BIOETHANOL

An increasing search for renewable feedstock for biofuel production, is ongoing in the past few decades with prime reason of; 

1. Reducing global reliance on fossil fuels
2. Lowering the greenhouse gas emission.
3. Expensive fossil fuel (petroleum) supply.

Ethanol is the most common sustainable, alternate fuel for gasoline in passenger cars, produced on a fair scale.

COUNTRY	FEED STOCK USED (Mostly agricultural commodities)	PRODUCTION
Brazil	Sugar cane molasses	6, 267 million gallons in year 2013
United States of America	Corn, a starch crops

13, 300 million gallons in year 2013

India

Sugar cane

Thailand

Cassava

Philippines

Cassava

Sweden

Softwood

France

Sugar beet

China

Corn

Canada

Wheat

CHALLENGES FOR FOOD PROCESSORS AND SCIENTISTS

Insufficient agricultural commodities used for feed stock.

SCOPE

Search for technological breakthrough is on the high, aiming to develop technologies for effectively converting agricultural and forestry lignocellulosic residues to fermentable sugars.

BIOFUEL

fuel that derives from organic material such as;

1.      Energy crops (corn, wheat, sugar cane, sugar beet, cassava)

2.      Crop residues (Rice straw, rice husk, corn stover, corn cobs)  

3.      Waste biomass (food waste, livestock waste, paper waste, construction-derived wood residues).

Of all biofuels, ethanol has been trusted as an alternate fuel for the future

ETHANOL

• biodegradable nature
• cheap fuel
• Is flexible transportation fuel
• Used as a blending agent in anhydrous form at 99.6 gay lussac (Gl)  in ethanol-gasoline blends( directly or indirectly i.e. In the form of ethyl tert-buthyl ether - etbe).
• Used as a primary fuel in neat hydrous form (95.5 gl).
• An excellent motor fuel (research octane number (ron) = 109, motor octane number (mon) = 90, which are higher compared to gasoline (ron of 91 to 98 and a mon of 83 to 90).
• Has a lower vapour pressure than gasoline (its reid vapor pressure is 16 kpa versus 71 for gasoline), resulting in less evaporative emissions.
• Flammability in air (1.3 to 7.6% v/v) is also much lower compared to gasoline (3.5 to 19% v/v), reducing the number and severity of vehicle fires.
• When used as a neat fuel, ethanol has a lower energy density than gasoline (ethanol has lower and higher energy density values of 21.2 and 23.4 mj/l, respectively; for gasoline the values are 30.1 and 34.9 mj/l) and cold-start problems exist as well.

STARCH HYDROLYSIS FOR ETHANOL PRODUCTION

Starch

• Is major dietary source of carbohydrates
• Is most abundant storage polysaccharide in plants
• occur as granules of size 1 to 100 μm.
• composed of a mixture of two kinds of polyglucans - Amylose and Amylopectin.

AMYLOSE	AMYLOPECTIN
linear component	Highly branched polymer
mostly comprised of α-1,4-linkages	consisting of short α-1,4-oligomers linked by α-1,6-bonds
Average degree of polymerization (DP) up to 6,000	average DP of 2 million (largest molecules in nature)
Molecular mass of 105 to 106 g/mol	107 to 109 g/mol
Depending on the botanical source, the amylose content varies from 0 to 70%

GLUCOSE : a ring-shaped molecule with six atoms in the ring

Source of starch: maize, potato, wheat, tapioca and rice.

The cereal crops, maize, waxy maize and wheat are grown in America and Europe

Potato is largely derived from the cooler climes of northern Europe.

Tapioca starch is sourced from Brazil, Thailand and Indonesia, while rice originates mainly from Asia.

Native starch granules have semi-crystalline regions formed by amylopectin and less ordered amorphous regions of amylase.

FEED STOCK

• Use of energy crops for ethanol production is a common practice.
• Most recently, crops residues and industrial by-products have been used as potential substrate for fermentation.

