Optimization of Saccharomyces cerevisiae culture in alginate–chitosan–alginate microcapsule

Biochemical Engineering Journal 25 (2005) 151–157

Optimization of Saccharomyces cerevisiae culture in alginate–chitosan–alginate microcapsule Qi Wen-tao a,b , Yu Wei-ting a , Xie Yu-bing a , Ma Xiaojun a,∗ a

Laboratory of Biomedical Material Engineering and Department of Science and Technology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China b Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China Received 9 March 2005; received in revised form 20 April 2005; accepted 21 April 2005

Abstract Microencapsulation is one of the promising methods for microorganism immobilization. The parameters of microencapsulation have impact on the growth and performance of microencapsulated microorganism. In this paper, the effects of microcapsules core state (solid or liquid), initial cell density (1.5 × 107 , 3 × 106 and 3 × 105 cells/ml of microcapsules), microcapsule diameter (200, 500, 600 and 700 ␮m) and membrane formation times (0, 5, 15 and 30 min) on cell growth, including proliferating capacity, metabolic activity and product secretion of Saccharomyces cerevisiae, cultured in alginate–chitosan–alginate (ACA) microcapsule, were investigated. The results showed that there was no significant difference in cell growth of microencapsulated cells, whether the core of the microcapsules was solid or liquefied. Increase in inoculate cell density shortened the lag phase time of cell growth, while cell density obtained 25 times of the initial density of 3 × 106 cells/ml of microcapsule, which was the highest. Increase in microcapsules size had no significant impact on cell proliferation, metabolism and product secretion, but the leakage of the bacteria was the least when the microcapsule size was 600 ␮m in diameter. The increase in membrane formation time reduced the leakage of cells. It was demonstrated that the optimized parameters for microencapsulated S. cerevisiae culture were initial cell density of 3 × 106 cells/ml of microcapsule, microcapsule size of 600 ␮m in diameter, and membrane formation time of 15 min. This study provides useful information for intestinal delivery of therapeutic agents from genetically modified food-grade microorganisms, using microencapsulation. © 2005 Elsevier B.V. All rights reserved. Keywords: Microencapsulation; ACA microcapsule; Saccharomyces cerevisiae; Cell growth

1. Introduction Over the past 20 years, there has been an increased interest in the role of recombinant microorganisms in human health. And immobilization of microorganisms is increasingly applied, since it has several advantages compared with suspension culture, including higher growth rate and higher biomass in a bioreactor, higher dilution rate without washout in the continuous process, easier collection and purification of bio-products, and better catalytic stability of biocatalysts as well as the tolerance against high concentrations of toxic compounds and toxic loadings [1]. ∗

Corresponding author. Tel.: +86 411 84379139; fax: +86 411 84379139. E-mail address: [email protected] (M. Xiaojun).

1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.04.019

Among the available techniques for immobilized living cells, entrapment in microcapsules has been frequently used especially for the production and intestinal delivery of therapeutic agents from genetically modified food-grade microorganisms. For example, microencapsulated genetically engineered bacteria Escherichia coli DH5 cells have been evaluated in uremic rats for urea and creatinine removal by oral administration [2,3]. And similar report can be found while the polyvinyl alcohol (PVA) microcapsules were used as carriers [4]. A variety of polymers, such as chitosan, polyacrylates, alginate, polyamino acids, and polyamides, have been used to make microcapsules [5,6]. Alginate–poly-lysine–alginate (APA) microcapsule is one of the most widely studied microcapsules for its good biocompatibility and good

