International Journal of Bio-Technology

& Research (IJBTR)

ISSN 2249-6858
Vol. 3, Issue 1, Mar 2013, 37-48
TJPRC Pvt Ltd.

RESPONSE SURFACE METHODOLOGICAL APPROACH TO OPTIMIZE PROCESS

PARAMETERS FOR THE BIOMASS PRODUCTION OF CHLORELLA PYRENOIDOSA
RAJASRI YADAVALLI1, RAMGOPAL RAO S2 & C. S. RAO2
1

Associate Professor, Department of Biotechnology, Sreenidhi Institute of Science and Technology (Autonomous)
Affiliated to Jawaharlal Nehru Technological University, Hyderabad, Andhra Pradesh, India

Department of Biotechnology, Sreenidhi Institute of Science and Technology, Hyderabad, Andhra Pradesh, India

ABSTRACT
Biomass concentration and overall lipid productivity hold the key for economic feasibility of algal oil for
biodiesel production.

For achieving higher yields of Chlorella pyrenoidosa, we employed Plackett-Burman design

followed by Response surface methodology using a Central-Composite design to derive the functional relationship
between the algal growth and process parameters in this study. Preliminary experiments revealed that light intensity, mode
of operation (Batch/Fed batch), sodium nitrate concentration had influence on biomass production of C. pyrenoidosa.
These factors were identified by using Plackett-Burman design and further optimized by response surface methodology.
The optimal process parameters obtained for achieving the maximum yield from C. pyrenoidosa were light intensity =
130.77 mol m-2s-1, and NaNO3 = 1.78g/L, respectively. The maximum predicted value of biomass obtained 2.956 g/L was
about 1.3-fold higher than using the original medium. We conclude that RSM approach eventually helps in bulk production
of C. pyrenoidosa for future industrial applications.

KEYWORDS: Light Intensity, Nitrogen, Chlorella Pyrenoidosa, Response Surface Methodology

INTRODUCTION
Global warming caused by increasing concentrations of greenhouse gases resulting from human activity such as
fossil fuel burning and deforestation has become one of the most serious environment problems. In recent years, many
attempts are being made to reduce the quantity of CO2 in the atmosphere. Studies on photosynthesis, CO2 fixation and
utilization of micro algae biomass have been carried out. Chlorella strains known widely for their high valued potential
substances such as chlorophyll, beta-carotene, protein, lipid content, can also be used as potential biomass albeit the
function of CO2 fixation 1.
Microalgal mass culture has been utilized for nearly six decades as a potentially important source of many
products, such as biofuels (biodiesel, biohydrogen), aquaculture feeds, human food supplements and pharmaceuticals

2-3

Even though biodiesel production from algal biomass is pertinent, the relatively high cost is a major obstacle for
commercial production. Biomass concentration, increasing lipid content, and overall lipid productivity hold the key for
economic feasibility of algal oil for biodiesel production. While the overall lipid productivity determines the costs of the
cultivation process, biomass concentration and lipid content significantly enhance the downstream processing costs. In this
context, process optimization that can maneuver the algal biochemical production and fast growth helps to achieve
environmental and economic sustainability.
Currently, Chlorella sp is cultivated extensively in photoautotrophic conditions for enhancing biomass
productivity. As microalgae growth by autotrophic means produce lower biomass, process optimization efficiently

