Bio-reaction of nitrobenzene with Microcystis aeruginosa: Characteristics, kinetics and application

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 9 0 e2 2 9 8

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Bio-reaction of nitrobenzene with Microcystis aeruginosa: Characteristics, kinetics and application Zhiquan Liu a, Fuyi Cui a,*, Hua Ma a, Zhenqiang Fan a,b, Zhiwei Zhao a, Zhenling Hou a,c, Dongmei Liu a a

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (SKLUWRE, HIT), PO Box 2650, Harbin 150090, China b School of Environmental and Municipal Engineering, North China Institute of Water Conservancy and Hydroelectric Power, Zhengzhou 450011, China c Changchun Institute of Urban Planning & Designing, Changchun 130021, China

article info

abstract

Article history:

The bio-reaction of nitrobenzene (NB) with Microcystis aeruginosa was investigated at

Received 10 October 2011

different initial algal densities and NB concentrations by performing static experiments.

Received in revised form

The results showed that the elimination of NB was enhanced by the bio-reaction, and the

26 December 2011

reaction rate varied as a function of the reaction time. Moreover, the reaction rate was

Accepted 29 January 2012

significantly affected by the algal density and NB concentration. A kinetic analysis showed

Available online 6 February 2012

that the elimination of NB in a solution without algae appeared to be pseudo-first-order with respect to the NB concentration, whereas a first-order model was too oversimplified

Keywords:

to describe the elimination of NB in a solution with algae. Assuming that different algal

Bio-reaction

cells have the same effect on the bio-reaction under the same conditions, the bio-reaction

Nitrobenzene

n can be described as dCNB ¼ k0Cm A ANBdt (where k0 is the reaction rate constant, CA is the

Microcystis aeruginosa

algae density and CNB is the concentration of NB). When the growth of algae was not

Kinetic model

considered, the value of k0, m and n were 8.170  104, 0.5887 and 1.692, respectively. Alternatively, when algae were in the exponential growth phase, the value of k0, m and n were 1.6871  105, 0.7248 and 2.5407, respectively, according to a nonlinear fitting analysis. The kinetic model was also used to elucidate the effect of nutritional limitation on the bio-reaction. ª 2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Due to industrial and agricultural growth, a large number of synthetic organic compounds have been discharged into the aquatic environment, and the transformation and degradation of these compounds have been highlighted by various environmental groups. Microorganisms in the aquatic environment play an important role in the elimination of synthetic

organic compounds. These bio-reactions have been well studied in bacteria, fungi and yeast (Ayed et al., 2009; Yang et al., 2009; Liao, 2010; Ding et al., 2008); however, only a few studies have been performed on algae. According to the results of previous studies, bio-reactions of synthetic organic compounds with algae include bioaccumulation and biodegradation. Bioaccumulation occurs via physical adsorption and is often associated with the

* Corresponding author. Tel.: þ86 451 86282098. E-mail address: [email protected] (F. Cui). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2012.01.049

