production from oil palm frond juice by Cupriavidus

Research Journal of Chemistry and Environment

___________________________________ Vol.17 (7) July (2013) Res. J. Chem. Environ.

Scaling up of poly (3-hydroxybutyrate) production from oil palm frond juice by Cupriavidus necator (CCUG52238T) Zahari M.A.K.M. Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, Lebuhraya Tun Razak, Kuantan, Pahang, MALAYSIA [email protected]

xyalkanoates (PHAs) have been drawing much attention because their physical properties are close to that of conventional plastics. In addition to that, PHA has been found to be superior compared to petrochemical-derived plastics in several aspects that include biocompatibility, biodegradability, and both environmental and human compatibility. However, industrial production of PHA is hindered due to their high production costs compared with chemically synthesized polymers that possess similar material characteristics. Consequently, much effort has been devoted to the development processes for biopolymer production by optimizing the upstream to downstream engineering strategies including the metabolic and cellular engineering of host cells, efficient fermentation and recovery process, and post-production modification of the PHA obtained.1

Abstract It has been reported that oil palm frond juice (OPF) can be used as the sole renewable, alternative and cheap carbon source for the production of poly(3hydroxybutyrate), P(3HB) at lab scale study. In order to further investigate the potential of OPF juice for P(3HB) production at larger scale, we scaled up the fermentation process from 500 ml shake flask to 20 L bioreactor based on constant volumetric mass transfer coefficient (kLa) of oxygen at both scale. It was interesting to note that the P(3HB) content obtained in this study was almost similar as reported in the previous study both in shake flask and 2 L bioreactor. The P(3HB) content for shake flask fermentation was 45 wt.%, while 44 wt.% and 42 wt.% in 2 L and 20 L bioreactor respectively.

One of the major bottlenecks facing many researchers to produce P(3HB) in large scale is the problem of scaling up the process for industrial scale production. For instance, most of the fermentation process performance could not sustain at larger scale compared to the lab scale. This was due to the difficulty in maintaining homogeneity in large systems, changes in surface to volume ratios, and changes in the cultures themselves due to the increased length of culture time.2 Therefore, it is important to scale up the fermentation process from shake flasks to industrial scale in stages. For instance, the scaling up process should be done from shake flasks to 2 L bioreactor, then from 2 L to 20 L bioreactor and so on. This is due to fact that the main objective of scaling up process is to obtain a similar total production per unit volume at both scales in the same cultivation time.3

Based on the current finding, it can be concluded that the scaling up method based on constant kLa was successful due to the almost similar P(3HB) content obtained in shake flask and bioreactor scale. Overall, it can be postulated that OPF juice can be successfully used as a non-food fermentation feedstock for the production of P(3HB) both in small and large scale fermentation. Keywords: Oil palm frond, renewable sugars, non-food fermentation feedstock, poly(3-hydroxybutyrate), volumetric mass transfer coefficient

Introduction During recent years, a variety of biopolymers have become available for use in many applications that are not only compatible with human lifestyle but also are friendly to the environment. As our standing of the biosynthesis of biopolymers and fermentation process development has been advanced, it has become possible to produce an increasing number of biopolymers, in adequate quantities from renewable resources.1 Today, some of these biopolymers are produced by bacterial fermentation and are used commercially in a wide range of applications such as foods, pharmaceuticals, plastics and agriculture. Not only refined carbohydrates but also agricultural and dairy byproducts can be used as substrates for the production of these biopolymers by fermentation process.1

Oxygen transfer rate (OTR) is the most important parameter implied on the design and operation of aeration and agitation of bioreactors and in scale-up.4-6 Efficiency of aeration depends on oxygen solubilization, diffusion rate into broths, and bioreactor capacity to satisfy the oxygen demand of microbial population. However, the DO in the broths is limited by its consumption rate on cells or the oxygen uptake rate (OUR) as well as by its OTR. The OTR could be affected by several factors such as geometry and characteristics of the vessels, liquid properties (viscosity, superficial tension etc.), the dissipated energy in the fluid, biocatalyst properties, concentration, and morphology of microorganisms. The OTR value depends on the air flow rate, the stirrer speed, mixing etc.

Some important biopolymer, for example polyhydro (18)


___________________________________ Vol.17 (7) July (2013) Res. J. Chem. Environ. glucose and sucrose was diluted from stock (55 g/l) to 16 – 17 g/l of total initial sugars and used as carbon sources throughout the study period.

