Effect of furfural and acetic acid in the fermentation performance of immobilized kluyveromyces marxianus

T N ĐỀ TÀI: Effect of Furfural and Acetic Acid in the Fermentation Performance of Immobilized Kluyveromyces marxianus... Increase in furfural level 0 – 5g/L or acetic acid content 0 – 8

ĐẠI HỌC QUỐC GIA TP HCM

TRƯỜNG ĐẠI HỌC BÁCH KHOA

CÔNG TRÌNH ĐƯỢC HOÀN THÀNH TẠI TRƯỜNG ĐẠI HỌC BÁCH KHOA–ĐHQG -HCM Cán bộ hướng dẫn khoa học : GS TS ăn iệt Mẫn

Cán bộ chấm nhận x t 1 : TS Ho ng im nh

Cán bộ chấm nhận xét 2 : TS h n Ngọ Ho

Luận văn thạ sĩ được bảo vệ tại Trường Đại học Bách Kho , ĐHQG Tp HCM ngày 5 tháng 1 năm 2016

Thành phần Hội đồng đánh giá luận văn thạ sĩ gồm:

(Ghi rõ họ, tên, học hàm, học vị của Hội đồng chấm bảo vệ luận văn thạ sĩ)

ĐẠI HỌC QUỐC GIA TP.HCM

TRƯỜNG ĐẠI HỌC BÁCH KHOA

CỘNG HÒA XÃ HỘI CHỦ NG Ĩ VI T NAM

Độc lập - Tự do - Hạnh phúc

NHI M VỤ LUẬN VĂN T ẠC SĨ

Họ tên học viên: Thị ệ Quy n MSHV: 13111026

Ng y, tháng, năm sinh: 23/06/1990 Nơi sinh: Tp Hồ Chí Minh Chuyên ngành: Công nghệ Thực phẩm Mã số: 60 54 01 01

I T N ĐỀ TÀI: Effect of Furfural and Acetic Acid in the Fermentation

Performance of Immobilized Kluyveromyces marxianus

II NHI M VỤ VÀ NỘI DUNG:

Nhiệm v

 So sánh hả năng háng hịu str ss ur ur l, ti i ủ nấm m n ố định tr n lá nướ v nấm m n tự o th ng qu hả năng sinh trưởng, sử ng ơ hất v sinh t ng hợp sản phẩm

 hảo sát ảnh hưởng ủ ur ur l, ti i đến th y đ i th nh phần i

o trong m ng tế o hất ủ nấm m n ố định tr n lá nướ v nấm m n tự o

tự o trong đi u iện tá động ủ ur ur l, ti i

III NGÀY GIAO NHI M VỤ : 19/01/2015

IV NGÀY HOÀN THÀNH NHI M VỤ: 04/12/2015

V CÁN BỘ ƯỚNG DẪN :

GS TS ăn iệt Mẫn

ACKNOWLEDGEMENTS

I wish to express my heartfelt gratitude to my scientific supervisor,

ro ssor ăn iệt Mẫn for his guidance and encouragement regardless place and time He is the one who gave me a lot of useful and valuable advises My study

actually could not be finished without his criticism and assistances

I would like to thank all the faculty members of Department of Food Technology, Ho Chi Minh City University of Technology that they give the best

opportunities for my study and all of their help with my project

I am also indebted to my classmates who always stand by me, encourage and

support to my work in night time

I gratefully acknowledge my parents for their encouragement, prayers, magnificent support, and love patience through my eyes of education I am so

blessed to have you as family I love you

Once again, I am grateful to everybody that involved directly or indirectly in helping me during the researching progress

