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Process Biochemistry 40 (2005) 21732182 www.elsevier.

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Statistical media optimization studies for growth and PHB production by Ralstonia eutropha
Shilpi Khanna, Ashok K. Srivastava*
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India Received 1 April 2004; received in revised form 30 June 2004; accepted 20 August 2004

Abstract Poly-b-hydroxybutyric acid (PHB) is a natural, biodegradable polymer, which is accumulated as an energy reserve material by a large number of bacteria when, nutrients such as nitrogen or phosphorous sources are available in limiting concentrations in the presence of excess carbon source. The major problem associated with the industrial production of PHB is its high production cost. In the present study, efforts were made to optimize the growth of Ralstonia eutropha NRRL B14690 in the presence of nutrients, which would not only decrease the production cost of PHB but also help in increasing the productivity. Using fructose and ammonium sulphate as carbon and nitrogen source (with the media reported in literature), R. eutropha exhibited a maximum biomass of 3.25 g/L with a PHB concentration of 1.4 g/L in 48 h. To determine the possibility of growth potential of R. eutropha, it was grown in different carbon sources of which fructose, lactic acid, sucrose and glucose yielded good growth and PHB production. In order to incorporate cheaper nitrogen source and growth factors in media, ammonium sulphate was substituted by ammonium nitrate, urea and ammonium chloride. Urea featured highest PHB accumulation of 3.84 g/ L after 60 h of growth. In place of yeast extract as a source of minerals and vitamins, corn steep liquor was used which yielded a PHB concentration of 2.66 g/L. Statistical media optimization design was then used to optimize the composition of culture medium for maximizing the productivity of PHB. A maximum of 6.65 g/L residual biomass and 6.75 g/L PHB was obtained using optimized concentrations, representing 94 and 83% validity of the predicted models for residual biomass and PHB production, respectively. A signicantly higher maximum biomass of 20.73 g/L with a PHB content of 9.35 g/L was obtained in a 7 L lab scale bioreactor thus giving a yield of 0.24 g PHB/g fructose consumed. Batch kinetics can be used for model development, which will facilitate simulation of nutrient limited cultivation(s) for over accumulation of PHB. # 2004 Elsevier Ltd. All rights reserved.
Keywords: Polyhydroxybutyrate; Ralstonia eutropha; Plackett Burman; Batch cultivation; Central composite design

1. Introduction Poly-b-hydroxybutyric acid (PHB) is a natural, biodegradable polyester, which is accumulated in the form of intracellular granules by a large variety of bacteria. These granules act as energy reserve materials when nutrients such as nitrogen and phosphorous source are available in limiting concentrations in the presence of excess carbon source [1,2]. They can be completely degraded to water and carbon dioxide under aerobic conditions and to methane under anaerobic conditions by microorganisms in soil, sea,
* Corresponding author. Tel.: +91 11 26591010; fax: +91 11 26582282. E-mail address: ashokiitd@hotmail.com (A.K. Srivastava). 0032-9592/$ see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.08.011

lake water and sewage [3]. The main factor preventing the large-scale production and commercialization of PHB is their high cost of production as compared with that of plastics based on petrochemicals. One of the major factors adding to the cost of PHB is the cost of substrates used for production. Therefore, less expensive substrates, improved cultivation strategies and easier downstream processing methods are required for reducing the cost [4,5]. Thus, utilization of media containing cheaper carbon and nitrogen sources should be used to reduce the production costs of PHB. Ralstonia eutropha is the most widely studied bacterium due to its ability to accumulate large amount of PHB from simple carbon sources [6]. Utilization of cheaper substrates

