Bioethanol Production and Dilute Acid Pretreatment of Lignocellulosic Materials:a Review*

Arthur Redding Chen Yuan-cai Fu Shi-yu Zhan Huai-yu Jay J．Cheng?

(1．Department of Biological and Agricultural Engineering，North Carolina State University，Raleigh NC 27695－7625，USA;2．State Key Laboratory of Pulp and Paper Engineering，South China University of Technology，Guangzhou 510640，Guangdong，China)

0 Introduction

Liquid fuelsmade up 37．2 quadrillion Btu(40%)of the total energy consumed in the United States(U．S．)in 2010．Consumption of liquid fuel for the transportation sector in 2010 was 27．6 quadrillion Btu，72%of the total liquid fuel consumption in USA，and is projected to increase to 38．0 quadrillion Btu(73%)by 2035．During the same period of time，it is projected that the consumption of liquid fuel for transportation will increase from 19．2 to 20．1 million barrels a day［1］．World demand for oil has grown over the last 20 years and is projected to continue to grow through the next 20 years．This is causing the price of oil to climb at increasing rate．It is projected that the price will increase by 25% from January 2009 to January 2011［1］．Given that oil production has already peaked in USA and is expected to peak worldwide within the next one to five decades，it is understood that the supply is limited．The price is only expected to increase further as a result of limited supply．

There are also environmental implications to using petroleum derived fuels．Oil is the result of natural processes converting organic matter(sequestered carbon)deposited millions of years ago and therefore burning oil releases carbon into the atmosphere which had been removed from the contemporary carbon cy－cle．Without a counterbalance，this carbon in the form of gasses builds up and has been linked to a variety of environmental impacts including increasing acidity in the oceans，melting of polar ice，and higher average temperatures globally［2］．In 2007，23% of the total carbon dioxide emitted by USA came from burning liquid fuels in the transportation sector［1］．Using biological sources to produce fuels minimizes the global warming impact by creating a cycle where the carbon released while burning the fuel is equal to the carbon taken in by the next generation of biofuel crop［3］．

2 Potential Gasoline Replacements

The liquid transportation fuel replacement candidates with the most potential include biomethanol，biobutanol，and bioethanol where the“bio”prefix establishes these fuels are produced from a biomass source．

2．1 Biomethanol

Biomethanol is one carbon alcohol historically generated as a byproduct of the conversion of wood into charcoal，earning it the nickname “wood alcohol”．Currently，biomethanol is produced from biomass through a gasification process to generate synthesis gas followed by a reaction in the presence of a catalyst to produce methanol．However，this process is not economically feasible．Additionally，methanol mixes well with water，is toxic to humans through ingestion and skin absorption，and it burns with an invisible flame making it potentially hazardous in comparison to other bio－alcohols［4］．

2．2 Bioethanol

Bioethanol is two carbon alcohol most notably ge－nerated from the six carbon sugar glucose by the yeast Saccharomyces cerevisiae as metabolic product．Five carbon sugars，like xylose，can also be converted to ethanol by genetically modified yeast strains and certain bacteria［5－7］．Additionally，synthesis gas(syngas)，a mixture of hydrogen，carbon monoxide，and carbon dioxide，generated from gasifying biomass can be converted to ethanol either through fermentation using bacteria or by chemical reaction using a catalyst［8－9］．As a combustion fuel，ethanol burns cleaner than gasoline and when blended with gasoline it provides oxygen，which aids in a more complete combustion．Because of these aspects，ethanol has been used as a gasoline additive(volume fraction of ethanol is 10%)in the US rather than the more toxic chemical fossil fuel derived methyl tertiary butyl ether(MTBE)［10］．The main di－sadvantages to using ethanol are that it is hygroscopic and has about 66%of the energy as the same volume of gasoline．Despite this lack of comparable energy density，ethanol can still transport a vehicle 80%of the distance an equal volume of gasoline would be able to transport thanks to efficiencygains from more complete combustion［11］．In the end，the combination of a large scale production technology that is more mature than the alternative bio－alcohols，surmountable disadvantages，and the current use in gasoline blends in USA makes bioethanol the ideal candidate for a near term gasoline replacement．

