Economics and environmental impact of bioethanol
production technologies: an appraisal (Part -1)

Anuj Kumar Chandel1,2, Chan ES3, Ravinder Rudravaram1, M. Lakshmi Narasu2, L.Venkateswar Rao1 and Pogaku Ravindra3*

Biotechnology and Molecular Biology Review Vol. 2 (1), pp. 014-032, February 2007Available online at http://www.academicjournals.org/BMBR ISSN 1538-2273 © 2007 Academic Journals
Standard Review

1Department of Microbiology, Osmania University, Hyderabad-500 007, India.
2Department of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad-500 007, India.
3School of Engineering and Information Technology, Universiti Malaysia Sabah,Kotakinabalu, 88999, Malaysia.
Accepted 25 January, 2007

Contemporary industrial developments and rapid pace of urbanization have called for an environmentallysustainable energy sources. Ethanol made from biomass provides unique environmental, economic strategic benefits and can be considered as a safe and cleanest liquid fuel alternative to fossil fuels. There is a copious amount of lignocellulosic biomass worldwide that can be exploited for fuel ethanol production. Significant advances have been made at bench scale towards the fuel ethanol generation from lignocellulosics. However there are still technical and economical hurdles, which make the bioethanol program unsuccessful at commercial scale. This review provides a broad overview on current status of bioethanol production technologies in terms of their economic and environmental viability. These technologies include pretreatment of biomass, the use of cellulolytic enzymes for depolymerisation of carbohydrate polymers into fermentable constituents and the use of robust fermentative microorganisms for ethanol production. Among all the available technologies, dilute acid hydrolysis followed by enzymatic hydrolysis by less expensive and more efficient cellulases has been found more promising towards the potential economics and environmental impact.

INTRODUCTION
In 1925, Henry Ford had quoted ethyl alcohol, ethanol, as"the fuel of the future.” He furthermore stated, "The fuel of the future is going to come from apples, weeds, sawdust – almost anything. There is fuel in every bit of vegetable matter that can be fermented." Today Henry Ford’s futuristic vision significance can be easily understood. In the current time, the importance of alternative energy source has become even more necessary not only due to the continuous depletion of limited fossil fuel stock but also for the safe and better environment, with an inevitable depletion of the world’s energy supply, there has been an increasing worldwide interest in alternative sources of energy (Wyman, 1999; Lynd, 2004; Herrera, 2004; Herrera, 2006; Lin and Tanaka, 2006, Schubert, 2006; Chandel et al., 2006a; Vertes et al., 2006, Dien et al., 2006). Keeping in view all the above said advantages, biomass based fuel development technologies should rapidly gain momentum and the barriers imposed earlier should be removed for successfully attempting the production of bioethanol at the commercial level. It is welcome to understand that the use of bioethanol as a source of energy would be more than just complementing for solar, wind and other intermittent renewable energy sources in the long run (Lin and Tanaka, 2006). During the last two decades, advances in technology for ethanol production from biomass have been developed to the point that large-scale production will be a reality in next few years (Yu and Zhang, 2004; Moiser et al., 2006). Ethanol production from biomass can be summarized briefly into following steps: depolymerization of holocellulose polymer into monomeric fermentable substrate, fermentation of depolymerized substrates, and the distillation of the fermentation broth to obtain dehydrated ethanol.
The ethanol yields and processes economics along with the technical maturity and environmental benefits of using ethanol blend fuel are the key parameters that determine the feasibility of bioethanol production (Nguyen and Saddler, 1991). The burning fossil fuel at the current rate is likely to create an environmental crisis globally. Use of fossil fuel generates carbon dioxide, methane and a significant quantity of nitrous oxide. Most of these harmful gases are formed due to incomplete combustion of fossil fuel; since ethanol contains 35% oxygen that may result in a more complete combustion of fuel and thus reduces tailpipe
emissions.
*Corresponding author. E-mail: dr_ravindra@hotmail.com. Phone: 006 088 320000 ext 3048. Mobile: 006 013 87666634. Fax: 006 088 320348.
Moreover, biomass energy can play an important role in reducing green house gas emissions. Ethanol
production process only uses energy from renewable energy sources. Hence no net carbon dioxide is added to the atmosphere, making ethanol an environmentally bene-ficial energy source (Bull et al., 1992; Kheshgi et al., 2000). Furthermore, fuel ethanol from lignocelluloses may also open new employment opportunities in rural areas, and thus make a positive socio-economic impact (Wyman, 2003; Bevan and Franssen, 2006). Developing ethanol as fuel, beyond its current role as fuel oxygenates will require developing lignocellulosic biomass as a feedstock because of its abundantly available and low cost. The world ethanol production in 2004 was estimated to be 40 giga litres (GL) (Berg, 2004; Kim and Dale, 2004). Brazil and the US are the world leaders, which together accounted for about 60% of the world ethanol production exploiting sugarcane and corn respectively. In India, lignocellulosic biomass (crop residues, forestry and fruit and vegetable waste and weeds) is available in plenty. Renewable fuels particularly ethanol should get more and more attention all over the world. The important issue that we wish to address affirmatively here is that the bioethanol production, without doubt, needs an economical approach to address the global fuel needs. Research efforts are needed to design and improve the process, which would produce sustainable and economically feasible transportation fuel. Improvement in process economics using new designed cellulases enzyme cocktail are important factors in establishing a cost effective technology, besides the low cost of feedstock (Mojovic et al., 2006; Gray et al., 2006). For the long haul, it is very important to understand bioethanol production technologies in terms of their economic viability, environmental feasibility and empowering employment opportunities before implementing a fuel ethanol policy.