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such as carbon dioxide [7] or methanol [8] has been reported for the production of PHB. Some amino acids have also been shown to stimulate PHB production in resting cells of R. eutropha [9,10]. Productivities of more than 1 g/L h have been achieved by cultivation of Azotobacter beijerinckii in media containing 20 g/L casein peptone [11]. Batch cultivation of PHB using starch and lactose as carbon source has also been carried out by Azotobacter chroococcum [12] and Pseudomonas cepacia [13], respectively. Growth of Alcaligenes latus using urea has been reported but it was found to be non satisfactory [14]. Till date no reports are available for production of PHB by R. eutropha using urea in the production media. Generally, for PHB production yeast extract is used as a source of vitamins and growth factors but it cannot be used in largescale reactors, as it would add to the cost. Utilization of cheaper and easily available corn steep liquor might help in removing this problem. Thus, using cheap substrates and low cost nutrients at an optimized concentration can feature an improvement in the productivity of PHB at economical cost. Optimization of fermentations has long been used to enhance the yield and productivities of many bioprocesses. Conventionally, fermentations were optimized by implementing the variation of one component at a time. This approach is time consuming, assumes that the process variables do not interact and that process response is function of a single parameter, which is varied. In the recent years, this approach has been replaced with statistical optimization methods that take into account the interaction of variables in generating the response. One of the statistical designs for the screening of the independent variables is PlackettBurman [15]. This design offers the screening of a large number of independent factors (N) in a small number of experiments (N + 1). This step is generally followed by a process optimization tool, e.g. Response Surface Methodology (RSM), BoxBehnken, Modied distance, etc. However, Response Surface Methodology is the most commonly used where the optimum concentration values of the factors studied, are determined. It is a 2-level factorial design where contour plots are generated by linear or quadratic effects of key variables and a model equation is derived that ts the experimental data to calculate the optimal response of the system. In the present study, bacterial growth was initially carried out in a medium reported in literature [16,17]. Thereafter, different carbon, mineral and nitrogen sources were tested to analyze their effect on growth and PHB production. By taking the best carbon, mineral and nitrogen source, statistical media optimization was carried out by Plackett Burman and Response Surface Methodology. Optimized media was eventually used to study the kinetics of growth in shake asks and in a bioreactor (7 L) under pH-stat conditions to investigate the improvement in growth and/ or production of PHB by R. eutropha.

2. Materials and methods 2.1. Microorganism R. eutropha NRRL B14690 was used in all experiments. The culture was maintained on nutrient agar slants at 5 8C, and sub cultured monthly. 2.2. Media 2.2.1. For initial shake ask study A mineral salt medium which consisted of: 2.0 g/L (NH4)2SO4, 2.0 g/L KH2PO4, 0.6 g/L Na2HPO4, 0.2 g/L MgSO47H2O, 20 mg/L CaCl2, 10 mL/L trace metal solution, 0.1 g/L yeast extract was used [16,17]. The trace metal solution consisted of: 1.3 mg/L ZnSO47H2O, 0.2 mg/ L FeSO47H2O, 0.6 mg/L (NH4)6Mo7O244H2O and 0.6 mg/ L H3BO3. Fructose was used as carbon source in concentration of 40 and 10 g/L for production media and inoculum development, respectively. Fructose, yeast extract and salt solutions were sterilized separately at 121 8C and then aseptically reconstituted at room temperature prior to inoculation. The pH of the resulting broth was adjusted to 7.0 with 2N NaOH/2N HCl. 2.2.2. For comparison of different carbon and nitrogen sources The media used for these experiments was same as described above. However, for comparison, different carbon (20 g/L) and nitrogen (2 g/L) sources were taken. 2.3. Methods 2.3.1. Study of growth kinetics of the culture Mineral salt medium containing 10 g/L fructose was used for inoculum development. The organism was cultivated at an agitation speed of 150 rpm and 30 8C for 24 h in a 250 mL Erlenmeyer ask containing 50 mL of the medium described above. Initial pH was set to 7.0. For production of PHB, 100 mL of media (containing 40 g/L fructose) was taken in a 500 mL ask and inoculated with 5 mL of inoculum. The ask was kept under shaking conditions for 48 h at 150 rpm and 30 8C. Samples were withdrawn at regular intervals and analysed for biomass, PHB and residual nutrients. 2.3.2. Effect of different carbon sources on growth and PHB production The PHB production phase was carried out using fructose, glucose, lactic acid, xylose, sucrose, molasses, sorbose, acetic acid, starch, sodium acetate, glycerol, lactose and propionic acid. Inoculum was prepared as described above. After 24 h, the cells were harvested under aseptic conditions by centrifugation at 10,000 rpm at 4 8C for 20 min. The cells were washed and resuspended in 50 mL saline (0.9%). One millilitre of this saline was added to

