is the bacterium Zymomonas mobilis which has a higher growth rate, is more ethanol- tolerant than yeasts but still only metabolizes glucose, fructose and sucrose.
crops are difficult to grow, lignocellulose is an attractive option. Lignocellulose can be obtained from trees species, wood residues, sawdust chips, construction residues, municipal wastes, paper, sewage sludge, maize stover, straws and grasses such as Miscanthus, switchgrass, sorghum, reed canary grass, bagasse, sugarbeet pulp, soft- wood, wheat straw, rice straw, pulp and paper mill residue, forest thinnings, munici- pal solid waste, winter cereals, and recycled paper.
Unfortunately, there are no ethanol-producing yeasts, other than genetically modified (GM) strains, that are capable of metabolizing starch and lignocellulose and only a few bacteria are capable of metabolizing lignocellulose. To make lignocellulose and starch suitable for fermentation both need to be converted into sugars in an inexpensive process.
Fig. 6.9. The production of ethanol via the Entner–Doudoroff pathway.
Glucose
Glucose-6-phosphate
6-Phospho-gluconolactone
6-Phospho-gluconate
2-Oxo-3-deoxy-6-phosphogluconate
Pyruvate ATP
NADP ADP
NADPH
Acetaldehyde
Ethanol
Carbon dioxide NADH
NAD
Lignocellulose
Lignocellulose consists of three polymer types, cellulose, hemicellulose and lignin, which are the main constituents of plant cell walls (Fig. 6.4). The primary cell wall consists of cellulose fibres embedded in a polysaccharide matrix of hemicellulose and pectin. Cellulose is the most abundant plant compound and the second most abundant is lignin which provides mechanical support and protection in plants. The composition of some lignocellulose sources is given in Table 6.7 in terms of lignin, cellulose and hemicellulose. Cellulose is a polymer of glucose linked together by 1,4-glycosidic bonds. Hemicellulose is heterogeneous polymers with a backbone of 1,4-linked xylose residues but contains short side chains containing other sugars such as galactose, ara- binose and mannose. Xylose is the predominant sugar in hardwoods and arabinose in agricultural residues.
Lignin is a highly branched polymer of phenyl-propanoid groups such as con- iferyl alcohol (Fig. 6.4). Cellulose represents 40–50% of dry wood, hemicellulose 25–35% and lignin 20–40% depending on the plant type. Although lignocellulose is abundant, it cannot be metabolized by yeast and therefore needs to be broken down to its constituent sugars before it can be used. In addition not all yeast can metabolize the pentose sugars derived from the hemicellulose. The composition of agricultural lignocellulose sugars is shown in Table 6.8. Saccharomyces cerevisiae can ferment
Table 6.7. The composition of various biomass and waste materials. (From Hamelinck et al., 2005; Ballesteros et al., 2004; Champagne, 2007.)
Substrate Cellulose Hemicellulose Lignin Hardwood eucalyptus 49.5 13.1 27.7 Softwood pine 44.6 21.9 27.7
Switchgrass 32.0 25.2 18.1
Wheat straw 35.8 26.8 16.7
Cattle manure 27.4 12.2 13.0
Pig manure 13.2 21.9 4.1
Poultry manure 8.5 18.3 4.9
Table 6.8. Sugar composition as a percentage of some agricultural lignocellulose materials. (Adapted from van Maris et al., 2006.)
Sugar Maize stover Wheat straw Bagasse Sugarbeet pulp Switchgrass Fermented by yeast
Glucose 34.6 32.6 39.0 24.1 31.0
Mannose 0.4 0.3 0.4 4.6 0.2
Galactose 1.0 0.8 0.5 0.9 0.9
Not fermented
Xylose 19.3 19.2 22.1 18.2 0.4
Arabinose 2.5 2.4 2.1 1.5 2.8
Uronic acids 3.2 2.2 2.2 20.7 1.2
glucose, mannose and galactose, but not the other sugars. Depending on the source of lignocellulose the sugar produced will change. Glucose and xylose are the main sugars in the agricultural lignocellulose except in switchgrass. Investigations are under way to find organisms which can ferment these other sugars and produce etha- nol or to genetically manipulate yeasts to be able to metabolize these sugars.
Lignocellulose is difficult to break down into sugars, but a number of technolo- gies are under investigation including enzymes. Because of the presence of hemicel- lulose and lignin and the crystalline nature of cellulose in lignocellulose some form of pretreatment is required before enzymatic or chemical hydrolysis. These pretreat- ments are shown in Fig. 6.10, and include carbon dioxide, steam and ammonia explo- sion, mechanical grinding, acid, white rot fungi treatment and ozonolysis.