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 9 0 e2 2 9 8

octanol/water partition coefficient (Kow) (Wright, 1978; Swackhamer and Skoglund, 1993; Skoglund et al., 1996; Daneshvar et al., 2007; Khataee et al. 2009), while biodegradation is a biochemical process in which xenobiotics are transformed into other forms of organic matters or are mineralised (Cerniglia et al., 1980; Lima et al., 2004; Kapanen et al., 2007; Yan and Pan, 2004). The mechanism of biodegradation has also been studied, and previous investigations have shown that xenobiotics can be biodegraded by both phase I (cytochrome P-450) (Thies et al., 1996) and phase II (conjugation) enzymes (Warshawsky et al., 1990; Tang et al., 1998) of selected species of algae to obtain energy and build tissue. Previous studies have also indicated that both the type of organic compound and species of algae have a significant effect on the bio-reaction. Papazi and Kotzabasis (2007) showed that the removal of phenolic compounds by Scenedesmus obliquus increased according to the following sequence: chloro- < bromo- < iodophenols. Moreover, 2,4dichlorophenol could not be biodegraded by Chlorella sp. (Kleknera and Kosaricb, 1992) but could be biodegraded by the diatom Skeletonema costatum at a rate of 30% in 4 days (Yang et al., 2002). However, due to the complexity of synthetic organic compounds, a limited number of publications on the bio-reaction of nitro-aromatic compounds with algae have been published. The industrial synthesis and incomplete combustion of fossil fuels produces nitro-aromatic compounds. Large areas of soil, ground and surface water have been contaminated by these xenobiotics (Kulkarni and Chaudhari, 2007; Gao et al., 2008), which originate from facilities used for the manufacturing, processing and disposal. Nitrobenzene (NB), which is the simplest nitro-aromatic compound, is widely distributed in Chinese aqueous environments (Gao et al., 2008) and exhibits considerable toxicity to human beings, fish, algae and microorganisms (Kulkarni and Chaudhari, 2007). Obviously, the transformation and elimination of NB in the aqueous environment must be studied. Kinetic analysis is a common analytical method in the field of chemistry and is often used to study reaction mechanisms and predict reaction processes. Due to the growth of algae, which may be affected by environmental factors (pH, temperature, substrate concentration, illumination, etc.) and is usually independent of the reaction time, kinetic analyses of the xenobiotic bio-reactions with algae are difficult. In previous studies (Yan et al., 1995; Huang et al., 1999), xenobiotic bio-reactions were considered first-order or second-order reactions. However, the bio-reaction of xenobiotics with algae is a complex process that includes bioaccumulation and biodegradation. The liquid/cell transfer efficiency, enzyme activity, photosynthetic rate, respiration rate and other factors may affect the bio-reaction. Thus, pre-set integer reaction orders may not be appropriate. In the present study, the characteristics of the bio-reaction of NB with Microcystis aeruginosa (a species of cyanophytes that causes water blooms) were investigated by monitoring the concentration of NB in solution under pre-set conditions. According to the experimental data, an empirical kinetic model was developed and examined. The model was also used to analyse the effect of nutritional limitation on the bioreaction.

2.

Materials and methods

2.1.

Algal culturing and determination

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A purchased stock culture of M. aeruginosa (from the Institute of Hydrobiology at the Chinese Academy of Sciences) was treated according to a method adopted from Semple and Cain (1996) to obtain axenic M. aeruginosa. The axenic stock culture of M. aeruginosa was incubated according to our laboratory method, which has been described by Liu et al. (2011). All of the samples and algal stock cultures were incubated in an illuminating incubator at a temperature of 25  1  C and a light intensity of 55 mmol m2 s1 in a 14-h light/10-h dark cycle. The algal density was monitored by spectrophotometry at 680 nm (UV759S, Shanghai Precision & Scientific instrument Co. Ltd.), and cell number counting with a microscope (Olympus BX41) was performed according to the method adopted by Ma et al. (2009). Photosynthetic and respiration rates were obtained by monitoring the difference in the dissolved oxygen content (WTW, pH/Oxi 340i) in the presence or absence of illumination under algal culturing conditions (25  1  C, 55 mmol m2 s1) according to a method adopted from Burris (1977). The photosynthetic or respiration rate was expressed as mg O2 per 108 algal cells per hour. Duplicate measurements were obtained, and the arithmetical mean (SD) was obtained and used as the final value.

2.2.

NB determination

The sample was centrifuged for 20 min at 6790 g, and the supernatant was used for the NB analysis. The concentration of NB (Analytical reagent, CAS: 98-95-3, Tianjin Kermel Chemical Reagent Co. Ltd.) was analysed by a high-pressure liquid chromatograph (HLPC) (Waters e2695) equipped with an ODS reversed-phase column (Symmetry C18 5 mm 4.6  150 mm column), and detection was performed at 263 nm using a variable wavelength detector (Waters 2489). The mobile phase consisted of a mixture of water and methanol (43:57) at a flow rate of 1 mL/min. The injection volume was equal to 100 mL and the temperature was set to 30  C.