Table: kinetics of bioethanol production from wheat milling by-products using Zymomonas mobilis in batch fermentation.

RAW MATERIAL	ETHANOL PRODUCTION
low-grade wheat flour	51.4 g/L
wheat flour	68.1 g/L
wheat bran	18.1 g/L

CELLULOSIC ETHANOL

An alternative to the use of energy crops as feedstock for ethanol production

Production of ethanol from cellulosic material is relatively low compared to sugar or starch crops.

There is need to develop fermentation processes that can convert energy crops (such as grasses, and agricultural by-products - straw and corn stover) into bioethanol, allowing high conversion of both hexoses and the difficult to ferment pentoses into ethanol at high yields.

Utilize rougher and woodier parts of plants.

lignocellulosic biomass is a potential source for ethanol that is not directly linked to food production .

The conversion of lignocellulosic material to ethanol is generally more complex, compared to starch hydrolysis and fermentation

More drastic hydrolysis steps are necessary for achievement of high conversion yields, due to the presence of various amounts of other sugars, such as xylose and arabinose.

The most expensive part of making ethanol from lignocellulosic feedstock: pre-treating the biomass to make it accessible to the enzymes that will then cut the sugars from the polymers so that they can be fermented.

Pretreatment : mechanical and physical actions to clean and size the biomass, and destroy its cell structure to make it more accessible to further chemical or biological treatment.

Lignocellulosic materials consist primarily of three components;

1. cellulose (40-50%),
2. hemicelluloses (20-30%)
3. lignin (20-30%).

The soluble sugar products are primarily xylose, and further mannose, arabinose and galactose.

A small portion of the cellulose may already be converted to glucose. However, the cellulose bulk will be converted in a separate step.

The product is filtered and pressed, solids (cellulose + lignin) go to cellulose hydrolysis, and liquids (containing the sugars) go to the fermentation step.

The choice of a pretreatment technology heavily influences cost and performance in subsequent hydrolysis and fermentation.

The present production costs of ethanol show a broad range;

Projected cellulosic ethanol production costs in Europe lie between $34 and $45/gigajoule, and in the US between $15 and $19/gigajoule (Hamelinck et al. 2003).

Some of the most commonly used chemical, physical and biological pretreatment methods are discussed in detail:

Chemical pretreatment

Common chemical pretreatment methods comprise;

• dilute acid
• alkaline
• ammonia
• organic solvent
• SO₂
• CO₂

Acid catalyzed pretreatment of biomass prior to fermentation;

• provide a near-term technology for production of fuel-grade ethanol from cellulosic biomass
• relatively low yields of sugars from cellulose and hemicelluloses (c.a. 50% to 60% of the theoretical yield)
• typical of dilute acid systems still have to be increased somehow, in order to be competitive with existing fuel options in a free market economy (Wyman et al. 1993).

Concentrated acid or halogen acids achieve high yields (essentially, 100% of theoretical).

However, because low-cost acids (such as H2SO4)  must be used in large amounts while more potent halogen acids are relatively expensive, recycling of acid by efficient, low-cost recovery operations is essential to achieve economic operation (Wyman et al. 1993).

Alkaline processes;

•  use bases as NaOH or Ca(OH)₂.
• All lignin and part of the hemicellulose are removed.
• Cellulose reactivity is sufficiently increased and the reactor costs are lower than those for acid technologies (Hamelinck et al. 2003).
• Alkaline-based methods are generally more effective at solubilising a greater fraction of lignin, while leaving behind much of the hemicellulose in an insoluble, polymeric form (DOE 2007).

Enzymatic hydrolysis (EH)

• One of the most extensively investigated pretreatment processes
• Uses fungal cellulolytic enzymes for conversion of cellulose onto of the biomass to glucose, which is then fermented to ethanol (Szengyel 2000; Varga et al. 2002).

