anahita deh khoda

8/12/2019 Anahita Deh Khoda

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Performed at: SEKAB E-Technol

Published by: Hgskolan i Bo

Anahita Dehkhoda

Concentrating lignocellulosichydrolysate by evaporation and

its fermentation by repeated fedbatch using flocculatingSaccharom ces cerevisiae


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MASTER THESIS IN INDUSTRIAL BIOTECHNOLOGY

Concentrating lignocellulosic hydrolysate byevaporation and its fermentation by repeated fed-

batch using flocculating Saccharomycescerevisiae

Anahita Dehkhoda

Performed at SEKAB E-Technologyrnskldsvik, Sweden

September 2007-March 2008

Supervisors:Dr. Tomas Brandberg, Prof. Mohammad Taherzadeh

This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Sciencewith a Major in Chemical Engineering, Industrial Biotechnology, 181 300 ECTS credits

No. 3/2008


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Title: Concentrating lignocellulosic hydrolysate by evaporation and its fermentation byrepeated fed-batch using flocculating Saccharomyces cerevisiae

Publication: Scientific paper / Submitted

AUTHOR:Anahita Dehkhoda

Master thesis

Series and Number Chemical engineering majoring in industrial biotechnology 3/2008

University College of BorsSchool of EngineeringSE-501 90 BORSTelephone +46 033 435 4640

Examiner: Prof. Mohammad Taherzadeh

Supervisors: Dr. Tomas Brandberg, Prof. Mohammad Taherzadeh

Client: SEKAB E-Technology, rnskoldsvik, Sweden

Keywords: Concentrating, lignocellulosic hydrolysate, evaporation, flocculatingeast, fermentation, ethanol.


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ACKNOWLEDGEMENT

The completion of this thesis project and my graduate education are indebted to thesupport of industry professionals and very experienced supervisors.

I am especially appreciative of Dr. Tomas Brandberg for choosing me and giving me theopportunity to do my diploma work in SEKAB E-Technology. Thanks for being patientwith me always and teaching me so much from your knowledge and never hesitating inrepeat yourself. Thanks for being like a friend to me, and helping me in everything fromgetting samples late at night to fixing disasters! You have been more than a supervisor tome, I will never forget you.

I would like to thanks to Prof. Mohammad Taherzadeh, first for informing your studentsabout this opportunity, second for your suggestions during the experiments which helpedus in improving them. Writing a scientific paper would not have been possible withoutthe considerable time and effort invested by you.

I take this opportunity to thank my assistant supervisor Annika Hgglund. Every time Ineeded help, you rushed to give me a hand and fix the sudden problems in lab, and thankyou for sharing your experiences with me. I want to extend special thanks to Torbjrnvan der Meulenfor giving me the opportunity to work at SEKAB.

Thanks to Carl-Axel Lalanderin helping with quick preparation of any equipment whichI ran out of. I would also like to recognizeStaffan Magnusson, Robert Selling, BirgittaLundgren and other people at MoRe Research for the processing and measurement ofsamples.

Thanks to my family for supporting me from such a far distance, and thanks to my fianc,whos presence and care kept me going. This work was supported by SEKAB E-Technology rnskoldsvik, Sweden and all experimental works was performed inlaboratory scales at SEKAB E-Technology.


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Master thesisIndustrial BiotechnologyBors University andSEKAB E-Technology, Sweden

ABSTRACT

In order to obtain a sugar concentration of more than 100g.l -1of fermentable sugars, aspruce wood hydrolysate was subjected to high pressure and vacuum concentration andthe fermentability of each hydrolysate was assessed by fermentation experiments withflocculating S. cerevisiae. The hypothesis that high pressure evaporated hydrolysate(evaporation carried out at 108C and 1.3 bar) would be more difficult to ferment than

vacuum evaporated hydrolysate (evaporation carried out at 80C and 0.5 bar) was notconfirmed by the results. Minor amount of cells lost their flocculating ability afterfermentation which their ratio and their viability and vitality was assessed.

By vacuum and high pressure concentration, the fermentable sugars (defined as theconcentration of glucose, mannose and galactose) in the hydrolysates reached to 120g.l -1

and 129g.l-1 respectively. Compared to the initial hydrolysate the concentration factorrepresented a 3-fold increase of fermentable sugars. Furfural was evaporated in both trialsand its concentration reached to 0.03g.l-1 and 0.1g.l-1 after vacuum and high pressureevaporation respectively. Fermentation with both 0.14h-1and 0.22h-1 initial dilution rateswas possible, while more than 96% of furfural and to less extent formic and acetic acids

disappeared from the hydrolyzates. However, HMF and levulinic acid remained in thehydrolyzates and concentrated proportionally. More than 84% of the fermentable sugarspresent in VEH were fermented by fed-batch cultivation using 12g.l-1 yeast and initialdilution rate (ID) of 0.22h-1, and resulted into 0.400.01g.g-1ethanol in 21h.

Fermentation of HPEH was as successful as VEH and resulted into more than 86% of thesugar consumption at the corresponding conditions. With an ID of 0.14h-1, more than97% of the total fermentable sugars were consumed, and ethanol yielded 0.440.01g.g-1.A viability and vitality determination from the supernatant of fermentation liquorrepresented that about 76% of the cells which lost their flocculating ability kept theirvitality. Cultivation of yeast with beet molasses was tricky in both batch and fed-batch

cultivation as the concentration more than 50g.l

-1

in batch cultivation prevent from yeastgrowing.

Keywords: concentrating, lignocellulosic hydrolysate, evaporation, flocculating yeast,ethanol, fermentation.


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PUBLICATION

The following scientific publication was prepared from this thesis work.

Anahita Dehkhoda, Tomas Brandberg and Mohammad J Taherzadeh.2008.Concentrating lignocellulosic hydrolyzate by evaporation and its fermentation byrepeated fed-batch using flocculating Saccharomyces cerevisiae (Submitted)


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CONTENTS

Chapter 1: Introduction..1

1.1 Background of ethanol production......................................................................11.2 Outline of the thesis .............................................. ..2

Chapter2: Bioethanol...3

2.1 Brief history of ethanol production..4

2.2 What is ethanol and where can be used?..................................................................4

2.3 Why ethanol as a fuel?.............................................................................................42.3.1 Environmental impact.........................................................52.3.2 Depletion of crude oil .............................................................52.3.3 Good Properties of fuel ethanol. ..................................................... 5

2.4 Disadvantages of ethanol.. ...............................................................6

2.5 Lignocelluloses materials, good sources for ethanol production.............................6

2.6 Characteristic of lignocellulosic materials...............................................................72.6.1 Celluloses............................................................72.6.2 Hemicelluloses................................................................72.6.3 Lignin..............................................................82.6.4 Extractive and ash...........................................................9

2.7 Pretreatment, first step for ethanol production ........................................................9

2.8 Hydrolysis......................................................10

2.8.1 Acid hydrolysis.....................................................102.8.2 Enzymatic hydrolysis............................................11

2.9 Inhibitors........................................................122.9.1 Organic acids. ...........................................................132.9.2 phenolic compounds .........................................................132.9.3 Furan compounds..........................................................13


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2.10 Inhibition control.. .......................................................14

2.11 Fermentation.. ..........................................................152.11.1 Fermentation of dilute acid hydrolysate .................................................... 15

2.11.2 Fermentation of enzymatic hydrolysyate (SSF and SHF) .........................16

2.12 Fermentation techniques......................................................162.12.1 Batch process.. ...........................................................162.12.2 Fed batch process...........................................................172.12.4 Continuous process........................................................17

2.13 Overall rocess of ethanol roduction from lignocellulosic materias..182.14 Fermentation s microorganism. .......................................................19

2.14.1 Yeast (Saccharomyces cerevisiae)............................................................192.14.1.1 Dissolved oxygen............................................................19

2.14.1.2 Carbon dioxide............................................................202.14.1.3 Hydrogen ion concentration........................................................202.14.1.4 Temperature ............................................................202.14.1.5 Required nutrients by yeast.........................................................212.14.1.6 Life cycle of Saccharomyces.cereviseae.....................................222.14.1.7 Metabolisms of S.cerevisise........................................................22

2.14.1.7.1 Glucose catabolism .................................................... 222.14.2 Bacteria.. ...........................................232.14.3 Filamentous fungi.. ................................................... 24

Chapter 3: Materials &methods ...253.1 Chemical and reagents...................................................253.2 preparation of dilute acid hydrolysate................................25

3.2.1 Initial hydrolysate... .............................................. 253.2.2 Concentrated hydrolysate..26

3.3 Yeast strain.............................................................27

3.4 Yeast start culture medium ............................................................27

3.5 Pre-culture..........................................................283.5.1 Beet moalsses, pre-culture nutrition .............................................................283.5.2 Batch cultivation of yeast..........................................................29

3.5.3 Fed-batch cultivation of Yeast....................................................30

3.6 Experiments methodology. ............................................................30


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3.7 Experiments Type 1 (A, B, C).. .........................................................313.7.1 Yeast cultivation . . ................................................... 313.7.2 Hydrolysate feeding......................................................313.7.3 Resting & airing........................................................31

3.8 Experiments Type 2 (D, E, F)....................................................32

3.9 Experiments Type 3 (G, E)........................................................32

3.10 Experiments Type 4 (I, J).....................................................33

3.11 Analysis........................................................343.11.1 Metabolic analysis. ........................................................343.11.2 Dry weight.. .......................................................343.11.3 Determination of cell vitality.........................................................343.11.4 Determination of cell viability.......................................................35