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50 mL of production medium. The production medium in each ask consisted of the mineral salt medium and a different carbon source (20 g/L). The asks were incubated at 150 rpm at 30 8C. Biomass and PHB were estimated in the culture broth after 60 h. 2.3.3. Comparison of growth in different nitrogen sources Different nitrogen sources investigated were: ammonium chloride, ammonium nitrate and urea. Cells washed and resuspended in saline (0.9%) were added to mineral salt medium (in duplicate) containing fructose (40 g/L) as carbon source and different nitrogen sources (2 g/L). After incubation at 30 8C and 150 rpm, biomass and PHB were estimated in cell broth after 60 h. 2.3.4. Effect of corn steep liquor on growth Relatively cheap corn steep liquor (CSL) was used in place of expensive yeast extract as a source of vitamins and minerals. To check the effect of CSL on growth and PHB production, the same media as used for initial studies containing fructose and ammonium sulphate as carbon and nitrogen sources respectively, was taken (in duplicate). Biomass and PHB were determined after 60 h. 2.4. Analytical methods Cell growth was monitored by measuring the absorbance of the culture broth at 600 nm on a Specoll 1200 (Analytik Jena, Germany) after suitable dilution with distilled water. Cell mass concentration was determined by standard plot between OD600 nm and dry cell mass. The cell pellet obtained after centrifugation was dried at 90 8C till constant weight was obtained. The supernatant obtained by centrifugation of the culture broth at 10,000 rpm for 10 min at 4 8C was used for residual substrate analysis. Residual fructose was estimated by a dinitrosalicylic acid (DNS) method [18]. The content of residual ammonia nitrogen was determined according to a Kjeldahl method [19]. Organic nitrogen in the samples was estimated following its mineralization with hot sulphuric acid [19]. Residual phosphate in the media was determined by the method of Murphy and Riley [20]. PHB content in the dried cells was determined by gas chromatography (Nucon gas chromatograph 5765, AIMIL, India) with benzoic acid as an internal standard [21]. Residual biomass (X0 ) was dened as the total cell dry weight minus PHB concentration. 2.4.1. Media optimization using PlackettBurman optimization design The carbon, nitrogen and mineral sources, which had been screened earlier, were used along with other components for media optimization studies. A total of eight factors were tested, which were: fructose (A), urea (B), KH2PO4 (C), MgSO47H2O (D), CSL (E), Na2HPO4 (F), CaCl2 (G) and trace metal solution (H). A design of 12 experiments was formulated for eight factors using the

software. Each parameter was tested at two levels, high (+1) and low (1). Concentration range for the variables was decided on the basis of literature reports for PHB production by R. eutropha. The experiments were done in Erlenmeyer asks containing 50 mL media at 150 rpm for 60 h in duplicate. Response was measured in terms of residual biomass (I) and PHB (J) production. The residual biomass and PHB obtained in these 12 experiments was subjected to compatible analysis, which yielded t-values. The components giving higher positive t-value were taken up for further studies. Six factors (fructose, urea, KH2PO4, MgSO47H2O, CSL, Na2HPO4), screened from Plackett Burman design, were studied for determining their optimum concentration values for residual biomass and PHB production. 2.4.2. Response Surface Methodology Once the relevant factors having high t-values were selected, RSM was used to determine the optimum concentration of these factors affecting cell growth, rest of the factors being kept at a constant level. A 2n factorial Central Composite Design (CCD) developed using Design Expert (version 5.0.9) software (Stat-Ease Corporation, USA) was used to optimize the concentration of the factors selected. Factors yielding high positive t-value were selected and an experimental design of 30 experiments was formulated using the Design Expert software. The remaining parameters, which were found not to be inuencing the residual biomass and PHB production (CaCl2 (0.02 g/L), trace metal solution (10 mL/L)) were maintained at a constant level. Experiments were conducted in 250 mL Erlenmeyer ask containing 50 mL of media (pH 7.0) prepared according to the design. The asks were kept in incubator shaker maintained at 30 8C and 150 rpm. Responses studied were residual biomass (X0 ) (g/L) and PHB (g/L) at the end of 60 h. Contour plots (2D) were generated to understand the interaction of various factors and then used to nd the optimized concentration of the media components majorly affecting the response. A special feature of the software, point-prediction was used to conrm the above obtained optimized values. To check the validity of optimized media predicted by the Design Expert software, growth kinetics of the culture was studied in shake ask. Five percent inoculum (containing 10 g/L fructose) was added to 200 mL of media taken in 1 L Erlenmeyer ask. The experiment was carried out in duplicate for 60 h. Samples were withdrawn at regular intervals and were analysed for biomass, residual nutrients and PHB content. 2.5. Batch kinetics in a 7 L bioreactor Seed culture was prepared in a 1 L Erlenmeyer ask containing 200 mL media. Batch cultivation was carried out at 30 8C in a 7 L LF-1523 Bioengineering (Bioengineering AG, Switzerland) bioreactor containing 4 L of media.