2．3 Biobutanol

Biobutanol is a four carbon alcohol that is a metabolic product of a number of microorganisms．Historically，industrial production has utilized Clostridium acetobutylicum because a starch feedstock could be used to produce acetone，butanol，and ethanol in a mass ratio of 3∶6∶1 with acetone being the primary product of interest initially［12］．Biobutanol has a number of advantages that make it an ideal gasoline replacement including energy content similar to gasoline，it can be used pure or as a mixture with gasoline in unmodified engines，and it separates from water［13］．Some disadvantages researchers are currently trying to address are low limits of butanol tolerance in the production organisms，expensive downstream processing to separate the butanol from metabolic coproducts，and the ability to use cellulosic feedstocks instead of starches［12－13］．

3 Feedstock Options for Bioethanol Production

As described above，bioethanol can be generated from syngas or sugars．While syngas production provides the luxury of being nearly feedstock independent，the process currently has many issues keeping it from economic production with current technology［14］．This leaves bioethanol production from su－gars．In general，there are three major sources of sugars which each requires different process steps to first convert them into fermentable sugars and then into bioethanol．

3．1 Sugar Feedstocks

The simplest option is to just use sugar．Example feedstocks are sugar cane，sweet sorgum，and sugar beets．Sugars from these sources are already available in a fermentable state and as a result only mechanical processing is necessary to extract the sugars for fermentation by yeast．Brazil uses this platform very successfully and by coupling this technology with the appropriate infrastructure，Brazil is able to offer up to 100%ethanol fuels as a gasoline replacement inexpensively．There are some parts of the U．S．that can use sugar feedstocks for ethanol production，but the wider adoption of this sugar source is largely limited by climate constraints．Because of this，sugar feedstocks will not be able to meet the full requirements of gasoline replacement by bioethanol in USA．

3．2 Starch Feedstocks

Starch is a polymer of glucose molecules connected by alpha－1－4 and alpha－1－6 glycosidic bonds．This configuration makes the polymer appear like a set of stairs with each step a glucose molecule．This type of bonding between glucose molecules makes it easier for enzymes(amylases)to access and break these bonds．Therefore，starches are an ideal feedstock because after mechanical processing starches can be saccharified in a mixture of hot water and amylases．Examples of major starch feedstocks include food crops like barley，corn，potatoes，rice and wheat depending on locale．In USA，corn is currently the leading source for producing bioethanol［15］．

Although corn is easy to convert into bioethanol，there are limitations associated with using it that will keep it from being able to supply enough bioethanol to replace gasoline．One of the most obvious problems with corn and many other starch sources is that they compete as food．This can negatively impact corresponding markets with inflated prices of the feedstock［16］．Another issue concerns the central locations where feedstocks are produced．In the case of corn in USA，the majority of production occurs in the central part of the country requiring transportation of the fuel to the coasts．Since ethanol is hygroscopic，it cannot be transported by pipeline like oil and must be transported by a more costly method in tankers［17］．Corn also requires arable land and a significant amount of nitrogen to grow．Nitrogen fertilizer is produced using large amounts of natural gas and often much of the nitrogen in fertilizer is lost through run－off and volatilization nulli－fying much of the environmental benefit［18－19］．Overall，corn is a good short－term source for bioethanol production，but it barely addresses environmental concerns and it will not be able to meet the supply requirements to replace gasoline alone．

3．3 Lignocellulosic Feedstocks

Lignocellulosic feedstocks offer a possible solution to the constraints faced with the sugar and starch feedstocks．Lignocellulose is the shorthand term for any bio－mass containing the three polymers hemicellulose，cellulose，and lignin．Hemicellulose is mostly made up by a backbone of the five carbon sugar xylose with side chains of other five and six carbon sugars like arabinose，glucose，and galactose．Cellulose is，like starch，a polymer of glucose molecules except that in cellulose the glucose bonds are beta－1－4 glycosidic bonds．This means that instead of stairs，cellulose appears to be a straight line making it much more stable(crystalline)and more difficult to degrade into glucose monomers．Lignin is a polymer made up of a variety of aromatic subunits and in a plant the general purpose of lignin is to prevent access to the structural carbohydrates(cellulose and hemicellulose)of the plant．Fig．1 below contains a simplified schematic of how lignin，hemicellulose，and cellulose are organized in lignocellulosic biomass．Example lignocellulosic feedstocks include agricultural，food processing and municipal wastes，perennial grasses，and woody biomass［20］．