3.11.5 Calculations........................................................35

Chapter 4: Results ..36

4.1 Two types of evaporated hydrolysates....36

4.2 Cultivation in bioreactor.....37

4.3 Fed-batch fermentation with vacuum evaporated hydrolysate, Ex 1 (A, B, C)...38

4.3.1 Fermentable sugars (mannose, glucose, and galactose)....384.3.2 Ethanol, biomass and glycerol yield......40

4.4 Fed-batch fermentation with HPEH, Ex 2(A, B, C) ..414.4.1 Fermentable sugars (mannose, glucose, and galactose)....414.4.2 Ethanol, biomass and glycerol yield..43

4.5 Fed-batch fermentation with double biomass and high pressure evaporatedhydrolysate;Ex, type 3 (A, B) ...444.5.1 Fermentable sugars (mannose, glucose, and galactose)....444.5.2 Ethanol, biomass and glycerol yield..46

4.6 Fed-batch fermentation lower dilution rate, Ex 4 (A, B)...474.6.1 Fermentable sugars (mannose, glucose, and galactose)....474.6.2 Ethanol, biomass and glycerol yield47

4.7 Comparison of results49

4.8 Inhibitors....50


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4.9 Cell viability.51

4.10 Cell vitality.53

Chapter 5 (Discussion & Conclusion remarks)....54

5.1 Discussion................................................................................................................54

5.2 Conclusion...55

5.3 Future work..55

Appendix A....56

Appendix B....58

Nomenclature.....59

References..59


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LIST OF FIGURES

1. Cellulose structure...7

2.Hemicellulose structure...8

3. Monomers of lignin.9

4. Inhibitors scheme.. 15

5. Schematic picture of ethanol production...18

6. Fermentation process.23

7. Inoculums culture for yeast cultivation..27

8. Fermentor (Belach BR 0.4 bioreactor, AB Teknik, Solna, Sweden) ....29

9. Aerobic fed-batch cultivation process with molasses solution..30

10. Diagram of volume versus time. Yeast production (aerobic) lasted 48 hours, and then

the resulting yeast culture was used for (anaerobic) fermentation in two cycles, with 2

hours of aeration between them. The feed during the fermentation consisted in VEH and

HPEH.............31

11. Fed-batch fermentation with dilute- acid high pressure evaporated hydrlosate and

double amount of yeast at experiment 3 (A, B).33

12. Volume changes versus time in fed-batch fermentation with high pressure evaporated

hydrolysate with a regular amount of yeast and lower dilution rate..33

13. Glass tubes containing centrifuged yeast solutions for dry measurement...34

14. Concentration of glucose in experiment 1 (A - C). Fed-batch fermentation with VEH

by S. cerevisiea .39

15. Concentration of mannose in experiment 1 (A-C). Fed-batch fermentation with

vacuum evaporated hydrolysate by S. cerevisea3916. Concentration of mannose from experiments type1 (A-C). Fed-batch fermentation

with vacuum evaporated hydrolysate by S. cerevisea...39

17.Concentration of glucose in Experiment 2(A-C), with feed consisting of high pressure

evaporated hydrolysate..42


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18. Concentration of mannose in Experiment 2(A-C), with feed consisting of high

pressure evaporated hydrolysate42

19. Concentration of galactose in Experiment 2(A-C), with feed consisting of high

pressure evaporated hydrolysate42

20. Glucose concentration during fermentation with higher (32g.l 1 ) initial yeast

concentration with feed consisting in HPEH.... 45

21. Mannose concentration during fermentation with higher (32g.l 1 ) initial yeast

concentration with feed consisting in HPEH.... 45

22. Galactose concentration during fermentation with higher (32g.l 1 ) initial yeast

concentration with feed consisting in HPEH.45

23. Glucose concentration during fermentation with lower dilution rate and feed

consisting of HPEH....47

24. Mannose concentration during fermentation with lower dilution rate and feed

consisting of HPEH... 47

25. Galactose concentration during fermentation with lower dilution rate and feed

consisting of HPEH47

26.Ethanol, biomss, glycerol yielde comparison .....49

27. Sample of CFU measurement with colonies52

28. Vitality determination of samples stained by methylen blue and analyzed by light

microscope. ...53

29. HMF, formic acid, levulinic acid, and acetic acid concentration in experiment 4(A, B)

with lower dilution rate + regular amount of yeast and HPEH.....56

30. HMF, formic acid, levulinic acid, acetic acid concentration in experiment 3(A, B)

with double amount of yeast and HPEH56

31.HMF, formic acid, levulinic acid, acetic acid concentration in experiment 2 (A, B)

with regular amount of yeast and HPEH...5732. HMF, formic acid, levulinic acid, acetic acid concentration in experiment 1(A, B, C)

with regular amount of yeast and VEH..57


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LIST OF TABLES

1. Production of ethanol in world based on billion gallons per year...4

2. Hardwood and softwood composition.. ..6

3. Comparison between dilute-acid and concentrated acid hydrolysis .........11

4. Comparison of enzymatic and acid hydrolysis. 12

5.Composition of beet molasses...28

9.Comparison of dilute-acid spruce hydrolysate after and before evaporation37

10. Yeast cultivation results up to the start-culture from experiments with VEH and

HPEH (The numbers are averages from three experiments).37

11. Average consumption of fermentable sugars in each stage of three experiments...38

12.Yield of ethanol, biomass, and glycerol per g of consumed sugar at the end of each

experiment..40

13. Average glucose, mannose, galactose consumption from three experiments in

fermentation with high pressure evaporated hydrolysate..41

14. Yields of ethanol, biomass, and glycerol per g of added hexoses during both

fermentation cycles....4315. Average sugars consumption from two experiments in fermentation with high

pressure evaporated hydrolysate+ double amount of yeast...44

16. Yields of ethanol, biomass, and glycerol per g of consumed hexoses at the end of

each experiment (Added sugars)46

17. Average glucose, mannose, galactose consumption from two experiments in

fermentation with HPEH and lower dilution rate..46

18. Fraction of ethanol, biomass, and glycerol per g of consumed sugar at the end of each

experiment..48

19. Comparison of important results in experiment (1-4)..49

20. Inhibitors concentration in vacuum and high pressure concentrated hydrolysate,

before fermentation50


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21.Inhibitors conversion rate after fermentation in Experiments 1-4. For experiments1-3,

Samples were taken during the two cycles of fermentation, at the end of each stage. For

experiment 4, more samples were taken even in the middle of stages..50

22.Results from dry weight and CFU measurements for experiments 1- 4..51

23.Performance condition of experiments58


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CHAPTER 1

Introduction

1.1 Background of ethanol production

Ethanol from renewable resources has been of interest in recent decades as an alternativefuel or oxygenated additive to the current fossil fuels. Its market grew from less than abillion liter in 1975 to more than 39 billion liters in 2006 and is expected to reach 100billion liters in 2015 (Licht et al, 2006). Fuel ethanol contributes relatively little to netcarbon dioxide emissions to the atmosphere (Bergeron et al, 1989). Besides, depletion ofcrude oil in the near future makes bioethanol an important fuel, not least because it iseasily used as an additive to gasoline.

Lignocellulosicmaterial is renewable and abundantly available for the production of fuelethanol. It can be obtained at low cost from a variety of resources, e.g. forest residues,municipal waste, paper, and crop residue recourses (Wyman, 1996).

Acid or enzymatic hydrolysis of lignocellulosic material can be used to convert celluloseand hemicellulose to monomeric sugars. These fermentable sugars can be anaerobicallyconverted into ethanol by microorganisms. A number of by-products are however formed

during the hydrolysis, and these compounds, e.g. furfural, hydroxymethylfurfural mayinhibit yeast metabolism (Larsson et al, 1999b; Taherzadeh et al, 1997b; Sanchez andBautista, 1988; Chung and Lee, 1985; Banerjee et al, 1976). The Inhibition of theseinhibitors can be avoided either by different detoxification methods prior to fermentation(e.g. by overliming) or by in situ detoxification by yeast.

Chemical detoxification has, however, its drawbacks. Overliming can be performedefficiently at low cost, but is known to cause sugar loses(Martinez et al, 2000). In situ


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detoxification was applied to develop a fed-batch process for cultivation of severelyinhibiting hydrolysates (Taherzadeh et al, 2000; Taherzadeh et al, 1999). Although thismethod has been proved potentially successful it has showed a drastic decrease in cellviability as soon as the feed rate or toxicity of the hydrolysate was increased (Nilsson, etal, 2001). Nevertheless, the effectiveness of a detoxification method depends on the types

of the hemicellulose hydrlysate because each type of hydrolysate has a different degree oftoxicity (Larsson et al, 1999).

An increase at the initial hydrolysate sugar concentration provides an increased ethanolconcentration which has a major effect on the energy demand; especially atconcentrations below 4 wt % of ethanol. The increased ethanol concentration in the feedto the distillation reduces the production cost considerably.

Thus, more than 100g.l 1 sugar concentration is needed for industrial ethanologenicfermentation, since huge costs are associated with equipment required for transportationand storage of large volume of water and costs for ethanol recovery. Increasing the sugar

concentration in the water-soluble fraction can be achieved either by evaporation of wateror less addition of water to the hydrolysis process. The dramatic evaporation (by a factor3) that was performed in this work is not industrially relevant, but should partly be seenas a simulation of a hydrolysis process where less water is added. Also, some evaporationmay be used on industrial scale, but the comparison of vacuum evaporation and highpressure evaporation is important.