2.3.

Experiments

The experiments were divided into three parts: A. Effect of algal density on the bio-reaction of NB with M. aeruginosa. Experiment A was performed at an initial NB concentration of 160 mg/L, and the initial algal densities ranged from 0.34  0.022  108 cells/L to 4.39  0.057  108 cells/L (OD680 0.0148  0.0010e0.1917  0.0022). The concentration of the substrate in the mixture was approximately 1% of full strength BG11 medium to limit algal growth and maintain algal survival. The algal density was determined by spectrophotometry at 680 nm (OD680). At the end of experiment A, the final OD680 was approximately 50% of the initial value, except in the last sample (the initial OD680 was 0.1917), where the OD680 was nearly

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a

0 0.0295 0.0965

CNB( g/L)

70

0

b

60

40

20

0 0

All of the experiments were performed in triplicate and the arithmetical mean (SD) was used as the final value.

3.

Results and discussions

3.1.

Characteristics of the bio-reaction

3.1.1.

Effect of the algal density on the bio-reaction

Experiments A and C were performed to study the effect of the algal density on the bio-reaction of NB with M. aeruginosa. In both experiments, the NB concentration was plotted as a function of the reaction time, and curves with similar shapes were observed. The reaction rate was not constant and varied as a function of the reaction time. In the first 24 h, significantly difference between the experimental and control groups was not detected; however, the reaction rate increased over the next 72 h and slowed down after 96 h of incubation, as shown in Figs. 1(a) and 2(a). The relative removal rate, r ¼ ððCNBt  C0NBt Þ=CNB0 Þ, (where r is the relative removal rate, CNBt is the concentration of NB in the experimental group at a reaction time of t, C0NBt is the concentration of NB in the control group at a reaction time of t and CNB0 is the initial concentration of NB), was used to describe the effect of algae on the bio-reaction. Under these conditions, the influence of algae on the volatilisation of NB was not considered. In both experiments, the effect of algae on the bio-reaction was enhanced as the algal density increased, as shown in Figs. 1(b) and 2(b). At the reaction time of 96 h, as the initial algal density increased, the relative removal rate increased from 5.5  4.03% to 43.8  3.24% in experiment A and from 27.6  8.59% to 30.6  7.43% in experiment C. The effect of the algal reproduction on the bio-reaction was also studied by comparing the elimination rates of NB in the two experiments. As shown in Fig. 2(a), algae in the

0.0148 0.0494 0.1917

140

Relative removal rate of NB(%)

three times greater than the initial value. A solution of NB at the concentration of 160 mg/L without algae was used as the control. B. Effect of NB concentration on the bio-reaction of NB with M. aeruginosa. In this part of the study, the initial algal density was set to 1.57  0.034  108 cells/L (OD680 was 0.0685  0.0015), and the initial NB concentration ranged from 40 mg/L to 200 mg/L. The same concentrations of NB in distilled water (without algae) were used as the control group. The mixture was incubated under the conditions described above. The growth rates of algae were similar, except for that of algae cultured at an initial NB concentration of 200 mg/L, which remained constant during the entire experimental period. C. Effect of algal growth on the bio-reaction of NB with M. aeruginosa. A mixture of algae and NB was incubated with half-strength BG11 medium with the same initial NB concentration (160 mg/L) and different initial M. aeruginosa densities (0.89  0.022  108 cells/L and 1.54  0.034  108 cells/L). During the experimental period, the algae were in the exponential growth phase, and their growth fit the Malthus model (Fig. S1, Supplementary data). The control group consisted of the NB solution without algae at a concentration of 160 mg/L.

