Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium

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Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium Débora Jung Luvizetto Faccin a , Rosane Rech b , Argimiro Resende Secchi c , Nilo Sérgio Medeiros Cardozo a , Marco Antônio Záchia Ayub b,∗ a

Chemical Engineering Department, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil Food Science and Technology Institute, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil c COPPE-Chemical Engineering Program, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil b

a r t i c l e

i n f o

Article history: Received 26 October 2012 Received in revised form 14 January 2013 Accepted 5 February 2013 Keywords: Bacillus megaterium Poly(3-hydroxybutyrate) Biopolymer

a b s t r a c t Poly(3-hydroxybutyrate)—P(3HB)—is a natural biodegradable polyester synthesized by several bacteria, produced from renewable resources. The effects of oxygen transfer rate on the intracellular accumulation of P(3HB) was evaluated, aiming at increasing P(3HB) synthesized by Bacillus megaterium DSM 32T in bioreactor batch cultures. Bench-scale bioreactor cultivations were performed under different volumetric oxygen mass transfer coefficients, kL a, setting stirrer speed on specified values. The results of this work show that oxygen transfer is a key factor on P(3HB) accumulation by B. megaterium, increasing the P(3HB) intracellular mass fraction from 39% to 62% of CDW at kL a condition of 0.006 s−1 . © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Poly(3-hydroxybutyrate) (P(3HB)) is the most important and well characterized biopolymer belonging to polyhydroxyalkanoates [1,2]. Polyhydroxyalkanoates (PHAs) are polyesters synthesized by microorganisms and accumulated in the cell cytoplasm as water insoluble granules in order to store carbon and energy [2–5]. Biodegradability, biocompatibility, possibility of production from renewable carbon resources derived from agriculture or industrial wastes, and the fact of presenting properties similar to those observed in some petrochemical polymers are the main reasons for the growing interest in PHAs, its production and industrial applications [1,6–9]. Many bacteria, both Gram-positive and Gram-negative, are able to synthesize PHAs, but so far most of the industrial scale PHA production is done by Gram-negative bacteria such as Cupriavidus necator (formely Ralstonia eutropha), Pseudomonas oleovorans, and recombinant strains of Escherichia coli [10]. However, the production using Gram-negative bacteria can be disadvantageous for biomedical applications because they possess lipopolysaccharides (LPS), which can be extracted together with PHA in a purification step. LPS act as endotoxins and can cause immunological

∗ Corresponding author at: Food Science and Technology Institute, Federal University of Rio Grande do Sul State, Av. Bento Gonc¸alves, 9500, P.O. Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil. Tel.: +55 51 3308 6685; fax: +55 51 3308 7048. E-mail address: [email protected] (M.A.Z. Ayub).

reactions [10,11]. Among Gram-positive bacteria lacking such endotoxins, Bacillus strains have some interesting characteristics to be used in industrial scale production. This bacterium presents fast growth, can metabolize several substrates, and can also tolerate high osmotic pressures and high temperatures [10,12–20]. Several carbon sources have been used as substrates for Bacillus sp. in the accumulation of short chain length polyhydroxyalkanoate, including arabinose, xylose, glucose, galactose, fructose, sucrose, maltose, cellobiose, glycerol, and mannitol [21]. Notwithstanding these advantages, the relatively low productivity of P(3HB) obtained in cultures of Bacillus is still a limiting aspect for its industrial application [20]. Reports on the literature show the limitations of P(3HB) production by Bacillus strains under commonly applied culture conditions such as excess of carbon with nutrient limitation (nitrogen and phosphor), or low oxygen supply, which can induce cell sporulation, leading to P(3HB) consumption [14,20,22,23]. One condition of special importance reported to affect the production of P(3HB) by Bacillus is the level of oxygen supply. In a previous work [22] this influence was observed based on the significantly higher P(3HB) production obtained in shaker scale when comparing results obtained in shaker and bioreactor cultures. Since the main difference between the two processes is in the oxygen supply, which is lower in shaker, those results suggested that some level of oxygen limitation is required for achieving high levels of P(3HB) production. However, the use of low oxygen supply must be carefully controlled because Bacillus megaterium is a strict aerobe [12,24,25].