1.2 Outline of the thesis

The objective of current work was to assess the fermentability of evaporatedhemicelluloses hydrolysate rich in fermentable sugars and suitable in fermentationprocess without performing any chemical detoxifications also making a comparisonbetween of the fermentation results from these two hydrolysates which performed by FYS. cereviseae in a fed-batch mode. To attain this objective the sugar concentration inhysrolysate was brought to more than 100g.l 1 by vacuum and high pressureconcentrating methods in order to reach to more than 4% ethanol in fermentation broth inan industrial scale.

Chapter two is about the background of ethanol production; later on chapter three is thematerials and method of experiments and chapter four includes the results, and at lastchapter five is about the discussion and main conclusion remarks.


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CHAPTER2

Bioethanol

2.1 Brief History of ethanol production

Production of alcoholic beverages is in fact as old as human civilization; the productionof pure ethanol apparently begins in the 12-14th century along with improvement ofdistillation. During the middle Ages, alcohol was used mainly for production of medicaldrugs but also for the manufacture of painting pigments. The knowledge of using starchymaterials for ethanol production was first employed in the 12 th century in typical beermaking countries like Ireland. It was only in the 19 th century that this trade became anindustry with enormous production figures due to the economic improvements of thedistilling process (Roehr et al, 2000).Alcoholic beverages still represent a large portionof industrial alcohol, but other applications are becoming more important. Alcohol can beused for various purposes in the chemical industry and its role as a fuel is well known.

It was at the beginning of the 20thcentury that it had become known that alcohol might beused as fuel for various combustion engines, especially for automobiles. However,production of large amounts of alcohol requires large amounts of sugars, which tends tolimit the production. Still, during the 20th century various processes were developed,based on for instance sugar cane, beet molasses and industrial by products. Obviously,the ethanol industry is usually in close connection with agricultural production (Roehr etal, 2000).

Today, a dramatic expansion of bioethanol production takes place. In countries like theUnited States a big effort is being made in the construction of large scale production forfuel ethanol. Using primarily starch from enzynatically hydrolyzed corns as raw material,the United States has the second largest bioethanol program.


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In Brazil, the proacool program was launched in the 1970s as a response to the oilcrisis. While ethanol is used as a fuel additive in the US it is frequently used as thedominating fuel component in Brazil (Roehr et al, 2000).

Table 1. Annual production of ethanol in world based on m

3

(www.ethanolrfa.org/industry/statistics).

Country 2004 2005 2006

USA 13362300 15776800 18074500

Brazil 15078420 15639900 16616700

China 3643920 3795120 3844260

India 1746360 1697220 1897560

France 827820 907200 948780

Russia 748440 748440 646380

2.2 What is ethanol and where can be used?

Ethanol (C2H5OH) or ethyl alcohol is an important organic chemical which can be used

in various ways. It is colorless, flammable, volatile and soluble in water and non-polar. Itis an energy dense fuel, which can burn easily. It is widely used as a fuel, but is also usedfor other industrial purposes or as an ingredient in beverages.

It can be mixed with gasoline or can be used as the dominating component in flexi fuelengines. All traditional gasoline engines can be fuelled with a mixture of at least 10%ethanol (Ramanthan, 1988). The share of ethanol in mixture of fuel is usually referred toE, combined with a subscript that indicates the percentage of alcohol in the liquid. Forexample a mixture which contains 10 % ethanol is denoted E10.

2.3 Why ethanol as a fuel?

In recent years the production of fuel ethanol has expanded dramatically. Thisdevelopment is driven by a combination of economical and political mechanisms.


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2.3.1 Environmental impact

2CO is a socalled greenhouse gas, trapping thermal radiation which would otherwiseradiate into space and thus warming up the lower parts of the atmosphere (Dickenson andCiceron, 1986). During the last 150 years the 2CO content in the atmosphere hasincreased by about 30%. This has coincided with an increase in the average temperatureof the earth which has been partially rapid during the last 30 years. In the 50 years thiscould lead to an increase in the average temperature as much as 5C (Dickenson andCiceron, 1986).

A major part of the increase in the atmospheric 2CO is most likely due to the utilizationof fossil fuels and in contrast combustion of biofuels such as biogas and bioethanol donot increase the atmospheric 2CO content (Wingren et al, 2003) for instance emission of

2CO decreases by 20% for E10 (10% ethanol, 90% gasoline), but higher ethanol blenddont give further decrease. For E10, the emission of NOx increases by 30% for E85 and

E95 the emission decrease by 20%.(Delmer and Amor, 1995; Galbe and Zacchi, 2002)

2.3.2 Depletion of crude oil

Relatively soon the production of crude oil will no longer be able to meet the increasingdemand. While energy demand is predicted to increase constantly, the supply of crudeoil will start to decline after a peak that according to the different sources will occurbetween 2009 and 2020. Therefore, another source of fuel must be investigated, andbioethanol can perhaps be an appropriate substitute (Salameh, 2003).

2.3.3 Good Properties of fuel ethanol

Ethanol is relatively high oxygenated and during combustion reduces the emission of

2CO by 32.5% and non- combusted hydrocarbons by 14.5%. It has a lower flametemperature and larger gas production per energy unit of fuel combusted than gasoline. Itis an energy densed, easily burned fuel.

Ethanol has a high octane number about 112, which makes it suitable as an octanenumber enhancing additive (Baired, 1999); Due to the low octane number of gasoline it isnecessary to increase the octane number with additives. Ethanol can reduces the need fortoxic octane raising additives such as MTBE (Dickenson and Ciceron, 1986) which has

been shown to be toxic to the environment and it is therefore advantageous to avoid it(Bonjar, 2004).


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2.4 Disadvantages of ethanol

There are some concerns with fuel ethanol that have to be addressed. Ethanol increasesthe vapor pressure of the ethanol-gasoline mixture, potentially causing more evaporativeemissions, furthermore the use of ethanol in combustion engines can increase the

acetaldehyde and formaldehyde emissions, and as the energy density in ethanol is lowerthan in gasoline then 25-35 more ethanol compared to gasoline is needed to drive thesame distances(Wyman, 1996),another disadvantage is that, the catalysts which are usednowadays are optimized for petroleum based fuels(Wheals et al, 1999; Zaldivar et al2001; Galbe and Zacchi, 2002).

2.5 Lignocelluloses materials, good sources forethanol production

Lignocellulose (wood, grasses and municipal solid waste) is an attractive feedstock for

ethanol production because of its availability at low cost and at large quantities. Approximately 50% of the biomass in the world is lignocelluloses and it has an estimatedannual production of 10-50.10kg (Classen, 1999). There are many types oflignocellulosic materials that can be served as feed stocks for ethanol production likeAgricultural residues, municipal and industrial waste materials, papers and forestry byproducts, trees, grasses.

The dominant sources of lignocellulic materials in northern hemisphere are softwoodssuch as pine and spruce, softwood is an object of interest in Sweden, Canada and westernUnited States as renewable resources for ethanol production, because it is cheaper thanhardwood (Galbe et al, 2005). Besides its content of pentose-rich hemicelluloses is

significantly lower than in hardwood. This is advantageous, since important fermentingorganisms (such as native strains of S. cerevisiae) dont consume pentose.

Table 2. Hardwood and softwood composition.

HemicelluloseMaterial

Cellulose%

Hemicellulose%

Lignin%

Hardwood 45-51 23-28 19-24

Softwood 41-42 24-31 29-31


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2.6 Characteristic of lignocellulosic materials

Lignocelluloses consist mainly of cellulose, hemicellulose and lignin; these componentsbuild up about 90% of dry matter in lignocelluloses, with the rest consisting of .e g,extractive and ash.

2.6.1 Celluloses

Cellulose is the major component in the cell wall of living plant cells. The most obviouspurpose of cellulose is to provide strength to the plant structure. It is a linearhomopolymer of anhydroglucose units linked by (1-4) glycosides bonds, its basicrepeating units is disaccharide cellebiose (Delmar and Amor, 1995). The length ofmacromolecules varies greatly as to the source and degree of processing (DP) that it hasundergone. Newsprint, e.g., exhibits an average DP of about 1000 while cotton is foundto have a DP of approximately 10,000 (Roehr, 2000).

Its not the primary structure which makes cellulose a hydrolysis resistant molecule. Itrather seems to be the effect of secondary and tertiary configuration of the cellulose chainas well as its close association with other protective polymeric structure within the plantcell wall such as lignin, starch, pectin, hemicelluloses, proteins and mineral elements.The easily and quickly hydrolyzed degraded regions in cellulose structure are amorphousin nature and difficult parts are crystalline parts (Roehr, 2000).

Fig. 1.Cellulose structure(www.troy.k12.ny.us).

2.6.2 Hemicelluloses

Hemicellulose is a highly branched, low molecular weight heteropolymers of D-galactoses, Dglucose, and D-mannose, D-xyloses, L- arabinoses and various otherSugars as well as their uronic acid. It is bound covalently to lignin and through hydrogenbonds to cellulose (Sjstrm et al, 1993).


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Fig. 3. Monomers of lignin (www.emeraldinsight.com).

2.6.4 Extractive and ash

Extractives are noncell-wall materials which can be extracted by specific organicsolvent and can be divided into wood resin and phenolics extractives. Phenolicsextractives can be found at the inner part of the wood as well as in the bark, whereas resinis found in resin channel and pockets. Wood resin comprises fat and fatty acids, stroles,esters, terpenoids. The phenolic compounds represent lignans, flavonoids, tannins,stilbenes. Terepenoids and resin acids and phenolic substrates protect wood againstmicrobial damage and insect attack (Sjstrm et al, 1993). These compounds may beliberated during pretreatment of lignocelluloses and can be inhibitory to microorganisms

despite their low quantities.