100

200

300

Time(hour) Fig. 1 e Elimination of NB by repressed M. aeruginosa in experiment A. The numbers in the legend represent the initial OD680. (a) Concentration of NB in the aqueous phase. (b) Relative removal rate of NB.

exponential growth phase were more efficient in the elimination of NB than repressed algae. Although the initial algal densities in some of the groups in experiment A were higher than those in experiment C, the elimination rates of NB in experiment C were higher than those observed in experiment A, except for the group with an initial OD680 of 0.1917  0.0022. The group with an initial OD680 of 0.1917  0.0022 in experiment A provided higher elimination rates of NB than that of experiment C. However, we divided the entire experiment into several periods, and compare the results obtained in the same period at the similar NB concentrations. When the average algal density in experiment C was greater than that of experiment A, the elimination of NB was greater than that of experiment A (Table 1). Thus, a high reproduction rate leads to a high algal density and is favourable for the bio-reaction. Similar finding was also obtained by Huang et al. (1999), who researched the biodegradation of dibutyl phthalate by S. obliquus. The mechanism of the bio-reaction was not studied in the present investigation; however, according to our results, the bio-reaction includes bioaccumulation and biodegradation, and biodegradation may be the principal pathway of NB elimination in algal solutions (data not shown). Nevertheless, the biodegradation mechanism and the liquid/cell transfer efficiency of NB are not well understood, and further studies are required.

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a

0 0.0673 0.0965

180

had a significant influence on the elimination of NB. The elimination rate and the relative removal rate of NB increased with an increasing in the initial NB concentration, indicating that high concentrations of NB favoured the bio-reaction of NB with M. aeruginosa. Although the growth of algae was inhibited by high concentrations of NB (200 mg/L, Fig. S2, Supplementary data), the elimination rate reached a maximum, which suggested that the effect of the NB concentration on the bioreaction was more important than that of the algal density. In previous studies (Omar, 2008; Liu et al., 2006), a high concentration of xenobiotics resulted in a high absolute elimination rate and a low relative elimination rate. However, in the present study, the relative removal rate also increased at high concentrations of NB, which may be due to the molecular structure of NB and the species of algae employed in the present investigation.

0.0389 0.0494 0.1917

CNB(ug/L)

120

60

0

b Relative removal of NB(%)

40

20

100

200

Kinetics analysis

3.2.1.

Elimination of NB in the solution without algae

The elimination of NB in the absence of algae was attributed to photolysis (Wang et al., 2008) and volatilisation (Li et al., 2008), and the reaction rate depended on the concentration of NB. Assumption that NB elimination was a pseudo-first-order reaction with respect to the concentration of NB, the reaction can be described by equation (1):

0 0

3.2.

300

Time(hour)

dCNB ¼ kv CNB dt  kp CNB dt

Fig. 2 e Elimination of NB by M. aeruginosa in the exponential growing in experiment C. The numbers in the legend represent the initial OD680. (a) Concentration of NB in the aqueous phase. The solid line was obtained from a solution containing algae in the exponential growth phase, and the dotted line was obtained from a solution containing repressed algae. (b) Relative removal rate of NB.

(1)

where CNB is the concentration of NB in solution, kv is the volatilisation rate constant, kp is the photolysis rate constant, and t is the reaction time. When the reaction rate constant (K ) is defined as the sum of kv and kp, equation (1) can be integrated using the initial conditions to obtain equation (2): ln

CNB ¼ Kt CNB0

(2)

The ratio of CNB =CNB0 is a function of the reaction time (t), and the slope is the reaction rate constant (K ). t4/5 is defined as the time when the residual concentration of NB is equal to 80% of the initial value and is independent of CNB0 (equation (2)). The results showed that t4/5 ranged from 102.5 mg/L to 112.8 mg/ L at different initial concentrations of NB (Table S1, Supplementary data), and a linear correlation between t4/5 and the initial concentration of NB was not observed

3.1.2. Effect of the initial concentration of NB on the bioreaction The results of experiment B demonstrated the effect of the NB concentration on the bio-reaction. The observed variation in the reaction rate and the shape of the plot of the NB concentration versus the reaction time were similar to those obtained in experiments A and C (Fig. 3). The initial NB concentration

