Statistical modelling and optimization of substrate composition for bacterial growth and cadmium removal using response surface methodology

Ecological Engineering 37 (2011) 2076–2081

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Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng

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Statistical modelling and optimization of substrate composition for bacterial growth and cadmium removal using response surface methodology Divya Bhatia a , Rajender Kumar b,∗ , Rajesh Singh b , Rout Chadetrik b , Narsi R. Bishnoi b a b

Université Paris-Est, Laboratoire Géomatériaux et Environnement, EA 4119, 5 bd Descartes, 77454 Marne la Vallée Cedex 2, France Department of Environmental Science & Engineering, Guru Jambheshwar University of Science and Technology, Hisar 125001, India

a r t i c l e

i n f o

Article history: Received 21 May 2011 Received in revised form 14 July 2011 Accepted 7 August 2011 Available online 6 September 2011 Keywords: Substrate composition Operating variables A. xyloxidans Sorption Response surface methodology (RSM) Cadmium

a b s t r a c t In this study, substrate composition was optimized for the growth of Achromobacter xyloxidans and biosorption of Cd(II) from aqueous solution. Response surface methodology (RSM) was used to investigate the function of three independent operating variables, namely, peptone (2.5–10 g/L), beef extract (2.5–5.0 g/L) and incubation time (24–96 h), on dependent variables, i.e. sorption of Cd(II) ions, protein content and biomass growth of A. xyloxidans. The maximum Cd(II) removal efficiency of 69.2%, protein content 1.9 mg/L and growth 0.354 optical density was found at optimal conditions of peptone 10 g/L, incubation time 60 h and beef extract 2.5 g/L. The significance of independent variables and interactions between variables were tested by means of the analysis of variance (ANOVA) with 95% confidence limits and values of “Prob > F” less than 0.0500 indicate that model terms are significant. Fourier transfer infrared (FTIR) analysis was used to investigate sorption mechanism and involved functional groups in Cd(II) binding. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The increase of industrial activities has intensified environmental pollution problems and the deterioration of several ecosystems with the accumulation of many pollutants, such as toxic metals (Zouboulis et al., 2004). The retention of heavy metal at high concentrations in the environment exerts toxic effect on fauna and flora (Xue et al., 2010) such as loss of ecosystem and agriculture productivity, diminished food chain quality, and tainted water resources. Cadmium is among the heavy metals regarded as having high toxicity. On the basis of the toxicity, persistence and bioaccumulation, Cd(II) has been proposed in the red list of priority substances under the Department of Environment, UK (1991). Since microorganisms have developed survival strategies in heavy metal polluted habits, their different microbial detoxifying mechanisms such as bioaccumulation, biotransformation, biomineralization or sorption can be applied either ex situ or in situ to design economical bioremediation processes (Beveridge and Doyle, 1989; Lim et al., 2003; Umrania, 2006; Sasmaza et al., 2008; Kumar et al., 2008; Hanif et al., 2009; Lawal et al., 2010). Generally, the sorption mainly utilizes the various types of functional groups like carboxyl, phosphoryl, sulfhydryl, hydroxyl, amino, etc. (Sharma et al., in press)

which are present on the surface of living or dead microorganisms to remove the heavy metals from aqueous solutions. Response surface methodology is a combination of mathematical and statistical techniques used for developing, improving and optimizing the processes and used to evaluate the relative significance of several affecting factors even in the presence of complex interactions (Myers and Montgomery, 2002). Recently, this methodology is being used for the optimization of various biological processes such as enzyme production (Senthilkumar et al., 2005), hydrogen production (Ghosh and Hallenbeck, 2010), secondary metabolite production (Lofty et al., 2007) and dye decolorization by Nostoc linckia (Sharma et al., in press). Utilization of this tool for the optimization of process parameters for heavy metal removal from wastewater has also used to optimize the temperature, pH and metal concentration (Singh et al., 2010). In this study, three variables were optimized, i.e. peptone concentration, beef extract and incubation time, and used to evaluate the single and interactive effects of the variables on responses. FTIR study was carried out to understand surface properties and available functional groups involved in sorption mechanism. 2. Materials and methods 2.1. Experimental set up for batch study