2.7 Pretreatment, first step for ethanol production

The aim of pretreatment is to hydrolyze the hemicelluloses to monomer sugars andmaking the cellulose accessible to enzymatic attack.

Due to the structure of softwood, mild pretreatment conditions will not be sufficient toachieve a high overall sugar yield. This implies that harsher conditions are required in thepretreatment steps. The improved carbon recovery can be obtained by a two stagepretreatment, at the initial step a major part of the hemicelluloses is degraded and after

removal of the hemicelluloses hydrolysate the residual material is exposed to more severecondition.

A number of physical and/or chemical methods can be used to separate celluloses fromits sheath of lignin and increase the surface area of the cellulose crystallite by sizereduction and swelling. The pretreatment methods include milling, steam explosion, useof solvent, swelling agents, lignin consuming microorganisms, but the most investigatedand commonly used pretreatment method is called steam-explosion (steam pretreatment)


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with or without acid catalyst (Clark et al, 1989; Kaar et al., 1998; San et al, 1995; Scultzet al, 1983; Tanahashi et al, 1988).

2.8 Hydrolysis

The aim of the hydrolysis is cleaving the polymers of celluloses and hemicelluloses tomonomeric sugars which can be fermented to ethanol by microorganisms. In ethanolproduction industry the process of hydrolysis is very complicated, depending on severalparameters such as properties of the substrate, acidity, and rate of decomposition of theproducts during hydrolysis (Taherzadeh and Karimi, 2007). The hydrolysis can be carriedout either chemically or by a combined chemical and enzymatic treatment. Acids arepredominantly applied in chemical hydrolysis and Sulphuric acid is the most investigatedone, although other acid such as HCL have been used too.

2.8.1Acid hydrolysis

The solubility of cellulose in acid has been detected already in 1815. The first industrialprocess however was developed in 1942 and run in Italy (Roehr, 2000). The acidhydrolysis can be performed by high acid concentration at a low temperature or that oflow concentration at a high temperature(Lee et al, 1999).

2.8.1.1 Concentrated acid

In 1819 first discovered that cellulose can be converted to fermentable sugars byconcentrated acids. The concentrated acid process is a quiet old process normallyinvolving concentrated sulphoric or hydrochloric acid. Concentrated acid processes areoften reported to give higher sugar yield and consequently higher ethanol yield,

compared to dilute-acid processes. Furthermore it can be operated at low temperature(e.g. 40 C), however severe problems with corrosion of hydrolysis equipment renderhigh investment cost, and also the recovery of the acid are expensive and difficult (Jonesand Semarau, 1984).

2.8.1.2 Dilute acid

Dilute acid is an old method and it was operated during the World War II in Germany inthe former Soviet Union, Japan, Brazil and the USA.Compared to a concentrated acidprocess a dilute acid process will consume much less acid, however high temperaturerequired often lead to corrosion problems and sugar degradation, resulting in lower sugar

yield and inhibition of the fermentation, but this problem can be solved by a two stageprocess, in which the hemicellulose is mainly hydrolysed in the initial step at temperature150-190 C and the remaining cellulose subsequently hydrolysed at more severeconditions at 90-230 C (Faith , 1945).


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Table 3.Comparison between dilute-acid and concentrated acid hydrolysis (Taherzadeh

and Karimi, 2007).

Hydrolysis method Advantages Disadvantages

Concentrated acidProcess

-operated at lowtemperature- High sugar yield

-high acid consumption-high energy consumptionfor acid recovery-longer reaction time (e.g.2-6h)-equipment corrosion

Dilute acidProcess

-low acid consumption-short residence time

- operated at hightemperature-low sugar yield

-equipment corrosion

2.8.2 Enzymatichydrolysis

Enzymes can be used to cleave the cellulose and hemicellulose polymers at lowtemperature into simpler sugars. Enzymes with the ability of degrading these polymersare collectively called cellulases(Galbe and Zacchi, 2002).

Cellulases are microbial enzymes capable of cellulose hydrolysis that are in reality anumber of several different synergistic components. They are induced enzymes andproduced only when the organism is grown in the presence of cellulose, cellobiose andlactose or other glucane which contain -(1-4) linkages. Denaturation by shearing is acommon drawback of cellulose enzymes (Roehr, 2000).

Cellulases are produced by many genus of fungi e.g. Thricoderma, Penicillum andAspergillus. Thrichoderma is the most investigated fungi for production of complexmixtures of cellullases that are specialized in breaking -(1-4) glucosidic bonds. Theenzymatic hydrolysis step (in combination with pretreatment) results in higher sugaryields than dilute acid hydrolysis, since the enzymes catalyze only sugar generation andnot sugar degradation. The enzyme mixture consists of exoglucanases, endoglucanasesand - glucosidases (Gusakov and Sinitsyn, 1992).


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Table 4.Comparison of enzymatic and acid hydrolysis (Roehr, 2000).

Acid Enzyme

1. Non-specific catalyst therefore will

delignify material as well as hydrolyzecellulose.

Specific macromolecule catalyst, therefore

extensive physical and chemicalpretreatment is necessary to makecellulose available for degradation.

2.Decomposition of hemicellulose toinhibitory compounds

production of clear sugar syrup ready forsubsequent anaerobic fermentation

3.Harsh reaction condition thereforenecessary increased costs for heat andcorrosion resistant equipment

Run under mild conditions(50,atmospheric pressure ,pH4,8)

4.Relatively low yield of glucose High glucose yield

2.9 Inhibitors (by-products released during hydrolysis)

One of the factors that complicate the fermentation of lignocellulosic hydrolysates is theformation of a large number of organic compounds, some of which are inhibitory to theyeast during the pretreatment or dilutes acid hydrolysate of hemicellulose, cellulose andlignin, which inhibits cell growth and fermentation.

The reason for inhibitors formation can be as follows: Inhibitors may be present in the material as such as simply released during the

pretreatment/hydrolysis. Phenolic compounds originating from the lignin can bean example of this category.

Inhibitors may be present as side groups on the heteropolymers and maybecleaved off during hydrolysis processes. Acetic acid originating from acetylatedhemicellulose and also phenolic compounds are examples of inhibitors that arecleaved off during the pretreatment and.

Inhibitors may be formed in carbohydrate degradation; furfural and HMF are twoexamples of carbohydrate degradation.


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The by products which produced during the hydrolysis can be divided to: Organic acids,furan compounds, phenolic compounds.

2.9.1 Organicacids

A large number of alphatic acids are present in dilute acid hydrolysates originated from

wood extractives, lignin degradation and sugar degradation. Acetic acid is a major acidconstituent in hydrolysates and is mainly produced from degradation of the acetyl groupin the polysaccharides whereas levulinic acids and formic acids are products of sugardegradation. Levulinic formed from HMF and formic formed from both HMF andfurfural, but their concentration is relatively low after moderate hydrolysis conditions.

It is proved that the toxicity of acetic acid is pH dependent (Gottschalk, 1987).Concentration above of 5g.l 1 is toxic to S. cerevisieas this undisssociated form of it candiffuse inside of the cell and affect the intracellular pH, but also it has shown that theconcentration of 3.3g.l 1 of undissociated acetic acid resulted in an increase in ethanolyield by 20%(Taherzadeh et al, 19997b),but in model fermentation with these three

acids, it has been shown that the ethanol yield and volumetric productivity decreased withincreasing concentration of acetic acid, formic acid and levulenic acid (Larsson et al.,1999a).

2.9.2Phenolic compounds

phenolic compounds which have been recognized in lignocellulosic hydrolysates,include: 3-methoxy-hydroxybenzaldehyde, acetovanilone, 4-hydroxyacetophenone,ferulic acid, vaniline, syringaldehyde, vanilic acid and 4-hydroxybenzoic acid .Thesecompounds are mainly liberated from lignin degradation in addition to aromaticextractives. Phenolic compounds are considered to be important inhibitors due to their

inhibitory effect in fermentation of lignocellulosic hydrolysates(Nicholson, 2000)

It has been shown that low molecular weight phenolics which derived from lignin canpotentially be inhibiting to S. cereviseae and limit the cell growth (Clark et al, 1984;Larsson et al, 1999b). Furthermore, 4-hydroxybenzoic acid about 1g.l 1 has beenreported to cause a 30% decrease in ethanol yield compared to reference fermentation,vanillin which constitutes a large fraction of the phenolic compounds in hydrolyate ofspruce, has been found less toxic than 4-hydroxybenzoic acid.

Inhibition of fermentation has been shown to decrease when phenolic monomers andphenolic acids were specially removed from a willow hydrolysate (Jnsson et al., 1998)

and 4-hydroxybenzoic acid, vanillin and catecol were the major constituent in theuntreated hydrolysatefrom spruce (Palmqvist et al, 1999).

2.9.3Furan compounds

Furan compounds, in this context, include furfural and 5-hydoxymethyl furfural. Duringthe hydrolysis pentose yield furfural and hexoses yield HMF. Furfural has been reportedto be a strong inhibitor for S. cerevisiae (Taherzadeh et al., 1999). The furfural


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concentration above 1g.l 1 was found to decrease significantly the 2CO evolution rateand the cell multiplication also the total viable cell number in the early phase offermentation (Azhar et al., 1981; Banerjee et al., 1981; Boyer et al., 1992; Chung andLee, 1985; Sanchez and Bautista, 1988).

