Environ Sci Pollut Res (2015) 22:3784–3793 DOI 10.1007/s11356-014-3582-4

RESEARCH ARTICLE

Stress response of Chlorella pyrenoidosa to nitro-aromatic compounds Chang Xu & Ruihua Wang & Y. F. Zhang & P. Cheng & Martin M. F. Choi & Karen Poon

Received: 19 May 2014 / Accepted: 8 September 2014 / Published online: 1 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Handling of two nitro-aromatic compounds, 4nitroaniline (4NA) and 4-nitrophenol (4NP), simultaneously by Chlorella pyrenoidosa was investigated. Algae would secrete or degrade nitro-aromatic compounds depending on different environmental conditions, in which the mode of handling was determined by the relative formation and degradation rate of the compound. Repeated intermittent trigger with externally added 4NA would induce the continuous secretion of 4NA by algae. Simultaneous exposure of both 4NA and 4NP to algae at normal condition would induce the algae to secrete both compounds. An increase in 4NA exposure concentration would elevate both 4NA and 4NP secretion, and that would be inhibited by the

Responsible editor: Philippe Garrigues C. Xu : Y. F. Zhang : P. Cheng : K. Poon (*) Program of Food Science and Technology, Division of Science and Technology, BNU-HKBU United International College, 28 Jinfeng Road, Tangjiawan, Zhuhai, Guangdong, People’s Republic of China e-mail: [email protected] C. Xu e-mail: [email protected] Y. F. Zhang e-mail: [email protected] P. Cheng e-mail: [email protected] R. Wang Department of Gastroenterology, Shanghai Jiao Tong University affiliated Sixth People’s Hospital South Campus, 6600 Nanfeng Road, Fengxian District, Shanghai, People’s Republic of China e-mail: [email protected] C. Xu : M. M. F. Choi Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong, SAR, People’s Republic of China M. M. F. Choi e-mail: [email protected]

stress conditions of starving or lack of oxygen. Increased 4NA degradation per production rate induced by starving or lack of oxygen might explain the subsequent decrease in 4NA secretion in the presence of 4NP in algae. For 4NP in the presence of 4NA, secretion at normal condition was completely stopped and turned to degradation mode in stress conditions. The decreased formation and increased degradation of 4NP during starving for replenishing energy would explain the net degradation of 4NP in starving condition. The condition of lack of oxygen would inhibit the 4NP formation from 4NA via oxidative deamination, while the degradation of 4NP might not be significantly affected because alternative pathway of degradation via nitro-reduction was available. It may lead to the degradation rate exceeding the formation and explain the net degradation of 4NP in the condition of lack of oxygen. Keywords Stress response . Chlorella pyrenoidosa . 4-Nitroaniline . 4-Nitrophenol . Secondary metabolites Abbreviations TCP 2,4,6-Trichlorophenol 4NA 4-Nitroaniline 4NP 4-Nitrophenol ACN Acetonitrile TAP Tris-acetate-phosphate

Introduction Chlorella pyrenoidosa is unicellular green algae. It contained high value of bioactive ingredients, such as fatty acids, pigments, carotenoids, and xanthophylls (Spolaore et al. 2006), which have been used as nutrition supplement in market for a long time. For environmental applications, algae were used in wastewater treatment (Perez-Garcia et al. 2011), such as heavy metal and organic

