bioremediation, Environ. Int. 33 (2007) 406-413. [40] X. Meng, D. Yan, X. Long, C. Wang, Z. Liu, Z. Rengel, Colonization by endophytic Ochrobactrum anthropi Mn1 promotes growth of Jerusalem artichoke, Microb. Biotechnol. 7 (2014) 601-610. [41] M.T. Gomez-Sagasti, D. Marino, PGPRs and nitrogen-fixing legumes: a perfect team for efficient Cd phytoremediation?, Front. Plant Sci. 6 (2015) 81. [42] A. Sy, E. Giraud, P. Jourand, N. Garcia, A. Willems, P. de Lajudie, Y. Prin, M. Neyra, M. Gillis, C. Boivin-Masson, B. Dreyfus, Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes, J. Bacteriol. 183 (2001) 214-220. [43] M.E. Trujillo, A. Willems, A. Abril, A.M. Planchuelo, R. Rivas, D. Ludena, P.F. Mateos, E. Martinez-Molina, E. Velazquez, Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov, Appl. Environ. Microbiol. 71 (2005) 1318-1327. [44] J.L. Zurdo-Pineiro, R. Rivas, M.E. Trujillo, N. Vizcaino, J.A. Carrasco, M. Chamber, A. Palomares, P.F. Mateos, E. Martinez-Molina, E. Velazquez, Ochrobactrum cytisi sp.

23

nov., isolated from nodules of Cytisus scoparius in Spain, Int. J. Syst. Evol. Microbiol. 57 (2007) 784-788. [45] P.L. Gratão, C.C. Monteiro, M.L. Rossi, A.P. Martinelli, L.E.P. Peres, L.O. Medici, P.J. Lea, R.A. Azevedo, Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium, Environ. Exp. Bot. 67 (2009) 387-394. [46] M. Vaculik, C. Konlechner, I. Langer, W. Adlassnig, M. Puschenreiter, A. Lux, M.T. Hauser, Root anatomy and element distribution vary between two Salix caprea isolates with different Cd accumulation capacities, Environ. Pollut. 163 (2012) 117-126.

24

Figure captions Fig. 1. TCC biodegradation by MC22 under aerobic condition. (A) TCC-dependent growth of MC22 and time-course TCC degradation in MSM medium supplemented with 9.40 mg L -1 TCC. The residual TCC concentration in the test system with MC22 (), and that in abiotic control () was determined at time interval. TCC-dependent cell growth () was monitored. The transiently accumulated intermediates were detected at two different HPLC retention times (RT): 34DCA at RT 3.928 min (), and 4CA at RT 3.444 min (). (B) Effect of additional carbon or nitrogen source (each at 1 g L -1) on TCC degradation at 9.40 mg L-1. (C) TCC-dependent specific growth kinetics of MC22 fitted with Haldane model. (D) TCC biodegradation kinetics of MC22 fitted with Monod-type kinetic model. The data are mean with standard deviation from at least three replicates.

Fig. 2 Cytogenotoxicity study of triclocarban and its degraded metabolites in meristematic cells of Allium cepa. It was done under the following conditions: a control with distilled water (A1-A5); triclocarban treatment at 9.40 (B1-B5) and 15.67 mg L-1 (C1-C5); and treatment with the degradative metabolites obtained from aerobic- (D1-E5), and anaerobic degradation (F1-G5) collected at 3 and 14 days, respectively.

Fig. 3 Anaerobic degradation of triclocarban by MC22. (A) Effect of electron donor and (B) electron acceptor on TCC biodegradation (9.40 mg L-1) was investigated in comparison to the controls: one without any additional compound, and abiotic control. The transiently accumulated intermediates were detected at three different HPLC retention times (RT): 34DCA at RT 3.928 min (), 4CA at RT 3.444 min (), and aniline at RT 3.200 min (). The data are mean with standard deviation from at least three replicates.

25

Fig. 4 Plant characteristics of one-week grown Vigna radiata and Glycine max (L.) Merr. under normal growth condition (white column); or with TCC exposure at 9.40 mg L -1 (dark column); or with TCC exposure with MC22 (grey column). The data are mean with standard deviation from at least six replicates. In each set of data, the letter in each column indicates significant differences (P  0.05) in comparison to the data of the normal growth condition.