4th December, 2015

Thị ệ Quy n

ABTRACT

Lignocellulosic material is potential for bioethanol production However, acidic pretreatment of lignocellulosic material generate various toxic compounds including furfural and acetic acid for yeast fermentation The objective of this study was to evaluate the effect of furfural and acetic acid in the fermentation performance of

free yeast and immobilized yeast on Nypa fruiticans leaf sheath pieces

Increase in furfural level (0 – 5g/L) or acetic acid content (0 – 8g/L) in the medium significantly reduced cell growth and ethanol formation rate of both

immobilized and free yeast However, the immobilized yeast on Nypa fruiticans leaf

sheath pieces fermented sugar faster and produced more ethanol than the free yeast under furfural and acetate stresses Application of immobilized yeast was therefore potential for improvement in ethanol fermentation from lignocellulosic material

LỜI C M ĐO N CỦA TÁC GIẢ

T i xin m đo n rằng đây l ng trình nghi n ứu của tôi, có sự hỗ trợ t giáo

vi n hướng dẫn l GS TS ăn iệt Mẫn Các nội dung nghiên cứu và kết quả trong đ tài này là trung thự v hư t ng được ai công bố trong bất cứ công trình nghiên cứu n o trướ đây

Nếu phát hiện có bất kỳ sự gian lận nào tôi xin hoàn toàn chịu trách nhiệm trước hội đồng, ng như ết quả luận văn ủa mình

Tháng 12 năm 2015 Học viên

Thị ệ Quy n

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION 1

CHAPTER 2: LITERATURE REVIEW 3

2.1 Kluyveromyces marxianus 3

2.2 Carrier 4

2.2.1 Yeast immobilization 4

2.2.2 Nypa fruticans 5

2.3 Inhibitors in ethanol fermentation 5

2.3.1 Fufural 6

2.3.1.1 Introduction 6

2.3.1.2 Adaptive response of yeast to furfural 7

2.3.1.3 Inhibition effects of furfural on yeast growth and ethanol fermentation 10

2.3.2 Acetic acid 10

2.3.2.1 Introduction 10

2.3.2.2 Adaptive response of yeast to acetic acid 11

2.3.2.3 Inhibition effects of acetic acid on yeast growth and ethanol fermentation 12

CHAPTER 3: MATERIALS AND METHODS 14

3.1 Materials 14

3.1.1 Kluyveromyces marxianus 14

3.1.2 Carrier 14

3.1.3 Chemicals 14

3.1.4 Medium 14

3.2.1 Research content 15

3.2.2 Experiment design 17

3.2.3 Analytical methods 18

3.2.4 Calculation formula 19

3.2.5 Statistical analysis 20

CHAPTER 4: RESULTS AND DISCUSSION 21

4.1 Effect of furfural on ethanol fermentation by Kluyveromyces marxianus

21

4.1.1 Effect of furfural on yeast growth 21

4.1.2 Effect of furfural on substrate assimilation 24

4.1.3 Effect of furfural on ethanol formation 26

4.1.4 Effect of furfural on unsaturation degree of fatty acids of cellular membrane 28

4.2 Effect of acetic acid on ethanol fermentation by Kluyveromyces marxianus. 30

4.2.1 Effect of acetic acid on yeast growth 30

4.2.2 Effect of acetic acid on substrate assimilation 32

4.2.3 Effect of acetic acid on ethanol formation 34

4.2.4 Effect of acetic acid on unsaturation degree of fatty acids of cellular membrane 36

CHAPTER 5: CONCLUSION AND SUGGESTION 38

5.1 Conclusion 38

5.2 Suggestion 38

TABLE OF FIGURES:

Figure 2 1: Nypa fruticans 5

Figure 2 2: Main hydrolytic components of lignocellulose biomasses and generated inhibitory compounds 6Figure 2 3: Chemical structure of furfural 7Figure 2 4: Conversion of furfural by the action of alcohol dehydrogenase to furfuryl alcohol 7Figure 2 5: A schematic illustration of glucose metabolic pathways and