Fig．1 General structure of lignocellulosic biomass

Most of the benefits attributed to lignocellulosic biomass are a result of the feedstock options．Lignocellulosic biomass is nearly ubiquitous on earth．This opens the possibility of local bioethanol production，which could keep costs to transport the fuel low．Additionally，many of the potential feedstocks are wastes or low－input crops that can be grown on marginal lands．This keeps the feedstock prices much lower than corn and avoids fertilizer related environmental impacts．Since lignocellulosic feedstocks are not food resources nor do they require land used to grow food，there is no risk that either food or lignocellulosic feedstock prices will increase from competition．

The reason that lignocellulosic materials are not being widely used currently is because there are few barriers to using these feedstocks for bioethanol production．One barrier is that the enzymes(cellulases)needed to break down cellulose into glucose monomers are much more expensive than the amylases used to break down starches．The US Department of Energy(DOE)funded four private companies in 2008 as part of a project to lower the price of cellulases per gallon of ethanol．While some decrease in the price was achieved previously，research into further lowering the price as well as alternative cellulase sources is still being pursued［21］．Another barrier is that the xylose component of the biomass is not fermentable using the samemicroorganism (Saccharomycescerevisiae)used in current starch based ethanol production．Because hemicelluloses make up 30%～40% (mass fraction)of the biomass，fermenting the xylose can be important for ma－king bioethanol production from lignocellulosic biomass economically feasible［22］．Research has yielded some gains in this area through the development of yeast and bacteria genetically altered to ferment xylose in addition to the application of some naturally occurring strains［5－7］．The largest barrier keeping lignocellulosic biomass from being a feasible bioethanol feedstock is the recalcitrant nature of the biomass to grant access to the cellulose and hemicellulose components．A process step referred to as“pretreatment”is required to disrupt the biomass structure to make it accessible to hydrolysis by cellulases in order to release fermentable monomeric sugars the yeast or bacteria can use．

4 Pretreatment Options for Lignocellulosic Biomass

Pretreatment，enzymatic hydrolysis and fermentation are the three areas receiving the most attention through research as alluded to above．Of these，pretreatment is of significant importance because as a process step，it is upstream of both enzymatic hydrolysis and fermentation．A good pretreatment will disrupt the biomass enough to allow for the maximum hydrolysis of both the hemicellulose and cellulose components into monomeric sugars with minimal generation of enzymatic hydrolysis and fermentation inhibitors［23］．The most often studied pretreatment technologies fall into four main categories which include physical pretreatments，chemical pretreatments，physio－chemical(combination)pretreatments，and biological pretreatments［22］．

4．1 Physical Pretreatments

Physical pretreatments include various methods ofmechanicalalteration like hammer and ball mills［24］．Mechanical alteration is the action of grinding or chipping materials into smaller pieces which，in the case of lignocellulosic biomass，disrupts the biomass structure and increases surface area．The smallest particles are the most susceptible to enzymatic hydrolysis and although small particle sizes are achievable，the amount of energy increases greatly as the size of particles decreases．As a result，physical pretreatment alone is not economically feasible at larger scales［25］．

4．2 Chemical Pretreatments

Chemical pretreatments involve using alkali reagents，dilute acids，peroxide，organic solvents，or ozone to disrupt the structure of lignocellulosic biomass［22］．The alkaline reagents most often used in pretreatment applications are sodium hydroxide and calcium hydroxide(lime)．The major mechanism of alkaline pretreatments is the saponification of the ester bonds between lignin and hemicellulose leading to the delignification of the biomass［26］．Lime pretreatments can potentially lower costs because disruption of biomass structure can occur at or near ambient temperature，however these reactions require residence times on the order of hours to days［27－28］．Dilute acid acts to hydrolyze hemicellulose out of the solid biomass．Although dilute acid pretreatment greatly increases the success of enzymatic hydrolysis，disadvantages include the formation of inhibitory compounds，expen－sive equipment made from stainless steel to resist corrosion and downstream neutralization of the acid prior to fermentation because a low pH is inhibitory to the growth of yeast and other microorganisms［22，28］．Dilute pero－xide can be used to disrupt lignin and hemicellulose bonds resulting in the removal of hemicellulose from the solid biomass at near ambient conditions．This improves resulting glucose yields from enzymatic hydrolysis to above 90%，but only after at least 8 hours of pretreatment time at 30℃［29］．Organic solvents，like methanol，ethanol，or acetone can be coupled with an acid to disrupt the lignin and hemicellulose bonding in biomass．These solvents and acids need to be removed to allow for further downstream processing and should be recovered for economic benefit［25］．Ozone，like lime，can also be used to affect lignin and hemicellulose bonding at ambient conditions．However，the amount of ozone required to significantly treat the biomass is not cost effective［22］．