Fig. 5 Root anatomy of one-week grown Vigna radiata and Glycine max (L.) Merr. under normal growth condition (white column); or with TCC exposure at 9.40 mg L-1 (dark column); or with TCC exposure with MC22 (grey column). Root cross section area (A, D), vascular bundle area (B, E), and xylem vessel area (C, F) of both plants were quantitatively analyzed by Rootscan v2.0 image analysis software. The data are mean with standard deviation from at least six replicates. In each set of data, the letter in each column indicates significant differences (P  0.05) in comparison to the data of the normal growth condition.

Fig. 6 Scanning electron micrographs of colonization of MC22 on the surface of primary root of (A) Vigna radiata and (B) Glycine max (L.) Merr. grown for 7 days in a haft-strength Hoagland’s nutrient solution supplemented with 9.40 mg L -1 TCC.

26

Fig. 1

8

0.3

6 0.2

4 0.1

2

0

B Cell growth (OD600)

TCC or intermediate concentration (mg L-1)

0.4

0.0 1

2

3 4 Time (d)

5

a

0.0020 a

b

0.0015

a

b

a

a

a

0.0010 b

0.0005 0.0000

6

0 .0 0 8 0 .0 0 6 0 .0 0 4 0 .0 0 2 0 .0 0 0 0

5

10

15

20

25

T C C c o n c e n t r a t io n ( m g L

30 -1

)

m g c e ll p r o te in

0 .0 1 0

-1

0 .0 1 2

( m g s u b s tr a te h

D

) -1

S p e c ific g r o w t h r a te ( h

C

S p e c if ic d e g r a d a t io n r a t e

-1

)

0

0.0025

Specific degrdation rate (mg substrate h-1 mg cell protein-1)

10

A

0 .0 0 3 0 0 .0 0 2 5 0 .0 0 2 0 0 .0 0 1 5 0 .0 0 1 0 0 .0 0 0 5 0 .0 0 0 0 0

5

10

15

20

25

T C C C o n c e n tr a tio n (m g L

30 -1

)

Fig. 1. TCC biodegradation by MC22 under aerobic condition. (A) TCC-dependent growth of MC22 and time-course TCC degradation in MSM medium supplemented with 9.40 mg L-1 TCC. The residual TCC concentration in the test system with MC22 (), and that in abiotic control () was determined at time interval. The optical density at 600 nm was monitored representing TCC-dependent cell growth (). The transiently accumulated intermediates were detected at two different HPLC retention times (RT): 34DCA at RT 3.928 min (), and 4CA at RT 3.444 min (). (B) Effect of supplementation of additional carbon or nitrogen source (each at 1 g L -1) on TCC degradation at 9.40 mg L-1. (C) TCC-dependent specific growth kinetics of MC22 fitted with Haldane model. (D) TCC biodegradation kinetics of MC22 fitted with Monod-type kinetic model. The data are mean with standard deviation from at least three replicates.

27

Prophase (A2)

Metaphase Anaphase (A3) (A4)

Telophase (A5)

Triclocarban exposure 15.67 mg L-1 9.40 mg L-1

(B1)

(B2)

(B3)

(B4)

(B5)

(C1)

(C2)

(C3)

(C4)

C5)

Exposure to aerobic TCC degradative metabolites

(D1)

(D2)

(D3)

(D4)

(D5)

(E1)

(E2)

(E3)

(E4)

(E5)

(F1)

(F2)

(F3)

(F4)

F5)

(G1)

(G2)

(G3)

(G4)

(G5)

Distilled water control

Interphase (A1)

Exposure to anaerobic TCC degradative metabolites

Fig. 2

Fig. 2 Cytogenotoxicity study of triclocarban and its degraded metabolites in meristematic cells of Allium cepa under the following conditions: non-treatment (using distilled water as a

28

control) (A1-A5); triclocarban treatment at 9.40 (B1-B5) and 15.67 mg L-1 (C1-C5); and treatment with triclocarban degradative metabolites obtained from aerobic- (D1-D5, E1-E5), and anaerobic (F1-F5, G1-G5) degradation conditions collected at 3 and 7 days of degradation period, respectively.