During anaerobic fermentation, furfural is reduced to furfuryl alcohol, while furoic acid isproduced from oxidation of furfural during aerobic cultivation. HMF has the similarinhibitory effect as furfural, except that it has a lower conversion rate that it can bebecause of its lower membrane permeability. An addition of 4g.l 1 of HMF decrease the

2CO evolution rate about 32%, ethanol production rate 40%, specific growth rate 70 %.However this inhibitory effect is less than caused by the same amount of furfural,therefore HMF can not be considered as toxic as furfural for growth and fermentation ofS. cereviseae. It belongs to the picture that since S. cerevisiae has an in situdetoxification mechanism for both furfural and HMF, these compounds are lessinhibitory if they are supplied continuously at a rate that is lower than the detoxificationrate.

2.10 Inhibitioncontrol

Pretreatment employing chemicals and high temperatures result in the generation of awide range of by-products (Larsson et al, 1999). Several methods for detoxification areknown, but most are difficult to apply on industrial scale.

A well characterized method, however, is overliming with calcium hydroxide followedby removal of precipitant by filtration or centrifugation, which decreases the toxicity ofthe hydrolysate. Overliming has also been with bases like potassium hydroxide, sodiumhydroxide or ammonia and it was shown that calcium hydroxide or ammonia increased

the fermentability more than other compounds.

The mechanism behind the technique is not fully elucidated but it has been suggested thattoxic compounds are precipitated during this operation. It has also been observed that theconcentration of furans and phenolic compounds decreases. Generally, extendeddetoxification periods and higher pH allow for better fermentability of the hydrolysate,but this tends to lower the ethanol yield due to losses of sugar during the detoxificationprocess.In addition, treatment with calcium hydroxide would create a large quantity of auseless solid-by product that may even damage the equipment.

On balance, the most cost efficient (and simple) method to deal with inhibitors is

probably to use a continuous fermentation strategy and adapt the feed-rate to the in situdetoxification capacity of the fermenting organism. It belongs to the picture that somestrains are significantly more tolerant than others. (Personal communication, TomasBrandberg) This is in principle the approach that was used in this project.


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Fig. 4. Inhibitors scheme (Taherzadeh and Karimi, 2007).

2.11Fermentation

During fermentation monomeric sugars released in the hydrolysis are converted into thedesired product, by a microorganism, these microorganisms could be yeasts or bacteria.Anaerobic production of ethanol is a typical example of fermentation. The metabolicbasis for the conversion is the desire of the microorganism to use the sugars as carbonand energy source in order to maintain viability and growth. The saccharid releasedduring hemicelluloses and celluloses degradation have to be fermented to ethanol byyeast or bacteria.

2.11.1Fermentation of dilute acid hydrolysate

Dilute acid hydrolysis is a relatively cheap, fast and straightforward method forhydrolysis of lignocellulosic materials. However during the dilute acid hydrolysis manycompounds are formed, besides of the desirable monosaccharide, several of thesecompounds inhibit the microbial fermentation of the sugar to ethanol, but there areoptions to reduce the inhibition of fermentation like using of high cell biomass,decreasing the feeding rate or using a robust strain of yeast. From an industrial

Hemicellulose acetic acid(11-37%) pentoses furfural

Formicacid

hexoses HMF

Formic acidLevulinic acid

Lignin phenolic compounds(17-32%)

Cellulose(32-54%)

Glucose

HMF

Formic acid

Levulinic

Extractives (1-5%) phenolic compoundsand wood resin

Ash(0-2%) various inorganic

Compounds


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perspective, the fermentation can be carried out in different ways, to a large extentdepending on the hydrolysis method.

2.11.2Fermentation of enzymatic hydrolysyate (SSF and SHF)

The fermentation in a process based on enzymatic hydrolysis can be carried out in twoways: separate hydrolysis and fermentation (SHF) or simultaneous saccharification andfermentation (SSF).

There are two main advantages with SHF. Hydrolysis and fermentation have differenttemperature optima and the yeast can be recycled since the sugar solution can be filteredprior to fermentation. A problem, however, is that the sugar decreases the efficient of theenzyme due to product inhibition.

The main advantage with SSF is that sugar inhibition is avoided, since the fermentingorganism is mixed with the enzyme and the slurry. Disadvantages associated with SSF

are mixing/cooling problems; the optimal temperature for fermentation is approximately30 C, while for hydrolysis it is about 50 C, thus SSF must be operated at intermediatetemperature, and that the fermenting organism cannot be recycled.

2.12 Fermentation techniques

Ethanol can be produced by applying mainly four types of operations at industry: batch,continuous, fed batch and semi continuous (Balesteros et al, 1992).

2.12.1Batch process

In batch process substrate and separately grown cells slurry are charged into thebioreactor together with nutrients and enzymes required. Generally batch fermentation ischaracterized by low productivity, and it is labor-intensive. When a single cell likeSaccharomycesstrain is grown in a submerged culture, a plot of the logarithm of the dryweight of cells produced against time, gives characteristic curve dependent on strain andenvironmental condition. A typical growth curve composes of three distinct stages: (A)lag phase, (B) exponential and (C) stationary phase (D) zero growth (Tuite and Oliver,1991).

A lag phase represents the time period between inoculation of the culture with the

organism and a measurable increase in cell concentration, during this time cells areadapting with their new environment. The lag phase can be shortened by using a largeinoculums or an inoculums culture that is already growing exponentially under similarcondition. If the culture medium is near the optimum temperature for the yeast growthand contains all the essential nutrients requirements for the yeast, this will also decreasethe apparent lag phase(Tuite and Oliver, 1991). The exponential phase is the time periodduring which the specific growth rate () is constant and it is at a maximum ( max) for


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the given strain and the environmental conditions (Tuite and Oliver, 1991), and then azero growth period appear which is called stationary phase.

2.12.2Fed batch process

A fed batch process can be regarded as a combination of batch and continuousoperations; it is a very popular type of process in ethanol industry. In this operation feedsolution which contains substrate yeast culture, important minerals and vitamins are fedat constant intervals while effluent is removed discontinuously (Roehr, 2000).

The start up of fed-batch operation is similar to the batch process start-up. Subsequentlysubstrate fed into the bioreactor in a specified manner, after the growth limiting substrate(generally carbon source) which is given at the beginning of the process has beenconsumed. The concentration of substrate must be kept constant in the reactor while thefeeding is made, in this way the substrate inhibition can be kept at a minimum level infed-batch process by adding substrate at the same rate at which it is consumed. Substrate

concentration can be measured and feed controlled accordingly so the level can be keptlow. The substrate consumption rate can be calculated from measured factors such ascarbon dioxide (Roehr, 2000).

The basic concept behind the success of this technique is the capability of in situdetoxification by the cells. Since the yeasts have a limited capacity for the conversion ofthe inhibitors, the achievement of a successful fermentation strongly depends on the feedrate of the hydrolysate. At too high feed rate, using an inhibiting hydrolysate, bothethanol production and cell growth can be expected to stop whereas at a very low feedrate the hydrolyzate may still be converted but at a very low productivity, which it wasexperimentally confirmed (Taherzadeh et al., 1999).

2.12.3Continuous process

In a traditional continuous cultivation, nutrients are continuously supplied to thebioreactor and a product stream is continuously withdrawn at the same rate as the supply,resulting in a constant volume. In principle, continuous cultivations are efficient in termsof productivity per volume unit, but they are also sensitive to infections.

Since cells are continuously being washed out of the bioreactor, there must be a cellgrowth that corresponds to the dilution rate, otherwise washout occurs. This problem canbe circumvented by the use of cell retention (recirculation or immobilization), but there

must be at least some production of new cells, otherwise the culture will age and lose itsfermentative capacity (Brandberg, 2005).


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2.13 Overall process of ethanol production fromlignocellulosic material

A generally simplified of ethanol production process from lignocellulosic materials isshown in Fig 5. The lignocellulosic materials initially are milled for size reduction andthen are hydrolyzed to obtain fermentable sugars, several by-products may be released inthis stage, if it is highly toxic a detoxification step is necessary prior to fermentation. Thehydrolysates are then fermented to ethanol in the bioreactors. Ethanol gets distilled at the

end and if fuel ethanol is desired then further dehydration to 99% must be performed bymolecular sieves. Ethanol normally required about 95% wt/vol, which can be achieved bydistillation. Very pure ethanol can be obtained by extractive distillation and azetreopiccolumns and can be concluded in multiple-column stills for absolute alcohol Fig. 5(Taherzadeh and Karimi, 2007).

Fig. 5.Schematic picture of ethanol production.


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2.14Fermentation s microorganism

There are a number of microorganisms that are able to produce ethanol. Among of themthere are several types of bacteria, yeasts and filamentous fungi. The specific organismshave some advantages and disadvantages that will be discussed below.

2.14.1Yeast (Saccharomyces cerevisiae)

S. cerevisie is one of more than 1000 validated yeast species belonging to the fungikingdom. It is a unicellular eukaryote. Species of the genus Saccharomycesspecialized ingrowing on sugars. Typically yeast can be found on fruits, plants also in soil and inseawater (Rose and Harisson, 1993). Yeast cells are round to oval and they have adiameter about 5-10m, most yeasts are reproduced by budding, the maximum number ofbud scars found on growing cells is around 25, and the doubling time of cells can bearound 90 min in a favorable growth environment. S. cerevisie is also a facultative

anaerobe; i.e. it can grow under aerobic as well as anaerobic conditions (Walker, 1998).Most microorganisms are sensitive to high concentration of ethanol, whereas S. cerevisiecan easily withstand 10-15% ethanol (Cassey and Ingledew, 1986). It has a highproductivity and high ethanol production yield, it s resistant to inhibitors and is alsotolerant to low pH. Its robustness makes is a suitable organism for fermentation oflignocellulosic hydrolysate.

