By Dr Ku Syahidah Ku Ismail Adapted from Drapcho et al (2008) Biofuels Engineering Process Technology

 Properties of Ethanol  Bioethanol Production Process Platform  Pretreatments for sugar production  Ethanol production process  Microorganisms for ethanol production  Ethanol purification  Bioethanol limitations

 Molecular formulaCH 3 CH 2 OH  Molecular weight46.07 g/mol  Density at 20  C kg/L  Viscosity at 20  C1.2 cP  Freezing point  C  Boiling point78.4  C

Ethanol can be burned to form carbon dioxide and water, releasing energy: C 2 H 5 OH(l) + 3O 2 (g) 2CO 2 (g) + 3H 2 O (l)  G = kJ/mol Why is ethanol considered as a clean fuel?

Sugar-platformStarch-platformCellulose-platform

Extraction Sugar platform Sugarcane, sugar beet, sweet sorghum Saccharification Starch platform Corn, potato, rice, wheat, sweet potato Pretreatment Cellulose platform Wood, grass, agricultural residues SUGAR Ways to obtain sugar from various raw materials

 From sugarcane, sugar beet, sweet sorghum.  Rich in sugars mainly sucrose  Involves extraction process to obtain sugar for fermentation Sweet sorghum Sugar beet

Starch Mixed with water 60  C 5-10 min Starch dissolves in water to form a mash Liquefaction process  -amylase added  C 2 hours Saccharification Glucoamylase added  C 30 min Degrade starch into oligosaccharides and dextrin Convert oligosaccharides and dextrin into glucose

FeedstockGlucan (cellulose) (%)Xylan (hemicellulose) (%)Lignin (%) Corn stover Corn fiber Pine wood Poplar Wheat straw Switchgrass Office paper Chemical composition of biomass (adapted from Mosier et al., 2005).

Schematic of goals of pretreatment on lignocellulosic material (adapted from Hsu et al., 1980).Hsu et al., 1980

Cellulose is a long-chain homogeneous polysaccharide of D-glucose units, linked by β-1,4 glycosidic bonds. It is composed of over 10,000 glucose units.

Helps in separation of main biomass components (cellulose, hemicellulose and lignin) Increase available surface area Reduce particle size Ideally pretreatment: – Solubilizes hemicellulose – Increases enzymatic hydrolysibility of cellulose

Cheap Fast Robust Simple Catalyst recycling Minimal environmental impact Very good overall sugar recovery in hydrolysable and fermentable form – Generates minimum amount of degradation products

Pretreatment Methods PhysicalChemicalBiological

 Possible methods: ◦ Mechanical  Milling (wet, dry) wet disc refining  Time consuming, intensive, expensive  Irradiation (microwave heating)  Slow, substrate specific, expensive ◦ Chemical (acid and base) ◦ Biological (white rot fungi)  Lignin-solubilizing microorganisms  No chemicals  Slow  Consumption of cellulose and hemicellulose ◦ Combination (synergistic effects)  Chemical followed by biological  Physical followed by biological

 By chipping, grinding, milling to reduce cellulose crystallinity.  Size of material is usually cm after chipping, and 0.2 – 2 mm after milling or grinding.  Vibratory ball milling is more effective than the ordinary ball milling.

Wood chipper Cross section of a ball mill

 Most commonly used methods for lignocellulosic materials  Chipped biomass is treated with high-pressure saturated steam and then the pressure is swiftly released, which makes the materials undergo an explosive decompression.  Carried out at 160 – 260  C (0.69 – 4.83 MPa)  It causes hemicellulose degradation and lignin transformation due to high temperature, thus increases the potential of cellulose hydrolysis.  Factors to consider during steam explosion pretreatment; residence time, temperature, chip size, moisture content  Addition of H 2 SO 4 (or SO 2 ) or CO 2 in steam explosion can improve enzymatic hydrolysis, decrease inhibitory compounds and lead to more complete removal of hemicellulose.

 Optimal conditions of steam explosion pretreatment for sugarcane bagasse: temperature, 220  C; residence time, 30 s; water-to-solid ratio, 2; H 2 SO 4 dose, 1 g H 2 SO 4 per 100 g dry bagasse. Based on this set condition, they got 65.1 g sugar/100 g starting bagasse after steam explosion pretreatment.  Advantages of steam explosion pretreatment: a) low energy requirement compared to mechanical comminution b) no recycling or environmental costs associated with chemical pretreatment c) the most cost-effective pretreatment processes for hardwoods and agricultural residues, but less effective for softwoods.  Limitations of steam explosion: a)destruction of a portion of xylan fraction b)incomplete disruption of lignin-carbohydrate matrix c)generation of inhibitory compounds in downstream processing

 Lignocellulosic material are exposed to liquid ammonia at high temperature for a period of time, and then flashed to a lower pressure.  The concept is similar to steam explosion.  A typical AFEX process: Dosage of liquid ammonia used is 1-2 kg ammonia/kg dry biomass; temperature 90  C, residence time 30 min.  AFEX can significantly improve the saccharification rates of various herbaceous crops and grasses eg. alfalfa, wheat straw, corn stover, rice straw, switchgrass.  It was reported that over 90% hydrolysis of cellulose and hemicellulose was obtained after AFEX using bermudagrass and bagasse.  AFEX is not very effective for the lignocellulosic biomass with high lignin content such as newspaper (18-30% lignin) and wood chips (25-35% lignin).