38

Rajasri Yadavalli, Ramgopal Rao S & C. S. Rao

enhances the yield of a desired metabolic product in a microbial system 4. In a fermentation process, optimization of the
variables provides information on their significant effects on the microbial growth and also their interaction at varying
levels. Conventionally, one independent variable is usually studied whilst all the other factors are maintained at a fixed
level. This approach could lead to unreliable results and erroneous conclusions. Furthermore, it does not guarantee the
optimization of process conditions, and fails to detect the frequent interactions that invariably occur between two or
more factors.
On the other hand, Response surface methodology (RSM) is a novel statistical method employed to analyze
problems wherein the response is dependent on several independent variables with an objective to maximize the process
variables for achieving optimum response 5. RSM uses quantitative data from appropriate experiments to determine and
simultaneously solve multivariate equations 6-7. RSM reduces the number of experiments eventually saving time, chemicals
and labor. Furthermore, it offers a rapid and reliable prediction of response, making it a lucrative option for experimental
design. Research indicates that so far such experimental approach has not been reported for enhancing the Chlorella
biomass concentration. Thus, the main objective of this study is to optimize the process parameters for obtaining higher
biomass productivities of Chlorella pyrenoidosa employing Plackett-Burman design followed by Response surface
methodology using a Central-Composite design (CCD).

MATERIALS AND METHODS

Algal Strain and Inoculum Preparation
Chlorella pyrenoidosa sp. (NCIM NO: 2738) was obtained from National Centre for Industrial Microorganisms
(NCIM), Pune, India. Stock culture of Chlorella pyrenoidosa was grown photoautotrophically in BG11 media at 28o C
under continuous light illumination in four 100 ml borosil flasks. Basal medium was slightly modified for use in this
study. Each liter of the BG11 medium contained NaNO3-1.5g, K2HPO4-0.04g, MgSO47H2O-0.075g, CaCl22H2O-0.036g,
Citricacid-0.006g, NaCO3-0.02g, H3BO3-0.00286g, MnCl24H2O-0.00181g, ZnSO47H2O-0.00022g, Na2 MoO42H2O0.00039g,CuSO45H2O-0.00008g,Co(NO3)26H2O-0.00005g, (NH4)6Mo7O24.4H2O-0.003 g, Na2EDTA-0.00001 g.
The inoculum was prepared by transferring the cells from stock culture, and incubated aseptically in a 250 ml
flask containing 100 ml of fresh BG11 media under continuous illumination of 34 mol m-2s-1 at 28o C for four days on an
orbital shaker set at 120 rpm. A 4 day old culture was used as inoculum at 10% volume for the preparation of stock
cultures.
Culture was prepared in 250 ml flasks by inoculating 39 ml of the 4 day old seed culture into 8 flasks, each
containing 100 ml of sterilized, fresh BG11 media of varying concentrations of sodium nitrate, respectively. Both low and
high concentrations of sodium nitrate were tested and denoted from N-1 to N-8 (Table 1). The flasks were incubated for 4
days at 28oC in a continuous light illumination of 110 mol m-2s-1 and 135 mol m-2s-1 on an orbital shaker set at 120 rpm.
Biomass Dry Cell Weight (DCW) Measurement
Biomass content was determined by measuring the optical density of samples at 600 nm (OD600). The conversion
factor was established by plotting OD600 versus DCW of a series of samples of different biomass concentrations. Samples
were diluted by appropriate ratios to ensure that the measured OD600 values were in the range of 0.20.9. DCW of a sample
was determined gravimetrically after drying, the algal cells were collected from samples with centrifugation (3,000g, 10
min) and washed with water. Linear regression equation obtained was found to be Y = 1.038557658*10-1 X 7.295013686*10-4 and r = 9.83945870610-1 where y is DCW of algal cells and x is optical density at 600 nm.