Three major classes of enzymes may be used for EH pretreatment of lignocellulosic biomass for bioethanol production, as follows:

1. Endo-1,4-β- glucanases or 1,4-β-D-glucan 4-glucanohydrollases (EC 3.2.1.4), act randomly on soluble and insoluble 1,4-β-glucan substrates, commonly measured from carboxymethylcellulose (CMC).
2. Exo-1,4-β-D-glucanase;  Includes 1,4-β- D-glucan glucohydrolases (EC 3.2.1.74), which liberate D-glucose from 1,4-β-D-glucans and hydrolyze D-cellobiose slowly, and 1,4-β-D-glucan cellobiohydrolase. 
3. β-D-glucosidases or β-D-glucoside glucohydrolases (EC 3.2.1.21)act to release D-glucose units from cellobiose and soluble cellodextrins, as well as an array of glycosides (Sheehan and Himmel 1999).;

These three types of enzymes work together synergistically in a complex interplay, resulting in efficient decrystallization and hydrolysis of native cellulose.

The EH yield is governed by many factors, which include:

 Type of substrate pretreatment:

Cellulosic biomass is resistant to enzymatic attack.

A pretreatment step is required to overcome this resistance, in order to proceed at reasonable rate with the high yields.

The pretreatment step must facilitate conversion of both the cellulose and hemicellulose fractions into ethanol, while minimizing the degradation of these fractions into compounds that cannot be fermented into ethanol (Wyman et al. 1993).

Several options for biomass pretreatment using chemical or physical methods  includes;

• dilute acid or alkaline treatment,
•  steam-explosion, 
•  liquid hot water

Currently, most industrial processes are based on dilute acid hydrolysis, making use of the electricity co-produced from the non-fermentable lignin (Hamelinck 2005). In this process, about 0.5% H₂SO₄ is added to the milled feedstock, and the mixture is heated to 140-160ºC for 5-20 minutes.  Under these conditions, most of the hemicellulose is hydrolyzed to form xylose, which is then removed in solution, leaving a porous material of primarily cellulose and lignin that is more accessible to enzymatic attack (Wyman et al. 1993).

Most successful method, which has been evaluated for various lignocellulosic materials, is the steam pretreatment (Szengyel 2000).

Inhibition of enzymatic activity by the end-products of the biodegradation:

The overall activity of cellulases is contributed to by

• the efficiency of the active site,
• susceptibility to end-product inhibition and to nonspecific or dead-end binding to the substrate,
• ability to decrystallize cellulose.

The net effect of reducing end product inhibition and non-productive binding is to increase available sites for substrate hydrolysis (Sheehan

and Himmel 1999). An early report (Szengyel 2000) evidenced that enzyme solutions produced on steam pretreated spruce showed less sensitivity towards toxic compounds formed during steam pretreatment for bioethanol production from wood.

Thermostability of enzymes and effect of medium pH:

higher operating temperatures mean a benefit from increased diffusion and thermodynamics of catalysis. Nevertheless, at the present, the extent of the benefits obtained from enhancing the temperature tolerance, as well as cellulose decrystallization, of saccharifying cellulases is to some extent unclear.

One of the major problems related to the EH of lignocellulosic biomass for bioethanol production is the different optimal conditions, mainly pH and temperature, for the hydrolysis of cellulose and fermentation. Cellulases work in an optimal way at 40-50ºC and pH of 4-5 whereas

the fermentation of hexoses with S. cerevisiae is carried out at 30ºC and pH 4-5, and fermentation of pentoses is optimally performed at 30-70ºC and pH 5-7 (Cardona and Sánchez 2007).

Some other factors of relevance while using EH as pretreatment for bioethanol production may be considered as well, such as:

• enzyme concentration and adsorption on the substrate,
• duration of the hydrolysis,
• substrate concentration in the medium
• rate of agitation of the medium.