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characteristics in cell culture. It has been mainly used as the immunoprotective device in the treatment for neurodegenerative diseases and various endocrinal diseases, especially diabetes [7–10]. But the high cost of poly-lysine may limit the large-scale use of APA microcapsule in microorganism immobilization. With the advance in the study of chitosan as biomaterial, alginate–chitosan–alginate (ACA) microcapsule has been developed where the positively charged chitosan was used to substitute poly-lysine. It has attracted much attention for its good biocompatibility and the low cost of chitosan, which is about 2500 times lower than that of poly-lysine, owing to chitosan’s abundance in nature [11–15]. Especially, ACA microcapsule is more practical for immobilized microbial culture. Therefore, it was selected as the immobilized carrier for fermentation in this paper. Calcium alginate beads has been used in immobilized bacteria culture and the parameters that can influence the viability of bacteria has been optimized, such as alginate concentration, calcium chloride concentration, hardening time of gel beads in calcium chloride, which were 1.8% (w/v), 0.1 M and 30 min, respectively [16]. For microencapsulated bacteria culture more parameters need to be optimized. The aim of this work was to investigate the influence of encapsulation parameters, including microcapsules core status, initial cell density, microcapsule diameter and membrane formation time on the growth, metabolism and product secretion of microencapsulated microorganisms as well as the leakage of cells, in order to improve the microencapsulation method by optimizing these encapsulation parameters. Considering the safety and several advantages as host cells to digestive tract, such as, eukaryotic post-translational modifications as recombinant microorganisms, secreting product out of cells, easy to accept due to the odour of alcohol, the yeast Saccharomyces cerevisiae was selected as model cell in this work.

300 meshes. Sodium alginate was dissolved in 0.9% (w/v) NaCl solution to form certain concentration. Others were analytical reagents. 2.2. Encapsulation of S. cerevisiae in ACA microcapsule and culture Sodium alginate was dissolved in 0.9% (w/v) NaCl solution to form final concentration of 1.5% (w/v), and was sterilized by filtration through a 0.22 ␮m micro-filter. The solution was stored overnight at 4 ◦ C before use, in order to facilitate deaeration. S. cerevisiae, in a late exponential phase, were centrifuged and suspended in sodium alginate solution. Then the solution was extruded into 100 mM CaCl2 solution, using high voltage electrostatic generator (DICP, CAS, China). After being hardened for 30 min, the micro-gel beads containing S. cerevisiae were obtained. The modified chitosan was dissolved in 0.1 mol/L sodium acetate–acetic buffer solution to form final concentration of 0.5% (w/v), and was sterilized by filtration through a 0.22 ␮m microfilter. The chitosan solution was added into calcium alginate micro-gel beads at the volume ratio of about 1:5 (beads:solution) to form membranes, followed by rinsing with 0.9% (w/v) NaCl solution to remove the excess chitosan. Then, 0.15% alginate solution was added to counteract charges on the membranes. At last, the beads were liquidized with 55 mmol/l sodium citrate, in order to obtain the ACA microcapsules with liquid cores. And 0.8 ml ACA microcapsules were inoculated in 3.2 ml YPD medium in a flask and incubated at 28 ◦ C in a HZQ-F double-layer all temperature vibrator (Harbin, China). Triplicate samples were used for each time points. The results were expressed as mean ± S.D. Anaerobic condition was maintained in all the cultures. 2.3. Optimization of encapsulation parameters

2. Materials and methods 2.1. Microorganisms and materials S. cerevisiae was kindly provided by College of Enviromental and Biological Science and Technology, Dalian University of Technology (Dalian, China) and cultured in yeast peptone dextrose (YPD) medium, composed of 3.85 g/L yeast extract, 3 g/L bacto-tryptone, and 10 g/L glucose at 28 ◦ C. The culture was terminated when the glucose was exhausted after about 14 h of cultivation. Chitosan was modified from raw material (Ocean Biochemical Corporation, Zhejiang, China) by our laboratory (DICP, CAS, China) with a molecular weight of 7.5 kDa, and the degree of deacetylation is 96–98%. Sodium alginate was purchased from the Qingdao Crystal Salt Bioscience and Technology Corporation (Qingdao, China), whose viscosity was over 0.02 Pa s, when dissolved to form a 1% (w/v) aqueous solution at 20 ◦ C, and whose powder was less than