∗ Corresponding author. Tel.: +91 1662263321. E-mail address: [email protected] (R. Kumar). 0925-8574/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2011.08.014

The bacterial strain Achromobacter xyloxidans used for this study was isolated aerobically in 100 ml sterile nutrient broth medium at pH 7.0, temperature 35 ± 1 ◦ C for 48 h on shaking incubator

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Table 1 Experimental design and results of the responses. Experiments run

Incubation time (h)

Peptone (g/L)

Beef extract (g/L)

% Removal

Protein (mg/L)

Growth (OD 620 nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

96 96 60 60 60 24 24 96 60 24 60 24 60 60 96 60 60

2.5 6.25 2.5 6.25 2.5 2.5 6.25 6.25 10 6.25 6.25 10 6.25 6.25 10 6.25 10

3.75 2.5 2.5 3.75 5.0 3.75 5.0 5.0 2.5 2.5 3.75 3.75 3.75 3.75 3.75 3.75 5.0

24.07 48.2 26.93 65.95 23.87 19.67 37.65 45.33 69.17 42.85 65.65 52.98 64.73 64.35 68.12 64.89 61.45

1.69 1.62 1.39 1.74 1.43 1.23 1.37 1.45 1.87 1.31 1.74 1.4 1.76 1.87 1.81 1.87 1.73

0.226 0.115 0.098 0.265 0.116 0.074 0.094 0.101 0.354 0.093 0.265 0.098 0.267 0.336 0.323 0.336 0.243

ANOVA for RSM model: Cd(II) removal: Fmodel = 5005.96 (P > 0.0001, df = 9); FLack of Fit = 6.97 (P > 0.0713, df = 3); R2 = 0.9983; protein content: Fmodel = 0.68468 (P > 0.0093, df = 9); FLack of Fit = 0.05883 (P > 0.0985, df = 3); R2 = 0.8985; bacterial growth: Fmodel = 0.144294 (P > 0.00497, df = 9); FLack of Fit = 0.024519 (P > 0.0665, df = 3); R2 = 0.8257.

at speed 120 rpm from electroplating industrial soil. For isolation, inoculated plates were incubated at 37 ◦ C for 72 h. The pure colony was obtained and identified from microbial type culture collection (MTCC), Institute of Microbial Technology, Chandigarh. The experiments were conducted to optimize pH, temperature, Cd(II) ions concentration and incubation time for sorption of Cd(II) ions and growth of bacterial sp. A. xyloxidans in batch process. For pH optimization, 50 ml of nutrient media with varying pH range from 1.0 to 9.0 at 100 mg/L concentration of Cd(II) ions were incubated at 34 ◦ C for 48 h. The pH value of solution was adjusted using 0.1 M HCl or 0.1 M NaOH solution. Effect of temperature was studied from 25 to 45 ◦ C at initial metal ions concentration 100 mg/L for 48 h. Similarly initial metal ions concentration was optimized with varying concentration from 20 to 120 mg/L, keeping the optimum pH and temperature from above experiments. For incubation time, bacterial growth (optical density OD) was monitored at regular interval time 8, 16, 24, 32, 40, 48, 56, 64 and 72 h at 620 nm using UV-spectrophotometer. After a fixed interval of time, 10 ml culture sample was withdrawn and centrifuged for 20 min. Supernatant and pellets were taken separate. The pellets were kept in oven at 70 ◦ C till the constant weight was attained (Chubar et al., 2008). After centrifugation, supernatant was used for analysis of protein content. The total protein content in the solution was estimated by Lowry’s method (Lowry et al., 1951). After acid digestion (HNO3 + HCl), removal of Cd(II) ions was analysed using atomic absorption spectrophotometer (GBC-932 Plus). The experiments were performed in duplicate. Uptake capacity and % removal of metal ions were calculated according to equations given in previous study (Kumar et al., 2008). 2.2. RSM and statistical analysis Design Expert software (Stat Ease, 7.13 trial version) was used for experimental design towards construction of a quadratic model (Table 1). Three independent variables i.e. peptone (2.5–10.0 g/L), incubation time (24–96 h), and beef extract (2.5–5.0 g/L) were taken to obtain responses removal of Cd(II) ions, growth of bacterial strains and protein content secreted by bacterial strains at pH 7.0, temperature 35 ◦ C and Cd(II) ion concentration 100 mg/L. The quadratic equation model for predicting the optimal point was expressed according to equations, a three parameters were varied, 10 coefficients has to be estimated, i.e. coefficients for the three