Table 1 e Comparison of the OD680 and the elimination of NB in different growth phases. Period (h)

In the exponential growth phase (initial OD680 is 0.0673  0.0012) OD680a

0 e 24 24 e 96 96 e 168 168 e 240

0.0803 0.1496 0.3528 0.7229

 0.0184  0.0780  0.2164  0.3161

OD680a

Elimination of NB (mg/L) During this period 7.66  3.44 68.08  9.68 17.77  14.19 29.28  14.99

In the repressed growth phase (initial OD680 is 0.1917  0.0022)

b

Total 7.66 75.74 93.51 122.78

 3.44  9.89  10.94  10.94

Elimination of NB (mg/L) During this period

0.2076 0.2658 0.4168 0.5650

 0.0225  0.0673  0.1473  0.0654

a The value is the arithmetical mean of the OD680 during the specified reaction period. b The value is the sum of the elimination of NB from 0 h to the end of the specified reaction period.

10.93 80.33 26.16 21.03

 6.91  8.04  8.66  7.63

Totalb 10.93 91.25 117.42 138.45

   

6.91 4.33 7.62 1.42

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a

40ug/L C.G. 80ug/L C.G. 120ug/L C.G. 160ug/L C.G. 200ug/L C.G.

CNB(ug/L)

240

40ug/L E.G. 80ug/L E.G. 120ug/L E.G. 160ug/L E.G. 200ug/L E.G.

n dCNB ¼ k0 Cm A CNB dt

(3)

where CA is the density of M. aeruginosa (obtained as OD680), k0 is the reaction rate constant, m is the reaction order of M. aeruginosa, and n is the reaction order of NB.

3.2.2.1. Kinetic analysis without considering the growth of algae. Under actual conditions, the density of M. aeruginosa

160

varies over time. If the variation of algal density is insignificant, the algal density can be considered a fixed value. kobs was defined as the product of k0 and Cm A and was considered to be constant when the algal density and other environmental factors were stable. Thus, equation (3) was written as follows:

80

dCNB ¼ kobs CnNB dt

which is an nth order reaction. When n is equal to 1, the halflife of NB (t1/2) is a fixed value. When n is not equal to 1, t1/2 is a function of the initial concentration of NB ðCNB0 Þ, as shown in equation (5), and the slope of a plot of In t1/2 versus ln CNB0 is equal to 1  n (equation (6)).

0

Relative removal rate of NB(%)

b

45

30

t1=2 ¼

15

0

2n1  1 ðn  1Þkobs cn1 NB0

ln t1=2 ¼ ð1  nÞln CNB0 þ ln

0

60

120

180

Time(hour) Fig. 3 e Effect of the NB concentration on the elimination of NB. The numbers in the legend represent the initial NB concentration. C.G. represents the control group, and E.G. represents the experimental group. (a) Concentration of NB in the aqueous phase. (b) Relative removal rate of NB.

(r ¼ 0.1092, p ¼ 0.8612), which implied that the assumption was reasonable. According to the regression analysis of experiment B at different CNB0 values (40e160 mg/L), K was equal to 2.049  103(R2 ¼ 0.9733, Fig. S3, Supplementary data). To verify the kinetic model, the regression of K was used to predict the elimination of NB at an initial NB concentration of 200 mg/L (Fig. S4, Supplementary data). The predicted value was related to the actual value (r ¼ 0.9910, p ¼ 1.790  106), and the relative deviation was less than 4.0%, which indicated that the kinetic model was appropriate. The results of kinetic analysis indicated that the elimination of NB in distilled water was dependent on the concentration of NB and was a firstorder reaction.

3.2.2.

(4)

Bio-reaction in algal solution

The experimental results showed that the concentration of NB and algal density affected the bio-reaction rate; thus, assuming that each algal cell has the same effect on the bioreaction during the same period of time, the reaction rate could be described by the following equation. All of the kinetic analyses were performed based on this assumption.

