Anahita Dehkhoda

Concentrating lignocellulosic hydrolysate by evaporation and its fermentation by repeated fedbatch using flocculating Saccharomyces cerevisiae

Performed at: SEKAB E-Technology Published by: Hgskolan i Bors

i

MASTER THESIS IN INDUSTRIAL BIOTECHNOLOGY

Concentrating lignocellulosic hydrolysate by evaporation and its fermentation by repeated fedbatch using flocculating Saccharomyces cerevisiae

Anahita Dehkhoda

Performed at SEKAB E-Technology rnskldsvik, 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 Science with a Major in Chemical Engineering, Industrial Biotechnology, 181 300 ECTS credits No. 3/2008 ii

Title: Concentrating lignocellulosic hydrolysate by evaporation and its fermentation by repeated 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 Bors School of Engineering SE-501 90 BORS Telephone +46 033 435 4640

Examiner: Supervisors: Client: Keywords:

Prof. Mohammad Taherzadeh Dr. Tomas Brandberg, Prof. Mohammad Taherzadeh SEKAB E-Technology, rnskoldsvik, Sweden Concentrating, lignocellulosic hydrolysate, evaporation, flocculating yeast, fermentation, ethanol.

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ACKNOWLEDGEMENT
The completion of this thesis project and my graduate education are indebted to the support of industry professionals and very experienced supervisors. I am especially appreciative of Dr. Tomas Brandberg for choosing me and giving me the opportunity to do my diploma work in SEKAB E-Technology. Thanks for being patient with me always and teaching me so much from your knowledge and never hesitating in repeat yourself. Thanks for being like a friend to me, and helping me in everything from getting samples late at night to fixing disasters! You have been more than a supervisor to me, I will never forget you. I would like to thanks to Prof. Mohammad Taherzadeh, first for informing your students about this opportunity, second for your suggestions during the experiments which helped us in improving them. Writing a scientific paper would not have been possible without the considerable time and effort invested by you. I take this opportunity to thank my assistant supervisor Annika Hgglund. Every time I needed help, you rushed to give me a hand and fix the sudden problems in lab, and thank you for sharing your experiences with me. I want to extend special thanks to Torbjrn van der Meulen for giving me the opportunity to work at SEKAB. Thanks to Carl-Axel Lalander in helping with quick preparation of any equipment which I ran out of. I would also like to recognize Staffan Magnusson, Robert Selling, Birgitta Lundgren and other people at MoRe Research for the processing and measurement of samples. 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 ETechnology rnskoldsvik, Sweden and all experimental works was performed in laboratory scales at SEKAB E-Technology.

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Master thesis Industrial Biotechnology Bors University and SEKAB E-Technology, Sweden

ABSTRACT
In order to obtain a sugar concentration of more than 100g.l-1of fermentable sugars, a spruce wood hydrolysate was subjected to high pressure and vacuum concentration and the fermentability of each hydrolysate was assessed by fermentation experiments with flocculating 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 not confirmed by the results. Minor amount of cells lost their flocculating ability after fermentation which their ratio and their viability and vitality was assessed. By vacuum and high pressure concentration, the fermentable sugars (defined as the concentration 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 factor represented a 3-fold increase of fermentable sugars. Furfural was evaporated in both trials and its concentration reached to 0.03g.l-1 and 0.1g.l-1 after vacuum and high pressure evaporation respectively. Fermentation with both 0.14h-1and 0.22h-1 initial dilution rates was 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 the hydrolyzates and concentrated proportionally. More than 84% of the fermentable sugars present in VEH were fermented by fed-batch cultivation using 12g.l-1 yeast and initial dilution rate (ID) of 0.22h-1, and resulted into 0.400.01g.g-1 ethanol in 21h. Fermentation of HPEH was as successful as VEH and resulted into more than 86% of the sugar consumption at the corresponding conditions. With an ID of 0.14h-1, more than 97% 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 liquor represented that about 76% of the cells which lost their flocculating ability kept their vitality. 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 yeast growing. Keywords: concentrating, lignocellulosic hydrolysate, evaporation, flocculating yeast, ethanol, fermentation.

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 by repeated fed-batch using flocculating Saccharomyces cerevisiae (Submitted)

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CONTENTS
Chapter 1: Introduction..1
1.1 Background of ethanol production......................................................................1 1.2 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?.............................................................................................4 2.3.1 Environmental impact.........................................................5 2.3.2 Depletion of crude oil .............................................................5 2.3.3 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...............................................................7 2.6.1 Celluloses............................................................7 2.6.2 Hemicelluloses................................................................7 2.6.3 Lignin..............................................................8 2.6.4 Extractive and ash.. .........................................................9 2.7 Pretreatment, first step for ethanol production ........................................................9 2.8 Hydrolysis. .....................................................10 2.8.1 Acid hydrolysis .....................................................10 2.8.2 Enzymatic hydrolysis............................................11 2.9 Inhibitors ........................................................12 2.9.1 Organic acids. ...........................................................13 2.9.2 phenolic compounds .........................................................13 2.9.3 Furan compounds..........................................................13

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

2.11 Fermentation.. ..........................................................15 2.11.1 Fermentation of dilute acid hydrolysate ....................................................15 2.11.2 Fermentation of enzymatic hydrolysyate (SSF and SHF) .........................16 2.12 Fermentation techniques ......................................................16 2.12.1 Batch process.. ...........................................................16 2.12.2 Fed batch process...........................................................17 2.12.4 Continuous process. .......................................................17 2.13 Overall rocess of ethanol roduction from lignocellulosic materias..18 2.14 Fermentation s microorganism. .......................................................19 2.14.1 Yeast (Saccharomyces cerevisiae)............................................................19 2.14.1.1 Dissolved oxygen............................................................19 2.14.1.2 Carbon dioxide............................................................20 2.14.1.3 Hydrogen ion concentration........................................................20 2.14.1.4 Temperature ............................................................20 2.14.1.5 Required nutrients by yeast.........................................................21 2.14.1.6 Life cycle of Saccharomyces.cereviseae.....................................22 2.14.1.7 Metabolisms of S.cerevisise........................................................22 2.14.1.7.1 Glucose catabolism ....................................................22 2.14.2 Bacteria.. ...........................................23 2.14.3 Filamentous fungi.. ...................................................24

Chapter 3: Materials &methods ...25

3.1 Chemical and reagents.. .................................................25 3.2 preparation of dilute acid hydrolysate................................25 3.2.1 Initial hydrolysate... ..............................................25 3.2.2 Concentrated hydrolysate..26 3.3 Yeast strain.............................................................27 3.4 Yeast start culture medium ............................................................27 3.5 Pre-culture..........................................................28 3.5.1 Beet moalsses, pre-culture nutrition .............................................................28 3.5.2 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).. .........................................................31 3.7.1 Yeast cultivation . . ...................................................31 3.7.2 Hydrolysate feeding. .....................................................31 3.7.3 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........................................................34 3.11.1 Metabolic analysis. ........................................................34 3.11.2 Dry weight.. .......................................................34 3.11.3 Determination of cell vitality.. .......................................................34 3.11.4 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)....38 4.3.2 Ethanol, biomass and glycerol yield......40 4.4 Fed-batch fermentation with HPEH, Ex 2(A, B, C) ..41 4.4.1 Fermentable sugars (mannose, glucose, and galactose)....41 4.4.2 Ethanol, biomass and glycerol yield..43 4.5 Fed-batch fermentation with double biomass and high pressure evaporated hydrolysate; Ex, type 3 (A, B) ...44 4.5.1 Fermentable sugars (mannose, glucose, and galactose)....44 4.5.2 Ethanol, biomass and glycerol yield..46 4.6 Fed-batch fermentation lower dilution rate, Ex 4 (A, B)...47 4.6.1 Fermentable sugars (mannose, glucose, and galactose)....47 4.6.2 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

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. cerevisea39 16. 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...57 32. 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....43 15. 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 alternative fuel or oxygenated additive to the current fossil fuels. Its market grew from less than a billion liter in 1975 to more than 39 billion liters in 2006 and is expected to reach 100 billion liters in 2015 (Licht et al, 2006). Fuel ethanol contributes relatively little to net carbon dioxide emissions to the atmosphere (Bergeron et al, 1989). Besides, depletion of crude oil in the near future makes bioethanol an important fuel, not least because it is easily used as an additive to gasoline. Lignocellulosic material is renewable and abundantly available for the production of fuel ethanol. 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 cellulose and hemicellulose to monomeric sugars. These fermentable sugars can be anaerobically converted into ethanol by microorganisms. A number of by-products are however formed during the hydrolysis, and these compounds, e.g. furfural, hydroxymethylfurfural may inhibit yeast metabolism (Larsson et al, 1999b; Taherzadeh et al, 1997b; Sanchez and Bautista, 1988; Chung and Lee, 1985; Banerjee et al, 1976). The Inhibition of these inhibitors 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 performed efficiently at low cost, but is known to cause sugar loses(Martinez et al, 2000). In situ 1

detoxification was applied to develop a fed-batch process for cultivation of severely inhibiting hydrolysates (Taherzadeh et al, 2000; Taherzadeh et al, 1999). Although this method has been proved potentially successful it has showed a drastic decrease in cell viability as soon as the feed rate or toxicity of the hydrolysate was increased (Nilsson, et al, 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 of toxicity (Larsson et al, 1999). An increase at the initial hydrolysate sugar concentration provides an increased ethanol concentration which has a major effect on the energy demand; especially at concentrations below 4 wt % of ethanol. The increased ethanol concentration in the feed to the distillation reduces the production cost considerably. Thus, more than 100g.l 1 sugar concentration is needed for industrial ethanologenic fermentation, since huge costs are associated with equipment required for transportation and 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 water or less addition of water to the hydrolysis process. The dramatic evaporation (by a factor 3) that was performed in this work is not industrially relevant, but should partly be seen as a simulation of a hydrolysis process where less water is added. Also, some evaporation may be used on industrial scale, but the comparison of vacuum evaporation and high pressure evaporation is important.