main effects, three quadratic effects, three interactions and one constant (Reddy et al., 2008): Yi = a0 +



ai Xi +



aii Xii2 +



aij Xi Xj + e

(1)

where Yi (i = 3) is predicted response i.e. sorption of Cd(II) ions with bacterial strain, a0 is the constant coefficient, ai is ith linear coefficient, aii is ith quadratic coefficient and aij is different interaction coefficients of the model; Xi Xj are coded independent variables related to factors, and e is error of model. However, in this study, the independent variables were coded as A, B and C. Thus, the second order polynomial equation can be presented as follows: Y = a0 + ai A + ai B + ai C + ai A2 + ai B2 + ai C 2 + aij A ∗ B + aij A ∗ C + aij B ∗ C

(2)

In this study, Cd(II) removal, protein content and growth was preceded for Eq. (2) including ANOVA to obtain interaction between process variables and the responses. The quality of fit of polynomial model was expressed by the coefficient of determination (r2 ) and statistically significance by F-test in the programme. 2.3. Fourier transform infrared spectroscopy (FTIR) FTIR spectrum study was carried out to explain sorption mechanism for identifying the presence of functionalities of the bacterial biomass. The sample preparation, technical detail of the instrument has already been mention in our earlier study (Singh et al., 2010). 3. Results and discussion 3.1. Effect of operating parameters on bacterial growth and Cd(II) removal Growth curves of the bacterial isolate in different pH (1.0–9.0) were plotted and indicate that the cells were grown well at pH 7.0 and decrease at lower and higher pH range (Fig. 1(A)). Maximum removal of Cd(II) was observed 68.5% during the incubation time 48 h at pH 7.0. The medium pH affects the solubility of metals and the ionisation state of the functional groups (carboxylate, phosphate and amino groups) of the microbial cell (Bishnoi and Garima, 2005). The optimum temperature is 35 ◦ C at which maximum bacterial growth was noticed at pH 7.0, incubation time 48 h

D. Bhatia et al. / Ecological Engineering 37 (2011) 2076–2081

Absorbance (620 nm)

Absorbance (620 nm)

60 50 40 30

1.2 1 0.8 0.6

20 10 0

0.4 0.2 0 1

2

3

4

5

6

7

8

OD (620 nm)

1.4

% Removal

80 70

1.6

OD (620 nm)

B

% Removal

0.6

80

0.5

70

0.4

60 50

0.3

40

0.2

30 20 10

0.1

0

0

9

25

28

pH % Removal

D

Absorbnce (620 nm)

90 80 70 60 50 40 30 20 10 0

1.6 1.2 1 0.8 0.6 0.4 0.2 0 40

60

80

100

120

Cd Ions concentration (mg/L)

% Removal

1.4

20

33

35

38

40

45

Temperature ( 0C)

OD (620 nm)

% Removal

C

% Removal

% Removal

Protein content

70

2

60

1.8 1.6

50

1.4

40

1.2

30

1 0.8

20

0.6

10

0.4 0.2

0

8

16

24

32

40

48

56

64

72

Protein content (mg/ml)

A

% Removal

2078

0

Time (hr)

Fig. 1. Factors affecting on removal of Cd(II) ions and bacterial growth (A) at various pH, (B) temperature, (C) initial concentration of Cd(II) ions and (D) different incubation time.