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The oxygen supply can play an important role in the scale-up and economy of aerobic biosynthesis systems [26,27]. The volumetric oxygen mass transfer coefficient (kL a) is an important parameter, since it is related to the oxygen transfer rate (OTR) and is commonly applied as a criteria for scale-up [26–31]. In this context, the aims of this research were to clarify the influence of oxygen supply of bioreactor cultures on the biomass production and P(3HB) synthesis by B. megaterium, through the comparison of cultures performed under different kL a, obtained by variation of stirrer speed. 2. Materials and methods

0.03 g L−1 ). Sucrose (16 g L−1 ) and ammonium sulphate (2 g L−1 ) were supplemented as carbon and nitrogen sources, respectively [22]. 2.2. Bench-scale bioreactor culture Bench-scale bioreactor cultivations were performed in a 5 L vessel Biostat B (Braun Biotech) with 4 L of culture medium and 80 mL of a pre-culture inoculum. Temperature was kept at 30 ◦ C and initial pH was adjusted at 7.0. The culture was performed under uncontrolled pH condition. Air was supplied at flow rate of 4 L·min−1 . Bioreactor experiments were carried out as independent duplicates, with stirrer speed (N) set at 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, and 600 rpm in order to promote different oxygen transfer rates. The instant at which the dissolved oxygen (DO) concentration returned to its saturation value (100%) was taken as completion of each batch culture.

2.1. Microorganism and culture media

2.3. Analytical procedures

Bacillus megaterium DSM 32T was used in this study. The strain was preserved as frozen samples in mineral medium, with a volume fraction of 20% glycerol. Cultures were activated by 2 subsequent pre-cultures (18 h and 5 h) before cultivation. Culture mineral salts medium had the following composition [32]: KH2 PO4 1.5 g L−1 ; Na2 HPO4 • 12H2 O, 9 g L−1 ; MgSO4 • 7H2 O, 0.2 g L−1 ; CaCI2 • 2H2 O, 0.01 g L−1 ; citric acid, 0.1 g L−1 , supplemented with 1 mL of trace elements solution (FeSO4 • 7H2 O, 20 g L−1 ; MnCI2 • 4H2 O, 0.03 g·L−1 ; H3 BO4 , 0.3 g L−1 ; CuSO4 • 5H2 O, 0.01 g L−1 ; CoCI2 • 6H2 O, 0.2 g L−1 (NH4 )6 Mo7O24 • H2 O, 0.03 g L−1 ; ZnSO4 • 7H2 O, 0.03 g L−1 ; NiSO4 • 7H2 O,

Total biomass (X), measured as cell dry weight (CDW), was evaluated from 10 mL to 60 mL samples of culture. The cell suspension was centrifuged at 2 500 × g for 20 min at 4 ◦ C, washed with distilled water, transferred to pre-weighed flasks, and dried at 80 ◦ C until constant weight. PHB content was determined by gas chromatography as described by Riis and Mai [33] using a GC Perkin Elmer (FID detector, capillary column: PE-WAX 30 m × 0.25 mm, Perkin Elmer). Pure poly(3-hydroxybutyrate) (Aldrich) was used as standard. Residual biomass (R) was estimated by the difference between total biomass and P(3HB) content.

Fig. 1. Time course of total and residual biomass, biopolymer concentration, sucrose concentration and nitrogen concentration in the culture with kL a of (a) 0.002 s−1 , (b) 0.006 s−1 , (c) 0.013 s−1 , (d) 0.016 s−1 , (e) 0.027 s−1 and (f) 0.037 s−1 .