1.2 Outline of the thesis

The objective of current work was to assess the fermentability of evaporated hemicelluloses hydrolysate rich in fermentable sugars and suitable in fermentation process without performing any chemical detoxifications also making a comparison between of the fermentation results from these two hydrolysates which performed by FY S. cereviseae in a fed-batch mode. To attain this objective the sugar concentration in hysrolysate was brought to more than 100g.l 1 by vacuum and high pressure concentrating methods in order to reach to more than 4% ethanol in fermentation broth in an industrial scale. Chapter two is about the background of ethanol production; later on chapter three is the materials and method of experiments and chapter four includes the results, and at last chapter five is about the discussion and main conclusion remarks.

CHAPTER 2
Bioethanol

2.1 Brief History of ethanol production

Production of alcoholic beverages is in fact as old as human civilization; the production of pure ethanol apparently begins in the 12-14th century along with improvement of distillation. During the middle Ages, alcohol was used mainly for production of medical drugs but also for the manufacture of painting pigments. The knowledge of using starchy materials for ethanol production was first employed in the 12th century in typical beer making countries like Ireland. It was only in the 19th century that this trade became an industry with enormous production figures due to the economic improvements of the distilling process (Roehr et al, 2000). Alcoholic beverages still represent a large portion of industrial alcohol, but other applications are becoming more important. Alcohol can be used for various purposes in the chemical industry and its role as a fuel is well known. It was at the beginning of the 20th century that it had become known that alcohol might be used as fuel for various combustion engines, especially for automobiles. However, production of large amounts of alcohol requires large amounts of sugars, which tends to limit 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 et al, 2000). Today, a dramatic expansion of bioethanol production takes place. In countries like the United States a big effort is being made in the construction of large scale production for fuel ethanol. Using primarily starch from enzynatically hydrolyzed corns as raw material, the United States has the second largest bioethanol program.

In Brazil, the proacool program was launched in the 1970s as a response to the oil crisis. While ethanol is used as a fuel additive in the US it is frequently used as the dominating fuel component in Brazil (Roehr et al, 2000). m3

Table 1. Annual production of (www.ethanolrfa.org/industry/statistics). Country USA Brazil China India France Russia 2004 13362300 15078420 3643920 1746360 827820 748440

ethanol

in

world

based

on

2005 15776800 15639900 3795120 1697220 907200 748440

2006 18074500 16616700 3844260 1897560 948780 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. It is an energy dense fuel, which can burn easily. It is widely used as a fuel, but is also used for 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 fuel engines. 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 to E, combined with a subscript that indicates the percentage of alcohol in the liquid. For example 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. This development is driven by a combination of economical and political mechanisms.

2.3.1 Environmental impact

CO 2 is a socalled greenhouse gas, trapping thermal radiation which would otherwise radiate into space and thus warming up the lower parts of the atmosphere (Dickenson and Ciceron, 1986). During the last 150 years the CO 2 content in the atmosphere has increased by about 30%. This has coincided with an increase in the average temperature of the earth which has been partially rapid during the last 30 years. In the 50 years this could lead to an increase in the average temperature as much as 5C (Dickenson and Ciceron, 1986).
A major part of the increase in the atmospheric CO 2 is most likely due to the utilization of fossil fuels and in contrast combustion of biofuels such as biogas and bioethanol do not increase the atmospheric CO 2 content (Wingren et al, 2003) for instance emission of CO 2 decreases by 20% for E10 (10% ethanol, 90% gasoline), but higher ethanol blend dont 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 increasing demand. While energy demand is predicted to increase constantly, the supply of crude oil will start to decline after a peak that according to the different sources will occur between 2009 and 2020. Therefore, another source of fuel must be investigated, and bioethanol 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 CO 2 by 32.5% and non- combusted hydrocarbons by 14.5%. It has a lower flame temperature and larger gas production per energy unit of fuel combusted than gasoline. It is an energy densed, easily burned fuel. Ethanol has a high octane number about 112, which makes it suitable as an octane number enhancing additive (Baired, 1999); Due to the low octane number of gasoline it is necessary to increase the octane number with additives. Ethanol can reduces the need for toxic 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).

2.4 Disadvantages of ethanol

There are some concerns with fuel ethanol that have to be addressed. Ethanol increases the vapor pressure of the ethanol-gasoline mixture, potentially causing more evaporative emissions, furthermore the use of ethanol in combustion engines can increase the acetaldehyde and formaldehyde emissions, and as the energy density in ethanol is lower than in gasoline then 25-35 more ethanol compared to gasoline is needed to drive the same distances (Wyman, 1996), another disadvantage is that, the catalysts which are used nowadays are optimized for petroleum based fuels(Wheals et al, 1999; Zaldivar et al 2001; Galbe and Zacchi, 2002).

2.5 Lignocelluloses materials, ethanol production

good

sources

for

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 estimated annual production of 10-50.10kg (Classen, 1999). There are many types of lignocellulosic materials that can be served as feed stocks for ethanol production like Agricultural residues, municipal and industrial waste materials, papers and forestry by products, trees, grasses. The dominant sources of lignocellulic materials in northern hemisphere are softwoods such as pine and spruce, softwood is an object of interest in Sweden, Canada and western United States as renewable resources for ethanol production, because it is cheaper than hardwood (Galbe et al, 2005). Besides its content of pentose-rich hemicelluloses is significantly lower than in hardwood. This is advantageous, since important fermenting organisms (such as native strains of S. cerevisiae) dont consume pentose.

Table 2. Hardwood and softwood composition. Hemicellulose Material Hardwood Softwood Cellulose % 45-51 41-42 Hemicellulose % 23-28 24-31 Lignin % 19-24 29-31

2.6 Characteristic of lignocellulosic materials

Lignocelluloses consist mainly of cellulose, hemicellulose and lignin; these components build 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 obvious purpose of cellulose is to provide strength to the plant structure. It is a linear homopolymer of anhydroglucose units linked by (1-4) glycosides bonds, its basic repeating units is disaccharide cellebiose (Delmar and Amor, 1995). The length of macromolecules varies greatly as to the source and degree of processing (DP) that it has undergone. Newsprint, e.g., exhibits an average DP of about 1000 while cotton is found to have a DP of approximately 10,000 (Roehr, 2000). Its not the primary structure which makes cellulose a hydrolysis resistant molecule. It rather seems to be the effect of secondary and tertiary configuration of the cellulose chain as well as its close association with other protective polymeric structure within the plant cell wall such as lignin, starch, pectin, hemicelluloses, proteins and mineral elements. The easily and quickly hydrolyzed degraded regions in cellulose structure are amorphous in 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 Dgalactoses, Dglucose, and D-mannose, D-xyloses, L- arabinoses and various other Sugars as well as their uronic acid. It is bound covalently to lignin and through hydrogen bonds to cellulose (Sjstrm et al, 1993).

Its composition differs from softwood to hardwood, it means that in hardwood hemicellulose mainly consists of xylose but glucose and mannose are the dominating buildingblocks in softwood carbohydrates (Sjstrm et al, 1993). Hemicellulose has a low degree of crystallinity and microfibrils and it has more amorphous regions than cellulose; this means that hemicelluloses are more susceptible to hydrolysis than to the rigid structure of cellulose.

Fig. 2. Hemicelluloses structure (www.life.ku.dk).

2.6.3 Lignin
Lignin is an aromatic large cross-linked polymer synthesised from phenylpropanoid precursors that compose around 25% of lignocellulose. Lignins are divided into two classes, namely ``guaiacyl lignins'' and ``guaiacyl-syringyl lignins'', differing in the substituents of the phenylpropanoid skeleton, Guaiacyl-lignins have a methoxy-group in the 3-carbon position, whereas syringyl- lignins have a methoxy-group in both the 3carbon and 5-carbon positions. It is the main by-product when lignocellulosic materials are used for ethanol production, but it can be used as an ash free solid fuel for production of heat and electricity (Galbe and Zacchi, 2002). The lignin is formed by removal of water from sugars, these reactions are not reversible. There are many possible monomers of lignin, and the types and proportions depend on the source in nature. This molecule of phenolic character is the dehydration product of three monomeric alcohols: Trans-p-coumaryl alcohol, Trans-coniferil alcohol, transsinapyl alcohol linked together by ether bonds. The lignin component of cellulosic based biomass is responsible to a great extent for the difficulties inherent in cellulose hydrolysis; its matrix forms a protective sheath around the cellulose microfibrils, and can be accounted for some degree of protection to the microfibrils. The composition of lignin varies depending on the source of raw material. Softwood contains a higher amount of lignin (about 30%) than hardwood (about 20%).

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 organic solvent and can be divided into wood resin and phenolics extractives. Phenolics extractives can be found at the inner part of the wood as well as in the bark, whereas resin is 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 against microbial damage and insect attack (Sjstrm et al, 1993). These compounds may be liberated 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 and making the cellulose accessible to enzymatic attack. Due to the structure of softwood, mild pretreatment conditions will not be sufficient to achieve a high overall sugar yield. This implies that harsher conditions are required in the pretreatment steps. The improved carbon recovery can be obtained by a two stage pretreatment, 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 severe condition. A number of physical and/or chemical methods can be used to separate celluloses from its sheath of lignin and increase the surface area of the cellulose crystallite by size reduction and swelling. The pretreatment methods include milling, steam explosion, use of solvent, swelling agents, lignin consuming microorganisms, but the most investigated and commonly used pretreatment method is called steam-explosion (steam pretreatment) 9

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

2.8 Hydrolysis
The aim of the hydrolysis is cleaving the polymers of celluloses and hemicelluloses to monomeric sugars which can be fermented to ethanol by microorganisms. In ethanol production industry the process of hydrolysis is very complicated, depending on several parameters such as properties of the substrate, acidity, and rate of decomposition of the products during hydrolysis (Taherzadeh and Karimi, 2007). The hydrolysis can be carried out either chemically or by a combined chemical and enzymatic treatment. Acids are predominantly applied in chemical hydrolysis and Sulphuric acid is the most investigated one, although other acid such as HCL have been used too.

