Performed at: SEKAB E-Technology Published by: Högskolan i Borås Anahita Dehkhoda
Concentrating lignocellulosic hydrolysate by evaporation and its fermentation by repeated fed- batch using flocculating
Saccharomyces cerevisiae
MASTER THESIS IN INDUSTRIAL BIOTECHNOLOGY
Concentrating lignocellulosic hydrolysate by evaporation and its fermentation by repeated fed-
batch using flocculating Saccharomyces cerevisiae
Anahita Dehkhoda
Performed at SEKAB E-Technology Örnsköldsvik, 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
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 Borås School of Engineering SE-501 90 BORÅS
Telephone +46 033 435 4640
Examiner: Prof. Mohammad Taherzadeh
Supervisors: Dr. Tomas Brandberg, Prof. Mohammad Taherzadeh Client: SEKAB E-Technology, Örnskoldsvik, Sweden
Keywords: Concentrating, lignocellulosic hydrolysate, evaporation, flocculating yeast, fermentation, ethanol.
A CKNOWLEDGEMENT
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 Hägglund. 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 Torbjörn van der Meulen for giving me the opportunity to work at SEKAB.
Thanks to Carl-Axel Lalanderin helping with quick preparation of any equipment which I ran out of. I would also like to recognizeStaffan 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 fiancé, who’s presence and care kept me going. This work was supported by SEKAB E- Technology Örnskoldsvik, Sweden and all experimental works was performed in laboratory scales at SEKAB E-Technology.
Master thesis
Industrial Biotechnology Borås University and
SEKAB E-Technology, Sweden
A BSTRACT
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 108°C and 1.3 bar) would be more difficult to ferment than vacuum evaporated hydrolysate (evaporation carried out at 80°C 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 0.40±0.01g.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 0.44±0.01g.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.
P UBLICATION
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)
C ONTENTS
Chapter 1: Introduction………….……….1
1.1 Background of ethanol production...….1
1.2 Outline of the thesis……… ...……2
Chapter2: Bioethanol ……….………..
32.1 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
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 ………...
253.1 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
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 yield………47
4.7 Comparison of results………49
4.8 Inhibitors………....50
4.9 Cell viability……….51
4.10 Cell vitality……….53
Chapter 5 (Discussion & Conclusion remarks)………....
545.1 Discussion...54
5.2 Conclusion………..….55
5.3 Future work………..55
Appendix A……….…...56
Appendix B……….…...58
Nomenclature………...59
References……….….59
L IST 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. cerevisea………39
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
18. Concentration of mannose in Experiment 2(A-C), with feed consisting of high pressure evaporated hydrolysate………42 19
.
Concentration of galactose in Experiment 2(A-C), with feed consisting of high pressure evaporated hydrolysate………42 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 HPEH………47 26. Ethanol, biomss, glycerol yielde comparison ………...49 27. Sample of CFU measurement with colonies………52 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 HPEH………56 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……….….57L IST 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 evaporation…………37
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 fermentation………50
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 experiments………58
C HAPTER 1
I ntroduction
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
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.
C HAPTER 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).
Table 1. Annual production of ethanol in world based on m3 (www.ethanolrfa.org/industry/statistics).
Country 2004 2005 2006
USA 13362300 15776800 18074500
Brazil 15078420 15639900 16616700
China 3643920 3795120 3844260
India 1746360 1697220 1897560
France 827820 907200 948780
Russia 748440 748440 646380
2.2 What is ethanol and where can be used?
Ethanol (C2H5OH) or ethyl alcohol is an important organic chemical which can be used in various ways. It is colorless, flammable, volatile and soluble in water and non-polar. 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
CO2 is a so–called 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 CO2 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 5˚C (Dickenson and Ciceron, 1986).
A major part of the increase in the atmospheric CO2 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 CO2 content (Wingren et al, 2003) for instance emission of CO2decreases by 20% for E10 (10% ethanol, 90% gasoline), but higher ethanol blend don’t 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 CO2 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, good sources for ethanol production
Lignocellulose (wood, grasses and municipal solid waste) is an attractive feedstock for ethanol production because of its availability at low cost and at large quantities.