Please cite this article in press as: Faccin DJL, et al. Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.02.004

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P(3HB) production has growth-associated and non-growth associated components. The highest P(3HB) concentration, 3.3 g·L−1 , was attained under kL a of 0.006 s−1 . The length of exponential growth phase decreased proportionally with kL a, suggesting growth limitation under low oxygen supply, since B. megaterium is a strict aerobe. The P(3HB) mass fraction attained in the cultures under different kL a values are presented in Fig. 2, while the statistical analyses of the culture parameters are presented in Table 1. It could be observed a strong effect of kL a on P(3HB) cell content (p = 0.0003). The highest P(3HB) mass fraction (62% of CDW) was achieved for kL a of 0.006 s−1 . Intermediate values of the P(3HB) mass fraction were obtained for kL a of 0.002 s−1 and 0.013 s−1 , with no significant difference between the values obtained at these two conditions. Increase of kL a above 0.013 s−1 led to additional decrease in the P(3HB) mass fraction, with an apparent limiting value of this variable being achieved, since no significant difference was observed for the remaining values of kL a (0.018 s−1 , 0.027 s−1 , and 0.037 s−1 ). At the lowest value of kL a (0.002 s−1 ) the accumulation of polymer followed a slow but constant kinetics, while for higher values, above 0.013 s−1 , the accumulation was fast but followed by the loss of P(3HB) in the final stages of culture, probably due to cell disruption or endogenous consumption. The statistical analyses showed that kL a had a significant effect on P(3HB) yield on sucrose (p = 0.0008) and P(3HB) productivity (p = 0.0098), but not on biomass yield on sucrose (p = 0.11). The lowest P(3HB) productivity (PP(3HB) ) was obtained at the lowest kL a value (0.002 s−1 ), where P(3HB) production was slower (Fig. 1). All other cultures (kL a values from 0.006 s−1 up to 0.037 s−1 ) presented productivities around 0.16 g L−1 h−1 , with no significant difference among them, but significantly higher than the value obtained at the kL a of 0.002 s−1 . The highest P(3HB) yield (YP(3HB)/S ), 0.32 g g−1 , was obtained at kL a of 0.006 s−1 and there was no significant difference among the values of YP(3HB)/S obtained for the other kL a tested. However, the residual biomass yields on sucrose (YR/S ) were not affected by the kL a condition. Regarding the specific growth rate for total (X ) and residual biomass (R ) during exponential phase of cultures, at the lowest kL a (0.002 s−1 ) both X and R , were significantly lower (p < 0.0001) than the other values. The results indicate that there is a lower boundary for oxygen limitation (close to the lowest kL a condition used), under which the level of oxygen affects growth rate. Fig. 3 shows the behavior of dissolved oxygen (DO) concentration along the cultivations for different kL a values. It can be observed that the decrease in the available dissolved oxygen starts earlier for smaller kL a while the return to air saturation starts earlier for higher kL a. For smaller kL a values, zero dissolved oxygen concentration was reached, meaning that cells promptly consumed all oxygen supplied to the bioreactor. Values of the pH of the medium during growth at each kL a condition are shown in Fig. 4. A decrease in the pH was observed since all experiments were performed without pH control. At the beginning of the cultivation the pH was adjusted at 7.0, and during the cultivation the pH dropped and reached final values between 4 and

Fig. 2. Time course of P(3HB) content, mass fraction of CDW, in batch cultures with kL a of () 0.002 s−1 , () 0.006 s−1 , (䊉) 0.013 s−1 , () 0.018 s−1 , () 0.027 s−1 and (×) 0.037 s−1 . Sucrose was analyzed by HPLC Perkin Elmer series 200, using a Rezex-RHM (300 mm × 7.8 mm) column at 80 ◦ C and RI detector. Nitrogen was determined by the phenol-hypochlorite reaction [34]. The oxygen concentration was measured on line with a polarographic oxygen sensor (InPro 6800/12/320 manufactured by Mettler-Toledo) as the percent saturation of dissolved oxygen (DO). The kL a was determined using the dynamic method [35] at 2 h and 4 h of each batch culture. All analytical procedures were carried out as independent duplicates. Data are presented as the mean of independent repeats of batch cultures and were analyzed by ANOVA (analyses of variance) and Tukey’s test (5% probability). 2.4. Investigation of endospore formation The formation or not of endospores was performed following the spore stain technique [36]. The cells were fixed and stained with malachite green and after stained with safranin as a counter-stain dye. This procedure was carried out for samples of all tested conditions.