The choice of the best technology for lignocellulose to bioethanol conversion should be decided on the basis of overall economics (lowest cost), environmental (lowest pollutants) and energy (higher efficiencies)that is, comprehensive process development and optimization are still required to make the process economically viable. In reality, environmental considerations, energy and tax policies will determine the extent of fuel ethanol utilization in the future (Keim and Venkatasubramanian, 1989) and therefore the role of one and all is very crucial to identify the gravity of the situation associated with bioethanol production and use of it as an alternative fuel. The focus of this review is on the current status of available ethanol production technologies in terms of their 016 Biotechnol. Mol. Biol. Rev. practical cost economics and the desirable environmental impact they have for a whole generation of commuters across the globe.

Lignocellulosic biomass
Composition
Basically, the lignocellulosic biomass comprises of cellulose, hemicellulose and lignin (Hayn et al., 1993). Cellulose is a linear, crystalline homopolymer with a repeating unit of glucose strung together beta-glucosidic linkages. The structure is rigid and harsh treatment is required to break it down (Gray et al., 2006). Hemi-cellulose consists of short, linear and highly branched chains of sugars. In contrast to cellulose, which is a polymer of only glucose, a hemicellulose is a hetero-polymer of D-xylose, Dglucose, D-galactose, D-man-nose and L-arabinose (Saha et al., 2003). The composi-tion of holocellulose (cellulose + hemicellulose) varies with the origin of the lignocellulosic material. Table 1 shows the composition of the selected crop residues, woody materials, vegetable and fruit waste and municipal solid waste and their simulated ethanol production. Ethanol production has been taken into estimation depending upon the ratio of hexosans (glucan, galactan and mannan) and pentosans (xylan, arabinan) in each biomass source. The ethanol production from each bio-mass sources was calculated / ascertained from US Department of Energy website, which provide “Theoretical ethanol yield calculator” at http://www.eere.ene-rgy.gov/biomass/ethanol_yield_calculator.html

Ethanol production technologies

Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation and product separation/ distillation.

Pretreatment
Pretreatment is required to alter the biomass macroscopic and microscopic size and structure as well as its submicroscopic chemical composition and structure so that hydrolysis of carbohydrate fraction to monomeric sugars can be achieved more rapidly and with greater yields (Sun and Cheng, 2002; Moiser et al., 2005). Pretreatment affects the structure of biomass by solubilizing hemicellulose, reducing crystallinity and increase the available surface area and pore volume of thesubstrate. Pretreatment has been considered as one of the most expensive processing step in biomass to fermentable sugar conversion with cost as high as 30 cents/gallon ethanol produced (Moiser et al., 2005). To asses the cost and performance of pretreatment methods, technoeconomic analysis have been made recently (Eggerman and Elander, 2005). There is huge scope in lowering the cost of pretreatment process through extensive R&D approaches. Pretreatment of cellulosic biomass in cost effective manner is a major challenge of cellulose to ethanol technology research and development.Native lignocellulosic biomass is extremely recalcitrant to enzymatic digestion. Therefore, a number of thermochemical pretreatment methods have been developed to improve digestibility (Wyman et al., 2005). Recent studies have clearly proved that there is a direct correlation between the removal of lignin and hemi-cellulose on cellulose digestibility (Kim and Holtzapple, 2006). Thermochemical processing options appear more promising than biological options for the conversion of lignin fraction of cellulosic biomass, which can have a detrimental effect on enzyme hydrolysis. It can also serve as a source of process energy and potential co-products that have important benefits in a life cycle context (Sheehan et al., 2003). Pretreatment can be carried out in different ways such as mechanical combination (Cadoche and Lopez, 1989), steam explosion (Gregg and Saddler, 1996), ammonia fiber explosion (Kim et al., 2003), acid or alkaline pretreatment (Damaso et al., 2004; Kuhad et al., 1997) and biological treatment (Keller, et al., 2003).