Approximately 50% of the biomass in the world is lignocelluloses and it has an estimated annual production of 10-50.10¹²kg (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) don’t consume pentose.
Table 2. Hardwood and softwood composition.
Hemicellulose Material
Cellulose
%
Hemicellulose
%
Lignin
%
Hardwood 45-51 23-28 19-24
Softwood 41-42 24-31 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 β (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).
It’s 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 D- galactoses, D–glucose, 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 (Sjöström 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 building–blocks in softwood carbohydrates (Sjöström 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 3- carbon 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, trans- sinapyl 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 non–cell-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 (Sjöström 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)
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 ºC), 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 ºC and the remaining cellulose subsequently hydrolysed at more severe conditions at 90-230 ºC (Faith , 1945).
Table 3. Comparison between dilute-acid and concentrated acid hydrolysis (Taherzadeh and Karimi, 2007).
Hydrolysis method Advantages Disadvantages
Concentrated acid Process
-operated at low temperature
- High sugar yield
-high acid consumption -high energy consumption for acid recovery
-longer reaction time (e.g.
2-6h) -equipment corrosion
Dilute acid
Process
-low acid consumption -short residence time
- operated at high temperature -low sugar yield -equipment corrosion
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 β-(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 β-(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 β- glucosidases (Gusakov and Sinitsyn, 1992).
Table 4. Comparison of enzymatic and acid hydrolysis (Roehr, 2000).
Acid Enzyme
1. Non-specific catalyst therefore will delignify material as well as hydrolyze cellulose.
Specific macromolecule catalyst, therefore extensive physical and chemical
pretreatment is necessary to make
cellulose available for degradation.
2.Decomposition of hemicellulose to
inhibitory compounds
production of clear sugar syrup ready for subsequent anaerobic fermentation
3.Harsh reaction condition therefore necessary increased costs for heat and corrosion resistant equipment
Run under mild conditions
(50ºС,atmospheric pressure ,pH4,8)
4.Relatively low yield of glucose High glucose yield
2.9 Inhibitors (by-products released during hydrolysis)
One of the factors that complicate the fermentation of lignocellulosic hydrolysates is 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 hetero–polymers 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.
The by products which produced during the hydrolysis can be divided to: Organic acids, furan compounds, phenolic compounds.
2.9.1
Organicacids
A large number of alphatic acids are present in dilute acid hydrolysates originated from wood extractives, lignin degradation and sugar degradation. Acetic acid is a major 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 (Jönsson 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
concentration above 1g.l−1 was found to decrease significantly the CO2 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 CO2 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.
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
Hemicellulose → acetic acid
(11-37%) → pentoses→ furfural ↓
Formic acid
→ hexoses → HMF ↓ Formic acid Levulinic acid
Lignin → phenolic compounds (17-32%)
Cellulose (32-54%) ↓ Glucose ↓ HMF ↓
Formic acid ↓
Levulinic
Extractives (1-5%) → phenolic compounds and wood resin Ash
(0-2%) → various inorganic Compounds
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 ºC, while for hydrolysis it is about 50 ºC, 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 inoculum’s 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 (µ) is constant and it is at a maximum (µ max) for
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).
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.
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 5-10µm, 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 doesn’t 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:
6 12
2H O
C →
on) (respirati
aerobic
fully 6CO2+ 6H2O ∆G= - 686 kcal
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).
6 12
2H O
C →
ion) (fermentat
anaeroic
Fully 2C2H5OH + 2CO2 ∆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 shouldn’t 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 28-35°C. However the tolerance limit for S. cereviseae is 40°C and
manufacture of Saccharomyces yeast the temperature initially maintained at 25°C but is allowed to rise gradually to 30°C by the end of the fermentation. At current work the temperature kept on 30±0.1°C 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 ofH2PO4 (minus). KH2PO4 is normally used as the source of phosphorus as well as H3PO4 (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)2SO4 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,
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 Μm (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 α. 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-3- phosohate in the upper part of gycolysisis reoxidesied by the formation of ethanol (www.scq.ubc).
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.