3. Results The kL a increased in the range of stirrer speed studied (p < 0.0001). The experimental data were fitted to a power-law function, as showed in Eq. (1) (R2 = 0.997). kL a = 1.1 × 10−6 × N 1.6

3

(1)

where kL a is the volumetric oxygen mass transfer coefficient and N is the stirrer speed. The kinetics under different kL a conditions of sucrose and nitrogen consumption, P(3HB) production and biomass formation (residual and total), are shown in Fig. 1. No substrate limitation for both nitrogen and sucrose was observed. Regarding sucrose, it could be observed that half of initial sucrose concentration remained in the medium, with the exception for kL a of 0.006 s−1 , where sucrose consumption was almost complete. The P(3HB) production started at exponential growth phase and continued until the beginning of the stationary phase, suggesting that the

Table 1 The effect of kL a on P(3HB) mass fraction, P(3HB) productivity (PP(3HB) ), P(3HB) yield (YP(3HB)/S ), residual biomass yield (YR/S ) and specific growth rate for total (X ) and residual (R ,) biomass. kL a [s−1 ]

P(3HB) [% of CDW]

0.002 0.006 0.013 0.018 0.027 0.037

49b 62a 49b 40 39 39

± ± ± ± ± ±

5.6 1.5 1.4 1.2c 1.4c 2.7c

PP(3HB) [g L−1 h−1 ] 0.05a 0.14b 0.16b 0.17b 0.16b 0.15b

± ± ± ± ± ±

0.017 0.029 0.019 0.032 0.013 0.001

YP(3HB)/S [g g−1 ] 0.18a 0.32b 0.21a 0.20a 0.17a 0.17a

± ± ± ± ± ±

0.046 0.004 0.014 0.005 0.011 0.012

YR/S [g g−1 ] 0.23a 0.21a 0.25a 0.28a 0.31a 0.28a

± ± ± ± ± ±

0.050 0.011 0.005 0.017 0.043 0.015

X [h−1 ] 0.51a 0.71b 0.72b 0.67b 0.76b 0.70b

± ± ± ± ± ±

0.021 0.031 0.006 0.032 0.003 0.060

R [h−1 ] 0.50a 0.66b 0.72b 0.66b 0.73b 0.68b

± ± ± ± ± ±

0.030 0.034 0.007 0.028 0.017 0.056

Data are the mean from independent repeats. Means with different superscript letters (a–c) at same column are significantly different at the p ≤ 0.05 level (Tukey test).

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Fig. 3. Time course of dissolved oxygen, DO, in batch cultures with kL a of () 0.002 s−1 , () 0.006 s−1 , (䊉) 0.013 s−1 , () 0.018 s−1 , () 0.027 s−1 and (×) 0.037 s−1 .

Fig. 4. Time course of pH in batch cultures with kL a of () 0.002 s−1 , () 0.006 s−1 , (䊉) 0.013 s−1 , () 0.018 s−1 , () 0.027 s−1 and (×) 0.037 s−1 .