The microcapsules were prepared with liquefying step, using sodium citrate to form microcapsule with liquid core or without liquefying to form microcapsule with solid core, with initial cell density of 1.5 × 107 , 3 × 106 or 3 × 105 cells/ml of microcapsule, with microcapsule size of 200, 500, 600 or 700 ␮m, and with membrane formation time of 0, 5, 15 and 30 min, in order to investigate the effects of core status, initial cell density, microcapsule size and membrane formation times on the proliferating capacity, metabolic activity and product formation of encapsulated bacteria. 2.4. Scanning electronic microscopy of microcapsules Scanning electronic microscopy was used to study the structure of microbial-loaded ACA microcapsules with different cores. Microcapsules were embedded in paraffin, sectioned, de-paraffin, and sputter-coated with gold. Samples were observed using a Philips XL-30 scanning electron microscope (Philips, Holand). This method allowed the

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visualization of fractured microcapsules, as well as details about the entrapped cells. 2.5. Measurement of cell concentration and dry weight The concentration of microbial cells in microcapsules culture was measured as OD 600 nm units, using a UV-2550 visible spectrophotometer (Shimadzu Co., Kyoto, Japan), after the microcapsules were broke up using a chemical method, as described in [17]. The maximum dry weight of microbial biomass was also measured after being cryo-dried for 24 h. Cell count was determined by a haemacytometer. And the mean of three individual determinations was used to calculate the cell number. 2.6. Cell release from microcapsules To determine the degree of the bacteria that release from the microcapsules, the culture medium outside the microcapsules were collected as a function of time. And the concentration of bacteria cells in the medium was measured as OD 600 nm units, using the UV-2550 visible spectrophotometer. 2.7. Glucose consumption and ethanol production For extracellular metabolite analysis, concentration of glucose in the medium was measured using SBA-40C lactate–glucose bio-sensitive analyzer (Jinan, China). The concentration of ethanol in the medium was determined with gas chromatography (GC-14C, Shimadze Co., Kyoto).

3. Results and discussion 3.1. Effect of the core status of microcapsule on the growth of the bacteria In ACA microencapsulated culture, the status of the microcapsular core is one of the most important environmental variables, since it can be the limited factor on the

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mass transfer inside the microcapsules. The core of the ACA microcapsules can be solid gel omitting the step of sodium citrate treatment, or liquefied with sodium citrate treatment during microencapsulation. It had been found that in the case of solid core microcapsules, hybridoma cells created multiple small aggregates that become dark after encapsulation, as a consequence of poor mass transfer of oxygen to the interior of the aggregates. However, cells presented an improved growth profile in liquefied microcapsules [18]. Microbial cells S. cerevisiae were encapsulated and cultured in ACA microcapsules with solid cores as well as liquefied cores. In order to observe the cell organization and distribution in microcapsule, microcapsules loaded with S. cerevisiae after culture for about 10 h were cryo-sectioned and observed under SEM. Cells formed non-uniform aggregates with no obvious dividing lines in liquefied microcapsule due to the cells could move freely in it (Fig. 1(a)), while the cells were formed spherical aggregates with clear outlines and distributed uniformly in the microcapsules with solid core (Fig. 1(b)). The cryo-SEM images also revealed that the membrane of the microcapsules formed by chitosan kept integrated during the cell culture. And the volume of microcapsules became expanded after the core was liquefied. To compare the growth of S. cerevisiae in microcapsule with solid or liquefied core, cell density, metabolic activity and product formation were measured as a function of time. The cell density in ACA microcapsules with liquefied core was a little higher than that with solid core, but the difference was not significant (Fig. 2). Similarly, there was no significant difference in the maximum dry weight of microbial biomass grown in microcapsules with liquid or solid core (data not shown). Meanwhile, there was also no significant difference in either glucose consumption or ethanol formation, whether the cores of the microcapsules were solid or liquefied (Fig. 2). Since anaerobic condition was maintained in both cultures, the influence of mass transfer of oxygen to the interior of cell aggregate in microcapsules with either liquid or solid core is neglectable. Therefore, the liquefying step with sodium citrate treatment during microencapsulation can be omitted in microencapsulated S. cerevisiae culture. To simplify the

Fig. 1. Cryo-SEM of fractured alginate–chitosan–alginate (ACA) microcapsule loaded with Saccharomyces cerevisiae after cultured for about 10 h: (a) microcapsules with liquefied core; (b) microcapsules with solid core.

