DSpace at VNU: Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces

Appl Biochem Biotechnol DOI 10.1007/s12010-013-0411-z Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces Hoang Du Le & Pornthap Thanonkeo & Van Viet Man Le Received: 26 April 2013 / Accepted: 19 July 2013 # Springer Science+Business Media New York 2013 Abstract Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C) Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C Temperature of 45 °C restricted yeast growth and lengthened the fermentation The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress The free cells of K marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane For fermentation at 40 °C with immobilized cells of K marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level Keywords Fatty acid Immobilized yeast Kluyveromyces marxianus Thermal stress Introduction Saccharomyces cerevisiae has been considered as the best choice for ethanol fermentation in industrial scale However, the growth of S cerevisiae was slightly reduced even at 35 °C [1] Therefore, many attempts have been done to find other species that can assimilate sugar and produce ethanol at high temperature [2–4] Among yeast species, Kluyveromyces marxianus is highly potential due to its advantages over S cerevisiae in terms of high-temperature H Du Le : V V M Le (*) Department of Food Technology, Ho Chi Minh City University of Technology, Ho Chi Minh City, Viet Nam e-mail: lvvman@hcmut.edu.vn P Thanonkeo Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand Appl Biochem Biotechnol fermentation K marxianus was reported to have the ability to assimilate sugars and produce ethanol at temperature from 40 to 45 °C [3, 5] Besides the use of thermotolerant yeast, high-temperature fermentation could be achieved by using immobilized yeasts Immobilized yeast cells offer advantages over free cells in terms of high ethanol productivity [6] and high stability in metabolic activity during hightemperature fermentation [7, 8] However, thermotolerance of fixed yeast was varied and depended on nature of the carriers used From the last decade, high cellulosic content material has been a potential support because of its high porosity for cell adsorption [6] In addition, the carrier pretreatment before use and the yeast immobilization procedure were simple [9] Furthermore, the yeast immobilized on cellulosic material exhibited higher thermotolerance in comparison with the free yeast [9] The studies about thermotolerance of immobilized yeast on cellulosic material, however, were mostly conducted with S cerevisiae [6, 7, 9] Ethanol fermentation with K marxianus immobilized on cellulosic carrier has not been reported In this study, for the first time, K marxianus immobilized on banana leaf sheath pieces was used for ethanol fermentation Banana is the main fruit in international trade in terms of volume and the fourth most important staple crop in the world Banana is grown in about 120 countries worldwide and very popular in tropical countries The banana fruit is consumed by local population in Asia, America, and Africa as a major staple food [10] The banana leaf sheath, however, is not used for human consumption and usually discarded as waste [11] From the nineties of the last century, the banana leaf sheath was used as adsorbents for removal of metal ions from waste water due to their highly porous structure [12, 13] Yu et al [6] stated that cellulosic material with high porous structure could be used as support for yeast immobilization However, no research was done to investigate the immobilization of yeast cells on banana leaf sheath pieces Generally, the thermotolerance of immobilized yeast was explained through the protection of carrier [7] This explanation, however, was not completely satisfied because yeast generally responded to high temperature through changes in cellular components [14] Many studies have been done to investigate the temperature adaptation of yeast to thermal stress through membrane fluidity [14–16] Increase in temperature was reported to augment membrane fluidity and yeast responded to these changes by regulating membrane fatty acid composition [16] Researches about the effects of temperature on fatty acid composition of yeast were mostly carried out with the free cells of S cerevisiae In addition, no study has been done to compare the thermal response of the free and immobilized K marxianus cells through fatty acid composition The aim of this work was to clarify the effects of high temperature on the growth, sugar assimilation, and ethanol production of the free and immobilized cells of K marxianus In addition, this work also investigated the response of the free and immobilized yeast on banana leaf sheath pieces to high temperature The results obtained would give a clearer understanding about the improvement in fermentation performance of the immobilized yeast in high-temperature fermentation Materials and Methods Yeast and Media A strain of K marxianus originated from Department of Biotechnology, Khon Kaen University (Thailand), was used for ethanol fermentation For the inoculum preparation, Appl Biochem Biotechnol the yeast strain was cultivated in 10 mL of growth medium containing 30 g/L glucose, g/L yeast extract, g/L NH4Cl, g/L KH2PO4, and 0.3 g/L MgSO4·7H2O in a test tube (150×16 mm) The test tube was shaken at 30 °C, 150 rpm for 24 h Ten milliliters of the preculture was then inoculated into a 250-mL Erlenmeyer flask containing 90 mL of growth medium The flask was also shaken at 30 °C, 150 rpm for 24 h The preculture was subsequently centrifuged at °C and 5,000 rpm for 20 Yeast cells were collected and washed with sterile water The medium composition for yeast immobilization and ethanol fermentation was similar to that for preculture preparation except that the glucose concentration was adjusted to 120 g/L The initial pH of the media was 5.5 All media were sterilized at 121 °C for 20 before use Carrier Leaf sheath of banana (Musa acuminata) was used as carrier for yeast immobilization Firstly, the leaf sheath was washed with potable water and then cut into tubular shape with 20 mm in diameter and mm in height Secondly, the leaf sheath pieces (LSP) were treated with 0.01 N NaOH solution at 120 rpm for 30 for tannin removal and subsequently washed with distilled water three times Finally, the LSP were sterilized at 121 °C for 20 before use Yeast Immobilization on Banana Leaf Sheath Pieces Twenty grams of sterilized LSP and 100 mL of medium for yeast immobilization were added into a 250-mL shaking flask The yeast biomass was then introduced into the flask in order to reach a cell density of 3.0×107 CFU/mL The flasks were shaken at 30 °C, 120 rpm for 20 h The liquid fraction was decanted and the LSP with immobilized cells were washed with the fermentation medium twice The immobilized biocatalyst was sampled for cell quantification Ethanol Fermentation Ethanol fermentations were carried out in 500-mL flasks containing 250 mL medium under stationary conditions Immobilized yeasts were introduced into the medium with the inoculum size of 1.0×107 CFU/mL The fermentation temperature was various: 30, 35, 40, and 45 °C The fermentations lasted for 72 h During the fermentation, samples were taken at 12h intervals for analysis The fermentations with the free yeast were also performed under the same conditions Analysis Total Dietary Fiber of Banana Leaf Sheath Total dietary fiber of banana leaf sheath was determined by enzymatic-gravimetric method [17] Cell Density The yeast cell concentration in liquid sample was determined by plate count agar with glucose-peptone agar medium and the incubation was performed at 30 °C for 48 h [7] Appl Biochem Biotechnol The immobilized cells adsorbed on the LSP were quantified by the procedure proposed by Vasconcelos et al [18] with slight modification Ten grams of the LSP with the immobilized cells and 90 mL of distilled water were mixed and ground in a grinder at 3,500 rpm for Afterwards, the cell number was determined by plate count agar with glucose-peptone agar medium and the incubation was performed at 30 °C for 48 h Glucose Glucose concentration was quantified by spectrophotometric method with dinitrosalicylic acid reagent [19] Ethanol Ethanol concentration was quantified 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 determining the fatty acid composition, the lipid in yeast cell membrane was extracted using the method proposed by Beltran et al [15] 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 mixture of chloroform and methanol (2:1v/v) to the sonicated sample 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 using formula as described previously [20] Statistical Analysis All experiments were performed in triplicate Mean values were considered significantly different when P