Process optimization for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polymer production by Nostoc muscorum

Biochemical Engineering Journal 37 (2007) 125–130

Process optimization for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polymer production by Nostoc muscorum Nirupama Mallick a,∗ , Suneel Gupta a , Bhabatarini Panda a , Ramkrishna Sen b a

Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur 721302, West Bengal, India b Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India Received 10 August 2006; received in revised form 6 April 2007; accepted 15 April 2007

Abstract This study aims at process optimization for poly(3-hydroxybutyrate-co-3-hydroxyvalarate) [P(3HB-co-3HV)] co-polymer production by Nostoc muscorum with respect to variation in different parameters, viz. carbon concentration, time of incubation and pH using response surface methodology (RSM). Under pre-optimized condition, P(3HB-co-3HV) accumulation in N. muscorum reached up to 28.2% (w/w) of dry cells (dcw) in presence of 0.2% acetate + 0.4% propionate, when incubated for 14 days at pH 8.5. A five-level four-factorial central composite design was employed to find out the interactions of four variables, viz. concentrations of acetate and propionate, pH and days of incubation. Using RSM, a second order polynomial equation was obtained by regression analysis. An optimum co-polymer yield of 31.4% (dcw) was achieved at a reduced level of carbons, i.e. 0.11% acetate and 0.08% propionate at pH 8.1 and an incubation period of 16 days. Thus after optimization, though the product yield was increased only by 11%, acetate and propionate requirements were reduced by 45 and 80%, respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: Acetate; P(3HB-co-3HV); Propionate; Response surface methodology (RSM)

1. Introduction Bacterial polyhydroxyalkanoates (PHAs) have increasingly become of interest as a raw material for biodegradable plastics. Amongst the 150 different types of PHAs identified so far, the homopolymer of hydroxybutyrate, i.e. PHB is widespread in different taxonomic group of prokaryotes including cyanobacteria [1,2]. However, this homopolymer has poor physical properties as brittleness and low mechanical strength, thus its application is highly limited. In addition to this, its high melting temperature, i.e. about 170 ◦ C makes its processing a difficult task. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or [P(3HB-co-3HV)] co-polymer on the other hand, is less stiff and tougher. Incorporation of 3-hydroxyvalerate, i.e. 3(HV) units not only reduces the crystallinity and melting temperature of PHB homopolymer, but also give better tensile strength to the polymer. To date, commercial production of P(3HB-co-3HV) copolymer is being carried on with Wautersia eutropha, formerly

∗

Corresponding author. Tel.: +91 3222 283166; fax: +91 3222 282244. E-mail addresses: [email protected]/, [email protected]/ (N. Mallick).

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.04.002

known as Ralstonia eutropha, by Metabolix, Massachusetts, USA, under fermentative condition. However, the cost of fermentative polymer production, due to high carbon requirement and oxygen demand, is a major obstacle for presenting P(3HB-co-3HV) co-polymer as a commodity material [3]. In this context, cyanobacteria can be considered as an alternative host system due to their minimal nutrient requirements and photoautotrophic nature. Existing literature reveal that although a number of cyanobacterial species have the ability to accumulate the homopolymer of PHB under photoautotrophic condition, Anabaena cylindrica 10 C is the only cyanobactrium reported so far to accumulate the P(3HB-co-3HV) co-polymer under propionate-supplemented condition [4] with a maximum yield of 2% of the dry cell weight (dcw). Our preliminary study also showed the synthesis of P(3HB-co-3HV) co-polymer by a N2 -fixing cyanobactrium, Nostoc muscorum under propionatesupplemented condition. The optimization of co-polymer production through traditional method, i.e. one variable at-a-time is time consuming and interactions of different variables can also affect the yield. Unlike conventional optimization, statistical optimization methods can take into account the interactions of variables in generating the process response [5,6]. Response surface methodology (RSM),

