Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress.. The aim of this work was to clarify the effec
Trang 1Impact of High Temperature on Ethanol Fermentation
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 DOI 10.1007/s12010-013-0411-z
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
Trang 2fermentation 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 high-temperature 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 protec-tion 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,
Trang 3the yeast strain was cultivated in 10 mL of growth medium containing 30 g/L glucose, 5 g/L yeast extract, 1 g/L NH4Cl, 1 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 5 °C and 5,000 rpm for 20 min 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 min 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 5 mm in height Secondly, the leaf sheath pieces (LSP) were treated with 0.01 N NaOH solution at 120 rpm for 30 min for tannin removal and subsequently washed with distilled water three times Finally, the LSP were sterilized at 121 °C for 20 min 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 3.0×107CFU/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 inoc-ulum size of 1.0×107CFU/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
12-h intervals for analysis T12-he fermentations wit12-h t12-he free yeast were also performed under t12-he 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]
Trang 4The 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 5 min 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 Reflec-tometer 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 5 W/g for 1 min 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 2 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 5 min 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<0.05 One-way and multi-way analyses of variance were performed using the software Statgraphics Centurion XV
Trang 5Results and Discussion
Effects of Temperature on Yeast Growth
Table1shows the effects of temperature on maximum cell density and specific growth rate
of the free and immobilized yeasts Generally, the fermentation temperature from 30 to 40 °C did not change the yeast growth Similar results were obtained by Rodrussamee et al [5] who reported that growth of the free cells of K marxianus was not altered when temperature increased from 30 to 40 °C In addition, K marxianus was known as a thermotolerant strain with the ability to grow at 40 °C [21] At the temperature range of 30–40 °C, the maximum cells density and specific growth rate of the immobilized yeast were always higher than those of the free yeast Recently, Pacheco et al [9] investigated ethanol fermentation of S cerevisiae and reported that immobilized yeast grew faster and reached higher cell density than the free yeast However, no study has been done to compare the growth of the free and immobilized K marxianus cells
At 45 °C, on the other hand, the growth of both the free and immobilized yeasts slowed down Rodrussamee et al [5] reported that the growth of the free cells of K marxianus at
45 °C was greatly reduced compared to that at 40 °C According to Roukas [22], high temperature caused denaturation of the enzyme system of K marxianus cells In addition, reduction of yeast growth at high temperature was due to accumulation of intracellular ethanol which would modify the structure of the yeast cell membrane [23] Furthermore, ethanol could remove hydrate layers around yeast cells and that led to a reduction in yeast growth [23] In this study, the reduction in maximum cell density and specific growth rate of the free yeast were higher than those of the immobilized yeast When the fermentation temperature increased from 40 to 45 °C, maximum cell density of the free and immobilized yeasts decreased by 52.7 and 26.8 %, respectively Moreover, specific growth rate of the free yeast decreased by 32.4 % compared to 14.9 % of the immobilized yeast It was reported that carriers with high level of–OH group could protect the hydrate layers around yeast cells [24] In this study, the level of total fiber in banana leaf sheaths was 57 % of dry matter basis Therefore, banana leaf sheath could increase ethanol tolerance of the immobilized yeast
Effects of Temperature on Substrate Assimilation of K marxianus
The evolution of glucose concentration during the fermentation at different temperatures is shown in Fig 1 Increase in temperature from 30 to 40 °C did not significantly change glucose uptake rate of both free and immobilized yeast The fermentation time was 48 h
Table 1 Effects of temperature on maximum cell density and specific growth rate of the immobilized and free Kluyveromyces marxianus
Temperature (°C) Maximum cell density (107CFU/mL) Specific growth rate (1/h)
Immobilized cells Free cells Immobilized cells Free cells
Various superscripts in table indicate significant differences (P<0.05)