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contaminant biodegradation. One of the interesting characteristics of algae was stress response to different environmental triggers. In order to increase their survival, secondary metabolites (Newman and Cragg 2007) that were named as allelochemicals, including flavonoids, terpenoids, glucosinolates, and alkaloid, should be produced. Reactive oxygen species (ROS), such as superoxide (O2−), could be generated in their defense mechanism to prevent pathogenic infection (Doke 1983); for example, nitric oxide (NO) involved in the intercellular and intracellular signal transduction was associated with the defense responses in plant (Stamler 1994). Under stress condition, both O2− and NO were reported to be generated simultaneously (Kozak et al. 2005) and reacted with each other to form peroxynitrite (ONOO−) (Padmaja and Huie 1993) that had a strong nitration activity to react with biomolecules, such as proteins. Tyrosine nitration as a result of ONOO− generation was reported in previous works (Saito et al. 2006; Leitner et al. 2009). A lot of the allelochemicals were found to be useful in pharmaceutical, farming, food, and environmental industries. For example, lipid secretions from algae could be used as biofueler feedstocks (Kind et al. 2012). Environmental triggers were also found to modify the content of bioactive ingredient of algae, which would offer an engineering opportunity to optimize the production of useful chemicals in algae (Kind et al. 2012). In the previous studies, yield of bioactive ingredient from algae was found to be increased by modulating the environmental condition (Masojídek and Prášil 2010); therefore, the proper use of algae could potentially produce a very high economic value (Wilkie et al. 2011). Although Chlorella has been found to have a variety of beneficial uses, they also could produce potential environmental and food safety problems. Some marine algae were found to secrete brominated secondary metabolites for defense mechanism (La Barre et al. 2010). Toxic secondary metabolites secreted by algae in response to environmental triggers (LeFlaive and Loic 2007) might impose the potential threat to aquatic lives and human (LeFlaive and Loic 2007; Yu et al. 2012). In order to maximize the beneficial and minimize the harmful effects of algae to environment and human, in-depth understanding of the behaviors of algae in response to environmental triggers is important. In our previous studies, algae were found to have the ability to degrade and secrete organic chemicals in response to environmental triggers (Yu et al. 2012). C. pyrenoidosa would increase 4-nitroaniline (4NA) in the medium upon the exposure of the same compound to the amount higher than that of originally added. Algae were also found to respond to 4-nitrophenol (4NP), in which the mode of net secretion or absorption was modified by environmental conditions (Yu et al. 2012). The extracellular algal secretion was likely a dynamic process that was determined by the relative rate of production, accumulation, and degradation (Rajamani et al. 2011). 4NP and 4NA are nitro-aromatic compounds commonly used in industry for the manufacture of

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herbicides, insecticides, synthetic dyes, and pharmaceuticals (Alif and Boule 1991; Takahashi et al. 1994). 4NP was one of the 114 organic compounds listed by the U.S. Environmental Protection Agency with maximum allowed concentration in water at 20 ppb. Degradations of 4NP and 4NA were reported in different conditions (Azeem et al. 2009; Adrian 1999; Nevim et al. 2002) and also in removal method using microbe incubation (Gao et al. 2011; Semple and Cain 1996; Hirooka et al. 2010). However, most of the previous studies were focused on product degradation, and little on chemical secretion, in particular to secrete the same exposure compound, was reported. As nitro-aromatic compounds were toxic and explosive in nature, which might impose environmental concern, understanding of the chemical handling mechanism of algae could help us to fully utilize algal metabolism in the production of useful chemicals and avoid the potential harmful effect. As algae were found to secrete 4NP and 4NA under certain environmental conditions, 4NP was the metabolite of 4NA, which made them good study targets for the investigation of chemical handling by algae in the present study. In the present study, we explore the mode of chemical handlings of algae to two chemicals 4NA and 4NP exposed simultaneously under different environmental triggers that would alter the rate of formation and degradation of chemicals, which might provide a better understanding about the secretion of both compounds.

Material and method Chemical and culturing medium 4NA and 4NP were purchased from Sinopharm Chemical Reagent Co., Ltd. 2,4,6-Trichlorophenol (TCP) was bought from Sigma-Aldrich. High-performance liquid chromatography (HPLC) grade methanol obtained from Sigma-Aldrich and MiniQ water were used as HPLC mobile phase. Tris-acetatephosphate (TAP) medium (Gorman and Levine 1965) was used for the culture medium of algae. Solid medium was prepared by 1.5 % pure agar in TAP medium. Algae culture The green algae C. pyrenoidosa were isolated locally and were morphologically confirmed by local authority in Freshwater Algae Culture Collection of the Institute of Hydrobiology China (Yu et al. 2012). The algae were transferred to TAP solid medium and incubated in an environmental chamber at 25 °C with light (4,800 lx)/dark cycle of 16/8 h. The algae of green colony were transferred to TAP liquid medium. The flask was covered with a filter of pore size of 0.22 μm and continuously shaken with a shaker at about 120 rpm in the same environmental-controlled chamber. The cells at the late exponential growth phase (3 days) were harvested for the experimental

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uses. The cell density was determined at A680 nm, and the cell density of about 2×107 cell/ml would be used for the exposure experiments. Starved algae cells were prepared by covering the culture flask with aluminum foil at second days of culture for 24 h before harvest. Algal experiments 4NA secretion upon the repeated intermittent addition/removal of 4NA Of 4NA (35 ppm), 0.2 ml was added to 0.98 ml algae (5×106 cell/ml) or TAP medium and incubated in the environmental chamber of temperature at 25 °C for 30 min, before 0.2 ml was collected from the supernatant of the centrifuged samples. The collected sample would be used for the determination of 4NA concentration by HPLC, while the 0.2 ml of fresh 4NA (35 ppm)/TAP would be added back to the test samples and incubated for another 30-min incubation. The procedures would be repeated until the incubation time reached 180 min.