conversion of furfural by tolerant Saccharomyces cerevisiae 9

Figure 2 6: Chemical structure of acetic acid 11

Figure 2 7: Mechanisms of acetic acid stress response in S cerevisiae cells 11

Figure 4 1: Growth curves of the immobilized and free yeast in medium with different furfural concentrations 21 Figure 4 2: Change in glucose concentration in the immobilized and free yeast cultures with different furfural concentrations 24Figure 4 3: Ethanol formation in the immobilized and free yeast cultures with different furfural concentrations 26Figure 4 4: The unsaturated degree of fatty acid on the cell membrane 29Figure 4 5: Growth curves of immobilized and free yeast in medium with different acetic acid concentrations 30Figure 4 6: Change in glucose concentration in the immobilized and free yeast cultures with different acetic acid concentrations 32Figure 4 7: Ethanol formation in the immobilized and free yeast cultures with different acetic acid concentrations 34Figure 4 8: The degree of unsaturated fatty acid of the cell membrane 37

LIST OF ABBRIVIATIONS

ATPase : Enzyme catalyzes the decomposition of ATP into

ADP and free phosphate ion

ADH6, ADH7 : Genes encoded for aldehyde reductions activity ALD4, GRE3 : Genes encoded for aldehyde reductions activity

AFT1P : Genes encoded for intracellular metal metabolism

FPS1P : Genes encoded for vacuolar degradation

GND2, GND1, NQM1 : Genes encoded for NADH regeneration

HOG1P : Genes encoded for pathway for acetic acid

resistance at low pH HOG1P, SLT2P, MAP kinase: Genes encoded for phosphorylation kinase

HAA1P : Genes encoded for resistance to acetic acid

in glucose medium

NADPH : Nicotinamide adenine dinucleotide phosphate

TAC cycle : The tricarboxylic acid cycle

TPO2, TPO3 : Genes encoded for the plasma membrane

multidrug transporter

YGP1 : Genes encoded for the cell wall glycoprotein ZWF1, SOL3, RBK1: Genes encoded for NADH regeneration

CHAPTER 1: INTRODUCTION

Limited availability of fossil fuels has led to stronger attempt to investigate various renewable sources such as biomass, hydropower, solar, wind and marine energy which have been proved to be potential for fossil fuel replacement since they are more environmentally friendly [1]

Among those, biomass seems to be more alternative energy compared to other natural gases, sewer and geothermal heat [2] Moreover, ethanol from biomass is the most promising energy source It is an oxygenated fuel, and is easily blended with gasoline for transportive purposes or converted to electricity [3, 4] Besides, the ethanol combustion system was investigated to reduce the greenhouse effects from gas pollution [3, 5]

In ethanol production, bio-resources such as starch, cereals, sugarcane have been investigated and concluded as the main feedstock because of their following advanced property [6] Starch contains a large number of glucose units linked tog th r y β-1,4- glycosidic bond; therefore, it is easily to be broken down to produce high yield of ethanol without the pretreatment [7] Using these kinds of material can shorten the processing time; however it will lead to unbalance between food and fuel supplied sources, increasing the risk of food security

Currently, lignocellulosic biomass, such as wood, sugarcane bagasse, waste in food processing have been reported as an attractively available material for bioethanol production [8, 9] In general, the process of ethanol production from lignocellulosic biomass must have the pretreatment in which carbohydrate fraction

of plan cell is broken by physical and/or chemical methods [10] Comparing with physical method, chemical pretreatment with acidic hydrolysis is simple and also achieves high sugar yield from lignocellulose However, the product of the diluted acid pretreatment contains some toxic compounds, such as weak acids, furans and phenolic compounds, which strongly inhibit the biological reactions of yeast [11] Among the toxic compounds, furfural has been found as the strong inhibitor to yeast

growth and ethanol production, while acetic acid affects the cellular physiology by changing the functions of biological membranes [10, 12] To facilitate fermentation process, additional treatments are often needed to remove these inhibitors However, the procedure is complex and could generate another type of waste products, as the result the price of final product would be increased [13]

In recent years, immobilization of yeast has been considered as a technique to protect the yeast against unfavorable conditions as well as to improve yeast fermentation performance [14]