5, the higher the kL a the lower the pH at the end of the cultivation. The lowest pH value at each kL a condition coincide with the end of exponential growth phase, indicating that pH can be the cause of the growth stopping, since both sucrose and nitrogen were still available in the medium. Finally, the observation of cells for endosporespores formation, which was carried out for samples of cultures run under all tested kL a did not show any spores formation (results not shown). 4. Discussion The high value of R2 obtained for the power-law function (Eq. (1)) indicates that this model adequately describes the correlation between kL a and the stirrer speed. A power-law dependence of the kL a on stirrer speed has also been reported by Garcia-Ochoa et al. [37] in the production of xanthan gum by Xanthomonas campestris, with exponent around 2.0. The results presented in the previous section show a clear dependence of the parameters usually employed to quantify P(3HB) production (mass fraction, productivity and yield) on kL a. Although the importance of the available oxygen in the P(3HB) production has already been addressed in other works, the results reported in the literature on this subject are somewhat contradictory. In the

study of B. megaterium growth in batch culture at 30 ◦ C and pH 7.0 and using molasses cane as carbon source, Kulpreecha et al. [38] reported higher P(3HB) production at higher oxygen supply, i.e., an inverse behavior regarding the results of the present work. The authors have obtained mass fractions of 47% CDW at 40% DO saturation and around 60% of CDW at 60% and 80% DO saturation. On the other hand, Philip et al. [39] investigated the effect of stirrer speed on P(3HB) production by Bacillus cereus SPV in batch culture at 30 ◦ C using glucose as carbon source and no pH control. The P(3HB) content was higher at 125 rpm stirrer speed (34% of CDW) decreasing to any higher stirrer speed. However, the authors did not present kL a values for the studied stirrer speeds. López et al. [23] also reported a high P(3HB) mass fraction (60% of CDW), under conditions of low oxygen supply (DO at 20% of air saturation) in batch cultures of B. megaterium using glucose as carbon source, although this was the only condition tested in their work. The higher P(3HB) production by B. megaterium at low oxygen supply has also been indirectly observed in a previous work of our group [22], comparing the results of shaker-scale cultures and 4 L batch bioreactor cultivations and taking into consideration that the main difference between the two processes relies on the oxygen supply, which is lower in shaker. P(3HB) mass fractions of 70% and 34% of CDW were obtained in shaker and in bioreactor cultivations, respectively, both without pH control and no oxygen transfer optimization. In this work the cultivations were run without pH control, since in a previous work it was shown that for this strain of B. megaterium, uncontrolled pH cultures produced higher amounts of P(3HB) as mass fraction (34% of CDW), when compared with pH controlled cultures at 7.0 (28% of CDW) [22]. Similar results were found for B. cereus by Philip et al. [39], who reported 34% P(3HB) mass fraction under uncontrolled pH cultures, while controlled pH cultures at 6.8, 3.0, and 10.0, produced 23%, 21%, and 16% P(3HB) cell mass fraction, respectively. Valappil et al. [10,18], also working with B. cereus, reported that low pH inhibited the endogenous consumption of P(3HB). In order to find additional support to the results related to the dependence of the P(3HB) production on the oxygen supply, it is important to consider the biochemical pathway of P(3HB) formation by B. megaterium. Acetyl coenzyme A (Acetyl-CoA) is an important intermediate in P(3HB) pathway synthesis, being the precursor of the monomer (R)-3-hydroxybutyryl coenzyme A (3HB-CoA). However, Acetyl-CoA is also a key compound in the cell metabolism, and it is oxidized via tricarboxylic acid cycle (TCA cycle), being either dissimilated to generate biologically useful energy or assimilated for cell growth. Consequently, P(3HB) pathway synthesis concurs with the TCA cycle for assimilation of acetyl-CoA [40,41]. The oxidation via TCA cycle predominates under balanced growth conditions, with NADH being generated and used in biosynthesis or energy generation. When biosynthesis decreases due to lack of a nutrient, the TCA cycle activity decreases due to the high NADH concentration, resulting in decrease of acetylCoA oxidation via TCA cycle. Additionally, since the TCA cycle is endergonic in the absence of oxygen [40], under limitation of oxygen the reducing power (e.g., NADPH) generated are not oxidized via electron transport phosphorylation. Therefore, low oxygen supply can reduce the TCA cycle activity, due to both thermodynamic reasons and metabolic control by reducing power, and allow that part of the acetyl-CoA available can be shifted to P(3HB) pathway [4,40,42,43].Another important aspect to be considered is the fact that B. megaterium is a strict aerobe [12,24,25] and endosporeforming bacterium [20,42,44]. From this point of view, the use of extremely low oxygen supply can negatively affect both cell growth and P(3HB) production, by inducing sporulation. Slepecky and Law [44] studied the synthesis and degradation of P(3HB) in B. megaterium cultures, showing that the endogenous consumption of P(3HB) can serve as energy source for the sporulation process. Wu