 To reduce the cost and protect the environment, ammonia needs to be recycled after the pretreatment.  AFEX did not produce inhibitors for the downstream biological processes, so water wash is not necessary.  AFEX pretreatment did not require small particle size for efficacy.  Cost of AFEX process is higher than steam explosion.

 Similar to steam and ammonia explosion  It was hypothesized that CO 2 would form carbonic acid and increase the hydrolysis rate.  The yields are relatively low compared to steam explosion or AFEX, but high compared to the enzymatic without pretreatment.  CO 2 explosion was more cost-effective than AFEX and did not form inhibitory compounds.

 Lignocellulosic materials are treated at temperature higher than 300  C.  Cellulose would rapidly decompose to produce gaseous and tarry compounds.

 Ozone is used to degrade lignin and hemicellulose, while cellulose is hardly affected.  Advantages: a)it effectively removes lignin b)it does not produce toxic residues for the downstream processes c)the reactions are carried out at room temperature and pressure.  Disadvantage: A large amount of ozone is required in the process, making the process expensive.

 Using concentrated acids such as H 2 SO 4 and HCl  Dilute sulfuric acid pretreatment could achieve high reaction rates and significantly improve cellulose hydrolysis.  There are two types of dilute acid pretreatment process: High temperature (T > 160  C), continuous-flow process for low solids loading (5%-10% [w/w]) Lower temperature (T < 160  C), batch process for high solids loading (10%-40% [w/w])  Cost is higher than steam explosion or AFEX.  A neutralization of pH is necessary before enzymatic hydrolysis or fermentation process.

 Alkaline pretreatment can disrupt lignin structure and decrease crystallinity of cellulose and degree of sugar polymerization.  Compared with acid pretreatment, alkaline pretreatment has less sugar degradation and inhibitory compounds (furan derivatives) formation  NaOH and lime can be recovered or regenerated.  NaOH is very efficient in removing lignin from lignocellulosic materials at a temperature of 100  C for min.  Lime pretreatment of switchgrass at low temperature could significantly improve the sugar yield, but the time is longer (6 h).

Pretreatment of Cellulosic Materials Lignocellulosic hydrolysates inhibitors Furfural, Acetic acid, Formic acid Vanillin HMF Inhibitors can cause harmful effects to the yeasts resulting in lower yield.

 Lignin biodegradation could be catalyzed by peroxidase enzyme with the presence of H 2 O 2.  It was reported that about 50% lignin and most hemicellulose were solubilized by 2% H 2 O 2 at 30  C within 8 h.

 An organic or aqueous organic solvent mixture with inorganic acid catalyst (HCl or H 2 SO 4 ) is used to break the internal lignin and hemicellulose bonds.  The organic solvents used include methanol, ethanol, acetone, ethylene glycol, triethylene glycol and tetrahydrofurfuryl alcohol.  At high temperature (T > 185  C), the addition of catalyst is unnecessary.  Usually, a high yield of xylose can be obtained with the addition of acid.  Solvents used in the process need to be drained from the reactor, evaporated, condensed and recycled to reduce the cost.  Removal of solvents from the system is necessary because the solvents may be inhibitory to the growth of organisms.

 Microbes used: brown-, white-, and soft-rot fungi.  Brown rots mainly attack cellulose.  White and soft-rots attack both cellulose and lignin.  White-rot fungi are the most effective basidiomycetes for biological pretreatment of lignocellulosic materials.  The white-rot fungus Phanerochaete chrysosporium produces lignin-degrading enzymes, lignin peroxidases in response to carbon or nitrogen limitation.  Advantages of biological pretreatment: Low energy requirement and mild environmental conditions  Disadvantage of biological pretreatment: Rate of hydrolysis is very slow White rot fungi

 Using yeast or bacteria  In yeast fermentation, the glucose solution obtained from starch saccharification or cellulose hydrolysis is cooled to around 32  C.  Yeast culture is added into the solution under aseptic condition  Glucose in the solution penetrates into the yeast cells through a series of enzymatic reactions to eventually ethanol, CO 2 and energy.  Some of the released energy and glucose are utilized by the yeast cells to support their growth during fermentation.  The rest of the energy becomes heat to the fermentation broth and may increase the temperature if not taken out of the system.  Both ethanol and CO 2 penetrate out of yeast cells.  CO 2 readily dissolves in water, but can easily be saturated in fermentation broth.  The excess CO 2 bubbles out of the liquid and can be collected for food and soft drink preparation.