Kluyvermyces marxianus is highly potential in ethanol industry due to

thermo-tolerance and ability to ferment both hexose and pentose However, the effects of by-products from the lignocellulose pretreatment on the immobilized

Kluyveromyces marxianus have never been reported Therefore, the objective of this

study is to investigate the effects of inhibitors including Furfural and Acetic Acid on

the ethanol fermentation by immobilized yeast Kluyveromyces marxianus Nypa

fruticans leaf sheath pieces were selected as yeast carrier due to abundance in nature,

low cost and high porosity for cell adsorption Control samples with the free yeast were also performed under the same conditions in order to clarify the differences in fermentation performance between the immobilized and free cells

CHAPTER 2: LITERATURE REVIEW

2.1 Kluyveromyces marxianus

Kluyveromyces marxianus was firstly described in 1888 by E C Hansen after

isolating from grapes and named as Saccharomyces marxianus However, the morphology and fermenting capacity of S marxianus were different from those of other Saccharomyces species; in 1956, Van der Walt separated Saccharomyces

marxianus into the new genus Kluyvermyces [15]

Like Saccharomyces cerevisiae, Kluyveromyces marxianus is a

respiro-fermentative yeast which can utilize sugar by oxidative phosphorylation or ethanol

fermentation for energy synthesis [16] It is notable that Kluyveromyces marxianus

had the ability to grow up at high temperature, from 45oC to 50oC When growing

on 14% glucose (w/v), the maximum ethanol concentration at 45oC and 50oC was 7%

(w/v) and 5.5% (w/v), respectively [17] Kluyveromyces marxianus is demonstrated

as one of the yeast that has the fastest growth rate and the rapid fermentation rate under thermo-condition [18, 19]

Kluyveromyces marxianus has been proved to utilize various carbon sources,

such as hexose, sucrose, xylose, lactose; the last two sugars cannot be used by

Saccharomyces cerevisiae [15, 20] Kluyveromyces marxianus also has high

fermenting activity when Switchgrass is used as medium for ethanol fermentation at

40 – 50oC; the ethanol yield was nearly 78% of theoretical glucose yield [19] Zafar

et al (2006) also reported that Kluyveromyces marxianus is able to produce ethanol

from crude whey [21] or from the cheese whey powder [22]

The fermentation ability of immobilized Kluyveromyces marxianus was also

evaluated by recent researches Xuewu et al (2010) compared the ethanol

production rate of the free and immobilized Kluyveromyces marxianus in the

alginate gel when using cheese whey as carbon source Ethanol level produced by the immobilized cells (4.1% v/v) was higher than that of the free cells (3.8% v/v) Moreover, the sugar consumption rate of the free cells was not as fast as the

immobilized cells In temperatures range of 30oC – 40°C, the ethanol yield of the immobilized yeast was 13% higher than that of the free yeast [23]

In summary, the yeast Kluyveromyces marxianus shows high potential

application for ethanol industry, especially under thermo-condition

2.2 Carrier

2.2.1 Yeast immobilization

Yeast immobilization is one of the important techniques in fermentation industry The yeast immobilization may be defined as the physical confinement or localization of intact cells to certain defined region of space with the preservation of some desired catalytic activity [14] Based on the physical mechanism causing immobilization, the classification of immobilized cell systems can be divided into four major categories, including:

 Attachment to surface

 Entrapment within porous matrix

 Containment behind a barrier

 Self-aggregation [24]

In ethanol production, attachment to surface considered as a popular method for cell immobilization due to its simple technique, low cost and potential application in large scale

According to the Vucurovic et al (2008), cellulose material with porous structure, can be used as carrier for cell immobilization [25] In the recent years, this type of carrier has been used for yeast immobilization in various studies, as indicated in Table 2.1.