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The reactor was sterilized in situ at 121 8C for 20 min, cooled and then inoculated with 5% inoculum (v/v). Culture pH was maintained at 7.0 by automatic addition of acid or base by pHmV controller M 7832 N. Dissolved oxygen concentration was maintained at 30% saturation value by manually adjusting the speed of the agitator and/or airow rate.

3. Results and discussion 3.1. Shake ask studies using media reported in literature Shake ask cultivation was performed at 30 8C and at 150 rpm by using the media reported in literature for the same strain of R. eutropha [16,17]. Initial pH of the media was adjusted to 7.0. Maximum biomass obtained was 3.25 g/ L with PHB concentration of 1.4 g/L at 48 h. An amount equal to 19.4 g/L of fructose was consumed out of an initial value of 40 g/L, while 1.9 g/L of ammonium sulphate was consumed out of total initial value of 2.0 g/L (Fig. 1). The remaining unconsumed fructose may be due to uncontrolled pH conditions in the shake ask. The pH value measured at the end of the cultivation was found to be 5.26 as against an initial value of 7.0. It was presumed that decrease in pH resulted in unfavourable growth conditions that led to reduced growth and thus the consumption of nutrients eventually stopped. The decrease in pH value may be due to the formation of H2SO4 when ammonium ions of ammonium sulphate were consumed by the culture. The PHB yield based on the amount of fructose consumed was 0.035 g/g. 3.2. Comparison of different carbon, nitrogen and mineral sources The major obstacle in large-scale production of PHB is the high cost of production. The media reported in earlier studies [16,17] for this strain of R. eutropha contained many expensive substrates. Thus, in order to nd cheaper and better substrate different carbon, nitrogen and mineral

sources were tested so as to improve the productivity and at the same time reduce the production cost of PHB. Based on the experiment done above, a comparative study was then carried out by growing the culture in different carbon sources. It was thought that this would not only help in nding a cheaper carbon source but would also help in accessing the carbon source utilization capacity of the culture. The objective was to nd a carbon source, which would be cheaper than fructose and at the same time would give productivity comparable to or greater than that being obtained with fructose. Also no data are available in literature, which could inform about the carbon source utilizing capacity of this strain of R. eutropha. Thus, twelve different carbon sources were selected based on easy availability or cheaper cost. Table 1 shows the biomass (g/L) and PHB (g/L) content obtained with the different substrates tested. A maximum biomass of 3.5 g/L and PHB content of 1.4 g/L was obtained with 20 g/L of fructose after 60 h. Lactic acid, which has recently gained interest, for example in the production of biodegradable lactide polymers, turned out to be the second best carbon source, in terms of PHB production, among the twelve carbon sources tested (Table 1). However, as compared to fructose the PHB content was much less. Minimum biomass and PHB were obtained with 2% propionic acid. This may be due to the severe inherent inhibitory effect of high concentration of propionic acid on bacterial growth. Growth and polymer production by Alcaligenes eutrophus has been shown to be inhibited by a propionic acid concentration of 0.3% or higher [22]. The presence of even lower propionic acid concentration (0.1%, w/w) in the medium has also been reported to inhibit PHA production by A. eutrophus [1]. Molasses was used as a carbon source (sucrose and other carbohydrates) keeping in mind the fact that it is a byproduct in the sugar industry. However, it was seen that despite the presence of large amounts of carbohydrates sufcient biomass and product could not be produced. Most of the industrial by-products (including molasses) contain ions and minerals that have deleterious effects on microbial growth and product synthesis [23,24]. Thus, in order to use

Fig. 1. Biomass, PHB, residual fructose, residual nitrogen and residual phosphate concentration for shake ask ((& ) fructose; (~) phosphate; () biomass; (^) PHB; (*) nitrogen.