The main difficulty in solving the problem of enzymatic de-polymerization of the lignocellulosic materials is used to their complexity. Cellulose fibrils are embedded in an amorphous matrix of lignin and hemicelluloses, which render the plant tissue resistant to microorganisms and their enzymes (Szczodrak et al. 1996).

Developing accurate methods for measurement of enzymatic cellulose digestibility is crucial for evaluating the efficiency of lignocellulosic pretreatment technologies (Zhang et al. 2007).

Several advantages enzyme-catalyzed processes  are;

• Achieve high yields under mild conditions with relatively low amounts of catalyst.
• enzymes are biodegradable and thus environment friendly (Wyman et al. 1993).
• corrosion-related problems can be neglected, compared to other chemical processes, such as dilute acid pretreatment.

Disadvantage of enzyme-catalyzed processes  are;

• the cost associated to enzymatic production is relatively high, unlike chemical or physical methods , though cost is decreasing gradually due to the development of new engineered enzyme systems (Ehara and Saka 2002).
• End-product inhibition of the enzymes used to hydrolyze the cellulose and the remaining hemicellulose. This problem was reportedly minimized by performing the saccharification and fermentation processes simultaneously (Öhgren et al. 2006).

Alternatively, the utilization of immobilized enzymes and hollow-fiber membrane reactor has been recently revealed as a promising alternative for hydrolysis of lignocellulosic biomass (Cardona and Sánchez 2007). In this type of system, the enzymes are confined inside the reactor allowing the separation of substrate and hydrolysis products (e.g. glucose, arabinose, xylose, among others) enabling the reutilization of the enzymes while preserving their activity as free catalysts. For instance, an increase of 53% in substrate conversion was attained by Gan et al. (2002) using commercial Trichoderma reesei for saccharification of lignocellulosic feedstock, compared to 35% conversion in the case of traditional batch hydrolysis. This increased efficiency was likely due to the reduction in the inhibition effect of formed sugars on cellulases, and to the increase in productivity during continuous operation. Ultimately, significant cost reduction for EH of lignocellulose

to fermentable sugars can be forecasted, should the scientific community keep on track with development of new engineered enzyme systems. Most likely, these systems should include inhibitor-tolerant pentose-fermenting industrial yeast strains.

CURRENT BIOETHANOL PRODUCTION PROCESSES

Ethanol has been produced by anaerobic yeast fermentation of simple sugars since early recorded history.

These fermentations used the natural yeast found on fruits and the sugars of these fruits to produce wines.

Beer fermentations made use of the amylases of germinating grain to hydrolyze the grain starches to ferment sugars.

Current practices utilize bacterial and fungal amylases to efficiently hydrolyze grain or tuber starch to glucose for fermentation to ethanol (Klass et al. 1981).

Ethanol can be produced by BIOLOGICALLY CATALYZED REACTIONS.

RAW MATERIAL	ORGANISMS	AGENT FOR LIQUEFACTION	PROCESS	INTERMEDIATE PRODUCT	PRODUCT
Sugars extracted from sugar crops, such as sugar cane	yeast and bacteria	acids or amylases	Fermention		beverage ethanol
Cellulose		Acids or cellulase enzymes	Fermention	glucose	ethanol
Hemicelluloses		xylanases	Fermention	various sugars, e.g. xylose	ethanol

Starch crops such as wheat for bioethanol production resulted in considerable high ethanol concentration in reduced fermentation time (Montesinos and Navarro 2000a).

In that case, slurries containing 300 g/L of raw wheat flour were initially liquefied using 0.02 g α-amylase/g starch at 95°C for 2 h, followed by saccharification using two different levels of amyloglucosidase activity (270 U/kg starch and 540 U/kg starch) and simultaneous fermentation by Saccharomyces cerevisiae at 35°C for 21h, reaching a final ethanol concentration of 67 g/L.

As for the hydrolysis of lignocellulosic biomass, various levels of enzyme load have been reported in the literature: Wooley et al. (1999) obtained bioethanol yield of c.a. 250 liter/ton by simultaneous saccharification and co-fermentation (SSCF) of Hardwood Yellow Poplar.