The main advantages of cellulosic carrier in the ethanol production include low cost, abundance in nature, environmental friendliness, and high tolerance under strong mechanical strength [14]

Table 2 1: Cellulosic carrier used for ethanol formation by Saccharomyces cerevisiae

Carrier Carbon source Refrence

2.2.2 Nypa fruticans

Nypa fruticans, belongs to Arecaceae family, is known as the palm that has

grown well in tropical countries Conventionally this plant has been used for mats, baskets and other household items [29]

Figure 2 1: Nypa fruticans

Nypa fruticans has been known as a good adsorbent material for metal ions in the

wastewater treatment due to their porous structure and stable at pH 5.0 - 5.5 [30]

From the last few year, Nypa fruticans has been used as carrier for yeast

immobilization under ethanol stress [31]

2.3 Inhibitors in ethanol fermentation

Treatment of lignocellulose at high temperature and pressure under acid condition is commonly used in the industry due to low cost and high sugar yield, even if they lead to the liberation of a range of compounds that are potential inhi itors o y st, ll “str ss”[10, 12] Generally, depending on the chemical structure, the inhibitors can be divided into three main groups, which are weak acids, furan derivatives and phenolic compounds [11] as describe in Figure 2.2

Figure 2 2: Main hydrolytic components of lignocellulose biomasses and generated inhibitory

compounds

Biomasses are generated from wastes such as (A) maize cobs (B) saw dust (C) sugar cane bagasse (D)

fast growing grasses [32]

Among furans derivatives, furfural is found the strong inhibitor to yeast growth and ethanol fermentation, while weak acid, acid acetic, affects the cellular physiology by changing the functions of biological membranes [10, 12]

2.3.1 Fufural

2.3.1.1 Introduction

Fur ur l, lso ll “ ur l hy ” is r own pro u t rom p ntos in th browning reaction during hydrolysis in the presence of strong acids [33] The chemical formula is OC4H3CHO, includes a heterocyclic aldehyde, with the ring structure shown at right Furfural has shown significant effect on the multiple glycolytic enzymes which are essential to central metabolism of the cell, leading to longer lag phase during cell growth or cell death due to DNA damage [34, 35]

Figure 2 3: Chemical structure of furfural

2.3.1.2 Adaptive response of yeast to furfural

Furfural is metabolized under both aerobic and anaerobic conditions by yeast Under an aerobic conditions, yeast has the ability of reduce furfural to a less toxic compounds, furfuryl alcohol, by reduction reactions coupled by NADH and alcohol dehydrogenase (ADH) [36, 37]

Figure 2 4: Conversion of furfural by the action of alcohol dehydrogenase to furfuryl alcohol

The adaptive response yeast for furfural transformation was performed by multiple genes mediated NADH – dependent aldehyde reduction [13] The furan ring and associated alcohol functional groups had been known as non-toxic to the metabolism of yeast but not the aldehyde [38] As a consequence, NADH has the function as oxidative component, donates the proton to aldehyde functional group

of furfural to form alcohol functional group, leading to a less toxic compound to yeast [13, 38]

By using the transcriptome profiling analysis on microarray, Liu et al (2006) found approximately 300 genes belongs to the pentose phosphate pathway genes of

Saccaromyces cerevisiae that associated to allow fermenting metabolic kinetics in

the presence of furfural [39] Among the genes encoded for aldehyde reduction activities, ADH6, ADH7, ALD4 and GRE3 present over expression in the culture treated with 40mM of furfural When one of the genes is removed from the genome, the growth ability of yeast cannot be observed in the presence of inhibitor [39]

In another study, Gorsich et al (2006) proved that the expression of multiple genes led to an adaptive response of yeast in medium with furfural concentration of

25mmol/L Among the genes, the genes that encode for pentose phosphate pathway enzymes, especially ZWF1, have strong ability to catalyze glucose-6-phosphate for NADH regeneration [40] Th uthors xpl in th t th ur ur l’s r ox r tion requires the NADH, which is the important cofactor in biological activity, thereby resulting imbalance of energy The expression all of genes in the pentose phosphate pathway for NADH regeneration would lead to the maintaining proper levels of reducing equivalents This conclusion is similar with the finding of Liu et al (2009), who stated that the furfural tolerance of yeast involved up – regulating of ZWF1, SOL3, GND1, GND2, NQM1, RBK1 for NADH and NADPH regeneration [41]