S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 21732182 Table 1 Biomass and PHB produced by growing the culture in different carbon sources (nitrogen sourceammonium sulphate; mineral sourceyeast extract) Carbon source Fructose Lactic acid Sucrose Molasses Glycerol Glucose Sorbose Acetic acid Xylose Sodium acetate Propionic acid Starch Lactose Biomass (g/L) 3.5 1.188 2.179 1.960 2.879 2.332 0.302 0.151 0.263 0.095 0.055 0.117 2.251 PHB (g/L) 1.4 0.089 0.042 0.039 0.034 0.031 0.007 0.023 0.003 0.001 0.001 0.0 0.0 Table 3 Biomass and PHB obtained with different mineral source Mineral source Corn steep liquor Yeast extract Biomass (g/L) 6.23 5.80

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PHB (g/L) 2.70 2.26

molasses as a carbon source these components have to be removed from molasses, which would again lead to an increase in cost. Although carbon sources like sucrose, molasses, glycerol and glucose were utilized by the culture for biomass production but PHB obtained with all these sources was much less as compared to that obtained with fructose presumably because of the inability of the culture to use these sugars. So, in order to increase the yield and productivity, their high concentrations would be required which would again add to the cost. Thus, it was seen that maximum biomass and PHB were produced when fructose was taken as the carbon source. Naturally occurring strains of R. eutropha has been reported to utilize only fructose as carbon source [7]. Although recombinant strains have the capacity to use other carbon sources, their long-term stability is questionable. Also, this strain of R. eutropha (NRRL B14690) has the capability to accumulate high percentage of PHB in their biomass when grown on fructose as can be seen in this work and other literature reports [16,17]. Therefore, for further optimization studies, fructose was used as carbon source. Since nitrogen source is also an important factor for the accumulation of PHB, different salts of ammonium (Table 2) were tested and provided at amounts equivalent to the amount of ammonium sulphate taken in the media initially used [16,17]. Highest growth and PHB production was obtained with urea as a nitrogen source followed by ammonium chloride and ammonium nitrate. PHB production in a variety of complex nitrogen sources (sh peptone, protease peptone, yeast extract, phytone and tryptone) has been tried and it has been found that addition of complex
Table 2 Biomass and PHB obtained with different nitrogen sources Nitrogen source Ammonium sulphate Urea Ammonium chloride Ammonium nitrate Biomass (g/L) 5.80 7.92 6.73 2.54 PHB (g/L) 2.26 3.84 2.05 0.66 PHB content (%) 39.0 48.5 30.45 26.0

nitrogen sources increased the yield of PHB produced by Azotobacter vinelandii UWD strain [25]. The same was assumed to be applicable here. However, addition of ammonium nitrate leads to accumulation of only 0.66 g/L PHB. It may be that with ammonium nitrate, only the nitrogen of the ammonium was available for use, and enzymes for assimilation of nitrate were not synthesized, thus leading to a low PHB content. In another set of experiments, using fructose as a carbon source and ammonium sulphate as nitrogen source, addition of CSL as a mineral source led to the production of 2.70 g/L PHB as compared to 2.26 g/L, with yeast extract (Table 3). This increase may also be due to the high amounts of growth factors present in CSL, which favored growth and led to high biomass production. Thus, replacing ammonium sulphate and yeast extract with urea and CSL led to an increase of 41 and 15%, respectively, in the amount of PHB. 3.3. PlackettBurman design For most multivariable processes such as biochemical systems, in which numerous potentially inuential factors are involved, it is not always obvious to determine which are the most important ones. Hence, it is necessary to submit the process to an initial screening design prior to optimization. The methodology of PlackettBurman is a tool for this initial screening, since it makes it possible to determine the inuence of various factors with only a small number of trials. The design proves to be useful for picking up the relevant factors from a long list. The experimental design protocol used in the study was developed using DesignExpert Software (version 5.0.9) (Stat-Ease corporation, USA). Eight factors were selected for initial screening prior to optimization. Each parameter was tested at two levels, high and low (Table 4) whose concentration range was found by extensive literature survey. A nutrient recipe consisting of 12
Table 4 Range of different factors studied in the PlackettBurman design Factors Name Level 1 A (g/L) B (g/L) C (g/L) D (g/L) E (g/L) F (g/L) G (g/L) H (mL/L) Fructose Urea KH2PO4 MgSO4.7H2O CSL Na2HPO4 CaCl2 Trace metal solution 10.00 1.00 0.50 0.10 0.50 0.60 0.01 5.00 +1 40.00 3.0 3.0 1.0 2.0 4.0 0.03 15.00

