DSpace at VNU: Comparison of Alcoholic Fermentation Performance of the Free and Immobilized Yeast on Water Hyacinth Stem...
Appl Biochem Biotechnol (2014) 172:963–972 DOI 10.1007/s12010-013-0574-7 Comparison of Alcoholic Fermentation Performance of the Free and Immobilized Yeast on Water Hyacinth Stem Pieces in Medium with Different Glucose Contents Van Nguyen Tran & Van Viet Man Le Received: August 2013 / Accepted: 30 September 2013 / Published online: 15 October 2013 # Springer Science+Business Media New York 2013 Abstract Ethanol fermentation with Saccharomyces cerevisiae cells was performed in medium with different glucose concentrations As the glucose content augmented from 200 to 250 g/L, the growth of the immobilized cells did not change while that of the free cells was reduced At higher glucose concentration (300, 350, and 400 g/L), the cell proliferation significantly decreased and the residual sugar level sharply augmented for both the immobilized and free yeast The specific growth rate of the immobilized cells was 27– 65 % higher than that of the free cells, and the final ethanol concentration in the immobilized yeast cultures was 9.7–18.5 % higher than that in the free yeast cultures However, the immobilized yeast demonstrated similar or slightly lower ethanol yield in comparison with the free yeast High fermentation rate of the immobilized yeast was associated with low unsaturation degree of fatty acids in cellular membrane Adsorption of S cerevisiae cells on water hyacinth stem pieces in the nutritional medium decreased the unsaturation degree of membrane lipid and the immobilized yeast always exhibited lower unsaturation degree of membrane lipid than the free yeast in ethanol fermentation Keywords Fatty acid High density medium Immobilized yeast Saccharomyces cerevisiae Water hyacinth Introduction Ethanol has re-emerged as an alternative to petroleum-based liquid fuels due to energy crisis [1] Many studies have been performed in order to improve economic efficiency of ethanol production [2, 3] It has been reported that using media with high sugar concentration resulted in high ethanol level in the fermentation broth and that could lead to great savings in process water and energy requirements in ethanol industry [2] However, high sugar content in media inhibited yeast growth and lengthened the fermentation as a result of high V N Tran : V V M Le (*) Department of Food Technology, Ho Chi Minh City University of Technology, Ho Chi Minh City, Vietnam e-mail: lvvman@hcmut.edu.vn 964 Appl Biochem Biotechnol (2014) 172:963–972 osmotic pressure In addition, increase in osmotic pressure of media could lead to an incomplete fermentation [2, 4] Application of immobilized yeast could lower the inhibitory effects in media with high sugar content [5, 6] The yeast cells entrapped in different gel matrices [5] or adsorbed on sintered glass beads [6] fermented sugar faster than the free yeast in high-density media Nevertheless, the osmotolerance of the immobilized yeast varied from matrix to matrix From the last decade, using natural supports with high-cellulose content for yeast immobilization has attracted great attention [3] These supports are low in cost, environmentally friendly, and abundantly available in many world regions [7] Yeast cells adsorbed on different cellulosic supports were used in ethanol fermentation and the sugar concentration in the medium varied from 50 to 200 g/L [8–10] There have been so few studies on alcoholic fermentation performance of the immobilized yeast on cellulosic supports in high-density media In this study, water hyacinth stem pieces were used as new cellulosic support for yeast immobilization and the immobilized biocatalyst was then inoculated in the medium with different glucose concentrations for ethanol fermentation Water hyacinth (Eichhornia crassipers) is a fast-growing aquatic plant widely distributed throughout the world This plant has been used in the production of paper, crafts, rope, and furniture Water hyacinth stem has been known as a good adsorbent material with a large specific surface [11] According to Yu et al [12], cellulosic material with high porous structure could be used as support for yeast immobilization There has been no study on yeast immobilization on water hyacinth stem pieces In high-density media, yeast cells suffered different stresses including osmotic and ethanol inhibition [13] The survival and metabolism of yeast cells depended on their ability to adapt quickly to the changing environment Change in plasma membrane composition could be an adaptive response by the yeast since it was highly variable and clearly influenced by environmental factors [14] There have been many studies to investigate fatty acid levels in cellular membrane as the yeast was exposed to high ethanol concentration [4, 15–17] Nevertheless, the effect of high sugar concentration on fatty acid composition in cellular membrane of the immobilized yeast was only reported in a unique study of HilgeRotmann and Rehm [6] who used alginate gel and sintered glass beads as supports for immobilization of yeast cells The objective of this research was to compare the alcoholic fermentation performance of the free and immobilized yeast on water hyacinth stem pieces in the medium with different glucose contents In addition, fatty acid composition in cellular membrane of the immobilized and free yeast during the fermentation was also evaluated