Figure 2 5: A schematic illustration of glucose metabolic pathways and conversion of furfural by

tolerant Saccharomyces cerevisiae [38]

Black arrowed lines and letters indicate normal or near normal levels of reactions, expressions or pathways, green indicates enhanced, and red for repressed expressions, reactions, or pathways Bolded lines and letters indicate the levels of expression and pathways are statistically significant Key steps of enhanced NAD(P)H regenerations are circled in blue and significant aldehyde reductions circled in orange

2.3.1.3 Inhibition effects of furfural on yeast growth and ethanol fermentation

The redox reaction of furfural to less toxic compound and the formation of ethanol from acetaldehyde have the same enzyme, ADH, leading to the largely requirement of cofactor NADH; as a result both reactions can happen at the same time Moreover, the reaction rate strongly depends on the relative substrate concentration [41] According to the study of Horvath et al (2003), in the medium without furfural, the converted concentration of NADH/C-mol of glucose in the glycerol synthesis was lower than its concentration in the medium containing furfural and 26 mmol of NADH/C-mol of glucose [37] The concentration of

glyceraldehyde-3-phosphate in Saccharomyces cerevisiae culture has been reported

from 0.5 – 1.7mM [42], while the concentration of furfural medium from lignocellulosic biomass varied from 5 to 30mM [43] As a consequence of imbalance cofactor NADH, the enzyme ADH for specific ethanol production decreases rapidly in the presence of furfural

For metabolic activity, acetaldehyde has the trend to be converted into acetate by using cytosolic AlDH; however, in the presence of furfural, acetaldehyde accumulation is above 0.1mM, which is demonstrated to inhibit the AlDH [38] The availability of furfural in the medium do not lead to the acetate formation as well as the TAC cycle cannot produce enough energy ATP for cell growth; as a result, the yeast biomass decreases significantly [37]

2.3.2 Acetic acid

2.3.2.1 Introduction

Acetic acid is the primary product in the process of hemicellulose hydrolysis This weak acid is in undissociated form, diffuses across the plasma membrane and goes into the cytoplasm In the cytoplasm, the dissociation of acetic acid occurs due

to neutral intracellular pH, leading to reducing the internal pH of the cell, giving an acidified cytoplasm [44] The cytoplasm damage leads to inhibition of synthesis of

12] Since pH is one of crucial parameters of fermentation process, acetic acid toxicity is pH dependent

Figure 2 6: Chemical structure of acetic acid

2.3.2.2 Adaptive response of yeast to acetic acid

Recent report for acidic stress caused by acetic acid reveals that there is a reaction between AFT1P-regulated intracellular metal metabolism and resistance by applying genome-wide functional analysis and gene expression profiling [45] It highlighted the vacuolar acidification and the HOG1P pathway for acetic acid resistance at low pH More to the point, a sub-lethal growth inhibitory concentration

of acetic acid was demonstrated as promoting the phosphorylation of HOG1P and

SLT2P, two MAP kinases in Saccharomyces cerevisiae [46]

Figure 2 7: Mechanisms of acetic acid stress response in S cerevisiae cells [32]

Red arrows: adaption and acetate metabolic pathways Green arrows: acetic acid – program cell death pathways

The activation of HOG1P by acetic acid caused the subtraction of channel FPS1P from the plasma membrane and restricted the accumulation of the

protein-acid The transcription factor HAA1P was associated with resistance to acetic acid

in glucose medium, where the knockout mutant displayed an increased lag phase [47] Principally, this effect was attributed to the down regulation of genes coding for the plasma membrane multidrug transporters (TPO2 and TPO3) and for the cell wall glycoprotein (YGP1)