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Table 5 Experimental design and response of PlackettBurman study Expt. no. A (g/L) B (g/L) C (g/L) D (g/L) E (g/L) F (g/L) G (g/L) H (mL/L) Response Residual biomass (g/L) 1 2 3 4 5 6 7 8 9 10 11 12 10.0 40.0 40.0 10.0 40.0 10.0 40.0 10.0 40.0 40.0 10.0 10.0 3.0 3.0 3.0 1.0 1.0 1.0 3.0 3.0 1.0 1.0 1.0 3.0 0.5 0.5 0.5 0.5 3.0 3.0 3.0 3.0 3.0 0.5 0.5 3.0 1.0 0.1 0.1 1.0 0.1 0.1 1.0 1.0 1.0 1.0 0.1 0.1 0.5 0.5 2.0 2.0 0.5 2.0 2.0 0.5 0.5 2.0 0.5 2.0 4.0 4.0 0.6 4.0 4.0 4.0 4.0 0.6 0.6 0.6 0.6 0.6 0.03 0.01 0.03 0.01 0.03 0.03 0.01 0.03 0.01 0.03 0.01 0.01 15.0 15.0 5.0 5.0 5.0 15.0 5.0 5.0 15.0 15.0 5.0 15.0 2.53 6.3 5.85 2.71 6.29 3.24 10.42 2.91 6.93 5.73 2.73 1.93 PHB (g/L) 0.07 3.05 2.01 0.33 4.92 0.23 3.93 0.48 6.44 4.8 0.32 0.02

experiments was designed by the software. Table 5 shows the distribution of the factors in the experiment according to the design expert software and the response in the study (Columns I and J). Table 6 presents t-values for the effect of medium components on residual biomass and PHB production. It is apparent from the table that except CaCl2 and trace metal solution all other factors had a positive effect on residual biomass (X0 ) production presumably because they are needed for the growth of the cells. Growth factors present in CSL may be responsible for its positive effect on growth. Fructose, KH2PO4 and MgSO47H2O had a positive effect on PHB production, whereas rest of the components had a negative effect. This can be easily explained by the fact that the limitation of any amongst N or P will lead to production of PHB by the culture. Of the six factors (AF), except urea and CSL all others were taken for optimization. From Table 5, it can be seen that PHB production was a maximum when these two factors were minimum (Experiments 5 and 9). Also, their contribution for the production of residual biomass is less when compared to other factors (Table 5). The lower limit of these factors was, therefore, as their optimum value. 3.4. Response Surface Methodology Response Surface Methodology is a successive, exploratory approach to establish the relationship between more than one variable with the obtained responses. The analysis
Table 6 Result of data analysis (t-value coefcient) for the effect of medium components on growth and PHB production Factors A B C D E F G H Name Fructose Urea KH2PO4 MgSO47H2O CSL Na2HPO4 CaCl2 Trace metal solution Residual biomass (g/L) 5.57 0.51 1.28 1.07 0.48 1.18 0.98 0.93 PHB (g/L) 21.45 6.77 4.93 4.97 3.58 1.40 1.43 2.38

develops a model by tting the experimental data in a generalized smooth curve, from which a specic predicted response could be calculated. In addition, contour plots are also generated which delineates predicted response over a range in the design surface. Thus, a step-by-step approach for response surface analysis establishes a relation between variable and response more efciently than traditional design [26]. Based on the PlackettBurman design, four factors (fructose, KH2PO4, MgSO47H2O and Na2HPO4) that showed positive inuence on growth and PHB were selected and CCD was used to determine the optimum levels of these parameters. A total of 30 experiments with different combinations of fructose (A), KH2PO4 (B), MgSO47H2O (C) and Na2HPO4 (D) were performed (Table 7). The results were analysed by Design Expert Software and following quadratic regression equations were obtained in terms of the selected variables for growth and PHB production: Residual biomass = +5.93 + 0.059A + 0.422B 0.138C + 1.14D 0.002A 2 0.116B 2 1.04C 2 0.771D 2 + 0.115AB 0.166AC 0.127AD + 0.169BC + 0.514BD 0.147CD PHB = +5.94 + 0.019A + 0.975 B 0.087C + 1.81D 0.216A2 0.67B2 1.19C2 0.957D2 0.111AB 0.101AC 0.052AD 0.011BC + 1.04BD 0.007CD For determination of the optimum operating concentration and interaction of factors on growth and PHB production, the response surfaces were studied in detail for all possible combinations by keeping two parameters constant at a time. The contour plots (Figs. 2 and 3) between various factors were analyzed and optimized concentrations of the media components were found. The predicted values obtained were conrmed by point prediction feature of the software. This is a feature of Design-Expert Software, which allows the study of responses by variation of one parameter at a time. It was found by contour plots and point prediction that when KH2PO4 and MgSO47H2O were varied from higher to lower limit, they were found to converge at a point. Above and below which, value of both response decreased. The converging value for KH2PO4 and MgSO47H2O were found to be 1.5 and 0.51 g/L, respectively. The predicted