Response Surface Methodological Approach to Optimize Process

Parameters for the Biomass Production of Chlorella Pyrenoidosa

39

Experimental Design and Optimization

Plackett-Burman Design
Pilot experiments revealed that four factors, including light intensity, mode of operation (Batch/Fed batch),
Sodium Nitrate concentration (nitrogen concentration) and pH had influence on biomass production of Chlorella
pyrenoidosa. It is well known that the Plackett-Burman design could evaluate the main effects of factors. The
factors having significant effects on biomass production of Chlorella pyrenoidosa were identified using SigmaXL
Version 6.1 Workbook (trial version). Each factor was investigated at a high (+1) and a low (-1) level . The factors,
with more effect (more contribution) were considered to have greater effects on the biomass production of Chlorella
pyrenoidosa and it is further optimized by response surface methodology using Central-Composite design. The firstorder model used to fit the results of Plackett-Burman design was represented as
(1)
Central Composite Design
CCD is employed to fit a second-order model. The design was generated by commercial statistical package,
Design-Expert version 7.0 (Statease Inc.,Minneapolis, USA, Trial version). The levels were calculated and experiments
were performed using CCD. The two independent formulation variables selected for this particular study were light
intensity and nitrogen concentration (sodium nitrate).
The actual and corresponding coded values of different variables along with experimental data values were
summarized in Table 2. CCD is an efficient and proven design, especially for two factors. CCD is also rotatable, which
means that all the points in the design area are at equal distance from center.
This leads to distribution of errors among all points equally. The numbers of design points in CCD are based upon
a complete 2k factorial. The total numbers of experiments are
N = 2k+2k + m,

(2)

where N is the total number of experiments, k is the number of factors, and m is the number of replicates. The test
variables were coded according to the following equation:
xi=

(3)

Where, xi is the coded variable and Xi is the natural value of independent variable, Xi is the value of the variable
at the center point and X is the step change value.
Multiple linear regression analysis was used, and the data was fitted as a second-order equation. The general
equation that was fitted is
Y = 0 + iXi + ii Xi2 + ijXiXj +

(4)

Where Y is the response variable, 0 is the intercept term, ii is the squared effect and ij is the interaction between
Xi and Xj. The number of coecients in the above equation is 6. The redundancy factor of the experimental design was
derived from Rf = number of experiments/number of coecients.
For our current study k=2, the numbers of coecients are 6 and hence the numbers of experiment are 10.
Therefore, the redundancy factor is 1.667.

40

Rajasri Yadavalli, Ramgopal Rao S & C. S. Rao

RESULTS AND DISCUSSIONS

Screening of the Important Factors by Plackett- Burman Design
Plackett-Burman design offered an effective screening procedure and we evaluated the significance of a large
number of factors in one experiment, which was time-saving and maintained convincing information on each factor. Table
2 shows the high and low levels of factors chosen for trials in Plackett-Burman design. Table 3 represents the four
independent factors and their concentrations at different coded levels and the experimental responses for 8 runs.
The biomass productivity showed considerable variation depending on the four independent factors in the
medium, by applying the regression analysis on the experimental data. The corresponding first-order model equation fitted
to the data obtained from the Plackett-Burman design experiment has the formula
Yield = 1.942 + 0.15225A + 0.8575B-0.0765C- 0.01175D

(5)

The regression coefficients and determination coefficient (R2) for the linear regression model of biomass
productivity are presented in Table 4. The model was significant (P < 0.05) and R2= 98.7%, indicating that 98.7% of the
variability in the response could be explained by the model. Statistical analysis of the data showed that light intensity,
NaNO3 concentration and mode of operation had a significant effect on biomass yield from Chlorella pyrenoidosa, with
the confidence level above 98% (P < 0.05). The other factors having confidence levels below 95% were considered
insignificant. The Pareto chart of Coefficients for Yield (Fig 1) clearly indicates the ranking of the factors in biomass
production.
In the present study, we observed that Chlorella pyrenoidosa, failed to grow in the absence of nitrogen source and
the concentration of nitrate in the medium had a profound effect on algal growth. Fig 1 substantiates that NaNO3 has a
significant influence on the growth of Chlorella pyrenoidosa followed by light intensity. This was in accordance with
earlier studies which demonstrated a direct and linear relationship between low nitrate concentration and reduction in
biomass production
reported

12

8-11

. A decrease in Nannochloropsis sp biomass concentration in low nitrate concentration was also

. Further, increase in nitrogen concentration resulted in the enhancement of Chlorella pyrenoidosa biomass

suggesting that nitrogen is crucial for the survival and growth of this alga.