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in this context, is the most widely used method, where contour plots are generated by linear or quadratic effects of the key variables and a model equation is derived that fits the experimental data to calculate the optimal response of the system with a limited number of experiments. Thus, the aim of this study is to optimize the nutritional and process parameters statistically, which have been observed to play significant roles in enhancing the co-polymer yield of N. muscorum, individually. These critical variables are acetate, propionate, pH and days of incubation. 2. Materials and methods 2.1. Organism and growth conditions Established culture of N. muscorum Agradh was grown axenically in 150 ml Erlenmeyer flasks containing 50 ml of NO3 -free BG-11 medium [7] at 25 ± 2 ◦ C, pH 8.5 under a photoperiod of 14 h light (75 ␮mol photon m−2 s−1 PAR) and 10 h dark cycles. Cell dry weight was determined following the gravimetric method of Rai et al. [8]. 2.2. Extraction of polyhydroxyalkanoates (PHAs) Extraction of PHAs was done following Yellore and Desia [9] with certain modifications. Algal biomass was suspended in methanol overnight at 4 ◦ C. The pellet obtained after centrifugation was dried at 60 ◦ C. The PHAs was extracted in hot chloroform followed by precipitation with cold diethyl ether. The precipitate was centrifuged at 10,000 × g for 20 min to get the pellet. The pellet was washed with acetone and the same was dissolved in hot chloroform again. 2.3. Assay of P(3HB-co-3HV) co-polymer Gas chromatography was performed for the assay of P(3HBco-3HV) co-polymer using a Perkin-Elmer (Clarus 500) GC system equipped with Perkin Elmer’s Elite-1 dimethylpolysiloxane capillary column (30 m × 0.25 mm × 0.25 ␮m) and a flame ionization detector. Benzoic acid was used as the internal standard. The purified sample was propanolysed according to Riis and Mai [10]. The esterified sample (0.2 ␮l) was injected in split mode (1:50) to the GC. The detection was made by comparing the retention time of the standard PHB and P(3HB-co-3HV) co-polymer (Aldrich, USA) with the sample. 2.4. Experimental design

Table 1 Experimental range and levels of the independent variables Variable

Acetate (X1 ), % (w/v) Propionate (X2 ), % (w/v) Incubation period (X3 ), day pH (X4 )

−2

−1

0

+1

+2

0.0 0.0 10 6

0.02 0.02 14 7

0.1 0.06 18 8

0.2 0.1 22 9

0.3 0.15 26 10

pH and incubation period to get maximum P(3HB-co-3HV) copolymer yield. The range and the levels of the variables taken for this study are given in Table 1. In developing the regression equation, the test variables were coded according to the equation: xi =

(Xi − Xi∗ ) Xi

(1)

where xi is the coded value of the ith independent variable, Xi is the uncoded value of the ith independent variable, Xi∗ is the uncoded value of the ith independent variable at the centre point and Xi is the step change value. The regression equation was solved by the multi-stage Monte-Carlo optimization method [11]. 3. Results 3.1. Accumulation of P(3HB-co-3HV) co-polymer with reference to growth Gas chromatographic analysis of the polymer extracted from N. muscorum cells grown under propionate-supplemented condition showed two distinct peaks, respectively, at retention time of 4.8 and 6.3 min, corresponding to the peaks of 3hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) standards (data not shown). Accumulation of P(3HB-co-3HV) co-polymer with reference to growth was studied (Fig. 1), which depicted a maximum value of 28.2% (dcw) on day 14 of incubation. The comparative mole fractions of 3HB and 3HV units are shown in Table 2, which did not register any significant change with varying propionate concentration from 0.1 to 0.4%. Table 2 Accumulation of P(3HB-co-3HV) at varied concentration of propionate in N. muscorum on day 14 of incubation Treatment

A 24 full factorial central composite rotary design (CCRD) for four independent variables each at five levels was employed to fit a second order polynomial model which indicated that 30 experiments were required for this procedure [5]. The ‘Design Expert’ software (Version 7.0.2, Stat-Ease Inc., Minneapolis, USA) was used for regression and graphical analysis. Contour plots (2D) were generated to understand the interactions of various factors and then used to find out the optimum values of the desired factors, viz. concentrations of acetate, propionate,

Range and level

Control (photoautotrophic) 0.2% A 0.2% A + 0.1% P 0.2% A + 0.2% P 0.2% A + 0.4% P