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et al. [20] observed spore septa formation and low P(3HB) production at low oxygen supply (DO maintained at 10% for the first 17 h and afterwards DO was kept in 5%) in fed-batch cultures of Bacillus sp performed at 35 ◦ C and pH maintained at 7. On the light of the different aspects concerning the metabolism of B. megaterium toward oxygen, it is theoretically consistent to propose the existence of an optimal oxygen supply condition (around kL a of 0.006 s−1 ) suggested by the results in the present work. Oxygen transfer rates higher than the optimal value would increase the TCA cycle activity and limit P(3HB) synthesis, while lower rates would also lead to reduction in P(3HB) production due to either strong impairment of bacteria metabolism or sporulation. Regarding the P(3HB) yield and productivity on sucrose the highest values (0.32 g g−1 and 0.16 g L−1 h−1 , respectively) were also obtained in the batch cultures at kL a of 0.006 s−1 , which indicates that the optimal oxygen supply condition is actually around this value of transfer rate. However, the productivity obtained is still possibly low for industrial applications and additional studies focused in the development of adequate strategies to increase this parameter are required. An alternative is the use of fed-batch cultures to improve cell concentration and P(3HB) productivity. Pandian et al. [15] attained a P(3HB) concentration of 11.32 g L−1 using fed-batch cultivation of B. megaterium SRKP-3 growing in dairy waste. Finally, it is important to remark that the volumetric oxygen mass transfer coefficient (kL a) was used as basis of analysis for all the results reported in the present work about the dependence of the different P(3HB) production parameters on the oxygen supply, differently from most works available in the literature, in which the stirrer speed is the reference parameter. Therefore, these results are expected to be more easily extended to other scales of production, since kL a is an important factor in scaling-up aerobic bioprocesses [26–31]. 5. Conclusions The results obtained with experiments performed under different volumetric oxygen mass transfer coefficient, kL a, showed the importance of oxygen transfer rate on the P(3HB) production by B. megaterium DSM 32T , the strain used in this research. The existence of an optimal condition of oxygen availability was identified in terms of value of kL a, allowing the extension of the results for higher scales of production, aiming at improving the production of this important biopolymer. Acknowledgements The authors wish to thank FAPERGS, CNPq and CAPES (Brazil) for their financial support of this research. References [1] Keshavarz T, Roy I. Polyhydroxyalkanoates: bioplastics with a green agenda. Curr Opin Microbiol 2010;13:321–6. [2] Lee SY. Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol 1996;14:431–8. [3] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Mol Biol Rev 1990;54:450–72. [4] Dawes EA, Senior PJ. The role and regulation of energy reserve polymers in micro-organisms. Advances in microbial physiology, vol. 10. London: Academic Press; 1973. p. 135–266. [5] Sudesh K, Abe H, Doi Y. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 2000;25:1503–55. [6] Braunegg G, Lefebvre G, Genser KF. Polyhydroxyalkanoates, biopolyesters from renewable resources: physiological and engineering aspects. J Biotechnol 1998;65:127–61. [7] Lee SY. Bacterial polyhydroxyalkanoates. Biotechnol Bioeng 1996;49:1–14. [8] Reddy CSK, Ghai R, Rashmi Kalia VC. Polyhydroxyalkanoates: an overview. Bioresour Technol 2003;87:137–46.

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Please cite this article in press as: Faccin DJL, et al. Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.02.004