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secretion rate and absorption rate were calculated according to the method of Rowland and Tozer (1989). HPLC condition HPLC system used for analysis was Agilent Series 1200 liquid chromatography (Palo Alto, CA USA) coupled with a binary pump, an in-line degasser, an Agilent Eclipse XDBC18 (3.0×150 mm) column, a diode array detector, and an autosampler. The gradient elution program with water and methanol as mobile phases A and B, respectively, was employed for analysis. The gradient program was started with 45 % methanol (B), changed to 95 % in 10 min and held in 95 % methanol for 10 min, and decreased to 45 % in 5 min. The flow rate was 0.55 ml/min and injection volume was 10 μl. The multiple wavelengths of 210, 240, 250, 254, 280, and 310 nm were applied for detection, and 310 nm was selected for analysis.

Exposure of 4NA and 4NP to algae

Result

For the exposure experiment of individual compound to algae, 440 ppm 4NP or 4NA was added to 1 ml of the algal cells in 2ml vial to make the final concentration of 20 ppm 4NP or 4NA. The vials were capped and mixed, which were then followed by incubation in environmental chamber, with the above same condition of algae culturing, for 1, 15, 30, 60, and 120 min. For the exposure experiments with two compounds to algae, 440 ppm 4NP and different concentration of 4NA (dilute by deionized water) were added to 1 ml of the algal cells in 2-ml vial to make the final concentration of 20 ppm 4NP with 5, 10, 15, and 20 ppm 4NA. The vials were capped and mixed, which were then followed by incubation in environmental chamber, with the above same condition of algae culturing, for 60 min. The supernatant of each vial was collected by centrifugation at 9,000×g for 5 min and then extracted with acetonitrile (ACN) after addition of TCP as internal standard. Excess amount of sodium chloride was added to saturate the solution in order to separate ACN and water layer for assisting extraction process. After vortexes, the organic layer was collected for HPLC analysis. As algae without exposing to chemical was found not to produce any chemical, control samples without algae cell were prepared following the same procedure, which was used to ensure the interpretation of result was not caused by pipetting or operational error. Single exposure to 4NP or 4NA in TAP solution followed the same procedure as the two chemical samples, and algae samples in deionized water were prepared by washing with deionized water for three times before chemical exposure experiments. All the samples including controls were prepared in triplicate. The

HPLC chromatogram Figure 1 showed the chromatographic analysis of algae exposure medium after 20 ppm 4NA and 20 ppm 4NP were added in the exposure medium for 1 h at 310 nm. Three nice peaks were shown in Fig. 1, which represented the peaks of 4NA, 4NP, and the internal standard of TCP. When the algae were exposed to single chemical either 4NA or 4NP, no significant amount of degradation products was shown in the HPLC analysis. When the algae were exposed to two chemicals of 4NA and 4NP simultaneously, at the absorbance at 310 nm, no significant amount of degradation products of either 4NA or 4NP was observed. However, extra peak at the latter time at the absorbance of 240, 254, 250 nm was observed after 1 h incubation with algae. Similar results were observed in other samples. Using the peak area, the concentration of chemical was quantified with reference to the corresponding standard curve. 4NA secretion upon the repeated intermittent addition/removal of 4NA When fresh 4NA was continuously added to the algal solution, increasing amount of 4NA secretion compared with the control was observed (Fig. 2). The higher slope value in the continuous addition in algae group indicated the continuous secretion of 4NA from algae upon the repeated intermittent addition of 4NA. When 4NA was continuously removed from the algal solution, decreasing amount of 4NA compared with the control was observed (Fig. 2). The decreasing amount