Hydrolysis
After pretreatment there are two types of processes to hydrolyze the feed stocks into monomeric sugar constituents required for fermentation into ethanol. The hydrolysis methods most commonly used are acid (dilute and concentrated) and enzymatic. To improve the enzymatic hydrolytic efficiency, the lignin-hemicellulose net work has to be loosened for the better amenability of cellulases to residual carbohydrate fraction for sugar recovery. Dilute acid treatment is employed for the degradation of hemicellulose leaving lignin and cellulose network in the substrate. Other treatments are alkaline hydrolysis or microbial pretreatment with white-rot fungi (Phaenerochate chrysosporium, Cyathus stercoreus, Cythus bulleri and Pycnoporous cinnabarinus etc.) preferably act upon lignin leaving cellulose and hemicellulose network in the residual portion. However during both treatment processes, a considerable amount of carbohydrates are also degraded, hence the carbohydrate recovery is not satisfactory for ethanol production.

Acid hydrolysis
There are two types of acid hydrolysis process commonly used - dilute and concentrated acid hydrolysis. The dilute acid process is conducted under high temperature and pressure and has reaction time in the range of seconds or minutes. The concentrated acid process uses relatively mild temperatures, but at high concentration of sulfuric acid and a minimum pressure involved, which only creates by pumping the materials from vessel to vessel. Reaction times are typically much longer than for dilute acid Chandel et al. 017

Table 1. Chemical composition of raw materials and simulated ethanol production

Dilute acid hydrolysis
In dilute acid hydrolysis, the hemicellulose fraction is depolymerized at lower temperature than the cellulosic fraction. Dilute sulfuric acid is mixed with biomass to hydrolyse hemicellulose to xylose and other sugars. Dilute acid is interacted with the biomass and the slurry is held at temperature ranging from120 - 220°C for a short period of time. Thus hemicellulosic fraction of plant cell wall is depolymerised and will lead to the enhancement of cellulose digestibility in the residual solids (Nigam, 2002; Sun and Cheng, 2002; Dien et al., 2006; Saha et al., 2005). Dilute acid hydrolysis has some limitations. If higher temperatures (or longer residence time) are applied, the hemicelluosic derived monosaccharides will degrade and give rise to fermentation inhibitors like furan compounds, weak carboxylic acids and phenolic compounds (Olsson and Hahn- Hagerdal, 1996; Klinke et al., 2004; Larsson et al., 1999). These fermentation inhibitors are known to affect the ethanol production performance of fermenting microorganisms (Chandel et al., 2006b). In order to remove the inhibitors and increase the hydrolysate fermentability, several chemicals and biological methods have been used. These methods include overliming (Martinez et al., 2000), charcoal adsorption (Chandel et al., 2006b), ion exchange (Nilvebrant, 2001), detoxification with laccase (Martin et al., 2002; Chandel et al., 2006b), and biological detoxification (Lopez et al., 2004). The detoxification of acid hydrolysates has been shown to improve their fermentability; however, the cost is often higher than the benefits achieved (Palmqvist and Hahn- Hagerdal, 2000; von Sivers and Zacchi, 1996). Dilute acid hydrolysis is carried out in two stages- First-stage and two-stage.

First-stage dilute acid hydrolysis
The lignocellulosic material is first contacted with dilute 018 Biotechnol. Mol. Biol. Rev. sulfuric acid (0.75%) and heated to approximately 50°C followed by transferring to the first stage acid impregnator where the temperature is raised to 190°C. Approximately, 80% of the hemicellulose and 29% of cellulose are hydrolyzed in the first reactor. The hydrolysate is further incubated at a lower temperature for a residence time of 2 h to hydrolyse most of the oligosaccharides into monosaccharides followed by the separation of solid and liquid fractions. The solid material again washed with plentiful of water to maximize sugar recovery. The separated solid material is sent to second stage acid hydrolysis reactor
(Figure 1).