Trang 6According to Rodrussamee et al [5], glucose assimilation of the free cells of K marxianus was unchanged when fermentation temperature increased from 30 to 40 °C At the temper-ature range of 30–40 °C, the residual sugars level in the free and immobilized yeast cultures were insignificantly different Some studies reported that most gel-carriers would prevent the diffusion of substrates from the media to the immobilized yeast cells [6,25] Therefore, sugar assimilation of immobilized yeast was decelerated In the present work, however, cellulosic material had porous structure It can be suggested that the diffusion of substrates was not prevented by the support Our results agreed with those of Pacheco et al [9] who reported that residual sugar level in the free and immobilized S cerevisiae cultures was similar
At 45 °C, substrate uptake rate of both free and immobilized yeast declined After 48 h, the residual sugar level in the free yeast culture was higher than that in the immobilized yeast culture In order to clarify the improvement in fermentation performance of the immobilized yeast at high temperature, the fatty acid composition of yeast cell membrane was deter-mined Our preliminary investigation showed that the level of long-chain fatty acid (C16 and C18) was dominant in comparison with that of other fatty acids in cellular membrane of the free yeast K marxianus The weight ratio of palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linoleic acid (C18:2) in cellular membrane of the free yeast was approximately 42, 26,
21, and 6 % of the total fatty acid level, respectively The change in saturated and unsaturated fatty acid level of the free and immobilized yeast at 40 and 45 °C was shown
in Figs.2and3, respectively The weight ratio of saturated fatty acid (C16:0 and C18:0) in cellular membrane of the free yeast was always higher than that of the immobilized yeast In addition, weight ratio of saturated fatty acid in cellular membrane of both free and immobilized yeast at 40 °C was always lower than that at 45 °C except for that the weight ratio of palmitic acid in cellular membrane of the immobilized yeast did not change when the fermentation temperature increased from 40 to 45 °C Banat et al [16] noted that increasing temperature caused increase in fluidity of membranes and yeasts responded to this physical change by altering their fatty acid composition The changes in fatty acid composition in cellular membrane were observed in many previous studies [1,14,16] with different trends
0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0
Fermentation time (h) Fig 1 Effects of temperature on sugar assimilation of the free (square and solid line, 30 °C; diamond and solid line, 35 °C; triangle and solid line, 40 °C; circle and solid line, 45 °C) and immobilized (square and dashed line, 30 °C; diamond and dashed line, 35 °C; triangle and dashed line, 40 °C; circle and dashed line,
45 °C) K marxianus cells
Trang 7According to Banat et al [16], increasing temperature led to an increase in relative composition of saturated fatty acid (palmitic acid) and a decrease in relative composi-tion of unsaturated fatty acid (linoleic and linolenic acid) in cellular membrane of S cerevisiae However, linoleic was observed increasing with the increase in temperature when Suutari et al [14] investigated the effects of temperature on fatty acid composi-tion of S cerevisiae
In this study, K marxianus responded to high temperature by increasing relative percent-age of saturated fatty acids and by decreasing relative percentpercent-age of unsaturated fatty acids
In addition, the reduction level of unsaturated fatty acid in the immobilized yeast cell was observed less than that of the free yeast Moreover, the relative percentage of palmitic acid in immobilized yeast cells did not significantly change even at high temperature (45 °C) and this value remained stable during the fermentation We supposed that thermotolerance of immobilized yeast was improved by the protection of carrier; therefore, high-temperature adaptation of the immobilized yeast was better than that of the free yeast Different response
of the free and immobilized yeast could be an explanation for the difference in sugar assimilation rate between the free and immobilized yeast
35.0 36.0 37.0 38.0 39.0 40.0 41.0 42.0 43.0 44.0 45.0
Palmitic acid (%
Fermentation time (h)
10.0 15.0 20.0 25.0 30.0 35.0
Stearic acid (% w/w)
Fermentation time (h) Fig 2 Effects of temperature on the saturated fatty acid composition in cellular membrane of the free (square and solid line, 40 °C; triangle and solid line, 45 °C) and immobilized (square and dashed line, 40 °C; triangle and dashed line, 45 °C) K marxianus cells during the fermentation