and 100 mg/L of Cd(II) ions concentration (Fig. 1(B)). The bacterial growth and Cd removal was decreased at heigher or lower than optimum temperature 35 ◦ C. High temperature makes inhibition effect on the growth of microorganism, microbial death was observed as well as growth and bioaccumulation of Cd(II) ions decreased with increasing temperature. At low temperatures, the binding of heavy metal ions to microorganism is by passive uptake (Schiewer and Volesky, 2000). Maximum removal of Cd(II) was observed 85% at initial concentration of 20 mg/L in 48 h. A significant reduction in growth of A. xyloxidans was predicted at concentration 120 mg/L (Fig. 3(C)). This is fact that, as Cd increases in growth medium the toxicity increases which leads to the decrease the bacterial biomass available for metal accumulation. This toxicity effect of Cd2+ was expressed by the rate of bacterial growth suppression relative to a control of bacterial growth without cadmium for Enterobacter strains (Khleifat et al., 2006). Removal of metal ions was increased with increases contact time due to longer log phase. In the initial period of time no cells growth and lag phase was found 8 h at concentration 100 mg/L. Highest removal of Cd(II) ions was observed 66.24% during the stationary phase in 56 h (Fig. 1(D)). The relationship between crude protein contents and sorption of heavy metals, indicating certain protein is a key molecule to bind heavy metals (Nakajima et al., 1981). The protein content was investigated in liquid nutrient agar medium amended with 100 mg/L of metal ions at different time intervals (Fig. 1(D)). The higher amount of protein in the culture having Cd dose clearly indicated the expression of the metal binding proteins under the stress conditions. These results suggested that proteins of cell components played an important role in metal uptake.

3.2. Validation of response surface models and statistical analysis The present model and data analysis were not only allowed to define optimum condition for Cd(II) removal, protein and growth but also showed the interactive effect of independent variables on the responses. The relationship between the independent variables and responses were based on the quadratic polynomial equations (3a)–(3c): % Removal (Y1 ) = +65.11 + 4.07 ∗ A + 19.65 ∗ B − 2.36 ∗ C + 2.68 ∗ A ∗ B + 0.58 ∗ A ∗ C − 1.16 ∗ B ∗ C − 12.88 ∗ A2 − 11.03 ∗ B2 − 8.73 ∗ C 2

(3a)

Protein (Y2 ) = +1.80 + 0.16 ∗ A + 0.13 ∗ B − 0.026 ∗ C − 0.012 ∗ A ∗ B − 0.058 ∗ A ∗ C − 0.045 ∗ B ∗ C − 0.22 ∗ A2 − 0.048 ∗ B2 − 0.14 ∗ C 2

(3b)

Growth (Y3 ) = +0.29 + 0.051 ∗ A + 0.063 ∗ B − 0.013 ∗ C + 0.018 ∗ A ∗ B − 3.75E − 3 ∗ A ∗ C − 0.032 ∗ B ∗ C − 0.11 ∗ A2 − 5.775E − 3 ∗ B2 − 0.085 ∗ C 2

(3c)

The analysis of variance (ANOVA) for sorption of Cd(II) ions was used in order to ensure a good model, the test for significance of regression model and the results of ANOVA are shown in Table 1.

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b

1.9

61.0

0.29

1.7

47.4

0.23

33.8 20.2

Protein (mg/L)

0.36

10.0

0.16 0.10

10.00 96.0

8.1

Peptone

42.0 2.5

Peptone

Time

24.0

96

8.13

42

Peptone

Time

24

1.8

Protein (mg/L)

1.9

0.29

Growth (OD)

0.36

20.2

0.23 0.16 0.10

5.00

3.75 4.4

2.50

2.5

1.6 1.5

Beef extract

4.38

2.50

2.50

Beef extract

Peptone

h 0.29

1.7

47.4

0.23

96.0

4.38

78.0

3.75

Beef extract

60.0

3.13 2.50

42.0 24.0

Time

Protein (mg/L)

61.0

Growth (OD)

1.8

5.00

4.38 2.50

2.50

Peptone

i

0.36

20.2

6.25

3.13

74.7

33.8

8.13

3.75

6.25

3.13

Peptone

g

10.00 4.38

8.13

3.75

6.3

3.13

1.7

10.00 4.38

8.1

Beef extract

Time

24

5.00

10.0 4.38

42

f

61.0

33.8

60

4.38 2.50

74.7

47.4

78

6.25

e

5.00

%Removal

1.2

60

4.38 2.50

d

1.4

78

6.25

60.0 4.4

1.6

10.00

96 8.13

78.0 6.3

%Removal

c

74.7

Growth (OD)