Two-stage dilute acid hydrolysis
In two-stage dilute acid hydrolysis process, first, biomass is treated with dilute acid at relatively mild conditions during which the hemicelluose fraction is hydrolyzed and the second stage is normally carried out at higher temperature for depolymerisation of cellulose into glucose. The liquid phase, containing the monomeric sugars is removed between the treatments, thereby avoiding degradation of monosaccharides formed (Figure 1). It is very important to avoid monosaccharide degradation products for improving the ethanol yield. Sanchez et al. (2004) carried out the two-stage dilute acid hydrolysis using Bolivian straw material, Paja brava. In first stage, P. brava material was pretreated with steam followed by dilute sulfuric acid (0.5 or 1.0% by wt) hydrolysis at temperatures between 170 and 230°C for a residence time between 3 and 10 min. The highest yield of hemicellulose derived sugars were found at a temperature of 190°C, and a reaction time of 5 – 10 min, whereas in second stage hydrolysis considerably higher temperature (230 °C) was found for hydrolysis of remaining fraction of cellulose.

Concentrated acid hydrolysis
This method uses concentrated sulfuric acid followed by a dilution with water to dissolve and hydrolyse the substrate into sugar constituents. This process provides complete and rapid conversion of celluose to glucose and hemicellulose to xylose with a little degradation. The concentrated acid process uses 70% sulfuric acid at 40 - 50°C for 2 to 4 h in a reactor. The low temperatures and pressure will lead to minimize the sugar degradation. The hydrolyzed material is then washed to recover the sugars. In the next step, the cellulosic fraction has to be deploymerized. The solid residue from first stage is de-watered and soaked in 30 - 40% sulfuric acid for 50 min. at 100°C for further cellulose hydrolysis. The resulting slurry mixture is pressed to obtain second acid–sugar stream (approximately 18% sugar and 30% acid). Both the sugar steams from two hydrolysis steps are combined and may be used for subsequent ethanol production. Iranmahboob et al. (2002) performed the concentrated acid hydrolysis of mixed wood chips and found that maximum sugar recovery (78 - 82% of theoretical yields) was achieved at sulfuric acid concentration (26%) for 2 h of residence time. The primary advantage of the concentrated acid process is the potential for high sugar recovery efficiency, about 90% of both hemicellulose and cellulose fraction gets depolymerized into their monomeric fractions. The acid and sugar syrup are separated via ion exchange and then acid is reconcentrated through multiple effect evaporators. The remaining lignin rich solids are collected and optionally palletized for fuel generation (Figure 2).

Enzymatic hydrolysis
The acid, alkaline or fungal pretreated lignocellulosics can be saccharified enzymatically to get fermentable sugars (Ghose and Bisaria, 1979; Kuhad et al., 1997; Itoh et al., 2003; Tucker et al., 2003). Bacteria and fungi are the good sources of cellulases, hemicellulases that could be used for the hydrolysis of pretreated lignocellulosics. The enzymatic cocktails are usually mixtures of several hydrolytic enzymes comprising of cellulases, xylanases, hemicellulases and mannanases. In the last decade, new cellulases and hemicellulases from bacterial and fungal sources have continued been isolated and regular efforts have been made for the improved production of enzymetictiters (Aro et al., 2005; Foreman et al., 2003). However, the cellulases were produced at a concentration toolow to be useful. There is a group of microorganisms (Clostridium, Cellulomonas, Tricho-derma, Penicillium, Neurospora, Fusarium, Aspergillus etc.) showing a high cellulolytic and hemicellulolytic activity, which are also highly capable of fermenting monosaccharides. Genetic engineering is used to produce super strains, which are capable of hydrolysing cellulose and xylan along with fermentation of glucose and xylose to ethanol (Aristidou and Penttila, 2000; Lin and Tanaka, 2006). The utilization of cellulose by microorganisms involves a substantial set of fundamental phenomena beyond those associated with enzymatic hydrolysis of cellulose (Lynd et al., 2002).

Separate hydrolysis and fermentation (SHF)
Enzymatic hydrolysis performed separately from fermentation step is known as separate hydrolysis and fermentation (SHF) (Sreenath et al., 2001; Wingren et al., 2003). The separation of hydrolysis and fermentation offers various processing advantage and opportunities. It enables enzymes to operate at higher temperature for increased  performance and fermentation organisms to operate at moderate temperatures, optimizing the utilization of sugars (Figure 3).

Figure 1. Dilute acid hydrolysis (First-stage and Two-stages) and separate fermentation of pentose and hexose sugars.

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