On the other hand, OUR is limited by increase in viscosity resulting from polymeric property.7-10 Traditional scale-up is highly empirical and makes sense only if there is no change in the controlling regime during scale-up, particularly if the system is only reaction or only transport controlled. Common scale-up rules are the maintenance of constant power-to-volume ratios, constant kLa, constant tip speed, a combination of mixing time and Reynolds number, and the maintenance of a constant substrate or product level (usually dissolved oxygen concentration).2 To reduce the complication of various variables and factors based on the theory of models and the principles of similarity, scaling-up for biopolymer production should be studied by consideration of the oxygen transfer parameters. Fixing of kLa values has been commonly used criteria for scale-up of aerobic fermentations.11-13

Determination of dissolved oxygen tension (DOT) curve: The volumetric gas/liquid mass transfer coefficient (kLa) is an important variable in aerated biological and chemical processes. In most aerated biological processes, the oxygen transfer rate (OTR) is mainly considered to be directly related to the kLa coefficient, especially in conditions where it is a physical rate limiting process. The determination of kLa of a fermenter is essential in order to establish its aeration efficiency and to quantify the effects of operating variables on the provision of oxygen. In this study, the dynamic gassing out (non-fermentative) method was used to determine the value of kLa. To determine the kLa value, 200 ml of distilled water was filled in the 500 ml shake flask and used as a solution. The solution was sparged with nitrogen until the DOT value become zero. After that, the shake flask was shaken at 220 rpm and 30oC, which was the optimized condition for biosynthesis of P (3HB) using OPF juice in shake flasks as described earlier16. The experimental set-up is shown in figure 1. At the same time, the increases in DOT value were monitored using dissolved O2 electrode and were recorded until the value became constant. The DOT curves versus time were plotted at the end of the experimentation.

During scaling up based on constant kLa, there are three important factors to be taken into consideration, which are inoculum development, medium sterilization as well as aeration and agitation. In this study, the discussion will be focused on aeration and agitation. This is due to the fact that different aeration and agitation system was applied in shake flasks and fermentor. In shake flasks, aeration was supplied to the culture broth when the broth surface contacted with the air during agitation. On the other hand, in fermentor, aeration was supplied via air sparging and agitation is provided by an impeller or by the motion imparted to the broth (liquid phase) by rising gas bubbles.14

Above methods were repeated by using a 2 L bioreactor filled with 1.5 L of distilled water supplied with air at 1.5 L/min (1.0 vvm) flow rate to obtain an almost similar pattern of DOT curves which was previously obtained in shake flask. These were done by trials and errors for different agitation speed (rpm) at constant air flow rate i.e. 1.5 L/min (1.0 vvm). The agitation speed that produced the almost similar DOT curve as previously obtained in shake flask will be used in the fermentation for the preparation of seed culture using OPF juice in 2 L bioreactor.

Previously, we demonstrated that OPF juice can be used as an alternative, renewable and sustainable carbon source for P(3HB) production by Cupriavidus necator.15 Subsequent experiment on optimization of P(3HB) production in shake flask using the wild-type C. necator strain CCUG 52238T resulted a 40% increment on P(3HB) content compared to the P(3HB) produced under non-optimized condition.16 Further experiment in a 2 L bioreactor (1 L working volume) with 30 % DOT yielded CDW of 11.37 g/L and P(3HB) content of 44 wt.%.16 Based on the optimized condition obtained in shake flask and 2 L bioreactor by C. necator CCUG 52238T, further investigation on the potential of OPF juice for P(3HB) production was done at larger scale and elaborated in this paper . In order to achieve this, the production of P(3HB) using OPF juice was scaled up from 2 L to 20 L bioreactor based on constant kLa. Prior to that the kLa was determined in shake flask and bioreactor based on the method developed by Asis et al.17

In order to obtain the agitation speed for 20 L bioreactor that produced an almost similar DOT curve in 2 L bioreactor, previous methods were repeated by using 20 L bioreactor filled with 15 L of distilled water supplied with air at 3.75 L/min (0.25 vvm) flow rate. The DOT curve versus time in 20 L bioreactor that produced the almost similar kLa value in 2 L bioreactor was determined by using trial and error method by manipulating the agitation speed at constant air flow rate i.e. 3.75 L/min (0.25 vvm). The agitation speed that produced the almost similar DOT curve as previously obtained in the 2 L bioreactor will be used in the fermentation for biosynthesis of P(3HB) using OPF juice in 20 L bioreactor.