2.8.1 Acid hydrolysis

The solubility of cellulose in acid has been detected already in 1815. The first industrial process however was developed in 1942 and run in Italy (Roehr, 2000). The acid hydrolysis can be performed by high acid concentration at a low temperature or that of low 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 by concentrated acids. The concentrated acid process is a quiet old process normally involving concentrated sulphoric or hydrochloric acid. Concentrated acid processes are often 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 render high investment cost, and also the recovery of the acid are expensive and difficult (Jones and 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 in the former Soviet Union, Japan, Brazil and the USA. Compared to a concentrated acid process a dilute acid process will consume much less acid, however high temperature required 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 stage process, in which the hemicellulose is mainly hydrolysed in the initial step at temperature 150-190 C and the remaining cellulose subsequently hydrolysed at more severe conditions 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 Concentrated acid Process Advantages -operated at low temperature - High sugar yield Disadvantages -high acid consumption -high energy consumption for acid recovery -longer reaction time (e.g. 2-6h) -equipment corrosion - operated at high temperature -low sugar yield -equipment corrosion

Dilute acid Process

-low acid consumption -short residence time

2.8.2 Enzymatic hydrolysis

Enzymes can be used to cleave the cellulose and hemicellulose polymers at low temperature into simpler sugars. Enzymes with the ability of degrading these polymers are collectively called cellulases (Galbe and Zacchi, 2002). Cellulases are microbial enzymes capable of cellulose hydrolysis that are in reality a number of several different synergistic components. They are induced enzymes and produced only when the organism is grown in the presence of cellulose, cellobiose and lactose or other glucane which contain -(1-4) linkages. Denaturation by shearing is a common drawback of cellulose enzymes (Roehr, 2000). Cellulases are produced by many genus of fungi e.g. Thricoderma, Penicillum and Aspergillus. Thrichoderma is the most investigated fungi for production of complex mixtures of cellullases that are specialized in breaking -(1-4) glucosidic bonds. The enzymatic hydrolysis step (in combination with pretreatment) results in higher sugar yields than dilute acid hydrolysis, since the enzymes catalyze only sugar generation and not sugar degradation. The enzyme mixture consists of exoglucanases, endoglucanases and - glucosidases (Gusakov and Sinitsyn, 1992).

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

Acid
1. Non-specific catalyst therefore will delignify material as well as hydrolyze cellulose.

Enzyme
Specific macromolecule catalyst, therefore extensive physical and chemical pretreatment is necessary to make cellulose available for degradation. production of clear sugar syrup ready for subsequent anaerobic fermentation Run under mild conditions (50,atmospheric pressure ,pH4,8)

2.Decomposition of hemicellulose to inhibitory compounds 3.Harsh reaction condition therefore necessary increased costs for heat and corrosion resistant equipment 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 the formation of a large number of organic compounds, some of which are inhibitory to the yeast during the pretreatment or dilutes acid hydrolysate of hemicellulose, cellulose and lignin, 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 be an example of this category. Inhibitors may be present as side groups on the heteropolymers and maybe cleaved off during hydrolysis processes. Acetic acid originating from acetylated hemicellulose and also phenolic compounds are examples of inhibitors that are cleaved off during the pretreatment and. Inhibitors may be formed in carbohydrate degradation; furfural and HMF are two examples 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 Organic acids

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 acid constituent in hydrolysates and is mainly produced from degradation of the acetyl group in the polysaccharides whereas levulinic acids and formic acids are products of sugar degradation. Levulinic formed from HMF and formic formed from both HMF and furfural, 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. cerevisie as this undisssociated form of it can diffuse inside of the cell and affect the intracellular pH, but also it has shown that the concentration of 3.3g.l 1 of undissociated acetic acid resulted in an increase in ethanol yield 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 with increasing concentration of acetic acid, formic acid and levulenic acid (Larsson et al., 1999a).

2.9.2 Phenolic 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 .These compounds are mainly liberated from lignin degradation in addition to aromatic extractives. 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 can potentially 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 been reported to cause a 30% decrease in ethanol yield compared to reference fermentation, vanillin which constitutes a large fraction of the phenolic compounds in hydrolyate of spruce, has been found less toxic than 4-hydroxybenzoic acid. Inhibition of fermentation has been shown to decrease when phenolic monomers and phenolic acids were specially removed from a willow hydrolysate (Jnsson et al., 1998) and 4-hydroxybenzoic acid, vanillin and catecol were the major constituent in the untreated hydrolysate from spruce (Palmqvist et al, 1999).

2.9.3 Furan compounds

Furan compounds, in this context, include furfural and 5-hydoxymethyl furfural. During the hydrolysis pentose yield furfural and hexoses yield HMF. Furfural has been reported to 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 CO 2 evolution rate and the cell multiplication also the total viable cell number in the early phase of fermentation (Azhar et al., 1981; Banerjee et al., 1981; Boyer et al., 1992; Chung and Lee, 1985; Sanchez and Bautista, 1988). During anaerobic fermentation, furfural is reduced to furfuryl alcohol, while furoic acid is produced from oxidation of furfural during aerobic cultivation. HMF has the similar inhibitory effect as furfural, except that it has a lower conversion rate that it can be because of its lower membrane permeability. An addition of 4g.l 1 of HMF decrease the CO 2 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 of S. cereviseae. It belongs to the picture that since S. cerevisiae has an in situ detoxification mechanism for both furfural and HMF, these compounds are less inhibitory if they are supplied continuously at a rate that is lower than the detoxification rate.

2.10 Inhibition control

Pretreatment employing chemicals and high temperatures result in the generation of a wide range of by-products (Larsson et al, 1999). Several methods for detoxification are known, but most are difficult to apply on industrial scale. A well characterized method, however, is overliming with calcium hydroxide followed by removal of precipitant by filtration or centrifugation, which decreases the toxicity of the hydrolysate. Overliming has also been with bases like potassium hydroxide, sodium hydroxide 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 that toxic compounds are precipitated during this operation. It has also been observed that the concentration of furans and phenolic compounds decreases. Generally, extended detoxification 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 detoxification process. In addition, treatment with calcium hydroxide would create a large quantity of a useless 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 situ detoxification capacity of the fermenting organism. It belongs to the picture that some strains are significantly more tolerant than others. (Personal communication, Tomas Brandberg) This is in principle the approach that was used in this project.

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Ash (0-2%) various inorganic Compounds

Extractives (1-5%) phenolic compounds and wood resin

Cellulose (32-54%) Glucose HMF Formic acid Levulinic

Hemicellulose acetic acid (11-37%) pentoses furfural Formic acid hexoses HMF Formic acid Levulinic acid Lignin phenolic compounds (17-32%)

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

2.11 Fermentation
During fermentation monomeric sugars released in the hydrolysis are converted into the desired product, by a microorganism, these microorganisms could be yeasts or bacteria. Anaerobic production of ethanol is a typical example of fermentation. The metabolic basis for the conversion is the desire of the microorganism to use the sugars as carbon and energy source in order to maintain viability and growth. The saccharid released during hemicelluloses and celluloses degradation have to be fermented to ethanol by yeast or bacteria.

2.11.1 Fermentation of dilute acid hydrolysate

Dilute acid hydrolysis is a relatively cheap, fast and straightforward method for hydrolysis of lignocellulosic materials. However during the dilute acid hydrolysis many compounds are formed, besides of the desirable monosaccharide, several of these compounds inhibit the microbial fermentation of the sugar to ethanol, but there are options 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

15

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

2.11.2 Fermentation of enzymatic hydrolysyate (SSF and SHF)

The fermentation in a process based on enzymatic hydrolysis can be carried out in two ways: separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). There are two main advantages with SHF. Hydrolysis and fermentation have different temperature optima and the yeast can be recycled since the sugar solution can be filtered prior to fermentation. A problem, however, is that the sugar decreases the efficient of the enzyme due to product inhibition. The main advantage with SSF is that sugar inhibition is avoided, since the fermenting organism is mixed with the enzyme and the slurry. Disadvantages associated with SSF are mixing/cooling problems; the optimal temperature for fermentation is approximately 30 C, while for hydrolysis it is about 50 C, thus SSF must be operated at intermediate temperature, 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).

2.12.1 Batch process

In batch process substrate and separately grown cells slurry are charged into the bioreactor together with nutrients and enzymes required. Generally batch fermentation is characterized by low productivity, and it is labor-intensive. When a single cell like Saccharomyces strain is grown in a submerged culture, a plot of the logarithm of the dry weight of cells produced against time, gives characteristic curve dependent on strain and environmental 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 are adapting with their new environment. The lag phase can be shortened by using a large inoculums or an inoculums culture that is already growing exponentially under similar condition. If the culture medium is near the optimum temperature for the yeast growth and contains all the essential nutrients requirements for the yeast, this will also decrease the apparent lag phase (Tuite and Oliver, 1991). The exponential phase is the time period during which the specific growth rate () is constant and it is at a maximum ( max) for

16

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

2.12.2 Fed batch process

A fed batch process can be regarded as a combination of batch and continuous operations; it is a very popular type of process in ethanol industry. In this operation feed solution which contains substrate yeast culture, important minerals and vitamins are fed at constant intervals while effluent is removed discontinuously (Roehr, 2000). The start up of fed-batch operation is similar to the batch process start-up. Subsequently substrate 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 been consumed. The concentration of substrate must be kept constant in the reactor while the feeding is made, in this way the substrate inhibition can be kept at a minimum level in fed-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 kept low. The substrate consumption rate can be calculated from measured factors such as carbon dioxide (Roehr, 2000). The basic concept behind the success of this technique is the capability of in situ detoxification by the cells. Since the yeasts have a limited capacity for the conversion of the inhibitors, the achievement of a successful fermentation strongly depends on the feed rate of the hydrolysate. At too high feed rate, using an inhibiting hydrolysate, both ethanol production and cell growth can be expected to stop whereas at a very low feed rate the hydrolyzate may still be converted but at a very low productivity, which it was experimentally confirmed (Taherzadeh et al., 1999).

2.12.3 Continuous process

In a traditional continuous cultivation, nutrients are continuously supplied to the bioreactor 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 terms of 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 cell growth that corresponds to the dilution rate, otherwise washout occurs. This problem can be 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 its fermentative capacity (Brandberg, 2005).