(5)

2n1  1 ðn  1Þkobs

(6)

The result presented in Fig. S5 (according to the data obtained in experiment B, supplementary data) showed that ln t1/2 decreased with an increasing in ln CNB0 , and a negative linear correlation was observed (r ¼ 0.9623, p ¼ 0.03772), indicating that the elimination of NB was altered by the presence of M. aeruginosa and could not be described by a pseudo-first-order kinetic model with respect to the concentration of NB. Based on the slope shown in Fig. S5, the reaction order of NB was 1.692. The reaction order of NB was substituted into equation (4), and the resulting expression was integrated to obtain equation (7): c0:692 c0:692 NB0 NB ¼ kobs t þ 0:692 0:692

(7)

Thus, c0.692 /0.692 should be linearly correlated with the NB reaction time (t), and the slope of equation (7) should be equal to kobs. Using the data obtained in experiment A, the linear /0.692 and t at different initial regressions between c0.692 NB OD680 values were obtained, and the results are listed in Table 2. Because kobs¼k0Cm A , ln kobs should be linearly correlated to ln CA. Moreover, the slope should be equal to the reaction order of M. aeruginosa, and the intercept should be equal to ln K0, as shown in equation (8). ln kobs ¼ ln k0 þ mln CA

(8)

The linear fitting results showed that the reaction order of M. aeruginosa (m) was 0.5877, and the reaction constant k0 was 8.170  104 (Fig. S6, Supplementary data). Thus, the kinetic C1.692 equation is dCNB ¼ 0.0008170C0.5877 A NB dt, which indicates that the concentration of NB has a stronger effect on the bioreaction than the algal density. Previous studies have shown that algae can bio-react with xenobiotics via bioaccumulation

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where CA0 is the initial OD680 of M. aeruginosa, and k1 is the growth rate constant. Equation (9) was substituted into equation (3), and the resulting expression was integrated to obtain:

Table 2 e The kobs at different initial algal densities (according to the results of experiment A). kobs

CA Value 0.0148 0.0295 0.0494 0.0965 0.1917

    

0.0010 0.0013 0.0008 0.0015 0.0022

8.339  9.816  1.116  1.780  3.887 

Statistics

Standard error Adj. R-square 5

10 105 104 104 104

2.972 4.629 4.997 5.596 1.494

    

106 106 106 106 105

 CNB ¼

0.9850 0.9739 0.9765 0.9883 0.9826

The interval of regression was from 0 h to 168 h to reduce the interference of algal growth.

and biodegradation (Skoglund et al., 1996; Daneshvar et al., 2007; Schulte et al., 2008; Ogola et al., 2009); however, few researchers have demonstrated that the kinetic model changes when algae are present. In the current study, the presence of M. aeruginosa significantly altered the elimination of NB, and a first-order kinetic model with respect to the concentration of NB was not appropriate. However, the regression results (Fig. S6, Supplementary data) were barely acceptable when the effect of algal growth was neglected. The kinetic model that did not consider the growth of algae could not reflect the observed variation in the algal density and may not be precise enough to predict the elimination of NB in the algal solution. In particular, differences in the growth rates between experiment A and C were not considered by the model.

3.2.2.2. Kinetic analysis when algae are in the exponential growth phase. The growth of algae may be influenced by many factors, such as illumination, temperature, pH, nutrient content and NB concentration. When the concentration of NB is not lethal, the growth of algae can be regarded as a function of the reaction time ( f(t)) and is independent of CNB. When the algae are in the exponential growth phase, the algal growth was well fitted to Malthus’s model (equation (9)): CA ¼ CA0 ek1 t

(9)

CNB(ug/L)

120

1 1n

(10)