Material and Methods Microorganism and cultivation conditions: The inoculum preparation, media and cultivation conditions for C. necator CCUG 52238T are similar as previously described by Zahari et al.15-16 OPF juice in this study was obtained by pressing the fresh OPF following the method described earlier. OPF juice which comprises of fructose,

Determination of kLa and kap: Scaling up of P(3HB) production from 500 ml shake flask to 2 L and 20 L bioreactor was done using the method of constant volumetric transfer coefficient (kLa) of oxygen. kLa values (19)



were derived by fitting the mass transfer equation to the data of dissolved oxygen tension (DOT) versus time. The mass transfer equation was obtained from Asis et al17 and is shown as follows: -

-

bioreactor Biostat Cplus (Sartorius, Germany) supplemented with OPF juice at 30% (v/v) dilution. The OPF juice and MSM medium were prepared similar as previously explained for the preparation of seed culture in 2 L bioreactor. The cultivation condition were similar to the experiment conducted in 2 L bioreactor except for the air flow rate and stirring speed which were set at 3.75 L/min (0.25 vvm) and 230 rpm respectively. Samples were withdrawn every 5 h for the period of 50 h for the determination of CDW, P(3HB) concentration, residual sugars and ammonical nitrogen (NH3-N) content.

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where YR(t) is dissolved oxygen concentration calculated theoretically at time t, kLa is a volumetric transfer coefficient for oxygen and kap is an electrode mass transfer coefficient of oxygen. The mass transfer equation will be solved by using MATLAB software to get the kLa and kap values. The Fibonacci Min Search method was selected to calculate both the values.

Analytical procedure: Dry weight measurement, P(3HB) content and residual sugars concentration analysis were done as previously described.15-16 Residual ammonical nitrogen (NH3-N) content analysis was done using Nessler method as previously described.16

Figure 1: Experimental set-up for the determination of kLa in shake flask Biosynthesis of P(3HB) in 20 L bioreactor: Figure 2 shows the cultivation steps for inoculum preparation and biosynthesis of P(3HB) using OPF juice by C. necator CCUG 52238T in 20 L bioreactor. As shown in figure 2, the inoculum preparation and cultivation conditions for inoculum development 1 and 2 are similar as previously explained.15-16 For the preparation of seed culture for 20 L bioreactor, approximately 10% (150 ml) of pre-grown cells from inoculum development 2 was transferred into 1350 ml MSM in 2 L bioreactor Biostat A (Sartorious, Germany) supplemented with OPF juice at 30% (v/v) dilution. The stock of OPF juice with 55 g/l of initial total sugars concentration was autoclaved separately prior to addition with the MSM medium. The MSM compositions were prepared as previously described by Zahari et al.15,16 The temperature inside the bioreactor was set at 30oC while the air flow rate and stirring speed were set at 1.5 L/min and 300 rpm respectively. The pH value during fermentation was controlled at pH 7.0 ±0.05 by 2M NaOH/H2SO4. The culture was incubated for 30 h and used as a seed culture for 20 L bioreactor.

Figure 2: Cultivation steps for inoculum preparation and biosynthesis of P(3HB) using OPF juice by C. necator (CCUG52238T) in 20 L bioreactor.

Results and Discussion DOT versus time curves: Figures 3 (a) and (b) show the DOT versus time curve for experiment conducted in shake flask and 2 L bioreactor; and in 2 L and 20 L bioreactor, respectively. As shown in figure 3 (a) and (b), it can be observed that the agitation speed in 2 L bioreactor that produced the almost similar DOT curve obtained in the shake flask was achieved at 300 rpm, meanwhile, for the experiment conducted in 20 L bioreactor, the agitation speed that produced the almost similar DOT curve obtained in 2 L bioreactor was achieved at 230 rpm. Determination of kLa and kap values: The comparison of DOT curve versus time based on experiment and calculated value using MATLAB software in shake flask, 2 L and 20 L bioreactor was shown in figure 4 (a), (b) and (c) Respectively, meanwhile, 0.3195 min-1 and 0.0090 min-1, respectively for 20 L bioreactor. , the values of kLa and kap for shake flask were 0.3299 min-1 and 0.0065 min-1 respectively. The values for kLa and kap for 2 L bioreactor

For the biosynthesis of P(3HB) in 20 L bioreactor, seed culture (1.5 L) which was previously prepared in 2 L bioreactor was transferred into 13.5 L MSM in 20 L (20)



were 0.3120 min-1 and 0.0113 min-1 respectively and 0.3195 min-1 and 0.0090 min-1 respectively for 20 L bioreactor.

was 45 wt.% while 44 wt.% and 42 wt.% in 2 L and 20 L bioreactor respectively.

