Optimization of Nutrient Media Composition for Microalgae Biomass Production Using Central Composite Design

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1 Optimization of Nutrient Media Composition for Microalgae Biomass Production Using Central Composite Design Kong Siew Fung, Hannah Ngu Ling Ngee, Emily Liew Wan Teng Department of Chemical and Petroleum Engineering Curtin University, CDT 250, Miri, Sarawak, Malaysia Abstract Microalgae is promising feedstock for various renewable fuel productions. However, it is not commercially viable due to the high cost associated with microalgae biomass production. Hence, there is a need to improve the biomass productivity of microalgae to reduce the production cost. Previous research studies showed that by varying nutrient media composition and growth conditions of microalgae, quality and quantity of biomass can be enhanced. The objective of this research is to optimize the biomass productivity of microalgae Chlorella vulgaris by varying potassium bicarbonate and sodium nitrate concentration in culture medium. The cultivation of Chlorella vulgaris was carried out in flat panel photobioreactor aerated with 3.5 L/min of air supply. The light is supplied at 4500LUX for 6 hours daily. Optimization was performed with the aid of Design Expert using Central Composite Design (CCD). CCD was selected in this study since it is one of the most commonly used response surface designs in fermentation studies. Based on the predicted results, the optimum biomass productivity of 36 mg/l/day was predicted at 6 g/l of potassium bicarbonate and 0.36 g/l sodium nitrate. Keywords- microalgae; biomass productivity; optimization; potassium bicarbonate, sodium nitrate I. INTRODUCTION Conventional fossil fuels are facing depleting crisis which lead researchers and scientists to participate in biologically based alternative fuel development. The renewable and biodegradable characteristics of biofuel has gained much interest and caused rapid increase in biodiesel production. Microalgae is a promising feedstock for various renewable fuel productions [1] and is reported to be the only renewable biodiesel which is capable of meeting global demand for transport fuel [2]. Microalgae is widely utilized because it has low oxygen content, high calorific value, high H/C ratio [3], strong adaptability to local growing condition, rapid growth rate of 100 times faster than terrestrial plant and low agricultural input [2,4]. The major obstacle that affects the development of microalgae oil production is due to its low lipid productivity, labour and harvesting cost [5-7]. Studies done by Griffiths and Harrison (2009) [6] showed that Chlorella vulgaris is categorized as low lipid content microalgae. Nevertheless, numerous studies have proven that by varying nutrient media composition and culture conditions such as light source, light intensity, ph, temperature, oxygen removal [8], carbon source concentration and nitrogen source concentration, the quantity and quality of algae biomass can be enhanced [2]. Chlorella is a type of single cell green algae which lives in fresh water. Research has proven that Chlorella species such as Chlorella vulgaris is ideal for biomass optimization studies [5,9,10]. Chlorella sp. was reported to have high adaptability in three types of growth conditions, i.e. mixotrophic, heterotrophic and autotrotophic [3]. Among all these three growth conditions, Chlorella vulgaris cultivated in mixotrophic medium have the highest biomass and lipid productivities respectively. Carbon source is also an important parameter that affects microalgae growth. There are two types of carbon sources, which are organic and inorganic. Examples of organic carbon sources are glucose, sucrose, glycerine and glycerol. Inorganic carbon sources can be categorized as dissociated type (HCO 3 -, CO 3 2- ) and undissociated type (CO 2, H 2 CO 3 ). The use of organic carbon sources result in better biomass productivity of microalgae whereas inorganic carbon source facilitates chlorophyll biosynthesis, stimulate pigment synthesis and improve photosynthesis process [11]. Inorganic carbon source is also frequently used in research because it is cheaper and facilitates chlorophyll biosynthesis which leads to high growth rate [11]. Potassium bicarbonate is classified as dissociated inorganic carbon source. On the other hand, nitrogen source is also vital for microalgae cell physiology and growth [2]. It is an essential component that contributes to the biomass formed. Lack of nitrogen will cause chlorophyll reduction and increase in carotenoids, which lead to discolouration of cells [12]. Most microalgae favour nitrogen-deprived medium as compared to nitrogen-sufficient medium because under nitrogen-deprived condition, microalgae have the ability to produce nitrogen storage material. Nitrogen-deprived medium also enhances the accumulation of polyunsaturated oil (PUFAs) and organic