Figure 1: Effect on Variables on Biomass Production from Chlorella Pyrenoidosa

41

Response Surface Methodological Approach to Optimize Process

Parameters for the Biomass Production of Chlorella Pyrenoidosa

Algae can be cultivated under certain conditions of temperature, light and sufficient nutrients to produce biodiesel.
Light intensity directly affects the growth and photosynthesis of microalgae since light itself acts as carbon source for their
photoautotrophic growth

13.

It is well established that light intensities also play a major role in growth of microalgae

14

Similarly, mode of operation has a significant role in biomass productivity. Regulation of nutrient feed rates to increase
productivity can be performed by fed-batch cultivation. Researchers have reported that maximum lipid productivity was
obtained with urea as a nitrogen limitation in a semi-continuous culture when compared with those in the batch and fedbatch cultivations of Chlorella sp.

15- 16

production in Chlorella pyrenoidosa.

. In our previous study we had established that fed batch strategy enhances lipid

17

Hence, the concentration of NaNO3 and light intensity were selected for further

optimization in this study to achieve a maximum biomass yield under batch mode of cultivation.
Central Composite Design
Following screening, RSM using Central-Composite design was employed to determine the optimal levels of the
two selected factors that significantly affected biomass yield. The respective low, zero and high levels with the coded
levels for the factors were defined in Table 5. The experimental design and results are shown in Table 6. Based on a
regression analysis of the data from Table 7, the effects of two factors on biomass yield were predicted by a second-order
polynomial function, as
2
Biomass =2.49+0.18A+0.99B-0.011AB+0.081A2 -0.76B

(6)

Where, Y was the predicted response and A, B were the Light intensity and NaNO3 concentration, respectively.
The statistical significance of Equation (6) as checked by f-test, and the analysis of variance (ANOVA) for the
second-order polynomial model is shown in Table 7. It was evident that the model was highly significant, as suggested by
the model F value and a low probability value (P = 0.0051). The analysis of factor (f-test) showed that, the second-order
polynomial model was well adjusted to the experimental data and the coefficient of variation (CV) indicated the degree of
precision with which the treatments were compared. The precision of a model can be checked by the determination
coefficient (R2) and correlation coefficient (R). The determination coefficient (R2) was calculated to be 0.9625, indicating
that 96.25% of the variability in the response could be explained by this model.
Normally, a regression model with an R2 value higher than 0.9 was considered to have a very high correlation 18.
The closer the value of R to 1, better correlation is expected between the experimental and predicted values. Here, the
value of R (0.9824) for Equation (6) indicated a close agreement between the experimental results and the theoretical
values predicted by the model equation. Therefore, the quadratic model was selected in this optimization study.
The significance of the regression coefficients was tested by t-test. The regression coefficients and corresponding
P-values for the model are given in Table 8. The P-values were used as a tool to check the significance of each coefficient,
which is necessary to understand the pattern of the mutual interactions between the best factors. The smaller the p-value,
the significance of the corresponding coefficient will be greater 19-21. Our results showed that, among the two independent
factors NaNO3 had more significant effect on biomass productivity. The positive coefficient of them showed a linear effect
to increase biomass productivity.
Comparison of Observed and Predicted Biomass Yield
A regression model could be used to predict future observations on the response Y (Biomass Yield) corresponding
to particular values of the regressor variables. In predicting new observations and in estimating the mean response at a
given point, one must be careful about extrapolating beyond the region containing the original observations. It was very

42

Rajasri Yadavalli, Ramgopal Rao S & C. S. Rao

possible that a model that fitted well in the region of the original data would no longer fit well outside the region. The
observed biomass yield (the response) versus those from the empirical model equation (6) was illustrated (Fig 2). The
figure proved that, the predicted data of the response from the empirical model is in good agreement with the observed
ones in the range of the operating variables.