P(3HB-co-3HV) % (dcw) 8.5 12.9 25.7 26.9 28.2

± ± ± ± ±

Composition (mol%) 3HB

3HV

0.5a

100a

0.6b 1.2c 1.3c 2.3c

100a 71.1 ± 5.0b 72.0 ± 4.2b 74.8 ± 4.9b

0 0 28.9 ± 5.0a 28.0 ± 4.2a 25.2 ± 4.9a

All value are mean ± SD, n = 3, A = acetate, P = propionate; Values in the column superscripted by same letters are not significantly different from each other (Duncan’s new multiple range test). Separate analysis was done for each column.

N. Mallick et al. / Biochemical Engineering Journal 37 (2007) 125–130

Fig. 1. Accumulation of P(3HB-co-3HV) co-polymer in N. muscorom with reference to growth in presence of 0.2% acetate + 0.4% propionate. Growth () and P(3HB-co-3HV) co-polymer().

3.2. Optimization of P(3HB-co-3HV) yield in N. muscorum by RSM The design matrix and the responses, both actual and predicted, for P(3HB-co-3HV) yield by CCRD are presented in Table 3. This showed yield ranging from 2.2 to 29.9% (dcw) in the actual terms, whereas the predicted values varied from 4.3 to 28.9% (dcw). Application of response surface to these values yielded the following regression equation in coded form. Y = 29.05 + 0.65x1 + 1.02x2 + 0.37x3 − 7.48x4 − 5.66x12 − 0.76x22 − 3.91x32 − 5.03x42 − 1.37x1 x2 − 0.39x1 x3 − 0.93x1 x4 + 0.59x2 x3 − 0.77x2 x4 + 0.16x3 x4

(2)

where, Y is the response, i.e. the P(3HB-co-3HV) yield, and x1 , x2 , x3 and x4 are the coded terms for the four test variables, i.e. acetate, propionate, incubation period and pH, respectively. The Fisher F-test [F0.01(14,15) = Sr2 /Se2 = 8.33 >

Fig. 2. 2D contour plot of interactive effects of acetate and propionate for P(3HB-co-3HV) yield keeping pH and incubation period at zero level.

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Fig. 3. 2D contour plot of interactive effects of propionate and pH for P(3HBco-3HV) yield keeping acetate and incubation period at zero level.

F0.01(14,15) tabular = 3.56] showed a very low probability value (Pmodel > F = 0.0001) for the regression model (Table 4). Regression analysis of the variables in their interactive conditions is presented in Table 5. The significance of each coefficient was determined by student t-test, where the interaction of acetate and propionate depicted the lowest P-value. Figs. 2–4 represent the 2D contour plots of the test variables. Each contour curve represents an infinite number of two test variables with the other two maintained at their respective zero levels. The interactions between propionate with other variables demonstrated elliptical contour plots (Figs. 2–4), which were however, more prominent for the interaction of propionate with acetate (Fig. 2). The interactions of incubation period with acetate and pH showed parallel contour lines (data not shown). Similar response was also observed for the interaction between acetate and pH (data not shown). The normal (%) probability plot of the residuals (Fig. 5) depicted that the errors were normally distributed and insignificant.

Fig. 4. 2D contour plot of interactive effects of propionate and incubation period for P(3HB-co-3HV) yield keeping acetate and pH at zero level.

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Table 3 Full factorial central composite design matrix for four variables along with the observed and predicted responses Obs 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

Variable

Response

Factor 1

Factor 2

Factor 3

Factor 4

Total PHA in dcw (%)

Acetate (%, w/v)

Propionate (%, w/v)

Incubation period (day)

pH

Actual value

Predicted value

0.02 0.2 0.02 0.2 0.02 0.2 0.02 0.2 0.02 0.2 0.02 0.2 0.02 0.2 0.02 0.2 0.0 0.3 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

0.02 0.02 0.1 0.1 0.02 0.02 0.1 0.1 0.02 0.02 0.1 0.1 0.02 0.02 0.1 0.1 0.06 0.06 0.0 0.15 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06