Conclusion

Growth and P(3HB) production profile in 20 L bioreactor: Figure 5 (a) and (b) show the profile change of CDW, P(3HB) content, residual sugars and ammonical nitrogen concentration during batch biosynthesis by C. necator CCUG 52238T in 20 L bioreactor (15 L working volume) supplemented with OPF juice at 30% (v/v) dilution. During this fermentation, the cells were found to grow steadily during 15 h of fermentation period. After that, it was increased rapidly and achieved maximum at 45th h of fermentation period.

Current findings showed that scaling up of P(3HB) production from 500 ml shake flask to 2 L and 20 L bioreactor using OPF juice by C. necator CCUG52238T was successfully done based on constant volumetric transfer coefficient (kLa) of oxygen at both scale. The rationale of fixing the kLa value is to ensure a certain mass transfer capability that can cope with the oxygen demand of the culture and often serves to compare the efficiency of bioreactors and mixing devices as well as being an important scale-up factor. Scaling up using the method of constant kLa of oxygen will produce P(3HB) content comparable in shake flask and both in 2 L and 20 L stirred tank bioreactor.

The highest CDW obtained at this point was 11.32 g/l. P(3HB) accumulation were found to increase rapidly during the 30th h fermentation and reached a maximum of 42 wt.% at 45 h of growth. As shown in figure 5 (b), it was observed that the sugars concentration began to decrease with the onset of P(3HB) formation and decreased from 16.80 to 1.92 g/l by the 45th hour. The biomass yield (Yx/s) obtained in this study was 0.76 g biomass/g sugars consumed, meanwhile the P(3HB) yield (Yp/s) was 0.32 g P(3HB)/g sugars consumed. The maximum P (3HB) productivity was 0.11 g/L/h.

Almost similar P(3HB) content in shake flask and 20 L bioreactor will produce 45 wt.% and 42 wt.% respectively, indicating the success of the scaling up process. It is worth to mention that the data provided in this study show that the production of P(3HB) from OPF juice has a potential to be scaled up to commercial scale. Thus, this study shows that biosugars from OPF have a great potential to be utilized as a sustainable and cheap fermentation feedstock to support the biotechnology industries in the long term.

As a comparison, it was interesting to note that the P(3HB) content obtained in this study was almost similar as reported in previous study both in shake flask and 2 L bioreactor explained in our study.16 In addition to that, an almost similar cell growth and P(3HB) production profile was obtained in 20 L bioreactor study compared to 2 L bioreactor with 30% DOT level within the similar cultivation time. Based on the current finding, it can be concluded that the scaling up method based on constant kLa was successful. Almost similar P(3HB) content was obtained in shake flask and bioreactor scale.

Acknowledgement The authors would like to acknowledge the Ministry of Higher Education (MOHE) for funding this research (RAGS: RDU121406) and the Japan Society for the Promotion of Science (JSPS) for giving the technical support during the study period. Author’s heartiest gratitude also goes to Universiti Malaysia Pahang (UMP) for the provision of study leave.

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One plausible explanation for this success is that, in this study, the volumetric mass transfer coefficient (kLa) was selected to be the scale up criterion, so the kLa in shake flask and bioreactor has to be similar. In other words, it means that the kLa in shake flask has to be similar with the kLa in 2 L and 20 L bioreactor. This criterion is usually applied to aerobic systems where oxygen concentration is most important and affects metabolism of the microbial cell.

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Since the role of oxygen on microorganism growth and metabolism is important, the DO transfer due to oxygen transfer rate (OTR) into a system during fermentation needs to be investigated.2,7,12 For aerobic fermentations, it is equally important to satisfy both agitation rate and the oxygen supply requirements. Scale up using the method of constant kLa of oxygen will produce P(3HB) content comparable in both scale i.e. shake flask and bioreactor. In this study, the P(3HB) content for shake flask fermentation

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Time (min) (b) Figure 3: Dissolved oxygen tension (DOT) versus time curve (a) in shake flask and 2 L bioreactor (b) in 2 L and 20 L bioreactor

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(c) Figure 4: DOT versus time curve based on experimental and calculated value using MATLAB software (a) Shake flask at 220 rpm (b) 2 L bioreactor at 300 rpm (c) 20 L bioreactor at 230 rpm. ( Calculated value, experimental data)

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(b) Figure 5: Time course batch fermentation of C. necator CCUG 52238T using OPF juice in 20 L bioreactor (a) cell dry weight (g/L) formation, P(3HB) concentration (g/L) and accumulation (wt.%), (b) remaining mixed sugars (g/L) and NH3-N (mg/L). (23)



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(Received 29th March 2013, accepted 20th May 2013)

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