One of the disadvantages of S. cerevisiae is that it doesnt naturally ferment arabinoseand xylose (pentoses), despite of glucose and mannose. In agricultural residues andhardwood the amount of pentoses is high and therefore an efficient pentose fermentingmicroorganism is necessary if these raw materials are used.

2.14.1.1 Dissolved oxygen (DO) and substrate inhibition effects onSaccharomyces cereviseae.

For growth and maintenance of yeast cells oxygen is a necessity and yeast cells can notstay alive more than 4 or 5 generations without oxygen (Tuite and Oliver, 1991), unlessthe ergestrol and twin (as fatty acid sources) be added to the medium. Completeoxidation of the sugar to carbon dioxide and water will give optimum cell production.Under conditions of high dissolved oxygen concentrations, fermentation of the sugars to

ethanol are inhibited, this effect called Pasteur Effect. Respiration release more energythan fermentation and therefore is the preferred process.

Respiration of glucose by yeast represented below:

6122 OHC

on)(respirati

aerobicfully6 2CO + 6 OH2 G= - 686 kcal


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Many Saccharomycesspecies are sensitive to glucose and their respiration is repressed inthe presence of a concentration of glucose greater than 1.0g.l 1 , under such conditionbiomass yield decrease and ethanol will be produced. This is known as a Crabtree effector contre-pasture effect. In a study of the crabtree effect in various yeast strains, growing

on a medium containing 30g.l1

glucose, seven of eight Sacchromyces species testedgave a positive crabtree effect (Tuite and Oliver, 1991).

6122 OHC

ion)(fermentat

anaeroicFully2 OHHC 52 + 2 2CO G= -54 kcal

In the brewing industry the specific growth rate, viability and yield of the Saccharomycesspecies employed have been found to increase with the level of oxygen concentration inthe wort for the levels of up to 20% saturation, as the saturation level is necessary foryeast cell maintenance and growth, the higher dissolved oxygen levels do not affect the

fermentation (Tuite and Oliver, 1991).

2.14.1.2 Carbon dioxide

Carbon dioxide, a by-product of yeast growth and ethanol production, is inhibitory toboth processes under aerobic and anaerobic conditions (Chen and Gutmanis, 1976;Kunkee and Ough, 1966). It can affect the permeability and composition of yeast cellmembranes and can also shift the equilibrium in carboxylation/decarboxylation reactionin the metabolic pathways of the yeast. The inhibitory effects are greater in high ethanolconcentration and at low pH values (Tuite and Oliver, 1991).

2.14.1.3 pH

The general pH for yeast cultivation is lower than for fermentation. It has been advised tolower the pH to (3.5-4.5) in order to decrease the risk of bacterial contamination duringthe cultivation period, but the pH shouldnt be less than 3.5 because it values the color ofthe yeast produced and if sucrose is the carbon source, the yeast invertase activity maybeaffected (Tuite and Oliver, 1991).

During fermentation of inhibitory hydrolysates, a higher pH causes inhibitory acids todissociate, which makes them less prone to permeate cell membranes. The pH at the

current work was maintained among 4.5-5.0 during the cultivation and among 5-6 duringthe fermentation phase.

2.14.1.4 Temperature

The optimum temperature for maximum growth rate is strain dependent and liesgenerally around 28-35C. However the tolerance limit for S. cereviseae is 40C andgrowth around this temperature cause disruption of fatty acids synthesis. In a commercial


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manufacture of Saccharomycesyeast the temperature initially maintained at 25C but isallowed to rise gradually to 30C by the end of the fermentation. At current work thetemperature kept on 300.1C during the all time.

2.14.1.5 Required nutrients by yeast

For successful cultivation of yeast, the ratio and amount of some nutrients in the mediumis important. A defined media is a good complex of nutrients, but it is rather expensive.For industrial production, a medium based on molasses can be used, with addition ofsome nutrients which are explained here.

Nitrogen (N): Yeast have a nitrogen content of around 10% of its dry weight hencenitrogen is an important constituent of any growth medium many inorganic ammoniumsalts have been found to promote the growth of S. cereviseaewhich the most efficient oneis ammonium salt, totally around 50 Mm nitrogen is needed for an optimum growthmedium.

Phosphorus (P) Phosphorus is needed for synthesis of lipids and nucleic acids andmaintaining the integrity of the cell wall (Lalander, 2002; Tuite and Oliver, 1991).Therefore it is essential for yeast growth. The requirement of phosphor is about 3 Mm(Lalander, 2002). It can be supplied in the form of 42POH (minus). 42POKH is normally

used as the source of phosphorus as well as 43POH (Tuite and Oliver, 1991).

Sulphur (S): Saccharomyces species can obtain the sulfure they require from inorganicsulfate, sulfite or thiosulfite which are reduced to aminoacid methonin in the cell wall.Sulfur constitutes about 0.45 of the dry weight of yeast cells. Salts as 424 SO)(NH or

MgSO4.7H2O are generally chosen for industrial production on the basis of cost

(Madigan et al., 1997).

Potassium (K): It is required for the synthesis of many enzymes furthermore it has a rolein proton transport, thus having a role in the control of intracellular pH. 30 Mm is therequired concentration for an optimum growth medium (Dahlin, 2000; Lalander, 2002).

Magnesium (Mg): Many important enzymes of the glycolysis require magnesium as acofactor .7Mm of magnesium is required in an optimal medium.

Biotin: Vitamin biotin is usually added to the culture medium to assist assimilation.Biotin is required for biosynthesis of essential amino acids and the purine in RNA.

Increase of biotin in the medium increase the content of protein and total ribonucleicacids in the cells.

Inositol: Inositol is a key growth factor for Saccharomyces, and deficiency of this vitamincan lead to less cell division and morphological changes within the cell wall.

Additional vitamins: Pantothenate is essential for all strains of S. cereviseae (Wiliams etal., 1940), and some strains require thiamine, pyridoxine, p-aminobenzoic acid, niacin,


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folic acid, riboflavin and nicotinic acid (Ratledge and Kristiansen, 2005; Tuite andOliver, 1991).

Trace elements: Additional elements such as iron, manganese, cobalt, borom, cadmium,chromium, copper, iodine, molybdenum and vanadium are needed in concentrations of

0.1-100 m (Tuite and Oliver, 1991).2.14.1.6 Life cycle of S. cereviseae

S. cereviseaeis a unicellular eukaryote which can reproduce both sexually (mioses) andasexually by budding (mitoses). Yeast has two mating types, called a and . when grownon rich medium, two haploid yeast cells with opposite mating types merge to form adiploid cell. Meioses and spore formation can therefore be induced by alternation of theculture conditions. A culture media with a high level of acetate and low concentration ofdexterose and nitrogen induces meioses in which four haploid spores are created. Thewhole process takes around 24 hours to complete.

2.14.1.7 Metabolism of S.cerevisise

S. cerevisie is chemoheterotrophic, which means that it uses material both for drivingenergy and as building blocks for cellular components. Organic sources that can be usedby S. cerevisie for growth are mannose fructose, glucose, sucrose, organic acids, e.g.acetate, pyrovate and lactate, ethanol and glycerol. These sources are up taken byfacilitated transport controlled by a system involving 20 different genes.

2.14.1.7.1 Glucose catabolism

S. cerevisie favors aerobic fermentation over respiration in the presence of high

concentration of sugar and less oxygen (Cassey and Ingledew, 1986), while respiratorymetabolism tends to dominate in the presence of oxygen and low sugar concentrations.All microorganisms need energy for growth and maintenance and ATP is used as anintracellular energy transporter. The (reversible) reduction of ATP to ADP releases freeenergy. Cells obtain ATP from their controlled chemical breakdown of glucose to twopyruvate molecules, which under aerobic conditions can be a dominating source ofintracellular energy.

This process is referred to as glycolysis. Once pyruvate is formed it can be processed inseveral different ways like in TCA cycle (kerebs cycle); this is referred as an aerobicrespiration. However, when oxygen is limiting other othetr metabolic pathways must be

used to deal with pyruvate. The fermentative path from pyruvate begins withdecarboxylation by pyrovate decarboxylase producing acetaldehyde. Acetaldehyde isthen reduced to ethanol with NADH being oxidised to NAD+ by the action of alchoholdehydrogenase. Consequently, the overall pathway leading from glucose to ethanol isredox neutral, since NADH formed in connection to oxidation of glyceraldehyde-3-phosohate in the upper part of gycolysisis reoxidesied by the formation of ethanol(www.scq.ubc).


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Fig. 6. Fermentation process that can lead to the ethanol production(www.emc.maricopa.edu).

2.14.1.8 Other ethanol producing yeasts

Some other yeasts, such asPichia stipisandPachysolen tannophilus, Candida shehataeare able to ferment xylose naturally., the disadvantages of these yeasts is that they aresensitive to ethanol and inhibitors, and require carefully monitored microaerophileconditions and unable to ferment at low pH, also shown that P. stipisandP. tannophilusare more sensitive to furfural than S. cerevisie, which can be a problem under industrialconditions (where low pH is sometimes used to inhibit bacteria).

However xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from P. stipishave been successfully transferred to strains of S. cerevisieand then produced a strain of

S. cerevisiethat can also utilize xylose (Wahlbom et al, 2001).This kind of recombinantstrains has low ethanol yield and a tendency to relatively high by-product formation.


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CHAPTER 3

Materials & methods

3.1 Chemical and reagents

424 SO)(NH , 42POKH , MgSO4.7H2O, Peptone, Glucose, Agar, Yeast extract, Antifoam,Methylene blue, beet molasses solution, Vacuum evaporated hydrolysate, High pressureevaporated hydrolysate.