180 actual predicted

0.97182 Standard Error 1.90843 3.37456 2.05305E-4

120 C (ug/L)

Adj. R-Square Value m 0.72475 n 2.54066 k0 1.68704E-5

k0 Cm k0 Cm A0 mk1 t A0 e þ ð1  nÞ mk1 mk1

When the initial OD680 was 0.0673  0.0015 (experiment C), the growth of the algae could be described as CA ¼ 0.0673e0.01161t (Fig. S1 (b), Supplementary data). This expression was substituted into equation (10) and was nonlinearly fit to the experimental data. The results are shown in Fig. 4. Therefore, C2.5407 dt. the kinetic equation is dCNB ¼ 1.6871  105C0.7248 A NB The parameters obtained by nonlinear fitting were verified by predicting the elimination of NB at an initial OD680 of 0.0389  0.0010. The same analytical process described above was performed, and the algal growth could be described as CA ¼ 0.0389e0.01178t (adj. R2 ¼ 0.9972, Fig. S1 (a), Supplementary data). This expression was substituted into equation (10), and the following parameters were used: m ¼ 0.7248, n ¼ 2.5407 and k0 ¼ 1.6871  105. The predicted value was linearly correlated to the actual value (r ¼ 0.9842, p ¼ 7.498  109), and the relative deviation was less than 18.2% (Fig. 5). The bio-reaction rate was not constant, as mentioned in Section 3.1.1, and the reaction rate increased during the period from 24 h to 96 h, which was the main source of the deviation. Except for during this period, the relative deviation was less than 7.1%. This phenomenon indicated that the kinetic model could describe the general elimination of NB but could not fit the fast reaction period (24e96 h). These results also implied that the hypothesis that each algal cell has the same effect on the bio-reaction during the same time frame was not appropriate and suggested that another factor also affected the reaction rate, such as enzyme activity or metabolic activity in algae cells, as reported in prior studies (Yang et al., 2002; Schulte et al., 2008; Thies et al., 1996; Warshawsky et al., 1990; Tang et al., 1998; Papazi and Kotzabasis, 2008). The model could be improved if the mechanism of the bio-reaction is thoroughly studied.

CNB 160

C1n NB0  ð1  nÞ

80 60

40 0

50

100

150

200

250

300

Time(hour) Fig. 4 e Nonlinear fitting of the bio-reaction of NB by M. aeruginosa in the exponential growth phase at an initial OD680 of 0.0673 in experiment C.

0

0

100

200

300

Time(hour)

Fig. 5 e Predicted and actual elimination of NB in exponential growth algal solution at initial OD680 of 0.0389 in experiment C.

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3.3.

Application of kinetic model

The elimination of NB in experiments A and C was compared, and the results showed that the low reproduction rate in experiment A was unfavourable for the bio-reaction. However, it was difficult to distinguish whether the bioreaction ability of M. aeruginosa in experiment A also decreased due to the inhibited algal density. A kinetic analysis can help us solve this problem. In experiment A, the growth of algae was inhibited by the concentration of nutrition. When the growth of algae was fitted as a function of the reaction time, the elimination of NB could be predicted by the kinetic model, and the results could be regarded as the effect of algae in the exponential growth phase. The growth of M. aeruginosa at an initial OD680 of 0.1917  0.0022 in experiment A was fitted by linear regression (Fig. S7, Supplementary data), which could be described as CA ¼ 0.0019  t þ 0.1669 (R2 ¼ 0.9521). The formula and parameters (k0 ¼ 1.6871  105, m ¼ 0.7248 and n ¼ 2.5407) were substituted into equation (3), and the resulting expression was integrated to obtained: 0:6491  : CNB ¼ 0:008372  ð0:0019t þ 0:1669Þ1:7248 This formula described the predicted elimination of NB by algae in the exponential growth phase, which has the same growth rate as inhibited algae at an initial OD680 of 0.1917  0.0022 in experiment A. This result was compared to the actual value obtained when the algae were inhibited by nutrition, as shown in Fig. 6. The elimination trends of NB were similar, and a significant linear correlation was observed (r ¼ 0.9733, p ¼ 4.666  105), which indicated that nutritional limitation had little direct impact on the bio-reaction ability of M. aeruginosa. In other words, algal cells inhibited by nutrition had the same effect on the bio-reaction as algae in the