Figure 2: Comparison of the Observed Biomass Yield (g/L) and the Predicted Biomass (g/L)
Localization of the Optimum Condition
The 3D response surface plots described by the regression model were drawn to illustrate the effects of the
independent factors and the interactive effects of each independent factor on the response factors. It also shows the
optimum concentration of each component required for the biomass production (Fig 3). These 3D plots provided a visual
interpretation of the interaction between two factors and facilitated the location of optimum experimental conditions. The
model predicted that the optimal values of the light intensity and NaNO3 concentration were A = 130.77 mol m-2s-1, B =
1.78g/L, respectively. The maximum predicted value of biomass obtained was 2.956 g/L.

Figure 3: Response Surface Curve for Biomass Productivity by C. Pyrenoidosa vs Light Intensity and NaNO3
Concentration

Response Surface Methodological Approach to Optimize Process

Parameters for the Biomass Production of Chlorella Pyrenoidosa

43

Model Adequacy Checking

Usually, it is necessary to check the fitted model to ensure that it provide an adequate approximation to the real
system. Unless the model showed an adequate fit, proceedings with the investigation and optimization of the fitted
response surface will likely give poor or misleading results. The residuals from the least squares fit played an important
role in judging model adequacy. By constructing a normal probability plot of the residuals, a check was made for the
normality assumption, was given (Fig 4).

Figure 4: Normal Probability of Internally Studentized Residuals

The normality assumption was satisfied as the residual plot was approximated along a straight line. Fig 5 presents
a plot of residuals versus the predicted response. The general impression was that the residuals scattered randomly on the
display, suggesting that the variance of the original observation was constant for all values of Y. Both plots (Figs 4 and 5)
were satisfactory implying that the empirical model was adequate to describe the biomass yield by response surface.

Figure 5: Plot of Internally Studentized Residuals vs the Predicted Response

44

Rajasri Yadavalli, Ramgopal Rao S & C. S. Rao

Verification of the Predicted Activity in the Optimal Medium

Three additional experiments in shake flasks were performed in batch mode in order to verify the predicted
biomass under the optimal medium compositions. The mean value of biomass productivity was 2.81g/L, which was in
excellent agreement with the predicted value. The final process parameters optimized were: light intensity= 130.77 mol
m-2s-1, NaNO3 = 1.78g/L respectively .The maximum predicted value of biomass obtained was 2.956g/L.

CONCLUSIONS
In this study, we demonstrated that Plackett-Burman design and Response surface methodology using Central
Composite design effectively helps in optimizing biomass production by Chlorella pyrenoidosa. The maximum predicted
value of biomass (2.956g/L) obtained was increased by 1.3 times when compared with the original medium (2.276 g/L).
Validation experiments were also carried out to verify the adequacy and the accuracy of the model, and the results showed
that the predicted value agreed with the experimental values accurately. The optimized process parameters obtained in this
experiment has given a basis for further study with large scale fermentation in a photobioreactor for the production of
biomass from this strain.

ACKNOWLEDGEMENTS
The authors thank the Management of Sreenidhi Institute of Science and Technology (SNIST) for their financial
support in carrying out this in-house funded project. Special thanks to Mr.L.Saida Naik, Head, Centre for Biotechnology,
Institute of Science and Technology JNTU Hyderabad for his inputs.

REFERENCES
1.

Anondho W., Dianursanti., Muryanto., Josia S., Praswasti P D., Kencana W., Heri H., Misri G., & Roekmijati W
S. (2008). Biomass production in chlorella vulgaris buitenzorg using series of bubble column photo bioreactor
with a periodic illumination. Makara, Teknologi, 12, 27-30.

2.

Zhang X., Zhou B., Zhang Y., Cai Z., Cong W., & Ouyang F. (2002). A simple and low-cost air lifts
photobioreactor for microalgal mass culture. Biotechnology Letters,24, 17671771.

3.