Trang 8In addition, the degree of unsaturation of fatty acids in cellular membrane of the immobilized and free yeast was also calculated Figure4 shows that at the beginning of the fermentation, the immobilized yeast exhibited higher unsaturation degree than the free yeast It can be noted that the adsorption of yeast cells on banana LSP increased the unsaturation degree of fatty acids in cellular membrane of K marxianus cells At 40 °C, the degree of unsaturation of fatty acids for both immobilized and free yeast remained stable during the fermentation At 45 °C, however, the degree of unsaturation of fatty acids for both immobilized and free yeast decreased during the fermentation No study has been done to compare the change of unsaturation degree of fatty acids in cellular membrane of the immobilized and free K marxianus cells during ethanol fermentation Previously, Hilge-Rotmann and Rehm [26] compared the unsaturation degree of fatty acids in cellular membrane of the immobilized and free S cerevisiae cells at the end of ethanol fermentation The results showed that the free yeast exhibited higher unsaturation degree of fatty acids than the immobilized yeast The difference in our results and those of Hilge-Rotmann and Rehm [26] was possibly due to the difference in yeast species In our study, increase in unsaturation degree of fatty acids in cellular membrane improved substrate assimilation of the immobilized K marxianus cells
0.0 5.0 10.0 15.0 20.0 25.0
Fermentation time (h)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
Fermentation time (h)
Fig 3 Effects of temperature on the unsaturated fatty acid composition in cellular membrane of the free (square and solid line, 40 °C; triangle and solid line, 45 °C) and immobilized (square and dashed line, 40 °C; triangle and dashed line, 45 °C) K marxianus cells during the fermentation
Trang 9Effects of Temperature on Ethanol Formation by K Marxianus
As shown in Table 2, increase in temperature from 30 to 40 °C did not alter ethanol formation of both the free and immobilized yeast Similar results were obtained by Rodrussamee et al [5] who reported that ethanol production of the free cells of K marxianus was not affected when fermentation temperature increased from 30 to 40 °C According to Moreno et al [21], K marxianus was a thermotolerant strain with the ability to produce ethanol at 40 °C It can be noted that the final ethanol concentration and ethanol yield of the immobilized yeast on banana LSP was 15.3 and 15.8 %, respectively, higher than those of the free yeast
At 45 °C, however, ethanol production of both the free and immobilized yeast was inhibited The average ethanol formation rate of the free yeast decreased by 42.8 % compared to 29.0 % for the immobilized yeast when the fermentation temperature increased from 40 to 45 °C In addition, ethanol evaporation is another factor to be considered
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
Fermentation time (h)
Figure 4 Effects of temperature on the unsaturation degree of fatty acids in yeast cell membrane of the free (square and solid line, 40 °C; triangle and solid line, 45 °C) and immobilized (square and dashed line, 40 °C; triangle and dashed line, 45 °C) K marxianus cells during the fermentation
Table 2 Effects of temperature on final ethanol concentration, average ethanol formation rate, and ethanol yield of the immobilized and free Kluyveromyces marxianus cells
Temperature
(°C)
Final ethanol concentration
(g/L)
Average ethanol formation rate (g/L h)
Ethanol yield (g ethanol produced/g glucose assimilated) Immobilized cells Free cells Immobilized
cells
Free cells Immobilized cells Free cells
30 52.72 k ±0.55 45.78 j ±0.47 2.07 g ±0.03 1.79 f ±0.08 0.45 b ±0.01 0.39 a ±0.01
35 53.67 k ±0.55 45.44 j ±0.71 2.16 g ±0.04 1.80 f ±0.08 0.46 b ±0.01 0.39 a ±0.01
40 52.07 k ±1.26 45.62 j ±1.26 2.10 g ±0.06 1.80 f ±0.11 0.44 b ±0.01 0.39 a ±0.01
45 34.73 i ±1.97 28.28 h ±0.05 1.49 e ±0.03 1.03 d ±0.09 0.45 b ±0.01 0.48 c ±0.02 Various superscripts in table indicate significant differences (P<0.05)
Trang 10However, it had a minor effect at the temperature of 45 °C because efficient evaporation occurs at 50 °C [27]
The immobilized yeast demonstrated higher ethanol concentration in the culture and higher ethanol formation rate than the free yeast at all fermentation temperatures According
to Gough and McHale [3], the immobilized yeast in alginate gel exhibited higher ethanol productivities compared to the free yeast The change in fatty acid levels of the immobilized yeast (Figs 2, 3, and 4) could be an explanation for its high ethanol formation rate Nevertheless, the ethanol yield of the fixed cells at 45 °C was slightly lower than that of the free cells
Conclusions
High-temperature ethanol fermentation could be improved by using immobilized yeast In the temperature range of 30–45 °C, the immobilized yeast exhibited a faster substrate consumption and ethanol formation in comparison with the free yeast Increase in relative content of saturated fatty acids (C16:0 and C18:0) and decrease in relative content of unsaturated fatty acids (C18:1 and C18:2) in the cellular membrane were the response of
K marxianus to high temperature Adsorption of K marxianus cells on banana LSP augmented the unsaturation degree of fatty acids in cellular membrane and that led to an improvement in glucose uptake rate of the immobilized K marxianus cells
Acknowledgments This work was financially supported by Vietnam National University, Ho Chi Minh City (Project B2012-20-11TD/HD-KHCN)
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