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Fig. 1 The chromatogram of algae exposure medium after added with 20 ppm 4NA+20 ppm 4NP in normal algae cell culture for 1 h at 310 nm. Peak a 4NA. Peak b 4NP. Peak c TCP (internal standard)

indicated that 4NA was net absorbed by the algae, and so the amount of 4NA was lower than that of the control (Fig. 2). Exposure of 4NA or 4NP individually to algae Figure 3 showed the time concentration of 4NA or 4NP in normal or starved algae. The concentration of 4NA and 4NP was found to increase with time and reach a peak value at about 15 min, before the concentration gradually dropped with time. Using the method introduced in pharmacokinetics (Rowland and Tozer 1989), secretion/production rate of the increasing mode and the absorption/degradation rate of the decreasing mode were estimated. The secretion/production rate of 4NA in normal algae was slightly lower than that of 4NP (Table 1), while the absorption/degradation rates were comparable (Table 1). The secretion/production rate of 4NA was increased in starved algae, while that of 4NP was decreased (Table 1). Both the absorption/degradation rates of 4NA and 4NP were increased in starved algae (Table 1). When algae were exposed to either 4NA or 4NP under different conditions, different chemical handlings were

Fig. 2 4NA concentration (ppm) (mean±SE) in (1) algal solution upon the continuous addition (black diamond), (2) TAP solution upon the continuous addition (white diamond), (3) algal solution upon the continuous removal (black square), and (4) TAP solution upon the continuous removal, of 4NA with time (hours) (white square)

observed. Under normal condition in TAP culture medium, algae were found to degrade 4NA or 4NP after 1-h exposure (Fig. 4a, b). However, if the algae were put under starving condition, e.g., restricting from light for 24 h, algae were found to degrade 4NA faster but not 4NP. No significant degradation of 4NP by algae was observed (Fig. 4a, b). When algae were stressed by repeatedly washing with deionized water, the normal degradation process of 4NA or 4NP was altered. No degradation was observed but rather secretion of 4NA (Fig. 4a, b).

Exposure of 4NA and 4NP to algae simultaneously Figure 5 showed the concentration difference of the exposure chemicals 4NA and 4NP after chemicals were added into the normal algal cell for 1 h. The concentration difference of the exposure chemical was calculated by subtracting the initial concentration of the chemical with the corresponding after 1-h exposure experiment. The negative value of the concentration difference indicated there was a net secretion of exposure chemical by algae cell. Results indicated that when the normal algae cells were exposed to the two chemicals 4NA and 4NP

Fig. 3 Plot of log concentration (mean±SE) of (1) 4NA in normal algae (black diamond), (2) 4NA in starved algae (white diamond), (3) 4NP in normal algae (black up-pointing triangle), and (4) 4NP in starved algae, vs time (white up-pointing triangle)

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Table 1 Secretion rate and absorption rate for (1) 4NA in normal algae, (2) 4NA in starved algae, (3) 4NP in normal algae, and (4) 4NP in starved algae

4NA in normal algae 4NA in starved algae 4NP in normal algae 4NP in starved algae

Secretion/production rate (ppm/min)

Absorption/degradation rate (ppm/min)

0.3678 1.5519 0.5784 0.3209

0.0188 0.1105 0.0178 0.0434

simultaneously, algae were found significantly to net secrete both 4NA and 4NP (Fig. 5). The higher the concentration of

initial 4NA exposure concentration, the higher was the amount of 4NA and 4NP being secreted by algae (Fig. 5). Algae in nitrogen-bubbled medium behaved differently from that in normal medium. The secretion of 4NA in response to the same chemical exposure remained but in a lower magnitude (Figs. 5 and 6), while the secretion of 4NP observed in the normal algae disappeared (Figs. 5 and 6). The way of 4NP handling was found to change from secretion to degradation (Fig. 6). The amount of 4NP from degradation was not influenced by the increasing concentration of 4NA in the exposure medium (Fig. 6), which was different from algae in normal medium and condition (Figs. 5 and 6).

a

b

Fig. 4 a Concentration difference (initial concentration–concentration at 1 h) of 4NA in the algae exposure medium 1 h after the addition of 20 ppm 4NA. **P

Stress response of Chlorella pyrenoidosa to nitro-aromatic compounds.

Handling of two nitro-aromatic compounds, 4-nitroaniline (4NA) and 4-nitrophenol (4NP), simultaneously by Chlorella pyrenoidosa was investigated. Alga...
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