The feedstock was pretreated with cellulase at 15 FPU/g of cellulose (FPU, Filter Paper Unit is the unit utilized to express cellulase activity) at 30ºC for 7 days.

Öhgren et al. (2006) were able to reach final ethanol concentrations of about 25 g/L by simultaneous saccharification and fermentation (SSF) of corn stover pretreated by EH using cellulose at 65 FPU/g of cellulose at 40ºC for 4 days.

Basically, two different processes can be used to produce ethanol from starch crops:

1. Dry grind (feed material is ground mechanically and cooked in water to gelatinize the starch. Enzymes are then added to break down the starch to form glucose, which yeasts ferment to ethanol. In that case, a fixed amount of ethanol is produced, along with other feed products and carbon dioxide, and has almost no process flexibility.)
2. wet milling

wet milling;
Initially, Insoluble protein, oil, fiber, and some solids are removed except starch slurry.

has the capability to produce various end products

has higher process flexibility, compared to the dry milling (Fernando et al. 2006).

Currently, about 65% of the ethanol in the US is produced from dry grind corn processing plants (DOE 2007).

Biological processing offers a number of advantages for converting biomass into biofuels.

• the enzymes used in bio-processing are typically capable of catalyzing only one reaction
• No formation of unwanted degradation products and by-products(Schmidt 2002).
• material not targeted for conversion can pass through the process unchanged and be used for other applications.

Although the individual steps for converting biomass into ethanol can be conveniently isolated, these can otherwise be combined in various ways in order to minimize the production cost (Johansson et al. 1993). Some of these integrated processes are described below.

SEPARATE HYDROLYSIS AND FERMENTATION (SHF) PROCESS

The starch molecule is initially hydrolyzed by the action of amylolytic enzymes:

1. α-amylase (for liquefaction)
2. glucoamylase(for saccharification).

After complete hydrolysis, the fermentation is conducted as single step separately.

(Mojović et al. 2006) achieved ethanol yield : 80% (w/w) of the theoretical yield, and reduced fermentation time for 4 h using distinct process steps for starch hydrolysis and glucose fermentation.

ADVANTAGES

Minimum interactions between these steps due to separate starch hydrolysis and sugar fermentation.

DISADVANTAGES

Need considerable efforts to overcome α-amylases inhibition by the accumulation of sugars

SIMULTANEOUS SACCHARIFICATION AND FERMENTATION

was introduced by Gulf Oil Company, US and the University of Arkansas (Gauss et al. 1976; Huff et al. 1976).

sequence of steps for the SSF is virtually the same as for the separate process, except that saccharification and fermentation steps are combined in one vessel.

saccharification of sugars released during starch hydrolysis (mainly maltose) is conducted simultaneously with fermentation.

ADVANTAGES

• The presence of yeast or bacteria along with enzymes minimizes the sugar accumulation in the vessel,
• because the sugar produced during starch breakdown slows down α-amylase action, higher rates, yields and concentrations of ethanol are possible using SSF rather than SHF, at lower enzyme loading. 
•  Presence of ethanol makes the mixture less vulnerable to contamination by unwanted microorganisms, which is a frequent burden in case of industrial processes (Montesinos and Navarro 2000a; Roble 2003).

Various reports on bioethanol production have mentioned the superiority of ethanol yield and productivity using the SSF process, compared to the SHF process (Söderström et al. 2005; Neves et al. 2007).

Other promising integration alternative is the inclusion of pentose fermentation in the SSF, process known as Simultaneous Saccharification and Co-fermentation (SSCF) (Sheehan and Himmel 1999; Cardona et al. 2007).

In this configuration, it is necessary that both fermenting microorganisms be compatible in terms of operating pH and temperature.

A combination of Candida shehatae and S. Cerevisiae was reported as suitable for the SSCF process (Cardona and Sánchez 2007).