%Removal

a

2079

0.16 0.10

1.5 1.4 1.2

5.00

5.00

96

96 4.38

78

3.75

Beef extract

42 2.50

24

78

3.75

60 3.13

4.38

Time

Beef extract

60

3.13

42 2.50

24

Time

Fig. 2. Interactive effect of two variables, i.e. peptone and incubation time at constant beef extract concentration 3.75 g/L (A) % removal of Cd(II) ions, (B) bacterial growth, (C) protein content; i.e. peptone and beef extract at constant incubation time 60 h, (D) % removal of Cd(II) ions, (E) bacterial growth, (F) protein content; i.e. beef extract and incubation time at constant peptone concentration 6.25 g/L (G) % removal of Cd(II) ions, (H) bacterial growth, and (I) protein content.

The larger values of linear coefficient for peptone (Eqs. (3a) and (3c)) incubation time (Eq. (3b)) illustrated the positive and significant effect of the variables on Cd(II) removal, growth and protein content. The positive linear coefficient for incubation time and peptone indicated the increase in response with increases incubation time and peptone concentration. The negative quadratic coefficients were indicates the existence of maximum response to a point for all the variables beyond that entire variables shows an inhibitory effect on Cd(II) removal, growth and protein content.

3D resonse surface plots were constructed which were selected on the based of interactive effect of two independent variables with another variable being at fixed level. The quadratic regression was showed that model is significant and value of Prob > F is less than 0.0500 indicates the significance of the model terms. The value of correlation coefficient R2 is 0.9983, 0.8985 and 0.8257 for % removal of Cd(II) ions, protein and growth of cells, respectively. The value of R2 is closer to 1.0 indicates the better fitness of model. The value of coefficient variation was indicate a very high

2080

D. Bhatia et al. / Ecological Engineering 37 (2011) 2076–2081

Fig. 3. Fourier transform infrared absorption spectrum of sorption of Cd(II): (A) native biomass sp. A. xyloxidans and (B) Cd(II) ions loaded biomass.

degree of precision and a good deal of reliability of experimental values. 3.3. Interactive effect of peptone, beef extract and incubation time on responses Peptone significantly affects sorption of Cd(II) ions (P > 0.0001) and had a positive effect on all the responses, i.e. sorption of Cd(II), protein content and microbial growth (Eqs. (3a)–(3c)). It was observed that increases peptone amount increased sorption of Cd(II) ions. Peptone is a major source of protein, i.e. nitrogen, and it provides nutrients for the bacterial growth, which enhanced removal of Cd(II) ions. Beef extract showed negatively effect on all the three responses and inhibitory affect on sorption on Cd(II) (P > 0.0001). The insignificant effect was also observed for protein production and growth response. Incubation time also play an important role in sorption of Cd(II) (P > 0.0001) and had a positive effect on protein production and growth of bacterial cells. Fig. 2(A)–(C) shows the interactive effect of two independent variables peptone and incubation time and one variable beef extract 3.75 g/L at fixed level. The positive and larger value of interactive coefficient pointed out that the enhancing effect on the % removal of Cd(II) ions and growth but inhibitory effect was observed on protein production Eqs. (3a)–(3c). The study reported that maximum removal of Cd(II) ions, protein content and growth was observed 65.95%, 1.74 mg/ml and 0.265 at optimum concentration for peptone 6.25 g/L and incubation time 60 h. The 3D plot showed that optimum incubation time was 60 h which can be confirmed from the Cd(II) removal and growth pattern of the strain (Fig. 2(A) and (B)). After that, % removal of Cd(II) ions decreased due to decrease in growth. Protein secretion was not decreased beyond optimal incubation time, indicating that the secretion of protein was not stopped with growth (division) of cells i.e. the cell