14 14 14 14 14 14 14 14 22 22 22 22 22 22 22 22 18 18 18 18 18 18 10 26 18 18 18 18 18 18

7 7 7 7 9 9 9 9 7 7 7 7 9 9 9 9 8 8 8 8 6 10 8 8 8 8 8 8 8 8

16.9 29.2 27.7 23.3 21.0 22.4 23.9 27.4 3.4 6.1 3.4 3.1 2.2 4.2 5.9 3.9 21.4 3.6 23.3 27.7 9.8 15.5 13.0 3.4 27.6 29.4 29.3 28.8 28.2 29.9

16.7 22.8 21.8 22.4 16.7 21.2 24.2 23.3 4.3 7.8 6.4 4.4 4.9 6.9 9.3 5.8 19.7 5.2 25.4 27.7 12.4 14.1 23.7 6.1 28.9 28.9 28.9 28.9 28.9 28.9

By solving the Eq. (2) with the help of multi-stage MonteCarlo optimization method the optimal values obtained for the test variables for maximum polymer yield in coded terms were as fallows: x1 = 0.10, x2 = 0.65, x3 = −0.48 and x4 = 0.11 with the corresponding co-polymer yield of Y = 32.8% (dcw). Thus, the optimized values obtained by putting the respective values of xi in Eq. (1) were: acetate 0.11% (w/v), propionate 0.08% (w/v),

pH 8.1 and an incubation period of 16 days with the predicted co-polymer yield of 32.8% (dcw). 3.3. Verification of the RSM optimization model To verify the model, experiments were performed in triplicate using the above optimized conditions. A co-polymer yield of 31.4% (dcw) was recorded against the predicted yield of 32.8% (Table 6). Thus after optimization, co-polymer yield of 31.4% was achieved at a reduced level of carbon concentrations, i.e. 0.11% acetate + 0.08% propionate at pH 8.1 and an incubation period of 16 days as compared to the pre-optimized yield of 28.2% in 0.2% acetate + 0.4% propionate at pH 8.5 and an incubation period of 14 days. Table 4 Analysis of variance (ANOVA) of the quadratic model

Fig. 5. Normal (%) probability plot of the ‘studentized’ residuals.

Source of variation

Sum of square

Degree of freedom

Mean square

F-value

Prob. (P) > F

Model Residual

2941.11 378.34

14 15

210.08 25.22

8.33

0.0001

COR. total

3319.45

29

Root MSE = 5.02; CV = 29.26%; R2 = 0.8860; r = 0.941; Adj. R2 = 0.7796.

N. Mallick et al. / Biochemical Engineering Journal 37 (2007) 125–130 Table 5 Regression analysis of the experimental design in the interactive conditions of the variables Variable

Coefficient

t-value

P-value

x1 × x2 x1 × x3 x1 × x4 x2 × x3 x2 × x4 x3 × x4

−1.37 −0.39 −0.93 0.59 −0.77 0.16

−1.096 −0.312 −0.744 0.468 −0.611 0.127

0.2910 0.7577 0.4709 0.6466 0.5463 0.8987

Standard error of means = 0.92575.

4. Discussion Incorporation of propionate in the culture medium induced synthesis of the co-polymer, P(3HB-co-3HV) in N. muscorum, as reported for another N2 -fixing cyanobacterium Anabaena cylindrica 10 C [4]. Under photoautotrophic as well as acetatesupplemented conditions this test cyanobacterium however, primarily accumulated 3HB units only [12,13]. The purity of the polymer was confirmed by gas chromatographic analysis, where two distinct peaks of 3HB and 3HV monomer units, without any other impurities, were clearly evident. Maximum accumulation of the polymer on day 14 (Fig. 1) agrees well with the earlier reports of Stal [14] and Panda et al. [15], where accumulation of PHAs reached its maxima at the stationary phase of growth. Cultures incubated under carbonsupplemented conditions depicted significant rise in PHA pool as compared to the photoautotrophic condition (Table 2). The positive impact of acetate on PHB accumulation could be related to the involvement of the usual pathway operated in bacteria, so that acetate was directly utilized for the synthesis of the polymer [16]. Varying propionate concentration from 0.1% to 0.4% however, did not register any significant change either on the total PHA production or on the mole fraction of 3HB and 3HV units (Table 2). This was the turning point to opt for a statistical optimization process such as RSM to get maximum product yield under minimum nutrient supplementation. A high value of correlation coefficient (r = 0.941, Table 4) justifies the excellent correlation amongst the independent variables [17]. The determination coefficient (R2 ) is 0.886 (Table 4), which depicts that the model could explain about 88.6% of variations in the response. At the same time a moderate value of the coefficient of variation (CV = 29.2%, Table 4) indicates a better precision and reliability of the experiments carried out. Table 6 P(3HB-co-3HV) co-polymer yield before and after optimization Variable X