In the view of a proteomic analysis of S cerevisiae, cells treated with acetic acid

were discovered that proteins from amino-acid biosynthesis, transcription or translation machinery, carbohydrate metabolism, nucleotide biosynthesis, stress response, protein turnover and cell cycle were affected 28 transcription factors which were identified as required for acetic acid resistance were found to have the highest percentage of targets among the genes required for acetic acid tolerance The down regulated genes were associated with mitochondrial ribosomal proteins and with carbohydrate metabolism and regulation, whereas those related to arginine, histidine, and tryptophan metabolism were up regulated Data indicated that acetic acid disturbed mitochondrial functions at translation, electron transport chain and ATP production levels, interrupted reserve metabolism (glycogen and trehalose metabolism and glucan synthesis), and regulated the central carbon metabolism and amino acid biosynthesis in yeast [48]

2.3.2.3 Inhibition effects of acetic acid on yeast growth and ethanol fermentation

Acetic acid was not metabolized by glucose-repressed yeast cells and entered the cell in the non-dissociated form by simple diffusion Inside the cell, the acid dissociation was observed If the extracellular pH was lower than the intracellular

pH, this would lead to an intracellular acidification and to the accumulation of its dissociated form, affecting cellular metabolism at various levels [48, 49] Intracellular acidification caused by acetic acid led to trafficking defects, hampering vesicle exit from the endosome to the vacuole [48] Though acetic acid induced plasma membrane ATPase activation (50 mM, pH 3.5), this enhanced activity was

effects of the undissociated form of the acid also translate into an exponential inhibition of growth and fermentation rates [49]

In the studies on enzymatic activities, the glycolitic enzyme was affected by acetic acid, presumably resulting in a limitation of glycolysis [49] As revealed by the proteomic analysis of acetic acid-treated cells, carbohydrate metabolism was strongly affected, according to a decreased glycolytic rate

Yeast growth in batch cultures following cellular adaptation to acetic acid was linked not only with a decrease in the maximum specific growth rate and in the ATP yield, but also with a recovery in intracellular pH and an increase in the specific glucose consumption rate, indicating that metabolic energy was diverted from metabolism as well [50]

Using anaerobic chemostate cultures, it indicated that higher intracellular trehalose content induced by lower growth rate or by the presence of ethanol were

related to higher tolerance of S cerevisiae to acetic acid [51]

CHAPTER 3: MATERIALS AND METHODS

3.1 Materials

3.1.1 Kluyveromyces marxianus

Kluyveromyces marxianus used in this study was originated from the culture

collection of Food Microbiology Laboratory, Food Technology Department, School

of Chemical Engineering, HCM City – University of Technology

Cell immobilization medium

Ethanol fermentation medium

The initial pH of the all media was adjusted to 5.5 All medium were sterilized at

1210C, 1 atm for 20 min before use

Kinetics of ethanol formation

Kinetics of yeast growth Maximum biomass level

Growth rate

Residual sugar level Sugar assimilation rate

Ethanol level Ethanol formation rate Fatty acid composition

of yeast cell membrane Unsaturated degree

Kinetics of sugar assimilation Effect of acetic acid

on ethanol

fermentation by

Kinetics of yeast growth Maximum biomass level Growth rate

Residual sugar level Sugar assimilation rate

Ethanol level Ethanol formation rate Fatty acid composition

of yeast cell membrane

Unsaturated degree

Procedure of yeast immobilization

Inoculation prepararion:

- For the first step, pre-culture were inoculated from the agar slant and grown at

300C for 48 hour in 10 mL of growth medium

- For the second step, 10 mL pre-culture from the first step were used to inoculate

in 100mL of growth medium The inoculation conditions were similar to those

in the first step of the inoculation preparation

Carrier pretreatment:

- After harvesting, Nypa fruticans leaf sheath was cut into pieces (3 x 3 x 0.5) cm,

washed with water, then sterilized at 1210C, 1 atm for 20 min before use

Yeast immobilization:

- Yeast biomass was suspended in 150 mL of yeast immobilization medium in

500 mL flash and 10g of carrier was added The mixture was shaken at 150 rpm

Inoculum preparation

Centrifugation

Washing Sterilization

Separation

Liquid Supernatant

- Finally, the Nypa fruticans leaf sheath pieces with immobilized yeast were

separated, washed with the fermented medium and used for ethanol fermentation

3.2.2 Experiment design

Section 1: Effect of furfural on ethanol fermentation

Fermentation was performed in 500mL flashes containing 300mL of medium Furfural was added to the medium in ordered to achieve various concentrations 0, 1,

2, 3, 4 and 5 g/L The immobilized yeast was introduced into the medium with the initial inoculum size of 2×107 cell/mL Ethanol fermentation was carried out at 30°C The fermentation was lasted for 84h Control samples were also performed without furfural addition under the same conditions During fermentation, samples are taken at 12h intervals for analysis:

- Cell density in the medium

- Glucose concentration

- Ethanol concentration

At the beginning and the end of the fermentation, yeast biomass was sampled for analysis of fatty acid composition of cellular membrane

Section 2: Effect of acetic acid on the ethanol fermentation

Fermentation was performed in 500mL flashes containing 300mL of medium Acetic acid was added to the medium in ordered to achieve various concentrations 0,

2, 4, 6 and 8 g/L The immobilized yeast was introduced into the medium with the initial inoculum size of 2×107 cell/mL Ethanol fermentation was carried out at 30°C The fermentation was lasted for 84h Control samples were also performed without furfural addition under the same conditions During fermentation, samples are taken at 12h intervals for analysis:

- Cell density in the medium

- Glucose concentration

- Ethanol concentration

At the beginning and the end of the fermentation, yeast biomass was sampled for analysis of fatty acid composition of cellular membrane

3.2.3 Analytical methods

 Cell density in the yeast culture

For the immobilized yeast culture, carriers with the immobilized yeast were mixed with 100mL distill water and grounded in the blender at 3500 rpm for 5 min The suspension obtained was used for evaluation of the cell density by counting chamber [52] The result was calculated and expressed in number of cells per 1 mL

3,5-0, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL [53] Glucose concentration was expressed in g/L

 Fatty acid composition of yeast cell membrane

2g of the harvested yeast biomass was used for evaluation of fatty acid

lipid extraction was carried out by adding the chloroform and methanol (2:1 v/v), agitating at 200rpm for 2h, and following by 0.8% potassium chloride addition The mixture is then centrifuged at 250C, 3000 rpm for 5min The organic phase was then collected and used for determination of fatty acid composition [26, 54]

Fatty acid composition of yeast membrane was evaluated by gas chromatography using a Hewlett-Packard model 5890A (Hewlett - Packard, The United States) 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-1 and

h pt noi i m thyl st r (1 μg μ -1) 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-1 from 70 °C to

200 °C Peak areas were measured using a Hewlett-Packard model 3396A integrator

 Glucose consumption rate

(g/L.h) With:

Content of sugar assimilated by yeast during the fermentation (g/L)

Ethanol fermentation time (hours)

 Ethanol formation rate

(g/L.h)

With:

Content of ethanol produced by the yeast during the fermentation (g/L)

Ethanol fermentation time (hours)

 Glucose uptake efficiency:

The initial sugar concentration in the medium (g/L)

 Un-saturation degree of fatty acids in yeast cell membrane

Un-saturation degree of fatty acids in the yeast cell membrane is calculated from the fatty acid composition in cellular membrane using the following formula [54] Unsaturated degree = ( x1*1 + x2*2+……+xn*n)/100

With:

x1: percentage of fatty acid containing 1 double bond

x2: percentage of fatty acid containing 2 double bond

xn: percentage of fatty acid containing n double bond

3.2.5 Statistical analysis

All experiments were triplicated The value is not significantly different from the others at p<0.05 by using analysis of variance The results are expressed as means ± standard deviations Statistical analysis is performed with Stagraphic Centurion software