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2.13 Overall process of ethanol production from lignocellulosic material

A generally simplified of ethanol production process from lignocellulosic materials is shown in Fig 5. The lignocellulosic materials initially are milled for size reduction and then are hydrolyzed to obtain fermentable sugars, several by-products may be released in this stage, if it is highly toxic a detoxification step is necessary prior to fermentation. The hydrolysates 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 by molecular sieves. Ethanol normally required about 95% wt/vol, which can be achieved by distillation. Very pure ethanol can be obtained by extractive distillation and azetreopic columns 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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2.14 Fermentation s microorganism

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

2.14.1 Yeast (Saccharomyces cerevisiae)

S. cerevisie is one of more than 1000 validated yeast species belonging to the fungi kingdom. It is a unicellular eukaryote. Species of the genus Saccharomyces specialized in growing on sugars. Typically yeast can be found on fruits, plants also in soil and in seawater (Rose and Harisson, 1993). Yeast cells are round to oval and they have a diameter about 5-10m, most yeasts are reproduced by budding, the maximum number of bud scars found on growing cells is around 25, and the doubling time of cells can be around 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. cerevisie can easily withstand 10-15% ethanol (Cassey and Ingledew, 1986). It has a high productivity and high ethanol production yield, it s resistant to inhibitors and is also tolerant to low pH. Its robustness makes is a suitable organism for fermentation of lignocellulosic hydrolysate. One of the disadvantages of S. cerevisiae is that it doesnt naturally ferment arabinose and xylose (pentoses), despite of glucose and mannose. In agricultural residues and hardwood the amount of pentoses is high and therefore an efficient pentose fermenting microorganism is necessary if these raw materials are used. 2.14.1.1 Dissolved oxygen (DO) and substrate inhibition effects on Saccharomyces cereviseae. For growth and maintenance of yeast cells oxygen is a necessity and yeast cells can not stay alive more than 4 or 5 generations without oxygen (Tuite and Oliver, 1991), unless the ergestrol and twin (as fatty acid sources) be added to the medium. Complete oxidation 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 energy than fermentation and therefore is the preferred process. Respiration of glucose by yeast represented below: C 2 H12 O 6 6 CO 2 + 6 H 2 O fully aerobic (respiration)
G= - 686 kcal

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Many Saccharomyces species are sensitive to glucose and their respiration is repressed in the presence of a concentration of glucose greater than 1.0g.l 1 , under such condition biomass yield decrease and ethanol will be produced. This is known as a Crabtree effect or contre-pasture effect. In a study of the crabtree effect in various yeast strains, growing on a medium containing 30g.l 1 glucose, seven of eight Sacchromyces species tested gave a positive crabtree effect (Tuite and Oliver, 1991). C 2 H12 O 6 2 C 2 H 5 OH + 2 CO 2 Fully anaeroic (fermentation) G= -54 kcal

In the brewing industry the specific growth rate, viability and yield of the Saccharomyces species employed have been found to increase with the level of oxygen concentration in the wort for the levels of up to 20% saturation, as the saturation level is necessary for yeast 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 to both processes under aerobic and anaerobic conditions (Chen and Gutmanis, 1976; Kunkee and Ough, 1966). It can affect the permeability and composition of yeast cell membranes and can also shift the equilibrium in carboxylation/decarboxylation reaction in the metabolic pathways of the yeast. The inhibitory effects are greater in high ethanol concentration 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 to lower the pH to (3.5-4.5) in order to decrease the risk of bacterial contamination during the cultivation period, but the pH shouldnt be less than 3.5 because it values the color of the yeast produced and if sucrose is the carbon source, the yeast invertase activity maybe affected (Tuite and Oliver, 1991). During fermentation of inhibitory hydrolysates, a higher pH causes inhibitory acids to dissociate, 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 during the fermentation phase.

2.14.1.4 Temperature
The optimum temperature for maximum growth rate is strain dependent and lies generally around 28-35C. However the tolerance limit for S. cereviseae is 40C and growth around this temperature cause disruption of fatty acids synthesis. In a commercial 20

manufacture of Saccharomyces yeast the temperature initially maintained at 25C but is allowed to rise gradually to 30C by the end of the fermentation. At current work the temperature 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 medium is 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 of some nutrients which are explained here. Nitrogen (N): Yeast have a nitrogen content of around 10% of its dry weight hence nitrogen is an important constituent of any growth medium many inorganic ammonium salts have been found to promote the growth of S. cereviseae which the most efficient one is ammonium salt, totally around 50 Mm nitrogen is needed for an optimum growth medium. Phosphorus (P) Phosphorus is needed for synthesis of lipids and nucleic acids and maintaining 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 H 2 PO4 (minus). KH 2 PO 4 is normally used as the source of phosphorus as well as H 3 PO 4 (Tuite and Oliver, 1991). Sulphur (S): Saccharomyces species can obtain the sulfure they require from inorganic sulfate, sulfite or thiosulfite which are reduced to amino acid methonin in the cell wall. Sulfur constitutes about 0.45 of the dry weight of yeast cells. Salts as (NH4)2 SO4 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 role in proton transport, thus having a role in the control of intracellular pH. 30 Mm is the required concentration for an optimum growth medium (Dahlin, 2000; Lalander, 2002). Magnesium (Mg): Many important enzymes of the glycolysis require magnesium as a cofactor .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 ribonucleic acids in the cells. Inositol: Inositol is a key growth factor for Saccharomyces, and deficiency of this vitamin can lead to less cell division and morphological changes within the cell wall. Additional vitamins: Pantothenate is essential for all strains of S. cereviseae (Wiliams et al., 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 and Oliver, 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. cereviseae is a unicellular eukaryote which can reproduce both sexually (mioses) and asexually by budding (mitoses). Yeast has two mating types, called a and . when grown on rich medium, two haploid yeast cells with opposite mating types merge to form a diploid cell. Meioses and spore formation can therefore be induced by alternation of the culture conditions. A culture media with a high level of acetate and low concentration of dexterose and nitrogen induces meioses in which four haploid spores are created. The whole 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 driving energy and as building blocks for cellular components. Organic sources that can be used by 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 by facilitated 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 respiratory metabolism 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 an intracellular energy transporter. The (reversible) reduction of ATP to ADP releases free energy. Cells obtain ATP from their controlled chemical breakdown of glucose to two pyruvate molecules, which under aerobic conditions can be a dominating source of intracellular energy.
This process is referred to as glycolysis. Once pyruvate is formed it can be processed in several different ways like in TCA cycle (kerebs cycle); this is referred as an aerobic respiration. However, when oxygen is limiting other othetr metabolic pathways must be used to deal with pyruvate. The fermentative path from pyruvate begins with decarboxylation by pyrovate decarboxylase producing acetaldehyde. Acetaldehyde is then reduced to ethanol with NADH being oxidised to NAD+ by the action of alchohol dehydrogenase. Consequently, the overall pathway leading from glucose to ethanol is redox neutral, since NADH formed in connection to oxidation of glyceraldehyde-3phosohate 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 as Pichia stipis and Pachysolen tannophilus , Candida shehatae are able to ferment xylose naturally., the disadvantages of these yeasts is that they are sensitive to ethanol and inhibitors, and require carefully monitored microaerophile conditions and unable to ferment at low pH, also shown that P. stipis and P. tannophilus are more sensitive to furfural than S. cerevisie, which can be a problem under industrial conditions (where low pH is sometimes used to inhibit bacteria). However xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from P. stipis have been successfully transferred to strains of S. cerevisie and then produced a strain of S. cerevisie that can also utilize xylose (Wahlbom et al, 2001). This kind of recombinant strains has low ethanol yield and a tendency to relatively high by-product formation.

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2.14.2 Bacteria
Zymomonas mobilis is naturally able to produce ethanol with a high productivity but it has some draw backs, it has a narrow substrate range and can not consume mannose, galactose or xylose and also it s sensitive to inhibitors (Dien et al, 2003).
There is another bacteria, Escherichia coli that has a broad substrate range and is able to convert glucose, mannose, galactose, xylose and arabinose into pyrovate and then pyrovate change to ethanol, acetic acid, lactic acid and formate therefore the ethanol yield is much lower than S. cereviae because the product is not only ethanol. These native strains of E. coli are sensitive to inhibitors too, but there are a lot of engineered E. coli that are able to produce ethanol with a high yield (Dien et al, 2003).

2.14.3 Filamentous fungi

Filmentous fungi belonging to genera such as Rhizopus, Mucor are capable of assimilating hexoses and pentoses. Especially M. heimalis and M. indicus have been shown to be good ethanol producers (Millati et al, 2005), however their pattern of growth makes some problems; they cause increased viscosity by attaching to the growth medium.

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CHAPTER 3
Materials & methods

3.1 Chemical and reagents

(NH4)2 SO4 , KH 2 PO 4 , MgSO4.7H2O, Peptone, Glucose, Agar, Yeast extract, Antifoam, Methylene blue, beet molasses solution, Vacuum evaporated hydrolysate, High pressure evaporated 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 was built in 2003-2004 in rnskoldsvik, Sweden, the plant construction enables both weak acid hydrolysis and enzymatic hydrolysis, the daily capacity of the plant is 2 tones of dry mass and production of 400-500 liters of ethanol. The wood hydrolysate used for this work 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 high pressure evaporation.