2 carbon compound such as polysaccharides [12]. Light also plays an important role in enhancing the biomass productivity of microalgae. Parameters such as light source, light intensity and photoperiod have significant effects on biomass productivity of Chlorella vulgaris. Previous research studies showed that fluorescent light is suitable for microalgae growth [8]. Photoinhibition occur when light intensity exceeds 6000 LUX [13]. Cellular content of protein, carbohydrates and lipids can be varied through variation of photoperiod. Photoperiod is important because photosynthesis process is controlled by a photochemical period which depends on light and biochemical dark phase which is independent of light [13]. Studies showed that dark phase is beneficial for microalgae growth because certain enzymes of pentose cycle which is used in photosynthesis and carbon dioxide fixation are active during dark phase and inactive during light phase. Kong et al. (2011) [11] also stated that less biomass is depleted during dark phase. The optimum photoperiod for microalgae cultivation varies depending on species. Many studies had been done to investigate the effect of different growth parameters to enhance biomass productivity of Chlorella vulgaris. However, tthere is no significant research regarding the interaction of carbon and nitrogen sources using potassium bicarbonate and sodium nitrate on biomass productivity of Chlorella vulgaris. Therefore, this research serves to determine the interaction of potassium bicarbonate and sodium nitrate, as well as to study the effect of individual parameter to improve the biomass productivity of Chlorella vulgaris. These two parameters were selected as they are important growth factors that strongly control or affect the biomass production of microalgae if compared to other growth factors. Other growth factors such as light intensity, light cycle and other nutrients concentration were kept constant so that the effect of carbon and nitrogen source on biomass productivity can be investigated. This study serves as a stepping stone for pilot scale studies and eventually the industrial application. II. METHODOLOGY A. Microalgae strain and culture medium Chlorella vulgaris was obtained from Commonwealth Scientific and Industrial Research Organization (CSIRO), Perth, Australia. Microalgae cell were cultivated in Modified Bold s Basal Medium (BBM) consisted of (g/l): NaNO 3, 10; CaCl 2.2H 2 O, 10; MgSO 4.7H 2 O, 10; K 2 HPO 4, 10; KH 2 PO 4, 10; NaCl, 10; EDTA, 1; FeSO 4.7H 2 O, 1. 50ml of inoculums in a test tube were added to 200ml of medium, and left to grow for 14 days. The culture medium was then topped up to 500ml and left to grow for 10 days before cultured in 3 litre medium. Cell density calculation was performed before the microalgae was transferred to the 3 litre medium to ensure data consistency and to improve microalgae growth. During subcultivation in 250ml and 500ml medium, 24 hours of light was supplied for continuous illumination whereas in 3 litre medium, light: dark cycle,of 6:18 hours was used to enhance biomass productivity of microalgae. B. Operations of Photobioreactor A flat plate photobioreactor with dimension of 40cm 25cm 10cm was used. Fluorescent light (TL5) with light intensity of 4500 LUX were placed on both side of PBR to supply light for microalgae growth. The light intensity at reactor wall was measured using Light Meter. Light: dark cycle of 6:18 hours was used for 3L cultivation. Aeration was supplied by bubbling air at constant pressure using Sheng Zhe BS410 super pump. C. Design of Experiment Design Expert was used for experimental design. 2 2 level factorials with 2 replicates were used to identify the interaction between potassium bicarbonate and sodium nitrate with biomass productivity of Chlorella vulgaris. In a 2 2 level factorial designs, each factor is varied over 2 levels, +1 and -1; therefore there will be 6 sets of experiments include 2 replicates for factorial design study. Central Composite Design (CCD) was chosen as the experimental design in this study since it is suitable to fit second-order models in microbial cultivations [14]. There are a total of 12 experimental sets with 4 replicates. CCD helps to estimate curvature and it has 4 star points for 2 factors. Therefore, there will be 6 sets of experiments including 2 replicates for augmentation control study [15]. Based on the results generated, there will be a total of 12 experimental sets. Table I shows the range of parameters used for experimental design and optimization. TABLE I. OPTIMIZATION RESULTS Level Low Level High Level Factor Name (g/l) (-1) (g/l) (+1) (g/l) A KHCO B NaNO D. Harvesting and Drying The medium was centrifuged at 3500rpm speed for 12 minutes using Scientific Labofuge 400 centrifuge. After centrifugation, slurry was filtered using funnel filter and filter paper before placing in oven at temperature of 60 C for 24 hours. E. Determination of Specific Growth Rate The specific growth rate of microalgae was calculated using the following equation: µ= (ln N t / N 0 )/(T t - T 0 ) [16] (1) Where µ, N t, N 0, T t, T 0 specific growth rate (day -1 ), max cell count, initial cell count, day when max cell count is obtained and initial day respectively. F. Determination of Biomass Productivity The biomass productivity was calculated using the formula below:

3 Q v =µx [5] (2) Where Q v, µ and X represent volumetric biomass productivity (g/l/day), specific growth rate (day -1 ), biomass concentration (mg/l/day) respectively. III. RESULTS AND DISCUSSIONS A. The Effect of Sodium Nitrate on Biomass Productivity of Chlorella Vulgaris The effect of nitrogen source on biomass productivity of Chlorella vulgaris is studied by controlling potassium bicarbonate concentration and manipulating sodium nitrate concentration. Results show that, excess of nitrogen reduces biomass productivity of Chlorella vulgaris. The biomass productivity of Chlorella increase when 0.7g/L of sodium nitrate is used and decrease when concentration is further increased to 1.4g/L. This is illustrated in Fig. 1, whereby it shows that 0.7g/L is the optimum sodium nitrate concentration as potassium bicarbonate concentration is fixed at 3.5g/L. Figure 1. Biomass productivity of Chlorella vulgaris grown using different sodium nitrate concentration at fixed potassium bicarbonate concentration of 3.5g/L. The results obtained in this research have similar trend with Bhola et al. (2010) [2], whereby biomass productivity of Chlorella vulgaris decreases when excess of nitrate is used. Apart from that, this finding is further supported by Golueke et al. (1967), who stated that excess nitrate will cause toxic and provide adverse effects to the growth of microalgae [2]. The optimum nitrate concentration obtained in this research is in close proximity with the concentration achieved by Bhola et al (2010) [2], Lv et al. (2010) [7] and Chen et al. (2010) [17] where both Bhola et al. (2010) [2] and Lv et al. (2010) [7] obtained 0.5g/L and Chen et al. (2010) [17] obtained 0.65g/L. The slight differences might occur due to different photoperiod and the type of carbon source used by other research studies. B. The Effect of Potassium Bicarbonate on Biomass Productivity of Chlorella vulgaris The effect of carbon source on biomass productivity of Chlorella vulgaris can be studied by maintaining sodium nitrate concentration at a constant value and manipulate the concentration of potassium bicarbonate. Results show that potassium bicarbonate is beneficial for biomass productivity of Chlorella vulgaris but excess of carbon source will cause adverse effect. The result is plotted as Fig. 2 for experimental sets containing 0.7g/L of sodium nitrate. From the graph, it can be seen that 3.5g/L is the optimum concentration for biomass production of Chlorella vulgaris and further increment to 7g/L will lead to biomass reduction. This result exhibits similar trend with the research conducted by Chen et al. (2010) [17] and Yeh et al. (2010) [8], where biomass productivity of Chlorella vulgaris increased with increasing carbon source followed by slight decrease when excess of carbon source was supplied. The optimum value achieved in this case however is higher than the optimum value achieved by other researchers. Yeh et al. (2010) [8] and Chen et al. (2010) [17] studies show that the optimum sodium bicarbonate concentration for biomass productivity of Chlorella vulgaris was 1.2g/L and 1.5g/L respectively and further increment will lead to biomass productivity reduction. The results obtained in this research exceed and contradict with Yeh et al. (2010) [8] and Chen et al. (2010) [17] research. The contradiction might due to variations in photoperiod and presence of nitrogen source. This can be explained by comparing both researchers studies. This is because Chen et al. (2010) [17] and Yeh et al. (2010) [8] have different tolerance of carbon concentration although both researchers utilize similar growth parameter. The major differences between both researches are nitrogen utilization and photoperiod. Hence, it is suspected that nitrogen utilization and presence of dark phase increase carbon tolerance of Chlorella vulgaris. Additionally, dark phase reactions function to fix carbon [18]. Hence, dark phase required more carbon source compare to medium without dark phase. This explains the phenomenon in this study where high potassium bicarbonate concentration was required to achieve higher biomass productivity since this research had longer dark phase than light phase. Figure 2. Biomass Productivity of Chlorella vulgaris grown using different potassium bicarbonate concentration at fixed sodium nitrate concentration of 0.7g/L. C. The Combined Effect of Sodium Nitrate and Potassium Bicarbonate Concentration on Biomass Productivity of Chlorella vulgaris The effects of sodium nitrate and potassium bicarbonate concentration on biomass productivity of Chlorella Vulgaris are presented in Table II. Maximum biomass productivity of