 Initially, yeast cell concentration is low and yeast growth is dominant  Glucose is mainly utilized to support the growth of yeast cells, so little ethanol and CO 2 are produced and the glucose conversion rate is relatively low.  The length of initial stage depends on yeast inoculation ratio and the fermentation temperature.  At normal inoculation ratio (5-10%), 30  C, it takes approximately 6-8 h.  Then the yeast cells will tremendously increase to over 10 8 cells/mL.  The fermentation becomes very active, resulting in rapid ethanol, CO 2 and energy production, which is indicated by vigorous bubbling and heat production.  At this time, cooling is required to maintain the fermentation temperature at 30  C.  Active fermentation lasts about 12h, then the fermentation activity slows down because less glucose is available.  During the slow fermentation period, the yeast cells do not grow anymore, the biochemical reactions are limited by the substrate (glucose) concentration.

 Can metabolize glucose, fructose, mannose, galactose, sucrose, maltose and maltotriose.  C 6 H 12 O 6 + 2Pi + 2ADP  2C 2 H 5 OH + 2CO 2 + 2ATP + 2H 2 O Glucose  2 ethanol + 2 carbon dioxide + energy  Theoretical yield is g ethanol produced per gram of glucose consumed.  This yield can never be realized in practice since not all glucose consumed is converted to ethanol.

 Prefer glucose and sucrose  The presence of these sugars represses the uptake and metabolism of other sugars  Requires certain minerals eg. Ca, Mg, Mn, Co, Fe, Cu, K, Na, Zn for growth and ethanol fermentation  Is inhibited by its own product – ethanol

 A bacteria that can produce ethanol faster than S. cerevisiae  Metabolizes sugars via the Entner-Doudoroff (ED) pathway  High sugar uptake and ethanol tolerance  No requirements for aeration during fermentation process  Not used for industries because of its sucrose metabolism  Sucrose has to be converted to glucose and fructose by the enzyme levansucrase first.

S. cerevisiaeZ. mobilis Produces organic acid by-products Ratio of acetic acid:lactic acid is 8:1 Ratio of acetic acid:lactic acid is 16:1 pH of fermentation medium tends to drop to 3 pH tends to stabilize at 4.5 Media at pH 3 inhibits most contamination, thus reducing the need of sterilzation Needs sterilization of media Starting yeast culture can be purchased in many forms and easy handling Tight control of stock culture

Conventional Process vs SSF Saccharification Lignocellulosic Material Pre- treatment Fermentation Distillation Produces inhibitory compounds: Furfural, Acetic acid, HMF Normal yeast Strain ~ 30  C Requires cooling Higher recovery cost to increase temperature In the same tank Requires removal Optimum temp  C Conventional process Ethanol Simultaneous Saccharification & Fermentation Usage of inhibitor tolerant strain Usage of heat tolerant strain

 Strains that can metabolize other sugars than glucose and convert them to ethanol at high yields and rates.  Strains that can metabolize cellulose and hemicellulose directly, thus eliminating the need of enzyme for biomass hydrolysis.  Strains tolerant to inhibitory compounds formed during pretreatment process  Strains tolerant to high ethanol levels  No requirements of expensive nutrients

Pathways for Glucose and Xylose Metabolism Lignocellulose Lignin Cellulose Hemicellulose Glucose Xylose C6 C5 EthanolXylitol Xylulose Xylulose-5- phosphate Pentose Phosphate Pathway Glycolytic Pathway Genetically Modified S. cerevisiae Cellulase Hemicellulase XR XDH XK XR and XDH were obtained from S. stipitis XK overexpressed from S. cerevisiae

 Fermentation product is usually a dilute aqueous solution containing 3-12 wt% of ethanol  Separation of ethanol from fermentation broth is an energy-intensive process  It usually take up a large fraction of the total energy requirement for the whole biorefinery.  Fermentation – distillation system can generate a maximum of 95% pure ethanol  Distillation – membrane vapour permeation  Continuous fermentation-pervaporation process

 Conversion of lignocellulosic biomass is costlier than its crude oil counterpart.  Biomass harvesting and yield  Cost of enzymes Please read Reading Material 1 for more limitations on bioethanol production.

 1. What are the differences between hemicellulose and cellulose?  2. Which pretreatment is suitable for biomass such as Napier grass?  Suggest an economical process for ethanol recovery system.