continued to secrete the protein in the stationary phase under the stress conditions up to 72 h and after that decrease in secretion of protein was observed (Fig. 2(C)). The increase in Cd(II) removal was observed with increasing peptone dose. This trend can be confirmed from the growth and protein secretion, which are also increased with addition of peptone. Fig. 2(D)–(F) shows the interactive effect of beef extract and peptone and had negative interactive effect on all the response (Eqs. (3a)–(3c)). The 3D response plot showed the removal of Cd(II) ions increased with increase in peptone concentration whereas, the removal of Cd(II) ions decreased at beyond an optimum value of beef extract 3.75 g/L (Fig. 2(D)). The similar results were also obtained with the protein secretion and growth of bacterial sp. (Fig. 2(E) and (F)). It was observed that increase in growth of bacterial sp. released more protein content and more removal of Cd(II) occurs due to more available binding sites. Fig. 2(G)–(I) shows the interactive effect of beef extract and incubation time on the responses. The positive value of interactive coefficient for beef extract and incubation was pointed out the enhancing effect on the % removal of Cd(II) ions (Eq. (3a)) but the negative interactive value was observed for protein and growth (Eqs. (3b) and (3c)). The significant value for linear coefficient of incubation time for % removal of Cd(II) ions and protein production indicated that increased in the variable enhanced response whereas, high non-significant value of beef extract for linear coefficient and for all the response as a result of their interaction showed inhibitory effect. Fig. 2(G) shows the maximum Cd(II) removal was 65.5% at incubation 60 h and beef extract 3.58 g/L. Maximum growth was observed at 60 h and 3.63 g/L beef extract (Fig. 2(H)), whereas, protein secretion was 1.828 mg/L at incubation time 72 h and 3.58 g/L of beef extract (Fig. 2(I)). The high value of protein production clearly indicated protein secretion by cell at stationary phase, i.e. non-dividing phase.

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3.4. FTIR analysis FTIR spectral analysis was done before and after metal loading of Cd(II) ions to find out the involvement of functional groups in sorption with bacterial biomass A. xyloxidans (Fig. 3). Broad spectral bands in the region of 3500–3300 cm−1 are the characteristics of N–H or O–H stretching vibrations and other bands around 2920–2850 cm−1 are the characteristics of alkyl chains. 3308.9 cm−1 is representing bonded –OH, and –NH groups. 2925.0 cm−1 could be assigned to the –CH stretching vibration of –CH3 and –CH2 groups. A strong asymmetrical stretching band at 1654.4 cm−1 corresponding to the amide I and 1545 cm−1 is bonded to amide II groups. The peaks of 1377.9, 1025 cm−1 indicative –CO, –C–O, –COO− , P O and P–OH indicates of existing groups involved in sorption mechanisms. 4. Conclusion In this study, response surface methodology was used to provide a critical analysis of the simultaneous interactive effects of independent variables for a better understanding of the Cd(II) removal process with A. xyloxidans. The maximum responses were found to be 65.65% Cd(II) removal, 1.74 mg/ml of protein content and 0.265 for bacterial growth at optimum independent variables such as peptone 6.25 g/L, beef extract 3.75 g/L and incubation time 60 h. This model explained 99.83%, 89.85% and 82.57% removal of Cd(II) ions, protein content and growth of cells, respectively. FTIR spectral analysis was done and observed main participated functional groups are –OH, and –NH, –CH stretching, –C–O, –COO− , P O and P–OH in binding of Cd(II) ions. The proposed response surface methodology proved to be an important tool for process optimization, time saving and to be most realistic where a large number of variables influences the process. References Beveridge, T.J., Doyle, R.J., 1989. Metal Ions and Bacteria. Wiley, New York. Bishnoi, N.R., Garima, 2005. Fungus: an-alternative for bioremediation of heavy metal containing wastewater: a review. J. Sci. Ind. Res. 64, 93–100. Chubar, N., Behrends, T., Cappellen, P.V., 2008. Biosorption of metals (Cu2+ , Zn2+ ) and anions (F− , H2 PO4 − ) by viable and autoclaved cells of the Gram-negative bacterium Shewanella putrefaciens. Colloid Surf. B: Biointerfaces 65, 126–133. Ghosh, D., Hallenbeck, P.C., 2010. Response surface methodology for process parameter optimization of hydrogen yield by the metabolically engineered strain Escherichia coli DJT135. Bioresour. Technol. 101, 1820–1825.

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