Regular articleHigh cell density fed-batch fermentation for the production of a microbial lipase
Introduction
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are versatile enzymes that are widely used in production of fine chemicals and other industrial processes [1], [2], [3], [4], [5], [6], [7], [8]. All commercially relevant lipases are produced as extracellular enzymes via microbial fermentation processes. Microbial lipases have been discussed extensively in the literature [9], [10], [11].
For a given producer species, the lipase titer depends on the biomass concentration in the broth, the nutritional characteristics of the culture medium and the other production conditions. A high concentration of the microbial biomass is desired in a fermentation to maximize the volumetric productivity of the enzyme and attain a high titer. High cell density fermentations (HCDF) have the potential for enhancing lipase productivity and titer [12]. HCDFs do have drawbacks [13]. For example, the control of culture conditions (e.g. dissolved oxygen concentration) can be difficult in an HCDF operation and this may adversely impact the production [12]. Notwithstanding their shortcomings, high cell density operation is considered economically attractive overall [13].
A high cell density production process typically involves fed-batch fermentation [12]. The feeding is controlled to ensure that the substrate concentration does not build up to an inhibitory level [12], [14], [15]. Various feeding strategies have been expounded in the literature, including a constant rate feeding and specific growth rate control feeding [12]. In specific growth rate control, the feed rate increases exponentially with time so that the specific growth rate is maintained at some predetermined value. Other feeding strategies include pH-stat operation and foam-dependent feeding. In pH-stat operation, the culture is fed in response to a change in pH from a specified value. In foam-dependent feeding, the complete consumption of a foam-suppressor substrate such as a vegetable oil may lead to onset of foaming and this becomes the signal for further feeding. Prior work on high cell density fermentations for the production of lipases has been reviewed [12].
Several published works reported lipase production from Candida rugosa ATCC 14830 [16], [17], [18]. Various feeding strategies with oleic acid as carbon source were studied for highest lipase yield. Constant substrate feeding rate and constant specific growth rate control feeding were employed in these works [16], [17], [18]. Constant specific growth rate control feeding at low rate yielded high cell concentration and lipase production in this strain as shown by Kim and Gordillo [16], [18]. A relatively high cell concentration of 90 g L−1 and extracellular lipase activity of 23.7 U mL−1 were obtained from using this feeding strategy [16]. On the other hand, similar substrate feeding method resulted in a much lesser cell concentration at 6.9 g L−1 but high extracellular lipase activity at 117 U mL−1 [18]. Yet in another similar study, good lipase yield was obtained using constant substrate feeding strategy instead of constant specific growth rate control [17]. This clearly indicated that no single feeding strategy could be made universally applicable for producing high lipase yield even from similar microbial strain.
To date no other published work dealt with lipase production from Candida rugosa ATCC 10571. Likewise, even though the utilization of palm oil as carbon substrate for the production of lipase was reported in other microorganisms [20], [21], [22], [23], no literature is available on the use of palm oil in HCDF to produce microbial lipase, and its application as a supplied carbon source in fed-batch fermentations. While the physical behavior of palm oil in aqueous fermentation medium may be approximated with the behavior of other immiscible substrate such as pure oleic acid, its chemical composition as triglycerides that are made up of different types of fatty acids is in contrast to oleic acid. Thus, it is worthwhile to examine the behavior of fed-batch HCDF when palm oil is used as sole carbon and energy source for growth and lipase by C. rugosa ATCC 10571. This would certainly add to the comparison data for extracellular lipase yield for the particular yeast strain and the implemented feeding strategies.
Consequently, the present study attempted to compare three feeding strategies, i.e., the pH-stat operation, the foaming-dependent control of feeding and the specific growth rate control feeding, in HCDF of the yeast Candida rugosa ATCC 10571 for producing lipases using palm oil as the sole source of carbon and energy. C. rugosa lipases have been reviewed by Domínguez de María et al. [24]. Production of native and recombinant lipases by C. rugosa has been reviewed by Ferrer et al. [25].
Section snippets
Yeast strain and growth medium
The yeast Candida rugosa ATCC 10571 was obtained from the American Type Culture Collection (ATCC) and maintained on YM agar medium. The medium contained (per liter): yeast extract 3.0 g; malt extract 3.0 g; peptone 5.0 g; glucose 10.0 g; and agar 15.0 g. The same medium without the agar was used in shake flask cultures. The production medium used in the bioreactor contained (per liter): 5.74 g K2HPO4; 3.7 g KH2PO4; 1.0 mL of a trace element solution [18], 10 mL of 0.1 M MgSO4·7H2O; 2 g (NH4)2SO4; 4 × 10−4 g
Results and discussion
Shake flask cultures were used to identify the time required to achieve the peak biomass concentration for inoculation of the bioreactor. A maximum biomass concentration of 7.5 g L−1) was achieved at 28 h on glucose as the sole carbon source. The maximum specific growth rate of the culture on glucose was 0.38 h−1. At this point, palm oil (1%, v/v) was added and incubation was continued for a further 12 h to induce the production of lipase.
Conclusions
It is clear from this study and other published works that low specific growth rate control feeding consistently helped to enhance the production of extracellular microbial lipase in HCDF. This is irrespective of the nature of the immiscible substrate used for growth and lipase production i.e., either a pure fatty acid such as oleic acid or whole plant oil such as palm oil. The exact reason behind the increased lipase production when this specific feeding mode was used in HCDF is unclear at
Acknowledgement
The authors acknowledge the University of Malaya for funding this work through research grants PV036/2012A, RP024-2012A, FP012/2012A, ER010-2012A and UM.C/625/1/HIR/MOHE/05.
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