Acetate % (w/v) Propionate % (w/v) Incubation period (day) pH

P(3HB-co-3HV) yield (% dcw) Before

After

0.2 0.4 14

0.11 0.08 16

8.5

8.1

Before

28.2

After Predicted

Experimental

32.8

31.4

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The elliptical nature of the contour plots (Figs. 2–4) demonstrates that the interactions between propionate with other variables have significant impact on the product yield. The more prolate contour lines as observed in Fig. 2 further depicts a highly significant interaction of propionate with acetate as compared to interactions of propionate with pH/incubation period. This can also be affirmed from Table 5, where the interaction of acetate with propionate depicted the lowest P-value as compared to the other interactions. However, the parallel contour lines of incubation period versus acetate, incubation period versus pH and acetate versus pH (data not shown) demonstrate that increasing or decreasing values from the zero level will have negative impact on the product yield. The normal (%) probability plot (Fig. 5) of the ‘studentized’ residuals indicates no violation of the assumptions underlying the analyses [6]. It can be visualized from Table 6 that the predicted product yield, i.e. 32.8% and the experimental (31.4%) are well in agreement, i.e. the variation is not so significant. After optimization the product yield was increased only by 11%. Interestingly however, the requirement of acetate and propionate was reduced, respectively, by 45 and 80%, which signifies that the process, hence optimized, could be used for P(3HB-co-3HV) production by N. muscorum cost effectively. In this study, the co-polymer yield reached only up to the level of 31% (dcw). Although this seems rather low as compared to the 74% yield of the most acclaimed bacterium, W. eutropha, the carbon requirement is many fold higher (20 and 1.3 g l−1 , glucose and propionate, respectively) than that has been observed for the test cyanobacterium [18]. Thus, the use of cyanobacteria for low-cost P(3HB-co-3HV) production seems highly promising. However, propionate supplementation was found to affect the biomass yield. This necessitates a two-stage cultivation practice, where at the first stage the cells must be grown to a high density under proper condition, followed by a second stage, as optimized in this study, to allow efficient P(3HB-co-3HV) co-polymer accumulation. Acknowledgements Financial support from Council of Scientific and Industrial Research, New Delhi, India is thankfully acknowledged. We are also thankful to Mr. A.K. Singh and Mr. Y.B. Krishna for their kind help in preparing the artworks. References [1] A. Steinbuchel, Biodegradable plastics, Curr. Opin. Biotechnol. 3 (1992) 291–297. [2] M. Vincenzini, R. De Philippis, Polyhydroxyalkanoates, in: Z. Cohen (Ed.), Chemicals from Microalgae, Taylor and Francis, London, 1999, pp. 290–312. [3] P.A. Holmes, Biologically produced PHA polymers and co-polymers, in: D.C. Bassett (Ed.), Developments in Crystalline Polymers, vol. 2, Elsevier, London, United Kingdom, 1988, pp. 1–65. [4] L. Lama, B. Nicholaus, V. Calandrelli, M.C. Manca, I. Romano, A. Gambacorta, Effect of growth condition on endo- and exopolymer biosynthesis in Anabaena cylindrica 10 C, Phytochemistry 42 (1996) 655–659. [5] A.I. Khuri, J.A. Cornell, Response Surface: Design and Analyses, Marcel Dekker, Inc., New York, 1987.

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