29

Fig. 3

B

Triclocarban concentration (mg L-1)

14

Ferric

12

Abiotic

Nitrate

Sulfate

None

10 8 6 4

2 0 0

2

4

6 8 10 Time (d)

12

14

14

Triclocarban concentration (mg L-1)

A

Succinate Acetate None

12

Glucose Abiotic

10 8 6 4 2 0 0

2

4

6 8 10 Time (d)

12

14

Fig. 3 Anaerobic degradation of triclocarban at the initial concentration at 9.40 mg L-1 by Ochrobactrum sp. MC22. Effect of electron donor and electron acceptor on TCC biodegradation was investigated in comparison to the controls: one without any additional compound, and abiotic control. (A) Effect of electron donor provided at the concentration as described in Materials and methods; and (B) Effect of electron acceptor provided at 1 g L-1. The transiently accumulated intermediates were detected at three different HPLC retention times (RT): 34DCA at RT 3.928 min (), 4CA at RT 3.444 min (), and aniline at RT 3.200 min (). The data are mean with standard deviation from at least three replicates.

30

1200

a

a

800

b

400

Normal growth

TCC exposure

a 30

TCC exposure & MC22 B

a b

20 10 0 15

Normal growth

TCC exposure

TCC exposure & MC22 C

a 10

c b

5

Plant wet weight (mg)

A 1600

Plant lenght (cm)

Plant lenght (cm)

Soybean (Glycine max) Norrmal TCC TCC exposure growth exposure & MC22

1600

0 40

Root lenght (cm)

Mung bean (V. radiata) Norrmal TCC TCC exposure growth exposure & MC22

Root lenght (cm)

Plant wet weight (mg)

Fig. 4

0

a

1200

a

D

b

800 400 0 40 30

Normal growth

TCC exposure

a

TCC exposure & MC22 E

a b

20 10 0 15

Normal growth

a

TCC exposure

TCC exposure & MC22 F

c

10

b 5

0 Normal growth

TCC exposure

TCC exposure & MC22

Normal growth

TCC exposure

TCC exposure & MC22

31

Fig. 4 Plant characteristics of one-week grown mung bean (Vigna radiata) and soybean (Glycine max) under normal growth condition in half-strength Hoagland’s medium agar (white column); or with TCC exposure at 9.40 mg L-1 (dark column); or with TCC exposure at 9.40 mg L-1 with the bioaugmentation of Ochrobactrum sp. MC22 (grey column). The data are mean with standard deviation from at least six replicates. In each set of data, the letter in each column indicates significant differences (P  0.05) in comparison to the data of the normal growth condition.

32

Fig. 5 Mung bean (V. radiata)

3.0 2.0 1.0

0.0 Normal growth

0.30 0.25 0.20

B

a

0.15

b

a

0.10

0.05 0.00

TCC TCC exposure exposure & MC22

Normal growth

TCC exposure & MC22 Xylem vessel area (mm2)

A

4.0

TCC exposure Vascular bundle area (mm2)

Root cross section area (mm2)

Normal growth

0.020

C

0.015

a

a 0.010

b 0.005 0.000

TCC TCC exposure exposure & MC22

Normal growth

TCC exposure

TCC exposure & MC22

Soybean (Glycine max)

a

a

b

D

3.0 2.0 1.0 0.0 Normal growth

TCC TCC exposure exposure & MC22

0.30 0.25

E

a

a

0.20

b

0.15 0.10 0.05

0.00 Normal growth

TCC TCC exposure exposure & MC22

TCC exposure & MC22 Xylem vessel area (mm2)

4.0

TCC exposure Vascular bundle area (mm2)

Root cross section area (mm2)

Normal growth

0.020

a a

0.015

F

b 0.010 0.005

0.000 Normal growth

TCC exposure

TCC exposure & MC22

33

Fig. 5 Root anatomy of one-week grown mung bean (Vigna radiata) and soybean (Glycine max) under normal growth condition in half-strength Hoagland’s medium agar (white column); or with TCC exposure at 9.40 mg L-1 (dark column); or with TCC exposure at 9.40 mg L-1 with the bioaugmentation of Ochrobactrum sp. MC22 (grey column). Root cross section area (A, D), vascular bundle area (B, E), and xylem vessel area (C, F) of both plants were quantitatively analyzed by Rootscan v2.0 image analysis software. The data are mean with standard deviation from at least six replicates. In each set of data, the letter in each column indicates significant differences (P  0.05) in comparison to the data of the normal growth condition.