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Fig. 2. Effect of the core status of microcapsule on cell growth: (A) glucose consumption; (B) ethanol formation of S. cerevisiae cultured in ACA microcapsules with liquefied or solid core. The error bars represent standard deviation of mean (n = 3). Square points, liquefied core; circle points, solid core.

process, the following studies were carried out in all the microcapsules with solid cores.

3.2. Effect of the initial cell density on the growth of the bacteria The effects of initial cell density in microcapsule on cell proliferation, metabolic activity, and product formation of the microencapsulated cells were investigated. As shown in (Fig. 3(A)), the microencapsulated S. cerevisiae required about 6, 2.5 and 1 h to achieve a doubling in cell density and reached the maximum cell density about 23, 25 and 4 times of the initial cell density, when the inoculated density was 3 × 105 , 3 × 106 and 1.5 × 107 cells/ml of microcapsule, respectively. But there was the longest lag phase when the inoculate density was 3 × 105 cells/ml of microcapsule. Fig. 3(B) showed the distinctive profile of the glucose consumption and ethanol formation in microencapsulated cell culture with different initial inoculated cell density. It was clear that both had a good metabolic activity when the inoculated density was 3 × 106 and 1.5 × 107 cells/ml of microcapsule. The concentration of ethanol of microcapsule with initial cell density of 3 × 106 cells/ml of microcapsule increased slower than that with 1.5 × 107 cells/ml of microcapsule, but reached the similar level after 14 h of cultivation. Compared to the initial cell density of 1.5 × 107 cells/ml of microcapsule, initial cell density of 3 × 106 cells/ml of microcapsule reached similar level of ethanol production with less cells, when cultured for 14 h, which means the specific ethanol production rate of 3 × 106 cells/ml of microcapsule is higher. Initial inoculated density of 3 × 106 cells/ml of microcapsule was the best among the conditions being tested as far as cell proliferation, metabolism and product formation was concerned.

Fig. 3. (A) Effect of the initial cell density on cell growth; (B) glucose consumption and ethanol formation of microencapsulated S. cerevisiae. Hollow triangle, 1.5 × 107 cells/ml microcapsule; hollow circle points, 3.0 × 106 cells/ml microcapsule; solid square, 3.0 × 105 cells/ml microcapsules.

3.3. Effect of the membrane formation time on the growth of the bacteria The membrane of microcapsule created a barrier for the transport of nutrients from the medium to the encapsulated cells. The permeability of the membrane determined the rate of transport of the secreted product and metabolites (nutrients and waste products), which in turn dictated the concentration of biomolecules within the core, the nature of the intramicrocapsular environment, and ultimately the behavior and function of the microencapsulated cells [19]. Also, the permeability of the membrane determined the efficacy of cell release from the microcapsule, which is very harmful when the microcapsules were used as carrier in genetically engineered cell for implantation. So the proliferation, metabolic activity, product formation and the release of the microencapsulated S. cerevisiae cells were compared when the membrane formation time were 0, 5, 15 and 30 min, respectively, which affected membrane permeability, and the diameters of the microcapsules were all about 600 ␮m. There

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Fig. 4. Effects of the membrane formation time on the proliferating capacity of the S. cerevisiae cultured in microcapsule. Solid square, 0 min; hollow circle, 5 min; solid triangle, 15 min; hollow triangle, 30 min.

was no significant difference in microencapsulated bacteria proliferation (Fig. 4) and metabolism (data not shown) when the membrane formation time was different, because the membrane formation time has no effect on the diffusion of small molecular substrates such as certain nutrients, glucose and ethanol (unpublished observation). However, there was serious bacteria release from the microcapsules when the formation time was 0 min due to no membrane formation. With the increase of the membrane formation time the cell release was decreased, but there was no significant effect on the bacteria release when the formation time was longer than 15 min (Fig. 5). The results indicate that it is not necessary to increase the membrane formation time longer than 15 min in microencapsulation of S. cerevisiae.