3.2 Preparation of dilute-acid hydrolysate

The current work was performed at SEKAB E-technology, located in rnskoldsvik,Sweden. The research pilot plant for converting lignocellulosic materials to ethanol wasbuilt in 2003-2004 in rnskoldsvik, Sweden, the plant construction enables both weakacid hydrolysis and enzymatic hydrolysis, the daily capacity of the plant is 2 tones of drymass and production of 400-500 liters of ethanol. The wood hydrolysate used for thiswork was produced in the pilot plant by dilute sulfuric acid treatment and sent to Epcon(Trondheim, Norway) for two types of evaporation: vacuum evaporation and highpressure evaporation.

3.2.1 Initial hydrolysate

The raw material, wood chips from spruce, arrives in covered containers to the plant. Afan blows the raw material via a cyclone to a scale on the roof of the plant, and from thereit is fed into the first process step. The first step in the process is pre-steaming. Pre-steaming is used to release trapped air as well as for pre-heating of the raw material.Connected to the pre-steaming reactor was a pre-steaming scrubber, which is used toremove particles from the excess steam. Chemical substances in the gas phase can also


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be removed from the gas steam in the pre-steaming scrubber. If the chemicals are notremoved during this step, then they stay in the stream and get transported to the waterscrubber. After pre-steaming, the steamed raw material is transported to the first reactor,a horizontal plug flow reactor. In this reactor, diluted sulfuric acid is added and thetemperature is regulated between around 170-200C.

Under such conditions mainly hemicellulose is hydrolyzed, and the released sugars aredissolved in the liquid phase. This water-sugar solution is separated from the solidresidues by filtration and is then transported to the detoxification step. The solidresidues, containing most cellulose and lignin, were transported by transportation screwsto reactor 2. From this reactor there is a gas flow to the 2SO scrubber.

In reactor 2 the conditions are harsher. The temperature in this reactor is around 200-230C. Solid material is fed to the top of the reactor. Also the hold-up time can beregulated. While mainly hemicellulose is hydrolysed in reactor 1 also cellulose ishydrolysed to a large extent in reactor 2. The harsh conditions cause formation of by-

products to decomposition of lignin and reactions involving the sugars. This does notonly decrease the sugar yield, it also hampers the fermentation since several by-productsare inhibitory to fermenting organisms.

When the slurry of lignin and cellulose has passed the reactors most of the cellulose ishydrolyzed and the remaining slurry is transported to the membrane filter press. Thepurpose of the press is to separate the liquid hydrolysate (e.g. sugars) from the solidresidues. The mix of the hydrolysate and solid residues is pumped into the press. Whenthe press is full, water is pumped through the solid.

The wash water will press out the remaining hydrolysate in the solid residues. The water

is not a part of the process stream, and after it is squeezed out from the solid residues it istransported to the environmental tank (collection tank for the waste water).

The hydrolysate is then transported to the detoxification unit. The remaining material inthe press is mostly lignin material and is collected and later incinerated. This increase inpH reduces the problem of inhibitors in the fermentation process. After detoxification theneutralized hydrolysate is ready to be fermented (the composition of SEKAB hydrolysateis in results and discussion part).

3.2.2 Concentrated hydrolysate

One of the aims of the biotechnology group at SEKAB E-Technology is to achieve morethan 4% of ethanol in the fermentation broth. Therefore, the hexose concentration in thehydrolysate must be at least 100g.l 1 . This may be difficult to reach in the pilot plant, andpossibly also on industrial scale. Evaporation can then be used, both as a method tosimulate lower water addition and as a unit operation on industrial scale. During thevacuu evaporation the boiling temperature of the liquid was 80 with 0.5bar pressure.Water (and compounds with low boiling point) evaporated from the hydrolysate and theamount of the hydrolysate was reduced to 1/3 of the original amount. No foaming,


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incrustation, precipitation or odor was observed during the evaporation. The pH was keptabove 2.1. The high pressure evaporation was performed at a temperature 107.50.5with a 1.3 bar pressure, and the pH was kept above 2.1. The amount of hydrolysate wasreduced to 1/3 of the original amount. (The complete results of the evaporation areavailable in result and discussion part). A pH below 2.0 or a temperature higher than 120

tends to trigger decomposition of sugars.

3.3 Yeast strain

Flocculating yeast strain of S. cerevisieae isolated from an ethanol plant (DomsjFabriker AB, rnskldsviksSweden) and registered at the culture collection inuniversity of Gteborg (Sweden) as CCUG 53310 was used in all experiments. This yeastexhibits two advantages. It has a strong ability to flocculate which allows for quicksedimentation. It is also highly resistant to the inhibitors in the hydrolysate. Flocks hadaverage dimension of 2 mm. They forms aggregates while sinking in cultivation solution,however the big flocks could be broken by mixing into much smaller cell aggregates The

average sinking velocity was 98 12 mm/sec for flocks with diameter around 1 mm(Purwadi et al., 2007).The strain was maintained on an agar plate made from 10g.l 1 yeast extract, 20g.l 1 soy peptone, 20g.l 1 agar with 20g.l 1 D-glucose as additionalcarbon source.

3.4 Yeast start culture medium

The medium for the inoculums culture was the same for all experiments. It was preparedin 300-ml Erlenmeyer flasks, one of them containing 2.2 g of glucose in 50 ml water andthe other containing 0.342 g 424 SO)(NH and 0.3 g of yeast extract in 50 ml water. One

of the Erlenmeyer flasks including a rubber tube was attached to a syringe (Fig. 7). Theother one was closed with foil. The flasks were sterilized for 15 min at 121C. Aftersterilization the content of the two flasks were mixed, which resulted in 100ml culturemedium. Yeast colonies from an YPD plate containing flocculating yeast CCUG 53310was added to the flask. The flasks were placed in a linier shaker bath set at 30 C with170 rpm for 24 hours.

Fig. 7.Inoculums culture for yeast cultivation.


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3.5 Pre-culture

3.5.1 Pre- culture nutrition

Under laboratory conditions, all nutrients can be supplied in the form of pure defined

chemicals. In contrast, in large scale production, for economic reasons cheap andcomplex additives must be used. Molasses with additional nutrients can be a good optionfor yeast cultivation on large scale. Since this experiment was performed for an industrialcompany, a complex medium of beet molasses was used for yeast cultivation. The beetmolasses (supplied by Danisco, Denmark) contain about 45-50% sugar, the sugar ispredominantly consists in sucrose.

One of the disadvantages associated with beet molasses is that 0.5-3 % of the sugar israffinose, a thrisaccharid that consist of fructose, glucose and galactose. Since S.cereviseae lacks -galactosidase activity, the raffinose is only partially hydrolysed. Aninvertase, which is a cell wall bound enzyme, breaks down the raffinose between fructose

and glucose, the raffinose can only be utilized to one third of its energy content. Also beetmolasses is limited in biotin (V itamin H or B7) for cell growth; hence it may need to besupplemented with a biotin source. The non-sugar content of beet molasses includesmany salts as calcium, potassium, oxalate and chloride.

Table 5.Composition of beet molasses.

Dry matter 76%

Sucrose, glucose, fructose, 46%

Sugar 47% (DW) raffinose 0.5-3%Galactinol (inositol source) 0.2%

pyrrolidonecarboxylate 3%

Organic non-sugar 30% (DW) 12% glu/gln 0.3%betaine

D, L-lactic acid 4%Citric acid 0.5%

8% Malic acid 0.6%

Volatile acids 3%

M-inositol 1.3 g. kg 1 Salts 10% (DW) Vitamins CA-panthotenate 36 mg. kg 1

Biotin 20 g. kg 1


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3.5.2 Batch cultivation of yeast

The batch fermentations started with 600ml water plus 35g beet molasses, resulting in a50g.l 1 molasses solution. Approximately 2ml of antifoam was injected into thebioreactor and after calibration of all parts; it was sterilized in an autoclave at 121C for

15 minutes. 100ml salt solution containing 7.5g.l 1 424 SO)(NH , 3.5g.l 1 42POKH , and0.75g.l 1 MgSO4.7 H2O was prepared as an additional nutrient in an Erlenmeyer flask andwas autoclaved for 15 minutes at 121.

After sterilization the salt solution was injected into the bioreactor through a sterile filter.In addition a biotin solution composed of 100ml of water with 0.025g.l 1 of biotin wasprepared. 10ml of this biotin solution was injected into the bioreactor by a sterile filter(The biotin solution was not autoclaved).

After adjusting the pH to approximately 4.0 and the temperature to 300.1 C the

medium was inoculated with 10ml of yeast suspension from the shake flask. During thebatch cultivation 2l.min 1 air was sparged into the bioreactor. The pH was regulated at4.5-5.0 by addition of 2-M (NaOH), agitation rate regulated on 500 rpm (Fig. 8).

Fig. 8.Fermentor (Belach BR 0.4 bioreactor, AB Teknik, Solna, Sweden) from the type that could be used under sterile conditions in a laboratory work (a sterile tank

reactor).Temperature, pH probes were inserted for monitoring the fermentation. Portswere available for addition of alkali, air, inoculums culture. Agitation was achieved by

animpeller.The lowest possible volume was 0.7 l.