S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 21732182 Table 7 Experimental design as given by Response Surface Methodology S. no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 KH2PO4 (g/L) 0.50 3.00 1.75 3.00 1.75 1.75 3.00 1.75 3.00 3.00 0.50 1.75 3.00 1.75 1.75 3.00 0.50 1.75 4.25 0.5 1.75 1.75 1.75 0.50 0.50 0.75 1.75 3.0 0.5 0.5 Na2HPO4 (g/L) 0.60 4.00 1.10 4.00 2.30 2.30 0.60 2.30 0.60 0.60 0.60 2.30 4.00 2.30 5.70 0.60 4.00 2.30 2.30 0.60 2.30 2.30 2.30 4.00 0.60 2.30 2.30 4.0 4.0 4.0 MgSO47H2O (g/L) 0.10 1.00 0.55 0.10 0.55 0.55 0.10 0.55 1.00 0.10 1.00 0.55 0.10 0.55 0.55 1.00 0.10 0.55 0.55 1.00 0.55 0.55 1.45 1.00 0.10 0.55 0.35 1.0 1.0 0.1 Fructose (g/L) 10.0 40.0 25.0 10.0 55.0 25.0 10.0 25.0 40.0 40.0 10.0 25.0 40.0 5.0 25.0 10.0 40.0 25.0 25.0 40.0 25.0 25.0 25.0 10.0 40.0 25.0 25.0 10.0 40.0 10.0 PHB (g/L) 0.33 6.25 2.99 0.70 6.12 7.27 0.48 6.42 1.82 2.76 0.36 8.06 6.32 0.00 5.43 0.33 6.97 6.96 6.29 2.35 3.86 3.07 1.88 0.52 1.77 5.75 2.35 0.48 6.78 0.69

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Residual biomass (g/L) 2.705 6.415 5.635 3.305 6.080 6.195 2.845 6.080 1.735 4.930 2.750 6.090 5.610 0.130 5.810 2.945 6.125 6.800 6.650 4.255 5.425 4.990 1.855 2.920 3.895 5.710 2.180 2.930 5.665 2.865

values for fructose and Na2HPO4 for the response was found to be increasing for their entire range given (Figs. 1 and 2). The contours clearly show that if the upper limit of Na2HPO4 would have been increased more, then the contours would have converged at a point. Thus, the optimum values of the media components (Table 8) obtained in this way are as follows: fructose, 40 g/L; KH2PO4, 1.5 g/L; Na2HPO4, 4.0 g/L; MgSO4.7H2O, 0.51 g/L. Shake ask studies were

then carried out for experimental verication of the predicted media concentration. Fig. 4 depicts the kinetics of R. eutropha grown on 40 g/L fructose as carbon source and 1 g/L urea as nitrogen source. An amount equal to 0.5 g/ L of CSL was used as mineral source. An optimized residual biomass of 7.10 g/L and PHB concentration of 8.16 g/L was predicted by the Design Expert Software. A maximum of 6.65 g/L residual biomass and 6.75 g/L PHB was obtained

Fig. 2. Contour plot between Na2HPO4 and fructose when response is residual biomass (biomassPHB).

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Fig. 3. Contour plot between Na2HPO4 and fructose when response is PHB.

Fig. 4. Shake ask kinetics in RSM optimized media ((&) fructose; (^) PHB; (~) phosphate; () biomass; (*) nitrogen).

by using the concentrations specied by the Design Expert, representing 94 and 83% validity of the predicted models for residual biomass and PHB production, respectively. 3.5. Batch kinetics in a 7 L bioreactor system Growth kinetics of the culture was then studied in a lab scale bioreactor. The culture was grown in a 7 L LF-1523 Bioengineering (Bioengineering AG, Switzerland) bioreactor to study growth, PHB production and the nutrient utilization prole. Fig. 5 shows the growth kinetics of

R. eutropha on 40 g/L fructose as carbon source and 1 g/L urea as nitrogen source. 0.5 g/L of CSL was used as mineral source. After a lag phase of approximately 10 h, the biomass increased to 20.73 g/L in 60 h. In this period, 0.37 g/L nitrogen was consumed out of an initial value of 0.55 g/L whereas approximately 30 g/L of fructose was metabolized. The synthesis of PHB, product of interest, started after 15 h and reached a nal concentration of 9.35 g/L. The yield (YP/ S) of PHB to fructose was 0.24 g/g and productivity of 0.15 g/L h was obtained. This is a marked improvement in comparison to 3.8 g/L (productivity: 0.08 g/L h) obtained

Fig. 5. Bioreactor (7 L) batch kinetics ((&) fructose; (^) PHB; (~) phosphate; () biomass; (*) nitrogen).