3.2.1 Initial hydrolysate

The raw material, wood chips from spruce, arrives in covered containers to the plant. A fan blows the raw material via a cyclone to a scale on the roof of the plant, and from there it is fed into the first process step. The first step in the process is pre-steaming. Presteaming 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 to remove particles from the excess steam. Chemical substances in the gas phase can also

25

be removed from the gas steam in the pre-steaming scrubber. If the chemicals are not removed during this step, then they stay in the stream and get transported to the water scrubber. 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 the temperature is regulated between around 170-200C. Under such conditions mainly hemicellulose is hydrolyzed, and the released sugars are dissolved in the liquid phase. This water-sugar solution is separated from the solid residues by filtration and is then transported to the detoxification step. The solid residues, containing most cellulose and lignin, were transported by transportation screws to reactor 2. From this reactor there is a gas flow to the SO2 scrubber. In reactor 2 the conditions are harsher. The temperature in this reactor is around 200230C. Solid material is fed to the top of the reactor. Also the hold-up time can be regulated. While mainly hemicellulose is hydrolysed in reactor 1 also cellulose is hydrolysed to a large extent in reactor 2. The harsh conditions cause formation of byproducts to decomposition of lignin and reactions involving the sugars. This does not only decrease the sugar yield, it also hampers the fermentation since several by-products are inhibitory to fermenting organisms. When the slurry of lignin and cellulose has passed the reactors most of the cellulose is hydrolyzed and the remaining slurry is transported to the membrane filter press. The purpose of the press is to separate the liquid hydrolysate (e.g. sugars) from the solid residues. The mix of the hydrolysate and solid residues is pumped into the press. When the 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 is transported to the environmental tank (collection tank for the waste water). The hydrolysate is then transported to the detoxification unit. The remaining material in the press is mostly lignin material and is collected and later incinerated. This increase in pH reduces the problem of inhibitors in the fermentation process. After detoxification the neutralized hydrolysate is ready to be fermented (the composition of SEKAB hydrolysate is 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 more than 4% of ethanol in the fermentation broth. Therefore, the hexose concentration in the hydrolysate must be at least 100g.l 1 . This may be difficult to reach in the pilot plant, and possibly also on industrial scale. Evaporation can then be used, both as a method to simulate lower water addition and as a unit operation on industrial scale. During the vacuu 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 the amount of the hydrolysate was reduced to 1/3 of the original amount. No foaming,

26

incrustation, precipitation or odor was observed during the evaporation. The pH was kept above 2.1. The high pressure evaporation was performed at a temperature 107.50.5 with a 1.3 bar pressure, and the pH was kept above 2.1. The amount of hydrolysate was reduced to 1/3 of the original amount. (The complete results of the evaporation are available 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 (Domsj Fabriker AB, rnskldsviksSweden) and registered at the culture collection in university of Gteborg (Sweden) as CCUG 53310 was used in all experiments. This yeast exhibits two advantages. It has a strong ability to flocculate which allows for quick sedimentation. It is also highly resistant to the inhibitors in the hydrolysate. Flocks had average 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 additional carbon source.

3.4 Yeast start culture medium

The medium for the inoculums culture was the same for all experiments. It was prepared in 300-ml Erlenmeyer flasks, one of them containing 2.2 g of glucose in 50 ml water and the other containing 0.342 g (NH4)2 SO4 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). The other one was closed with foil. The flasks were sterilized for 15 min at 121C. After sterilization the content of the two flasks were mixed, which resulted in 100ml culture medium. Yeast colonies from an YPD plate containing flocculating yeast CCUG 53310 was added to the flask. The flasks were placed in a linier shaker bath set at 30 C with 170 rpm for 24 hours.

Fig. 7. Inoculums culture for yeast cultivation.

27

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 and complex additives must be used. Molasses with additional nutrients can be a good option for yeast cultivation on large scale. Since this experiment was performed for an industrial company, a complex medium of beet molasses was used for yeast cultivation. The beet molasses (supplied by Danisco, Denmark) contain about 45-50% sugar, the sugar is predominantly consists in sucrose. One of the disadvantages associated with beet molasses is that 0.5-3 % of the sugar is raffinose, a thrisaccharid that consist of fructose, glucose and galactose. Since S. cereviseae lacks -galactosidase activity, the raffinose is only partially hydrolysed. An invertase, 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 beet molasses is limited in biotin (V itamin H or B7) for cell growth; hence it may need to be supplemented with a biotin source. The non-sugar content of beet molasses includes many salts as calcium, potassium, oxalate and chloride.

Table 5. Composition of beet molasses. Dry matter 76%

Sugar

47% (DW)

Sucrose, glucose, fructose, raffinose Galactinol (inositol source)

46% 0.5-3% 0.2%

pyrrolidone carboxylate Organic non-sugar 30% (DW) 12% glu/gln betaine D, L-lactic acid Citric acid Malic acid Volatile acids

3% 0.3%

8%

4% 0.5% 0.6% 3%

Salts 10% (DW)

M-inositol Vitamins CA-panthotenate Biotin

1.3 g. kg 1 36 mg. kg 1 20 g. kg 1

28

3.5.2 Batch cultivation of yeast

The batch fermentations started with 600ml water plus 35g beet molasses, resulting in a 50g.l 1 molasses solution. Approximately 2ml of antifoam was injected into the bioreactor 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 (NH4)2 SO4 , 3.5g.l 1 KH 2 PO 4 , and 0.75g.l 1 MgSO4.7 H2O was prepared as an additional nutrient in an Erlenmeyer flask and was 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 was prepared. 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 the batch cultivation 2l.min 1 air was sparged into the bioreactor. The pH was regulated at 4.5-5.0 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. Ports were available for addition of alkali, air, inoculums culture. Agitation was achieved by an impeller. The lowest possible volume was 0.7 l.

29

3.5.3 Fed-batch cultivation of Yeast

Fed-batch fermentation started with the addition of 0.85 l (38g.l 1 ) autoclaved molasses solution after 24h of batch cultivation. The molasses solution was fed into the fermentor by 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 the fermentor; the agitation rate was kept on 500 rpm. After two days of cultivation the favorable amount of yeast was achieved and the volume of the fermentor reached to 1.55 l (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 the fermentability of the concentrated hydrolysate, allowing for comparisons between the fermentation 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 vacuum evaporated hydrolysate and high pressure evaporated hydrolysate respectively. In experiment 3 the initial amount of yeast was increased and in experiment 4 the amount of yeast was kept regular and the feed rate was decreased.

30

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

This experiment (carried out three times) was performed with the vacuum evaporated hydrolysate. 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 of air). The batch phase, which lasted 24 hours, started with 0.7 l molasses solution with 50g.l 1 concentration. The second day a fed-batch cultivation started with molasses solution (concentration: 38g.l 1 , 1 liter), which increased the volume of the bioreactor to 1.55 l (at 48h). Initial dilution rate: ID= 0.05h 1 , Initial volume: IV= 0.7 l). pH was kept between 5-6 by addition of 2-M NaOH. The temperature was kept at 30 , the stirrer worked 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 for 18h(ID= 0.22h 1 ), as the diagram shows, the volume at the end of this stage reached to 3.5 l. 3.7.3 Resting & airing: At the end of feeding the pump switched off and the bioreactor worked in a batch mode for 3h, thereafter the content of the bioreactor was sucked to 0.7 l and with the aim of biomass growth air was sparged into it (2l. min 1 ) for 2h. 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 the bioreactor switched off and by this way one experiment was done ( this experiment repeated for three times).

4.2 3.5 Volume (l) 2.8 2.1 1.4

batch 50 g/l fed-batch 38 g/l 2 h aeration 3 h resting 3 h resting 18 h feeding

18 h feeding

0.7 0 0 24 48

72 Time (h)

96

120

Fig. 10. Diagram of volume versus time. Yeast production (aerobic) lasted 48 hours, and resulting 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.
31

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

3.9 Experiment 3(A, B): Fermentation with higher initial biomass concentration.
In experiment 1 and 2 a large amount of sugars were not been utilized and remained in the bioreactor, there are two options for decreasing the remained sugar in the fermenter either increasing the amount of yeast or decreasing the feed rate, but the intention of an ethanol 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 the feed rate. In this group of experiments the yeast amount was doubled. These two experiments took 2 weeks. The methodology kept the same with experiments type1 and type 2, just for increasing the yeast amount a slightly changes were performed on the yeast cultivation part. The yeast batch cultivation with 100g.l 1 beet molasses and double 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 mode with 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 salt solution was injected in two loads, 100ml of that in batch part and the other 100ml in the second fed-batch.

32

4.2 3.5 Volume (l) 2.8 2.1 1.4 0.7 batch 50 g/l 0 0 24 48 72 Time (h) 96 120 144 2 h aeration fed-batch 38 g/l fed-batch 38 g/l 3 h resting 3 h resting

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 with lowered initial dilution rate.
After experiments with double amount of yeast, and no improvement in sugar consumption another series of experiments were defined which the feeding period was prolonged for about 50%. The methodology for yeast cultivation part was repeated like the experiments from type 1 and 2, with a regular amount of yeast about 12g.l 1 (one aerobic batch and fed-batch plus 100ml salt solution). For fermentation part, 2.8 l high pressure evaporated hydrolysate was fed into the fermentor in 27h, the initial dilution rate decreased to 0.14h 1 and then a 3h resting and 2h of aeration was repeated like the previous experiments (sucking of supernatant by syringe repeated at the end of yeast cultivation and aeration to 0.7 l volume).
4

3h resting
3 Volume (l)

3h resting 27 h feeding

27 h feeding
2

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

2h aeration
0 0 24 48 Tim e (h) 72 96 120

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.

33

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 ionexchange 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 HPLC using 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 is measured with spectrophotometer at 286.5nm after dilution. Indulin used as standard

3.11.2 Dry weight

The cells dry weight was determined via the first method that explained above. At the end of each stage the rotation rate changed from 500 rpm to 800 rpm and then duplicate 8-ml samples were taken two times during the cultivation and at least 5 times during the fermentation, the samples were centrifuged, washed with distilled water, and dried for 24h at 105 . Then the dried mass after 24h weighed. The yeast must be kept at a temperature bellower than 10 in order to prevent from their destabilization before dry weight measurement, but at current work the samples were measured immediately or were 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 the fermentation with these hydrolysates and they cant flocculate (sediment), this assumption came to mind when the pump and stirrer get switched off, and samples were taken from the supernatant and the results showed a little concentration of yeast in there (This shouldnt happen because cells are supposed to sediment after switching off the stirrer and not to be floating in the supernatant). Therefore the vitality determination processed

34

for supernatant liquor. The vitality, i.e. the fraction of metabolically active cells was estimated using methylene blue staining. An equal volume of a cell suspension and a methylene blue solution were mixed and the percentage of unstained cells (determined by light microscope examination) was taken as the fraction of vital cells (The analyses of vitality were performed by light microscope).

3.11.4 Determination of cell viability

For the same reason which it was mentioned in vitality part the viability of cells was analyzed in supernatant too. The viability, i.e. the fraction of cells able to divide was assessed by determination of colony forming units (CFU), colonies may be counted from a sample which is spread over a nutrient agar and inoculated before counting the number of colonies formed, either by naked eye (from a plate) or microscopically (from an agarcoated slide). For CFU measurements duplicate samples of 0.1ml of cell suspension were diluted to 10-106 times and spread on the agar plates which were then placed in an incubator with 30 for 48h. The colonies were counted with naked eyes. Plates with 30300 colonies were used for calculation of the viability. The results of viability measurements are not 100% right, because the flocculating yeasts tend to stick to each other so during counting few colonies look like a single colony.