Samira C., Bouziane M., Khaled C., Fethia A.,& Kamel A.(2011). Biohydrogen production using green
microalgae as an approach to operate a small proton exchange membrane fuel cell . Int J Hydrogen Energ, 36,
40894093.

4.

Pankaj S., Manpreet S., Ashwini LK., & Uttam CB.(2007). Response surface optimization of the critical medium
components for carbonyl reductase production by Candida viswanathii MTCC 5158. Bioresour.Technol , 98, 829833.

5.

Montgomery, DC.(2001).Design and Analysis of Experiments. John Willy & Sons (Asia):Singapore.

6.

Gorret N., Rosli SK., & Oppenheim SF. (2004). Bioreactor culture of oil palm (Elaeis guineensis) and effects of
nitrogen source, inoculum size and conditioned medium on biomass production. J. Biotechnol, 108, 253- 263.

7.

Tokcaer Z., Bayraktar E., & Mehmetoglu U. (2006). Response surface optimization of antidipteran deltaendotoxin production by Bacillus thuringiensis subsp. israelensis HD 500. Proc. Biochem, 41, 350-355.

8.

Subhasha N., Monika P R., & Rupali S.(2011). Effect of Nitrogen on Growth and Lipid Content of Chlorella
pyrenoidosa. Am. J.Biochem. Biotechnol , 7,126-131.

45

Response Surface Methodological Approach to Optimize Process

Parameters for the Biomass Production of Chlorella Pyrenoidosa

9.

Yeesang C., & Cheirsilp B.(2011). Effect of nitrogen, salt, and iron content in the growth medium and light
intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour.Technol, 102,
3034-3040.

10. Mandal S., Mallick N.(2009). Microalga Scenedesmus obliquus as a potential source for biodiesel production.
Appl. Microbiol. Biotechnol, 84, 281-291.
11. Gouveia L., & Oliveira A C. (2009). Microalgae as a raw material for biofuels production. J. Ind. Microbiol.
Biotechnol , 36, 269-274.
12. Hu H., & Gao K. (2005). Response of growth and fatty acid compositions of Nannochloropsis sp. to
environmental factors under elevated CO2 concentration. Biotechnol Lett , 28,987-992 .
13. Chisti Y. (2007). Biodiesel from microalgae. Biotechnol. Adv, 25, 294-306.
14. Rajasri Y., Rao C S., Chandrakanth D R., Sivasai K S R., & S.Ramgopal R. (2010). Effect of different culture
media on cell concentrations of Chlorella pyrenoidosa under photoautotrophic conditions. Int. J. Nat. Eng. sci, 4,
53-57 .
15. Takagi M., Watanabe K., Yamaberi K., & Yoshida T. (2000).

Limited feeding of potassium nitrate for

intracellular lipid and triglyceride accumulation of Nannochloris sp UTEX LB1999. Appl. Microbiol. Biotechnol,
54, 112117.
16. Hsieh C H.,& Wu W T. (2009). Cultivation of microalgae for oil production with a cultivation strategy of urea
limitation. Bioresour. Technol, 100, 3921-3926.
17. Rajasri Y., Ramgopal R S., & Rao C S. (2012). Lipid Accumulation Studies In Chlorella Pyrenoidosa Using
Customized Photobioreactor- Effect of Nitrogen Source, Light Intensity and Mode of Operation. Int J Eng
Res.Appl, 2,2446-2453.
18. Haaland, PD. (1989).Separating signals from the noise. In Experimental design in biotechnology. New York:
Marcel Dekker ,Inc.
19. Li Y., Cui FJ., Liu ZQ., Xu ZQ., Xu YY., & Zhao H. (2007). Improvement of xylanase production by Penicillium
oxalicum ZH-30 using response surface methodology. Enzyme Microb. Tech, 40, 1381-1388.
20. Liu JZ., Weng LP., Zhang QL., Xu H., & Ji LN . (2003). Optimization of glucose oxidase production by
Aspergillus niger in a benchtop bioreactor using response surface methodology. World J. Microbiol. Biotechnol,
19, 317-323.
21. Li Y., & Lu J. (2005). Characterization of the enzymatic degradation of arabinoxylans in grist containing wheat
malt using response surface methodology. J. Am. Soc. Brew. Chem, 63, 171-176.