Steam explosion
High-pressure and high-temperature steam can be used to treat the lignocellulose material where hemicellulose is hydrolysed by acids released during steam treatment.
Acid addition increases the sugar yields but sulfuric acid can yield sulfur dioxide which can be inhibitory to further treatment. Steam treatment is less energy intensive than mechanical disruption.
Mechanical Carbon dioxide explosion Steam explosion Ammonia explosion Acid
Conc acid Dilute acid Cellulase enzymes
Glucose
Lignin Hemicellulose
White rot fungi
Ozonolysis Alkali
Cellulose
Cellulose Lignin
Lignocellulose Pre-treatment
Pentoses e.g. Xylose Treatment
of cellulose
Fig. 6.10. The pretreatment of lignocellulose and subsequent hydrolysis prior to fermentation.
Mechanical disruption
The grinding of lignocellulose materials into small particles increases the surface area and allows subsequent enzyme or acid treatment to hydrolyse the cellulose. The pro- cess requires a considerable energy input and is not as effective as other treatments.
Ammonia explosion
The lignocellulose is milled and the ground lignocellulose, with a moisture content of 15–30%, is placed in a pressure vessel with ammonia (1–2 kg/kg biomass) at pressures of 12 atmospheres for 30 min. No sugars are released but the hemicellulose and cel- lulose are opened up to enzymatic digestion.
Acid treatment
Acid treatment can use sulfuric, hydrochloric, nitric and phosphoric acids although sulfuric is used most widely. Acid treatment converts the hemicellulose to sugar (xylose) (80–95%) and furfural, and increases cellulose digestibility. The treatment time is short (minutes), and depending on the substrate between 80 and 95% of the hemicellulose sugar can be recovered.
Alkaline treatment
Alkali treatment reduces the lignin and hemicellulose content and can be carried out at lower temperatures and pressures than acid treatment. The treatment also increases the surface area of the biomass.
Ozonolysis
Ozone can be used to degrade lignin and hemicellulose (Sun and Cheng, 2002). The advantages are it removes lignin, produces no toxic residues and it is carried out at room temperature and pressure.
Enzymes
Fungal enzymes from white and brown rot fungi, such as Sporotrichum pulverulentum and Pleurotus osteatus, can be used to pretreat lignocellulose. The brown rot fungal enzymes degrade cellulose whereas white rot fungal enzymes degrade lignocellulose.
Cellulose breakdown
After pretreatment the cellulose is suitable for hydrolysis to glucose and a number of methods can be used (Fig. 6.10).
Concentrated acid
Concentrated acid hydrolysis of cellulose gives a rapid and complete conversion to glucose using 70% sulfuric acid for 2–4 h. The problems are associated with the dif- ficulties of handling concentrated sulfuric acid and the cost of the acid, which requires recovery and reuse to be economical.
Dilute acid hydrolysis
The cellulose is broken down by dilute acid in complex reaction at a higher tempera- ture than the hemicellulose reaction and has a sugar recovery of around 50%.
Cellulase enzymes
Once the structure of the cellulose has been opened up by the pretreatment, enzym- atic hydrolysis can proceed. The crude cellulase enzyme is a consortium of enzymes, which operate under mild conditions, pH 4.8 and 45–50°C. Although cellulase is commercially available it is usually obtained from fungi such as Trichoderma reesei, and the yields are better than acid hydrolysis. Cellulases can be produced by both fungi and bacteria which can be grown both aerobically, and anaerobically. The bac- teria include Clostridium, Cellulomonas, Bacillus, Thermomonospora, Bacteroides, Erwinia, Acetovibrio, Microbispora and Streptomyces. Three enzymes are involved in the hydrolysis, endo-1,4-β-glucanases (endoglucanases), cellobiohydrolyases (exoglucanases), and β-glucosidases. The endoglucanases cleave the cellulose chain randomly and the exoglucanases hydrolyse the cellulose chain, releasing glucose and cellobiose. The β-glucosidases catalyse the conversion of cellobiose to glucose.
The fungi produce all three types of cellulase but the exoglucanases are the major enzymes with T. reesei. The Trichoderma sp. are considered the best of cellulase enzyme producers. Cellulose on hydrolysis liberates cellobiose which is cleaved into two molecules of glucose by the enzyme β-glucosidase. The disadvantage of the enzyme process is that both products glucose and cellobiose act as inhibitors of cel- lulase and β-glucosidase enzymes.