4 36.711mg/L/day was achieved when high potassium bicarbonate concentration (6g/L) is used together with low sodium nitrate concentration (0.2g/L). well. Linear graph indicates no transformation is needed and the model is satisfactory. The final equation in terms of actual factors is shown below: Block Block 1 Block 2 TABLE II. NaNO 3 Concentration (g/l) EXPERIMENTAL RESULTS KHCO 3 Concentration (g/l) Biomass Productivity (mg/l/day) Biomass Productivity= A B AB A B A 2 B (3) Where A is potassium bicarbonate concentration, B is sodium nitrate concentration. TABLE III. Sum of Mean Source Squares Square Block STATISTICAL ANALYSIS TABLE F value P-Value Prob>F Model A-KHCO B-NANO AB A B A 2 B Residual Lack of Fit Pure Error D. Statistical Analysis Factor A, B, AB, A 2, B 2, A 2 B and intercept were analysed as the function of the model, whereby A is KHCO 3 concentration and B is NaNO 3 concentration. F value represents the relative contribution of curvature variance to residual variance. Large F value is desirable as small value indicates similar variance and less contribution. Large F value indicates that more variance is considered in the model. The F value for the model is 20.19, which is considered as a large value and thus, the model is significant. There is only 0.59% that model F-value is large could occur due to noise. Apart from that, the factors of A, A 2, B 2 and A 2 B are analyzed to be significant as well except for B and AB. Further checking is performed by using P-Value Prob>F test, whereby this is the probability of F value which will be observed when this value is less than The results show that A, A 2, B 2 A 2 B and model are significant whereas B and AB have no significant effect on the model because Prob>F is more than Lack of fit is an undesirable factor for a model; hence it is favourable for the parameter to be insignificant. The lack of fit is expected to have small F value and large Prob>F, which is compatible with this case. The R-squared value for this model is , closer to 1, indicates that the model is reliable for biomass productivity prediction. The adequate precision is satisfactory, which is well above 4. Apart from that, normal probability graph as shown in Fig. 3 can be used to check the significance of the model as Cor Total Standard Deviation Mean C.V% PRESS R-Squared Adjusted R-Squared Predicted R-Squared Adequate Precision Figure 3. Normal Plot of Residuals

5 E. Optimization Analysis The potassium bicarbonate concentration is ranged from 1g/L to 6g/L whereas the concentration of sodium nitrate ranged from 0.2g/L to 1.2g/L. The optimized values were obtained from the numerical optimization method. Fig. 4 shows the 3D graph generated for the numerical optimization. Figure 4. Numerical Optimization Graph It is predicted that the optimum biomass productivity of mg/L/day is obtained when 6g/L potassium bicarbonate concentration and 0.2g/L sodium nitrate concentration is used. CONCLUSIONS AND RECOMMENDATION As a conclusion, the highest biomass productivity obtained from this study is mg/l/day for Chlorella vulgaris grown in culture containing 6g/L of potassium bicarbonate and 0.2g/L of sodium nitrate. Results show that carbon source and nitrogen source play an important role in biomass productivity of Chlorella vulgaris but excess of both sources will lead to biomass reduction. Hence, optimization is needed to attain the optimum carbon and nitrogen concentration which yields highest biomass productivity. The results generated by Design Expert shows that the optimum biomass productivity for potassium bicarbonate ranged from 0.2g/L to 1.2g/L and sodium nitrate ranged from 1.0g/L to 6.0g/L will be obtained when 6g/L of potassium bicarbonate and 0.36g/L of sodium nitrate is used. It is recommended to repeat the optimized parameters in pilot scale photobioreactor to verify the transferability of the findings to indutrial scale. References [1] K-L. Yeh, and J-S. Chang, Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31. Bioresour. Technol., vol. 105, pp , Doi: /j.biortech [2] B. Virthie, R. Desikan, S.K. Santosh, K. Subburamu, E. Sanniyasi, and F. Bux, Effects of parameters affecting biomass yield and thermal behaviour of Chlorella vulgaris. J. Biosci. 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