180

actual predicted

120 C (ug/L)

Similar to the kinetic model that did not consider algal growth, the kinetic model that considered algal growth in the exponential growth phase showed that variations in the NB concentration has a stronger effect on the bio-reaction than that of the algal density. However, significant differences in the values of the parameters between the two models were observed due to differences in the analytical methods. The two types of kinetic models showed that the reaction orders were not integers, which could be attributed to the complexity of the bio-reaction. Xenobiotics can be bioaccumulated into different organelles of algae (Jabusch and Swackhamer, 2004) and can be biodegraded by different enzymes (Yang et al., 2002; Schulte et al., 2008; Thies et al., 1996; Warshawsky et al., 1990; Tang et al., 1998). Thus, both the transfer efficiency and enzyme activity affect the bioreaction. Papazi and Kotzabasis (2008) indicated that the metabolism of S. obliquus was inhibited by nitro-phenols, which may affect the biodegradation rate of xenobiotics. These complex reaction processes were described by a onestep kinetic model, and the reaction order represented the total reaction order, which was affected by bioaccumulation and biodegradation. Therefore, in the present study, the reaction order can not be an integer, and a pseudo-first-order reaction model was too oversimplified to describe the reaction.

60

0 0

60

120

180

Time(hour)

Fig. 6 e Predicted and actual elimination of NB in an algal solution inhibited by nutrition at an initial OD680 of 0.1917 in experiment A.

exponential growth phase. However, the nutritional limitation inhibited the growth of algae, and the low algal density reduced the reaction rate. The metabolic rate expressed as the photosynthetic and respiration rates provided further explanation for the results of the kinetic analysis. The photosynthetic and respiration rates of M. aeruginosa which were inhibited by nutrition at an initial OD680 of 0.1917, were 423.9  64.5 mg/108 cells/h and 143.5  8.4 mg/108 cells/h, respectively, which was approximately the same as that of M. aeruginosa in the exponential growth phase (430.7  113.9 mg/108 cells/h and 137.3  27.0 mg/ 108 cells/h, respectively). Biodegradation is the principal pathway for the bio-reaction of NB with M. aeruginosa. Thus, the photosynthetic and respiration rates may determine the bio-reaction ability of M. aeruginosa. In experiment A, limited nutrition inhibited the reproduction of M. aeruginosa, however, algal survival was maintained. Because the photosynthetic and respiration rates were not significantly inhibited, algae inhibited by nutrition may have the same ability to biodegrade NB as that of algae in the exponential growth phase.

4.

Conclusions

In the present study, the bio-reactions of NB with M. aeruginosa at different initial algal densities and NB concentrations were investigated. According to the experimental data, an empirical kinetic model was developed and examined. The key finding from the present research are listed below:  The bio-reaction significantly accelerated the elimination of NB in aqueous solution. The reaction rate was not constant, and the highest reaction rate was obtained from 24 h to 96 h of incubation.  High initial concentrations of NB and high algal densities favoured the bio-reaction.  The kinetic analysis showed that a first-order reaction model fitted the elimination of NB in a solution without algae, but was not suitable for the elimination of NB from an algal solution.

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 9 0 e2 2 9 8

n  The model dCNB ¼ k0Cm A ANBdt described the elimination of NB in an algal solution.  The kinetic analysis showed that nutritional limitation did not inhibit the bio-reaction ability of M. aeruginosa. However, nutritional limitation inhibited the growth of algae and reduced the reaction rate.

Acknowledgments This work was supported by the Funds for Creative Research Groups of China (Grant No. 51121062), the National Natural Science Foundation of China (Number: 50778048), and State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2010TS08).

Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.watres.2012.01.049.

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