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3.5.3 Fed-batch cultivation of Yeast

Fed-batch fermentation started with the addition of 0.85 l (38g.l 1 ) autoclaved molassessolution after 24h of batch cultivation. The molasses solution was fed into the fermentorby an initial dilution rate of 0.05h 1 (35ml.h 1 feeding rate) during 24 hours and no

additional nutrient was added into the feed. 2l.min 1 oxygen was sparged into thefermentor; the agitation rate was kept on 500 rpm. After two days of cultivation thefavorable amount of yeast was achieved and the volume of the fermentor reached to 1.55l (Fig. 9).

Fig. 9. Aerobic yeast fed-batch cultivation process with molasses solution.

3.6 Experiments methodology

In this work, four types of experiments were defined in order to determine thefermentability of the concentrated hydrolysate, allowing for comparisons between thefermentation of hydrolysates which were concentrated at vacuum and high pressure.

The following experiments were carried out twice (A, B) or three times (A, B, C):Experiment type 1 (A, B, C): ~12g.l 1 yeast + VEH + ID 0.22h 1 .Experiment type 2 (A, B, C): ~12g.l 1 yeast + HPEH + ID 0.22h 1 .Experiment type 3 (A, B): ~32g.l 1 yeast + HPEH + ID 0.22h 1 .

Experiment type 4 (A, B): ~12g.l 1 yeast + HPEH + lower ID (0.14h 1 ).

Experiments 1 and 2 were identical except for the feed, which consisted of vacuumevaporated hydrolysate and high pressure evaporated hydrolysate respectively. Inexperiment 3 the initial amount of yeast was increased and in experiment 4 the amount ofyeast was kept regular and the feed rate was decreased.


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3.7 Experiment 1(A, B, C): Fermentation with vacuumevaporated hydrolysate

This experiment (carried out three times) was performed with the vacuum evaporatedhydrolysate. See Fig. 10 for an overview of the yeast production in this experiment.

3.7.1 Yeast cultivation:The yeast cultivation was performed aerobically (2l.min 1 ofair). The batch phase, which lasted 24 hours, started with 0.7 l molasses solution with50g.l 1 concentration. The second day a fed-batch cultivation started with molassessolution (concentration: 38g.l 1 , 1 liter), which increased the volume of the bioreactor to1.55 l (at 48h). Initial dilution rate: ID= 0.05h 1 , Initial volume: IV= 0.7 l). pH was keptbetween 5-6 by addition of 2-M NaOH. The temperature was kept at 30 , the stirrerworked with 500rpm.

3.7.2 Hydrolysate feeding: At the end of 48 h the volume of the bioreactor was

sucked to 0.7 l and the vacuum evaporated hydrolysate was fed into the bioreactor for18h(ID= 0.22h 1 ), as the diagram shows, the volume at the end of this stage reached to3.5 l.

3.7.3 Resting & airing: At the end of feeding the pump switched off and thebioreactor worked in a batch mode for 3h, thereafter the content of the bioreactor wassucked to 0.7 l and with the aim of biomassgrowth air was sparged into it (2l. min 1 ) for2h. After 72 h the first cycle was completed and the second cycle started for another 18h,this cycle repeated like the first one. At the end of the second resting, after 93h thebioreactor switched off and by this way one experiment was done ( this experimentrepeated for three times).

0

0.7

1.4

2.1

2.8

3.5

4.2

0 24 48 72 96 120

Time (h)

Volume(l)

batch 50 g/l fed-batch 38 g/l

3 h resting

18 h

feeding

18 h

feeding

2 h aeration

3 h resting

Fig. 10.Diagram of volume versus time. Yeast production (aerobic) lasted 48 hours, andresulting yeast culture was used for (anaerobic) fermentation in two cycles, with 2 hours

of aeration between them. The feed during the fermentation consisted of vacuum and high

pressure evaporated hydrolysate.


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3.8 Experiment 2(A, B, C): Fermentation with highpressure evaporated hydrolysate.

For these series of experiments the methodology was performed exactly the same withthe experiments type 1 just the hydrolysate changed. An aerobic fed-batch cultivation ofyeast was applied for 48h, then 2 cycles of anaerobic hydrolysate fermentation with fed-batch method was performed (18h feeding, 3h resting, 2h aeration). A regular amount ofyeast (12g.l 1 ) and high pressure evaporated hydrloysat were used for fermentation, pHwas controlled among 5-6 by addition of 2-M NaOH, temperature set on 30, androtation rate of stirrer was 500 rpm(Condition of experiments is available at appendix B).

3.9 Experiment 3(A, B): Fermentation with higher initialbiomass concentration.

In experiment 1 and 2 a large amount of sugars were not been utilized and remained inthe bioreactor, there are two options for decreasing the remained sugar in the fermentereither increasing the amount of yeast or decreasing the feed rate, but the intention of anethanol production project is to produce ethanol in an economical way and shorter time,therefore raising the amount of yeast can be a better option rather than decreasing thefeed rate. In this group of experiments the yeast amount was doubled.

These two experiments took 2 weeks. The methodology kept the same with experimentstype1 and type 2, just for increasing the yeast amount a slightly changes were performedon the yeast cultivation part. The yeast batch cultivation with 100g.l 1 beet molasses anddouble amount of salt solution was unsuccessful and ended up just to 2.5g.l 1 biomass,also cultivation with 50g.l 1 molasses in batch and 76g.l molasses in fed-batch modewith 200ml salt solution ended up again just to 12g.l 1 yeast, like before.

Double amount of biomass was gained by keeping the batch part concentration 50g.l 1 and performing the fed-batch part twice with 38g.l 1 molasses in each fed, 200ml saltsolution was injected in two loads, 100ml of that in batch part and the other 100ml in thesecond fed-batch.


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.

0

0.7

1.4

2.1

2.8

3.5

4.2

0 24 48 72 96 120 144

Time (h)

Volum

e(l)

batch 50 g/l

fed-batch 38 g/l

3 h res ting

fed-batch 38 g/l

3 h resting

2 h aeration

18 h

feeding

18 h

feeding

Fig. 11. Fed-batch fermentation with high pressure evaporated hydrlosate and double

amount of yeast at experiment 3 (A, B).

3.10 Experiments Type 4(A, B): Fermentation withlowered initial dilution rate.

After experiments with double amount of yeast, and no improvement in sugarconsumption another series of experiments were defined which the feeding period wasprolonged for about 50%. The methodology for yeast cultivation part was repeated likethe experiments from type 1 and 2, with a regular amount of yeast about 12g.l 1 (oneaerobic batch and fed-batch plus 100ml salt solution). For fermentation part, 2.8 l highpressure evaporated hydrolysate was fed into the fermentor in 27h, the initial dilution rate

decreased to 0.14h1

and then a 3h resting and 2h of aeration was repeated like theprevious experiments (sucking of supernatant by syringe repeated at the end of yeastcultivation and aeration to 0.7 l volume).

0

1

2

3

4

0 24 48 72 96 120

Time (h)

Volume(l)

fed-batch 38 g/l

batch 50 g/l

27 h

feeding

27 h

feeding

3h resting 3h resting

2h aeration

Fig. 12. Volume changes versus time in fed-batch fermentation with high pressure

evaporated hydrolysate with a regular amount of yeast and lower dilution rate.


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3.11 Analysis

3.11.1 Metabolic analysis

The metabolites were quantified using either HPLC or GC. Formic acid, acetic acid,

levulinic acid and ethanol concentrations were determined by HPLC using an ion-exchange column (Aminex HPX-87H, Bio-Rad, USA) with a refractive index (RI)detector at 55-65C, using 0.005 M sulfuric acid as the eluent at flow rate 0.6 ml/min.Furfural and HMF were detected by a UV detector. Sugars were quantified by the HPLCusing Aminex HPX-87P column (Bio-Rad) at 80-85C and pure water as the eluent.Glycerol and lactic acid were derivatized to silylesters and then analyzed using a GC-MS.Sulfate is measured with titration and the method SCAN-N 6:85. Dissolved lignin ismeasured with spectrophotometer at 286.5nm after dilution. Indulin used as standard

3.11.2 Dry weight

The cells dry weight was determined viathe first method that explained above. At the endof each stage the rotation ratechanged from 500 rpm to 800 rpm and then duplicate 8-mlsamples were taken two times during the cultivation and at least 5 times during thefermentation, the samples were centrifuged, washed with distilled water, and dried for24h at 105 . Then the dried mass after 24h weighed. The yeast must be kept at atemperature bellower than 10 in order to prevent from their destabilization before dryweight measurement, but at current work the samples were measured immediately orwere kept at refrigerator at temperature 5 for few hours.

Fig. 13. Glass tubes containing centrifuged yeast solutions for dry measurement.

3.11.3 Determination of cell vitality

There was an assumption, that some part of the cells lose their vitality during thefermentation with these hydrolysates and they cant flocculate (sediment), this assumptioncame to mind when the pump and stirrer get switched off, and samples were taken fromthe supernatant and the results showed a little concentration of yeast in there (Thisshouldnt happen because cells are supposed to sediment after switching off the stirrerand not to be floating in the supernatant). Therefore the vitality determination processed


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Table 9. Comparison of dilute-acid spruce hydrolysate after and before evaporation.

Component inhydrolyzate

g.l1

Originalhydrolyzate

g.l1

VEHg.l 1

concentrationfactor

HPEHg.l 1

concentrationfactor

Glucose 19.67 61.53 3.1 64.83 3.3

Galactose 3.42 10.90 3.2 11.33 3.3

Mannose 15.66 47.08 3.0 52.85 3.4

Xylose 8.74 27.07 3.1 28.71 3.3

Arabinose 2.11 6.71 3.2 6.88 3.2

HMF 1.46 4.54 3.0 4.93 3.4

Furfural 1.00 0.03 0.0 0

anahita deh khoda

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