S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 21732182 Table 8 Optimized value of media components obtained after PlackettBurman and RSM studies Constituent Fructose KH2PO4 Na2HPO4 MgSO47H2O CaCl2 Urea Corn steep liquor Trace metal solution Optimized concentration (g/L) 40.0 1.50 4.00 0.51 0.02 1.00 0.5 10 mL/L

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4. Conclusion Addition of urea and corn steep liquor to the media in place of ammonium sulphate and yeast extract led to a better and cheaper production of PHB (PHB = 6.75 g/L; biomass = 13.39 g/L in 60 h) as compared to media being used earlier (PHB = 1.4 g/L; biomass = 3.25 g/L in 48 h). The methodology of PlackettBurman was very useful for the short-listing of relevant variables for process optimization. Using the method of factorial design and response surface analysis it was possible to determine optimal operating conditions, which led to higher concentrations and productivity without any loss of yield. Optimized media obtained by Response Surface Methodology consisted of: fructose = 40 g/L; urea = 1.0 g/L; CSL = 0.5 g/L; KH2PO4 = 1.5 g/L; Na2HPO4 = 4.0 g/L; MgSO47H2O = 0.51 g/L; CaCl2 = 0.02 g/L; trace metal solution = 10 mL/L. A signicantly higher maximum biomass of 20.73 g/L with a PHB content of 9.35 g/L was obtained when batch cultivation was conducted in 7 L lab scale bioreactor thus giving a yield of 0.24 g PHB per fructose consumed (g/g). The data obtained by batch kinetics would be used for mathematical model development and simulation of nutrient feeding strategies in fed batch/continuous cultivation for over production of PHB.

earlier [16] in batch cultivation of Alcaligenes eutrophus with 40 g/L fructose. Perdos-Alio et al. [27] obtained only approximately 1.9 g/L of the polymer by A. eutrophus with fructose as carbon source but the polymer content of the cell dry matter was very high ($96%). The results obtained in the present study are higher than a maximum polymer content of 6.9 g/L obtained with R. eutropha, when fructose was used a carbon source, with a productivity of 0.14 g/L h [28]. P. cepacia when grown on xylose gave a PHB content of 1.55g/L in 60 h under batch cultivation [29]. The same culture when grown on lactose gave a PHB content of 2.0 g/ L in 120 h [13]. A total biomass of 1.17 g/L with a productivity of 0.014 g/L h was obtained in batch cultivation of A. chroococcum with starch as carbon source [12]. Thus, the results obtained in the present study are signicantly better than the literature reported investigations. This signicant improvement in PHB and biomass may be due to optimization of media done in the present study, which had been missing in the earlier studies. An important feature of the present study was relatively cheap CSL being used instead of expensive yeast extract as a source of vitamins and growth factors which can not be used for large scale cultivation. By carrying out fed batch cultivation, PHB content signicantly higher [3032] than that obtained in the present study had been reported in literature. However, the main aim of the present study was to nd out cheap media components, which would allow maximum growth and PHB production and then to carry out media optimization studies in order to nd the optimal concentration of all media components for increasing the product concentration. In the present study, although a carbon source better than fructose could not be obtained but it is expected that by carrying out further reactor operating strategies in the optimized media enough PHB would be produced which would justify the cost addition due to fructose. Batch cultivation studies were also carried out to understand the kinetic behaviour of culture under controlled conditions of pH, temperature and dissolved oxygen. These studies would eventually form the basis of mathematical modeling and designing of nutrient feed strategies in fed batch cultivatons for further increase in biomass and PHB production.

Acknowledgement One of the authors (Ms. Shilpi Khanna) is thankful to the Council of Scientic and Industrial Research (CSIR), Govt. of India, New Delhi, for providing fellowship during the work done.

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