The obtained results would give a clearer understanding about the improvement in fermentation rate of the immobilized yeast in medium with high sugar content Materials and Methods Yeast Saccharomyces cerevisiae TG1 from the culture collection of Food Technology Department, Ho Chi Minh City University of Technology was used Support Water hyacinth (E crassipers) stems were washed with potable water to remove adhering dirt, cut into cylindrical pieces, and subsequently sterilized at 121 °C for 20 After Appl Biochem Biotechnol (2014) 172:963–972 965 cooling, the support was ready for cell immobilization The height and diameter of the support were approximately and cm, respectively Media Medium A was used for yeast growth and immobilization The medium contained glucose (120 g/L), yeast extract (4 g/L), (NH4)2SO4 (1 g/L), KH2PO4 (1 g/L), and MgSO4 (5 g/L) Medium B was used for ethanol fermentation The chemical composition of medium B was similar to that of medium A except that the glucose concentration was adjusted to 200–400 g/L Yeast Immobilization Yeast cells were grown at 30 °C for 24 h in medium A and subsequently separated at °C in a refrigerated centrifuge (Sartorius, Switzerland) For yeast immobilization, the cells were resuspended in medium A to form a yeast suspension with the cell concentration of 3.5× 107 cfu/mL; 30 g support was then added into 500-mL shake flask containing 140 mL yeast suspension and the mixture was incubated in a thermostat shaker (Sartorius, Switzerland) at 30 °C for 20 h Finally, the support with immobilized cells was removed and washed with sterile water three times The cell density was 4.5×108 cfu/g wet support The immobilized yeast obtained was ready for ethanol fermentation Fermentation Static fermentation was conducted at 30 °C in L Erlenmeyer flasks containing 500 mL of medium B The inoculum size was 1.0×107 cfu/mL Control samples with free yeast cells were simultaneously carried out under the same conditions The fermentation was considered completed when the residual sugar concentration was unchanged during 12 consecutive hours Analytical Methods Cell Density in Yeast Cultures and Specific Growth Rate The cell density in the free yeast culture (colony forming unit per milliliter) was determined by plate count agar with glucose–peptone agar medium and the incubation was performed at 30 °C for 48 h [18] The immobilized cells on the support were quantified by the procedure as described previously [18] with slight modification Five grams of the support and 95 mL distilled water were ground in a grinder at 3,000 rpm for min; the cell number in the suspension obtained was determined by plate count agar with glucose–peptone agar medium and the incubation was performed at 30 °C for 48 h The cell density in the immobilized yeast culture was calculated and expressed as colony forming unit per milliliter fermented medium The specific growth rate of the free and immobilized yeast was calculated according to the formula described elsewhere [19] Glucose Glucose content was evaluated by spectrophotometric method using 3,5-dinitrosalicylic acid reagent [20] 966 Appl Biochem Biotechnol (2014) 172:963–972 Ethanol Ethanol concentration was determined by enzymatic method using ethanol kit with a reflectometer model 116970 (MercK KgaA, Germany) Under the catalytic effect of alcohol dehydrogenase, alcohol is oxidized by NAD to acetaldehyde In the presence of an electron transmitter, the NADH formed in the process reduces a tetrazolium salt to a blue formazan that is determined reflectometrically Fatty Acid Composition of Yeast Cell Membrane Prior to determine fatty acid composition, the lipid in yeast cell membrane was extracted using method proposed previously [21] with slight modification Yeast biomass was added into methanol and the mixture was subsequently treated with ultrasound by an ultrasonic probe model VC 750 (Sonics & Materials Inc., USA) at an ultrasonic power of W/g for to disrupt the cell wall The lipid extraction was then carried out by adding chloroform and methanol (2:1 v/v) to the sonicated mixture The weight ratio of material and solvent was 5:2 The extraction was performed at the agitation rate of 200 rpm for h The organic phase was then transferred into a glass screw tube containing 0.88 % KCl solution The mixture was centrifuged at 25 °C and 3,000 rpm for The organic phase was then collected and used for determination of fatty acid composition Fatty acid composition was determined by gas chromatography using a Hewlett-Packard model 5890A (Hewlett-Packard, USA) The extract was injected into an FFAP-HP column of 25 m×0.2 mm with an HP automatic injector Helium was used as carrier gas at 1.0 mL/min and heptadecanoic acid methyl ester (1 μg/μL) was added as an internal standard Column inlet pressure was 150 kPa The injector temperature was 250 °C Detector temperature was 250 °C The temperature program was 25 °C/min from 70 to 200 °C Peak areas were measured using a Hewlett-Packard model 3396A integrator Unsaturation Degree of Fatty Acids in Yeast Cell Membrane Unsaturation degree of fatty acids in yeast cell membrane was calculated from the fatty acid composition in cellular membrane using the following formula [22] =mol ẳ ẵ1mol % monoenesị ỵ 2mol % dienesị ỵ 3mol % trienesị=100 Statistical Treatment The presented results were the average of three independent experiments The obtained results were subjected to analysis of variance (p