APPENDICES
Table 1: Concentration of Nitrate as Nitrogen Source Used in the Present Study
S.No
1
2
3

Nitrogen Source
Concentration(g/L)
0.025
0.05
0.1

Medium Label
N-1
N-2
N-3

46

Rajasri Yadavalli, Ramgopal Rao S & C. S. Rao

Table 1 Contd.,
0.15
0.5
1.0
1.5
2.0

4
5
6
7
8

N-4
N-5
N-6
N-7
N-8

Table 2: Coded and Real Values of the Factors Tested in the Plackett-Burman Design
Factor

Code

Light Intensity(mol m-2s-1)

NaNO3(g/L)
Mode of Operation

A
B
C

pH

Levels of Factor
-1
+1
110
135
0.025
2.0
Fed-Batch
Batch
6.0

7.0

Table 3: Experimental Design and Results of the N = 8 Plackett-Burman Design

Run
1
2
3
4
5
6
7
8

Factor1A:
Light
Intensity
(mol m-2s-1)
-1
1
1
1
-1
1
-1
-1

Factor2
B:
NaNO3(g/L)

Factor3
C: Mode of
Operation

Factor
4
D: pH

Response
Biomass(g/
L)

1
1
-1
1
-1
-1
-1
1

-1
1
-1
-1
-1
1
1
1

1
1
1
-1
-1
-1
1
-1

2.715
2.846
1.295
3.061
1.003
1.175
0.865
2.576

Table 4: Regression Results of the Plackett-Burman Design

Term

Coefficient

SE Coefficient

P-Value

0.15225

0.01425

10.684

0.0049

B: nitrogen

0.8575

0.01425

60.175

0.0001

C: mode

-0.0765

0.01425

-5.368

0.0234

D: pH

-0.01175

0.01425

-0.824561

0.4854

Constant

1.942

A: Light

Table 5: Coded and Real Values of Factors in Central-Composite Experimental Design

Factor

Levels of Factor

Code
-1.414

-1

+1

+1.414

Light Intensity

104.82

110

122.5

135

140.18

NaNO3

-0.38

0.025

1.01

2.0

2.41

47

Response Surface Methodological Approach to Optimize Process

Parameters for the Biomass Production of Chlorella Pyrenoidosa

Table 6: Experimental Conditions of Central Composite Design

Run No

Light
Intensity

NaNO3

1
2
3
4
5
6
7
8
9
10

0
-1
1
0
1.414
0
-1.414
-1
0
1

0
-1
-1
0
0
1.414
0
1
-1.414
1

Biomass
Experimental
(g/L)
2.49
0.86
1.175
2.57
2.91
2.93
2.31
2.576
0.32
2.846

Table 7: Analysis of Variance (ANOVA) for the Second Order Polynomial Model
Source
DF
MS
F-Value
Model
5
1.56
22.17
Residual
4
0.071
Lack of Fit 3
0.093
29.08
Pure error
1
3.2x10-3 Total
9
R2
Coefficient of variation (CV) = 12.66%; correlation coefficient (R)

P>F
0.0051
0.1353
0.9652
= 0.9824; DF, degrees of freedom and MS,

mean square. Statistically significant at 95% confidence level (P < 0.05).

Table 8: Regression Result of Central Composite Design
Model term
Intercept
A-Light Intensity
B-NaNO3 concentration

Degree of
Freedom
1
1
1

Estimate

P-Value

2.49
0.18
0.99

0.1291
0.0008a

AB

-0.011

0.9366

0.081

0.5227

B
1
-0.76
0.0124a
Statistically significant at 95% confidence level (P < 0.05).