3.11.5 Calculations
A biomass composition of CH1.8O0.5N0.2 was used in the carbon balance calculations (Vandijken et al., 1990). A mass balance was calculated based on gram for the calculation of ethanol, glycerol, and biomass yields, the residual sugars were taken into account. The metabolite and biomass yields were calculated from the determined concentrations at the end of the exponential growth phase.

35

CHAPTER 4
Results

4.1 Two types of evaporated hydrolysates

The hydrolysate, extracted from the pilot plant, was evaporated with the main purpose of increasing the sugar content to more than 100g.l 1 . All sugar concentrations were more than tripled, while the concentration factor of other compounds varied (Table 9). Furfural was an exception among of inhibitors and its concentration decreased drastically to 0.03 in VEH and to 0.1 in HPEH. Formic and acetic acids were also partially evaporated. If no evaporation of acetic acid occurred, one would expect more than 9.0g.l-1 of the acetic acid in the concentrated hydrolyzates, while it is only available by 3.9g.l 1 in the hydrolyzates (Table 9). On the other hand, the evaporations had negligible effect on HMF and levulinic acid, and their concentrations were increased almost by the same proportion as the sugars in the hydrolyzates. The amount of just fermentable sugars at the initial hydrolysate from the pilot plant was about 38.6g.l 1 which after vacuum and high pressure evaporation increased to 119.5g.l 1 and 129g.l 1 respectively. The amount of sugars in high pressure evaporated hydrolysate was 8% higher than the sugar content in vacuum evaporated hydrolysate. The ratio between xylose and glucose in the hydrolyzates remained at 0.44g.g 1 after either of the evaporations. It indicates neither of the evaporations decomposed the carbohydrates. There was no indication of decomposition of sugars during either of the evaporations.

36

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

Component in hydrolyzate g.l 1 Glucose Galactose Mannose Xylose Arabinose HMF Furfural Formic acid Levulinic acid Acetic acid Lignin Sulfate Original hydrolyzate g.l 1 19.67 3.42 15.66 8.74 2.11 1.46 1.00 0.67 0.69 2.99 2.15 1.70 VEH g.l 1 61.53 10.90 47.08 27.07 6.71 4.54 0.03 0.89 2.13 3.90 6.80 5.80 concentration factor 3.1 3.2 3.0 3.1 3.2 3.0 0.0 1.3 3.1 1.3 3.2 3.4 HPEH g.l 1 64.83 11.33 52.85 28.71 6.88 4.93 0.10 1.34 1.46 3.91 8.20 6.00 concentration factor 3.3 3.3 3.4 3.3 3.2 3.4 0.1 2.0 3.3 1.3 3.9 3.5

4.2 Cultivation in bioreactor

As it s shown in Table 10 after 24 h batch cultivation about 2.8g and at the end of the fedbatch cultivation about 6.5 g yeast has been gained which gave us totally 8.84g yeast in 1.5 l, however at the end of yeast cultivation the ingredient of bioreactor was sucked by syringe to 0.7 l that ended-up to 12.6g.l 1 of yeast concentration.

Table 10. Yeast cultivation results up to the start-culture from experiments with VEH and HPEH (The numbers are averages from three experiments).
Cultivation method Flask Batch Fed-batch Totally Totally Time After 24h After 48h After 72h After 72h After 72h Amount of yeast ...... 2.8g 6.5g 8.8g 8.8g Volume 0.1l 0.7l 1.5l 1.55l 0.7l Concentration . 3.9g.l
1

ID 0.05h -1

4.3g.l 1 5.9g.l 1 before sucking 12.6g.l 1 after sucking

37

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

This experiment was repeated three times with identical methodology. The FY S. cereviceae was produced with fed-batch under aerobic conditions. Then the yeast culture was used to ferment vacuum evaporated hydrolysate in two (or in one case three) cycles. Complete methodology documentation of these experiments is available in the chapter 3.

4.3.1 Fermentable sugars (mannose, glucose, and galactose)

Glucose, galactose, and mannose are regarded as fermentable sugars. See Table 11 for sugar concentrations in experiments 1(A-C). Note that the third cycle was only performed in experiment 1(C). The average consumption of glucose over the whole process was about 98% of the added glucose. The consumption of glucose by yeast was about 17.5g.h 1 during the feeding time. Apparently, the sugar consumption declines from cycle one to cycle two, since the total amount of residual sugars increase from 23.9g.l 1 in first cycle to 42.7g.l 1 in second cycle. It is also generally noted that the sugar concentration decreases faster during aeration compared to the anaerobic post fermentation before emptying. This may depend on the aeration itself but also on the increasing of cell concentration. The concentration of mannose in the feed was about 47g.l 1 . The average consumption of mannose by yeasts was about 4.6g.h 1 during the feeding phase which is less than the rate of glucose consumption and also the consumption of mannose in second cycle is about 22% less than of the first cycle. The concentration of galactose was about 11g.l 1 at the inlet flaw of hydrolysate and its total consumption was about 60% which was less than the other sugars.

Table 11. Average consumption of fermentable sugars in each stage of three experiments.
Added 1st cycle (g) Glucose Mannose Galactos 172.2 131.8 30.5 Consumed 1st cycle (%) 95.4 76.4 60.1 Added total (g) 344.4 263.7 61.04 Consumed total (%) 92.62.5 64.30.1 600.0

Average yield of aeration 97 % Average yield of resting 76.6 %

38

15 12

Glucose (g/l)

9 2 6 3 0 0 20 40 60 80 1

Time (h)

Fig. 14. Concentration of glucose in experiment 1 (A - C). Fed-batch fermentation with vacuum evaporated hydrolysate by S. cerevisiea ( Ex 1A, Ex 1B, Ex 1C) .
30 25 3 4

Mannose (g/l)

15 10 5 0 0 20 40 60 80

Time (h)

Fig. 15. Concentration of mannose in experiment 1 (A-C). Fed-batch fermentation with vacuum evaporated hydrolysate by S. cerevisea( Ex 1A, Ex 1B, Ex 1C).
6 5 3 4

Galactose (g/l)

3 2

1 1 0 0 20 0

Time (h)

40

60

80

Fig. 16. Concentration of mannose from experiments type1(A-C). Fed-batch fermentation with vacuum evaporated hydrolysate by S. cerevisea ( Ex 1A, Ex 1B, Ex 1C).

39

Volume(l)

Volume (l)

20

Volume (l)

4.3.2 Ethanol, biomass and glycerol yield

Ethanol is a product of fermentation and is produced under anaerobic conditions. The ethanol yield during the two first cycles in experiment (A-C) was 0.4101.239g.g 1 (average number based on three experiments). The ethanol, biomass and glycerol concentrations of experiment 1 (A-C) are shown in Table 12. Low acid concentrations (<100mmol.l 1 ) have been shown to increase the ethanol yield at pH 5.5, whereas the yield decreased at higher concentrations (Palmqvist et al, 1999a). It has been observed that less carbon was consumed for glycerol production in the presence of furfural, which resulted in an increased ethanol yield in the presence of 29mmol.l 1 furfural compared with fermentation in the absence of furfural (Palmqvist, 1999a). HMF may have the same effect, since its structure and detoxification mechanisms are similar.

Table 12. Yield of ethanol, biomass, and glycerol per gram of consumed sugar at the end of each experiment.
g.g 1

Ex1A 0.420 0.015 0.029 0.535

Ex1B 0.395 0.016 0.024 0.564

Ex1C 0.415 0.015 0.022 0.546

Average yield 0.4101.239 0.0150.004 0.0250.003 0.5480.020

Ethanol Biomass Glycerol CO2

For growth and maintenance of yeast cells oxygen is a necessity and yeast cells can not stay alive more than 4 or 5 generations without oxygen (Tuite and Oliver, 1991), unless the ergestrol and twin (as fatty acid sources) be added to the medium. In the current work air was added for 2 hours between the fermentation cycles. The biomass yield was low (0.015g.g 1 per consumed hexoses). One possible explanation is that weak acids are liposoluble and can diffuse across the plasma membrane. The growth-inhibiting effect on microorganisms has been proposed to be due to inflow of undissosiated acid into the cytosol(Axe and bailey, 1995; Vandijken et al,1990) also compounds like formic acid, levilinic acid HMF, furfural, acetic acid can inhibit cell growth more than the ethanol fermentation and no net growth was observed at the initial 10g.l 1 yeast when 5g.l 1 acetic acid, 10g.l 1 formic acid, 1.2g.l 1 furfural, 1.3g.l 1 HMF and 23g.l 1 levulinic caid were present in fermentation broth (Larsson et al, 1998). Glycerol is a product of fermentation by S. cerevisiae. One direction of glucose goes to glyceraldehydes and pyrovate production which results in biomass and ethanol production. The other direction goes to dehydroxyacetate and glycrol production.

40

4.4 Fed-batch fermentation with high pressure evaporated hydrolysate. Experiment 2 (A, B, C)
In this series of experiments the condition was kept like in experiment 1, but the vacuum evaporated hydrolysate was switched to high pressure hydrolysate. The experiments were, like previous and designed to produce 12g.l 1 yeast for fed-batch fermentation.

4.4.1 Fermentable sugars (mannose, glucose, and galactose)

The hydrolysate is in principle the same as the one used in Experiment 1, but it was evaporated at high pressure. The total sugar concentration was slightly higher (129g.l 1 compared to119.5g.l 1 in Experiment 1, A-C).

Table 13. Average glucose, mannose, galactose consumption from three experiments in fermentation with high pressure evaporated hydrolysate.
Added 1st cycle (g) Glucose Mannose Galactose 181.5 147.9 31.7 Consumed 1st cycle (%) 99.3 84.4 16.1 Added total (g) 363.0 295.9 63.4 Consumed total (%) 97.60.0 74.30.0 16.00.0

Average yield of aeration 87 % Average yield of resting 70.6% Like in Experiment 1, the consumption of hexoses in the second cycles decreased drastically, compared to the first cycle. However, the drop in fermentation was smaller than in Experiment 1. The average residual hexose concentration after two cycles in Experiment 1 was 27.5g.l 1 with vacuum evaporated hydrolysate while it was 29.4g.l 1 with high pressure evaporated hydrolysate. The difference is not very large, but in conclusion these experiments do not confirm the assumption that vacuum evaporation would be better than high pressure evaporation for fermentability.