34

Fig. 6

A

B

Fig. 6 Scanning electron micrographs of colonization of Ochrobactrum sp. MC22 on the surface of primary root of (A) mung bean (V. radiata) and (B) soybean (Glycine max) grown for 7 days in a haft-strength Hoagland’s nutrient solution supplemented with 9.40 mg L-1 TCC.

35

Table titles Table 1 Mitotic index and chromosomal aberration index examined using root tips cells of Allium cepa treated with triclocarban, and degradative metabolites

Table 2 Plant-growth promoting activities of Ochrobactrum sp. MC22 with and without triclocarban.

36

Table 1 Mitotic index and chromosomal aberration index examined using root tips cells of Allium cepa treated with triclocarban, and degradative metabolites from aerobic and anaerobic triclocarban degradation by Ochrobactrum sp. MC22. Treatment

TCC concentration (mg L-1) -

Number of Number of cells with chromosome aberrations dividing ML MC MA AB cells 191a 0 0 0.63  0.09 0

9.4 15.7

98b 87b

4.08 ± 0.17 0

0 3.93 ± 0.45

0 2.47 ± 0.16

Aerobic degradative 9.4* metabolites* 15.7*

133c 121b

4.09 ± 0.51 0

0 0

Anaerobic degradative 9.4** metabolites** 15.7**

118b 110b

0 0

0 5.89 ± 0.33

Distilled (control) Triclocarban

water

AL

CB

Mitotic index (MI) (%)

Aberration (AI) (%)

0

0

18.2  0.93a

0.06 ± 0.03a

5.03 ± 0.22 0

0 7.52 ± 0.86

1.92 ± 0.13 0

9.4 ± 0.38b 8.3 ± 0.33b

1.05 ± 0.07b 1.27 ± 0.05b

0 2.01 ± 0.34

0 0

0 0

0 3.55 ± 0.41

12.6 ± 0.29c 11.5 ± 0.24b

0.39 ± 0.09c 0.53 ± 0.11d

7.14 ± 0.65 0

0 0

0 0

0 2.11 ± 0.31

11.3 ± 0.15b 10.5 ± 0.33b

0.68 ± 0.13d 0.76 ± 0.06d

* The aerobic degradative metabolites were collected at day 3 from the time-course degradation test of TCC at the initial concentration indicated. ** The anaerobic degradative metabolites were collected at day 14 from the time-course degradation test of TCC at the initial concentration indicated. ML: metaphase lagging chromosome; MC: metaphase cluster; MA: metaphase aberration; AB: anaphase bridges; AL: anaphase lagging chromosome, CB: chromosome breaks. Each value represents the mean ± SD of three replicates per treatment. Statistical analysis was conducted according to Dunnett’s test with significant differences at P ≤ 0.05 levels over the control of each column and is indicated by different letters.

index

37

Table 2 Plant-growth promoting activities of Ochrobactrum sp. MC22 with and without triclocarban. PGPB function

Synthesis of useful compound

Facilitating nutrient uptake from environment

Stress

or nutrient(s) PGPB activity

Ammonia

under each test

response

Indole acetic acid

Organic acid production

Phosphate

Siderophore production

EPS

production production

Gluconic acid Succinic acid

solubilization

SA

DHBA

production

condition

(g mL-1)

(g mL-1)

(g mL-1)

(g mL-1)

(g P solubilized mL-1)

(g mL-1)

(g mL-1)

(g mL-1)

Normal growth

846  25a

22.1  0.9a

4.3  0.1a

9.2  0.1a

137.7  0.3a

28.8  1.3a

10.1  3.6a

54.5  4.9a

978  51b

24.4  0.9b

4.9  0.1b

9.4  0.2a

130.6  3.3b

27.0  2.7a

9.2  0.4a

45.5  2.1b

754 34c

25.0  0.1b

4.4  0.3a

9.7  0.1b

121.9  10.5c

25.6  2.1c

7.8  2.4b

41.0  2.8c

(control) 9.4 mg L-1 TCC exposure 15.7 mg L-1 TCC exposure Values represent the mean (± SD) of at least three replicates per treatment. Statistical analysis was conducted according to Dennett’s multiple comparison with significant differences at P ≤ 0.05 levels over the control of each column and is indicated by different letters. Abbreviations: EPS: Extrapolymeric substances; IAA: Indole acetic acid; SA: Salicylic acid; DHBA: 2,3-Dihydroxybenzoic acid.