Fig. 5. Effects of the membrane formation time on the leakage of S. cerevisiae from the microcapsules. Solid square, 0 min; hollow circle, 5 min; solid triangle, 15 min; hollow triangle, 30 min.

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Fig. 6. Effects of the microcapsule size on the proliferating capacity of S. cerevisiae cultured in microcapsule. Hollow circle, diameter of 200 ␮m; hollow triangle, 500 ␮m; solid square, 600 ␮m; solid triangle, 700 ␮m.

3.4. Effect of the microcapsule size on the growth and leakage of the bacteria The effect of microcapsule size on the growth and leakage of S. cerevisiae cells was investigated and the membrane formation times of the microcapsules with different size were all about 15 min. There was no significant difference in cell growth (Fig. 6), glucose consumption and ethanol production (data not shown) in cells cultured in microcapsule of different size for the first 10 h of cultivation. The glucose can be exhausted in all the cultures. The density of S. cerevisiae reached the highest when cultured in microcapsules of 200 ␮m in diameter for 14 h. When cells grew in microcapsule, cells had the tendency to form multiple small aggregates that became dark as a consequence of poor mass transfer to the interior of the aggregates in microencapsulated culture. The more the aggregates formed, the poorer the mass transfer was in the center of the microcapsules, especially at the end of the culture. Microcapsules with smaller volume, such as 200 ␮m in diameter, have fewer aggregates and higher cell growth rate because of better mass transfer. As a result, the smaller the microcapsule volume was, the easier the microcapsules were full of the bacteria cells. However, the release of bacteria cells from these microcapsules was more serious than that of microcapsules with bigger size, such as 600 ␮m in diameter (Fig. 7(A)), because the fullness of bacteria cells reduced the strength of the microcapsule and made it easy to break up. However, it did not mean that the larger the microcapsule, the less the leakage of microcapsule. It was found that the membrane of the microcapsules without liquefying broke up easily once they were larger than 1000 ␮m for expansion of microcapsules’ volume (data not shown). There were also some cracked microcapsules with diameter of 700 ␮m, which had serious bacteria cell release from the microcapsules. This may be because that the mechanical stability of the membrane reduced with the increase in the

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formation. Initial cell density of 3 × 106 cells/ml of microcapsule is of advantage for better cell proliferation, metabolism and product formation. There is no significant effect on cell growth and leakage when the formation time was longer than 15 min. Microcapsules of 600 ␮m in diameter had the least cell leakage, while gave rise to higher cell proliferation. And further study is still needed to improve the preparation of microcapsules in order to keep the microencapsulated cells having the best proliferating capacity and metabolic activity with no leakage before the ACA microcapsules could be used as a safe and protective intestinal bio-micro-reactor to secret bio-drugs in vivo.

Acknowledgements This work was supported by the National Natural Science Foundation under grant number 20236040 and National 863 Project under grant number 2003AA205111. We would also like to thank many colleagues and collaborators who have contributed to the development of this work.

References

Fig. 7. (A) Time course of the leakage of S. cerevisiae from the microcapsules with different diameters; (B) effects of the microcapsule size on the leakage of S. cerevisiae from the microcapsules. Hollow circle, diameter of 200 ␮m; hollow triangle, 500 ␮m; solid square, 600 ␮m; solid triangle, 700 ␮m.

volume of microcapsule. Therefore, bacteria cells should be entrapped within a limited range of microcapsule size shown as (Fig. 7(B)). Interestingly, Microcapsules of 600 ␮m in diameter had the least cell leakage (Fig. 7), while gave rise to higher cell density (Fig. 6). It suggested that there existed a point of the volume of the microcapsule, which can not only provide a rich mass transfer but also offers a mechanical stability and friendly environment for cell growth. Our result showed that it was about 600 ␮m in diameter.

4. Conclusion The influence of core status, initial cell density, membrane formation time and microcapsule size on cell growth were investigated and these parameters were optimized in the term of better cell growth, more product formation and less cell leakage. The core status of microcapsule has no significant effect on S. cerevisiae proliferation, metabolic activity and product

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