41

10

8 3 Glucose (g/l) Volume (l)

Volume (l)

6 2 4 1 2

0 0 10 20 Tim e (h) 30 40 50

Fig. 17. Concentration of glucose in Experiment 2(A-C), with feed consisting of high pressure evaporated hydrolysate( Ex 2A, Ex 2B, Ex 2C).
25 4

20 3 Mannose (g/l) Volume (l) 15 2 10 1 5

0 0 10 20 Tim e (h) 30 40 50

Fig. 18. Concentration of mannose in Experiment 2(A-C), with feed consisting of high pressure evaporated hydrolysate( Ex 2A, Ex 2B, Ex 2C).
12 4

9 Galactose (g/l)

0 0 10 20 Tim e (h) 30 40 50

Fig. 19. Concentration of galactose in Experiment 2(A-C), with feed consisting of high pressure evaporated hydrolysate( Ex 2A, Ex 2B, Ex 2C).

42

4.4.2 Ethanol, biomass and glycerol yield

The average yield of ethanol in Experiment 2 was 0.404 2.8g.g 1 (based on consumed hexoses). Production of biomass (0.015g.g 1 ) and glycerol (0.03g.g 1 ) explains some of the deviation from the theoretical level (as well as some potential measurement errors), but apparently the hexose levels decrease significantly during aeration while the ethanol and biomass concentration remain nearly constant. Thus a significant loss of carbon is lost to respiration, i.e. for production of CO2. Also there is very little renewal of cells, which means that the system is not sustainable, which is also confirmed by the increasing levels of residual sugars.

Table 14. Yields of ethanol, biomass, and glycerol per gram of consumed hexoses during both fermentation cycles
g.g 1

Ex 2A 0.376 0.018 0.028 0.557

Ex 2B 0.405 0.009 0.026 0.558

Ex2C 0.430 0.018 0.037 0.514

Average yield 0.4042.715 0.0150.005 0.0300.004 0.5430.145

Ethanol Biomass Glycerol

CO 2

43

4.5 Fed-batch fermentation with concentration. Experiment 3 (A, B)

higher

biomass

The results from the previous experiments based on regular amount of yeast (12g.l 1 ) with two different types of hydrolysates showed that, there is a lot of sugars left in the fermentor at the end of the experiments (about 27.5g.l 1 in fermentation with vacuum evaporated hydrolysate and 29.4g.l 1 after fermentation with high pressure evaporated hydrolysate). It can be argued that higher cell density should allow for more efficient uptake of sugars and a more efficient intracellular detoxification of the inhibitors. Therefore the methodology in Experiment 3 was designed to produce a higher initial cell concentration by adding more carbon source. The yeast amount was doubled (it increased from 12g.l 1 to 32g.l 1 ). The biomass doubled in 3days cultivation with beet molasses (two fed-batches and one batch phase). The complete methodology of this experiment is available in page 32.

4.5.1 Fermentable sugars (mannose, glucose, and galactose)

Sugar consumption reached to 93% after one cycle (21h) which shows improvement compare to previous experiments, however total sugar consumption in these two experiments was about 82.4% and 81%, which doesnt show significant improvement from the experiments with regular amount of yeast. In one of these experiments the volume was once kept constant after the second cycle and a sample was taken at t = 65 h. The analysis shows that the glucose and mannose concentrations had decreased.

Table 15. Average glucose, mannose, galactose consumption from two experiments in fermentation with high pressure evaporated hydrolysate+ double amount of yeast.
Added 1st cycle (g) Glucose Mannose Galactose 181.5 147.9 31.7 Consumed 1st cycle (%) 99.1 96.5 48.2 Added total (g) 363.0 295.9 63.4 Consumed total (%) 95.80.0 74.70.0 33.30.0

Average yield of aeration 97.6 % Average yield of resting 94 %

44

10 8
Glucose (g/l)

3
Volume (l)
Volume (l)
Volume (l)

6 2 4 2 0 0 20 40
Tim e (h)

0 60 80

Fig. 20. Glucose concentration during fermentation with higher (32 g.l 1 ) initial yeast concentration with feed consisting in high pressure evaporated hydrolysate ( Ex 3A, Ex 1B).
30 4

24 Mannose (g/l)

18 2 12 1

0 0 20 40 Tim e (h) 60 80

Fig. 21. Mannose concentration during fermentation with higher (32g.l 1 ) initial yeast concentration with feed consisting in high pressure evaporated hydrolysate ( Ex 1A, Ex 1B).
12 4

9 Galactose (g/l)

0 0 20 40 Tim e (h) 60 80

Fig. 22. Galactose concentration during fermentation with higher (32g.l 1 ) initial yeast concentration with feed consisting in high pressure evaporated hydrolysate ( Ex1A, Ex 1B).
45

4.5.2 Ethanol, biomass and glycerol yields

The ethanol, biomass, glycerol yields of experiment 3(A, B) are shown in Table 16. Based on increased sugar consumption the ethanol increased after one cycle, but after two cycles there was no more ethanol produced. The sample taken at t = 65 h confirms that more fermentation time apparently allows for a better uptake of hexoses and a higher ethanol concentration.

Table 16. Yields of ethanol, biomass, and glycerol per gram of consumed hexoses at the end of each experiment.
g.g 1

Ex3A 0.407 0.002 0.030 0.564

Ex3B 0.415 0.022 0.030 0.531

Average yield 0.4110.375 0.0190.012 0.0300.012 0.5470.165

Ethanol Biomass Glycerol

CO 2

46

4.6 Fed-batch fermentation with lower initial dilution rate. Experiment 4 (A, B)
In this series of experiments the conditions were kept like Experiment 1 and 2. The flocculating yeast was cultivated with batch and fed batch methodology and the high pressure evaporated hydrolysate, was fermented in two cycles (each one composed of 18h feeding, 3h resting, 2h aeration). The goal amount of yeast from cultivation was12g.l 1 , just the dilution rate lowered (ID = 0.14, IV= 0.7 l).

4.6.1 Fermentable sugars (mannose, glucose, and galactose)

The results from two experiments with lower dilution rate shows an improvement in sugar consumption, the sugar consumption reached to 97% after one cycle of fermentation and to 93% after two cycles of fermentation in experiment 4(A, B) .

Table 17. Average glucose, mannose, galactose consumption from two experiments in fermentation with high pressure evaporated hydrolysate and lower dilution rate.
Added 1st cycle (g) Glucose Mannose Galactose 181.5 147.9 31.7 Consumed 1st cycle (%) 99.7 99.1 75.5 Added total (g) 363.0 295.9 63.4 Consumed total (%) 99.40.1 94.50.0 47.90.1

4.6.2 Ethanol, biomass and glycerol yield

By lowering the initial dilution rate the ethanol yield increased to 0.44g.g 1 after 21h or one cycle however after two cycles the ethanol production doesnt show improvement as it was 0.40g.g 1 . Compare to previous experiments biomass growth had a great improvement and it almost doubled. During the whole process in average about 0.2g.l 1 lactic acid was produced.

Table 18. Fraction of ethanol, biomass, and glycerol per gram of consumed sugar at the end of each experiment.
g.g 1

Ex4A 0.409 0.040 0.022 0.527

Ex4B 0.392 0.06 0.033 0.513

Average yield 40.1070.869 0.0500.010 0.0270.003 0.5200.007

Ethanol Biomass Glycerol

CO 2

47

4 Glucose (g/l)

3 Volume (l)
Volume (l)

3 2 2 1

0 0 20 40 Time (h) 60 80

Fig. 23. Glucose concentration during fermentation with lower dilution rate and feed consisting of high pressure evaporated hydrolysate ( Ex 4, Ex 4).
12 4

9 Mannose (g/l)

3 Volume (l)

0 0 20 40 Tim e (h) 60 80

Fig. 24. Mannose concentration during fermentation with lower dilution rate and feed consisting of high pressure evaporated hydrolysate ( Ex 4, Ex 4).
10 4

8 Galactose (g/l)

6 2 4 1

0 0 20 40 Tim e (h) 60 80

Fig. 25. Galactose concentration during fermentation with lower dilution rate and feed consisting of high pressure evaporated hydrolysate ( Ex 4, Ex 4).
48

4.7 Comparison of results

Table 19. Comparison of important results in experiments 1-4.
Sugar Sugar Residual Produced consumption consumption sugars ethanol After 1 After 2 after 2 after 2 cycle cycles cycles cycles 35.3g/l 84.7% 78% 27.5g

Type of experiments Ex 1(A,B,C)

Y x/s
(g.g
1

Ygly/s
) (g.g
1

Yp/s
) (g.g
1

0.015 0.025 41.021 (0.004) (0.003) (1.239) 0.015 0.030 40.392 (0.005) (0.004) (2.715) 0.019 0.030 41.113 (0.012) (0.012) (0.375) 0.050 0.030 40.107 (0.010) (0.003) (0.869)

Ex 2(A,B,C)

86.7%

81%

29.43 30 13.1

41.8g/l

Ex 3(A,B)

93.5%

82%

37.4g/l

Ex 4(A,B)

97.3%

93%

44g/l

60

A
50 40 30 20 10 0 60

B
50 40 30 20 10 0

1 8

21

24

42 Time (h)

45

48

66

69

24

27

30

33

48

52

55

60

63

Time (h)

Fig. 26. Black(ethanol), white(biomass) in fed-batch cultivation of (A) VEH with 12g.l 1 biomass and ID 0.22h-1, (B) HPEH with 12g.l 1 biomass and ID 0.22h-1, (C) HPEH with 32g.l 1 biomass and ID 0.22h-1, and (D) HPEH with 12g.l 1 biomass and ID 0.14 h-1.

49

4.8 Inhibitors
It is well known that lignocellulose hydrolysate contains inhibitory compounds. In this section the inhibitor concentrations before and after fermentation in the feed are presented. The water was evaporated in 67 minutes with 80 C from hydrolysate for vacuum concentration and in 75 minutes with118C for high pressure concentration.

Table 20. Inhibitors concentration in vacuum and high pressure concentrated hydrolysate, before fermentation.
Inhibitors (g.l 1 ) Furfural HMF Acetic acid Formic acid Levulinic acid

Before concentration After vacuum concentration After high pressure concentration

1.00 0.03 0.10

1.46 4.54 4.93

2.99 3.90 3.91

0.67 0.99 1.34

0.69 2.13 1.46

Table 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.
Types of
experiments Furfural HMF Levulinic acid Acetic acid Formic acid

Ex 1 A, B, C Ex 2 A, B, C Ex 3 A, B Ex 4 A, B

<0.005 <0.005 <0.005 <0.005

95.3 0.8 97.23 1.16 90.0 95.5

26.91 27 13.3 2.8 29.7 16.0

-1.533 4 12.73 3.06 23.9 43.0

46.5 19 46.1 17 56.54 74.0

Description about inhibitors consumption is available in discussion part (chapter5).

50

4.9 Cell viability

The purpose of cell viability counts was to assess the concentration of reproducing cells in the supernatant (Purwadi et al, 2007) assumed that cells that dont flocculate may actually be dead, or at least not reproducing. So, few minutes after the stirrer had been switched off (in experiments 1-4) cell samples were taken and diluted in isotonic water and spread on agar plates. Experiments type 1 and 2 These samples were diluted 1:106 (up to six times 1:10) and a volume of 0.1 ml of diluted samples (duplicates) was spread on agar plates. After 48 h the CFU were counted. Plates with 2-300 colonies were chosen for measurement. The results are averages taken from duplicate plates.

Table 22. Results from dry weight and CFU measurements for experiments 1- 4.
12g.l 1 yeast VEH+HPEH 32g.l 1 yeast HPEH 12 g.l 1 yeast+ lower ID HPEH

CFU Dry weight Viability

2.107 1.00g.l 1 0.02%

138.107 4.60g.l 1 0.35%

173.107 0.75g.l 1 1.75%

In experiments with regular 12g.l 1 yeast, just the plates with 10-10 3 were contained yeasts. As in these experiments the yeast was just spread in plates with dilution of 10 3 to was measured and the dry measurement from the same sample showed 1.3g.l yeast which according to the references 1g.l 1 dry weight is equal to 1. 1011 Cells and no bacteria were observed. Therefore after calculations 0.02% viable cells were concluded in supernatant. This means that 99.98 % of cells in supernatant lost their ability of flocculating and they are not viable but maybe they can metabolize, which the results of metabolization also was performed which it comes later.
1

106 then the cells were counted in plate with 10 3 dilution rate. 2.10 CFU.l
7

Experiments type 3 For experiments with double amount of yeasts the same samples were taken and both dry measurement and plate counting were performed. In these experiments all plates with different dilution rates were containing yeast. Like the previous experiments plates with 10 3 dilution rate were chosen and an average from two plates gave 138. 10 7 CFU. l 1 + 8. 10 7 CFU. l 1 bacteria and dry measurement gave 4.6g.l 1 yeast and according to the

51

references this much dry weight is equal to 4. 1011 yeast. Dividing the amount of yeasts in supernatant to total amount of yeast gives 0.35% viable yeast cells which means that 99.65% of cells in supernatant lost their ability to flocculate and 0.02% bacterial cells. There was not a big progress in raising the viability of yeast cells by doubling the amount of biomass.

Fig. 27. Sample of CFU measurement with colonies.

Experiments type 4 In experiments with lower initial dilution rate and regular amount of yeast, the same measurements were done. And the average CFU in 10 3 dilution rate was about 173. 10 7 CFU. l 1 yeast cells + No bacteria cells. Dry measurement showed a 0.75g.l 1 yeast cells which gives a 1.75% viable yeast cells in supernatant. This can be inferred as a good progress in keeping more cells alive by giving them more time to metabolize the toxic hydrolysate.

Conclusion

Totally we can say that the cells which loose their viability, they loose their flocculating ability too, but this is not for sure and more researches can be done in the future works. By lowering the initial dilution rate the smaller amount of yeasts loose their viability, and dry weight samples from supernatant shows a lower amount of yeasts too.

52

4.10 Cell vitality

The same samples which were taken from the supernatant for viability measurement were taken for vitality determination as well, and pictures were taken from the samples. The centrifuged samples were stained with methylene blue and analyzed by light microscope. Colored cells are considered as dead. An average of samples from four experiments indicted that about 76% of the yeast cells in the supernatant were vital according to this method, and they had intact membranes and metabolic activity. An important conclusion can be achieved from the result of both viability and vitality measurements which it is summarized here. 76% are vital 99% non-viable cells 23% are dead Thus, while only 1% of the cells in the supernatant were viable according to CFU counts, 76% maintained metabolic capacity according to counting of methylene stained cell samples.

Fig. 28. Vitality determination of samples stained by methylen blue and analyzed by light microscope. 500 cells were counted in each sample (Blue colored = dead cells, colorless=alive).

53

CHAPTER 5
Discussion & conclusion remarks

5.1 Discussion
The results of this work show a successful increase of the sugars concentration in lignocellulosic hydrolyzates followed by successful fermentation by repeated fed-batch operation using a flocculating strain of S. cerevisiae. Both evaporations under vacuum or pressure resulted in high-sugar and fermentable hydrolyzates by fed-batch operation. These results may be interesting industrially, since higher sugar concentrations in the hydrolyzates lead to less energy consumption in the distillation and downstream processes, while fermentation can be carried out successfully with no prior detoxification. Furthermore, the successful evaporation of the hydrolyzates under vacuum and pressure can lead in designing multi-effect evaporators, in which the hydrolyzate can be evaporated with low consumption of energy. The evaporations under either vacuum or pressure used in this work did not decompose the sugars, but were able to remove the volatile inhibitors partially or completely. As long as the evaporation is performed at pH higher than 2.1 and temperature lower than 120, the risk of sugars decomposition is negligible. The boiling points of the inhibitors reported in this work at normal pressure are 161.7, 118.1, 100.8 and 245C for furfural, acetic acid, formic acid and levulinic acid, while HMF is not volatile and boils at ca. 115C at 1 mbar. Comparing these boiling points with the results presented in Table 1 indicates that volatility is important, but probably not the only factor that governs decreasing the concentration of the inhibitors, since e.g. more furfural than formic acid disappeared from the hydrolyzate during evaporations. Furfural is a strong inhibitor for bakers yeast.

54

Carboxylic acids might inhibit or enhance fermentation, depending on the cultivation conditions . Presence of acetic acid in the cultivation media can result in higher ethanol yield from sugars , while more than 5g.l-1 undissociated molecules of this acid can severely inhibit the fermentation . During cultivation of flocculating yeasts in toxic dilute-acid hydrolyzates, some yeast cells lose their vitality as well as the ability to flocculate . This means that the viability and vitality are high with the flocculated cells, but probably not with the non-flocculated cells. In this work, the cells in the supernatants had 76% vitality, and thus were still able to produce ethanol.

5.2 Conclusions
Evaporation of lignocellulosic hydrolyzates with low sugar concentration and subsequent fed-batch cultivation by flocculating yeast may help to fulfill the demands for industrial production of ethanol from lignocellulosic materials. The evaporation increases the sugar concentrations in inverse proportionality to the volume. It does not decompose the sugars in either VEH or HPEH, but removes part of the toxic components. The results showed no significant difference between the vacuum and high-pressure evaporation of hydrolyzates. The flocculating yeasts tolerate the remaining inhibitors in fed-batch operation, and are able to consume most of the fermentable sugars and produce ethanol in high concentration, although with a low biomass yield.

5.3 Future work

Concentrating of lignocellulosic hydrolysate can fulfill the demand of high sugar concentration in industrial scale and also it is possible to ferment this hydrolysate with a fair yield of ethanol, but since after concentrating the amount of inhibitors increase in this hydrolysate as well, in the future a suitable way of detoxification which is economical and doesnt degrade the carbohydrates can be investigated.

55

Appendix A
Inhibitors figures
5 HMF, levulinic, acetic, formic acid (gl) 4

3 Volume (l)

3 2 2 1

0 0 20 40 Tim e (h) 60 80

Fig. 29. HMF, formic acid, levulinic acid, acetic acid concentration in experiment 1(A, B, C) with regular amount of yeast and vacuum evaporated hydrolysate. () HMF, () acetic acid, () formic acid, () levulinic acid.

4 HMF, formic acid, levulinic acid, acetic acid (g/l)

3 Volume (l)

0 0 10 20 Time (h) 30 40 50

Fig. 30. HMF, formic acid, levulinic acid, acetic acid concentration in experiment 2 (A, B) with regular amount of yeast and high pressure evaporated hydrolysate. () HMF, () acetic acid, () formic acid, () levulinic acid.
56

4 HMF, levulinic acid, formic acid, acetic acid (g/l)

3 Volume (l)

0 0 20 40 Tim e (h) 60 80

Fig. 31. HMF, formic acid, levulinic acid, acetic acid concentration in experiment 3(A, B) with double amount of yeast and high pressure evaporated hydrolysate. () HMF, () acetic acid, () formic acid, () levulinic acid.

4
HMF, levulinic acid, acetic acid, formic acid (g/l)

3
Volume (l)

0 0 20 40
Tim e (h)

0 60 80

Fig. 32. HMF, formic acid, levulinic acid, and acetic acid concentration in experiment 4(A, B) with lower dilution rate + regular amount of yeast and high pressure evaporated hydrolysate. () HMF, () acetic acid, () formic acid, () levulinic acid.

57

Table 23. Performance condition of experiments

Appendix B

Yeast cultivation Batch Total time ID IV Feed rate Feed volume Feeding time Resting Aeration 24h . 0.7 l . . Fed-batch 24h 0.05 1/h 0.7 l 35.5 ml/ h 0.85 l 24h . 24h

Hydrolysate fermentation Fed-batch(1st cycle) 24h 0.22, 0.14 1/h 0.7 l 155.5, 103* ml/h 2.8 l 18, 27* h 3h 2h Fed batch (2nd cycle) 24h 0.22, 0.14 1/h 0.7 l 155.5, 103* ml. h 2.8 l 18, 27*h 3h N.P

58

Not performed * Experiments 4(A, B) with lower ID.

Nomenclature
CFU Cf HPEH ID VEH V Y x/s Ygly/s Yp/s Colony forming unit Concentration factor High pressure evaporated hydrolysate Initial dilution rate Vacuum evaporated hydrolysate Vacuum Yield of biomass Yield of glycerol Yield of ethanol

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