4．3 Physio-Chemical Pretreatments

Combinations of physical and chemical pretreatments have also been investigated with typical examples being steam explosion，ammonia fiber explosion(AFEX)，and carbon dioxide explosion．The general idea shared by each of these variations is a pressurization and heating of the biomass，forcing an intermediate into the structure，followed by a rapid depressurization to ambient pressure，which causes the biomass structure to explode［22，25］．Steam has the added effect of heat which promotes autohydrolysis，or the formation of organic acids from the biomass，which then help to break up the structure of the biomass through the removal of hemicellulose．Dilute acid has also been coupled with steam explosion to improve the hydrolysis of hemicellulose［24，30］．AFEX is done at lower temperature and pressure than steam explosion，but takes advantage of ammonia which acts like an alkali reagent and removes lignin from the biomass structure without degrading the carbohydrates［24］．Carbon dioxide explosion is a similar notion to steam explosion and AFEX，except with lower yields［22，31］．Zheng et al［32］reported lower inhibitor levels for carbon dioxide explosion compared to steam explosion and AFEX．

4．4 Biological Pretreatments

Organisms which naturally break down lignocellulosic biomass have been researched to identify if they could be applied as a pretreatment in large scale bioethanol production．Varieties of white－rot fungus have been the most popular organism investigated for this application because of effective and preferential lignin degradation that has been observed．The main benefits of biological pretreatments include low energy requirements，lower equipment costs，and lower water requirements．Unfortunately，the fungi may also metabolize a portion of the carbohydrates and pretreatment times are on the order of days to weeks which makes this type of pretreatment unsuitable for industrial scale production［33－34］．

5 Dilute Acid Pretreatment:Benefits and Applications

Dilute acid pretreatment has a number of benefits that currently make it a better choice compared to the other pretreatment options outlined above and as a result，dilute acid pretreatment is first in line for commercial application in the production of bioethanol from lignocellulosics［35］．One benefit from a process standpoint is that dilute acid generates separable streams．There is a liquid pre－enzymatic hydrolysis stream containing a majority of xylose，a liquid postenzymatic hydrolysis stream containing a majority of glucose，and a solid stream containing a majority of lignin．This removes the need for complex or costly unit processes for separation and allows for product specific unit processes．Additionally，acids，like sulfuric acid and acetic acid，are less expensive compared to other chemicals， most specifically alkalis［24，27］．Eggeman et al［36］reported dilute sulfuric acid pretreatment as cheaper per gallon of ethanol produced than sodium hydroxide，lime，or AFEX．The last major benefit，as outlined in the examples below，is that researchers have shown that using dilute acid and dilute acid combined with steam explosion are effective pretreatments prior to enzymatic hydrolysis across many types of lignocellulosic feedstocks resulting in high yields of monomers from both the hemicellulose and cellulose components．

5．1 Agricultural Residues

Agricultural residues are ideal feedstocks because they are already associated with a cropping system and being wastes keeps the costs low．Corn stover is one of the most researched feedstocks in this category．In a summary of studies using different pretreatment technologies on corn stover，dilute acid stands out as the only pretreatment yielding great than 90%of both xylose and glucose［25］．The dilute acid pretreatment referenced is from a study done by Lloyd and Wyman［37］where at solid loading of 10% ，91%of theoretical glucose and xylose were recovered after a pretreatment of the corn stover at 160℃ and 0．49%sulfuric acid over a residence time of 20 minutes and an enzymatic hydrolysis step．Tucke et al［30］showed that combining dilute sulfuric acid with steam explosion to pretreat corn stover at a solid loading of 46%yielded greater than 90% xylose and glucose for a pretreatment at 190℃ and 1．1%(mass fraction)acid for a residence time of 90 seconds．Another agricultural residue example，wheat straw，was pretreated with 0．75%(volume fraction)sulfuric acid at 121℃ for 60 minutes at about a 7% solid loading and was shown to yield 74%of total sugars after enzymatic hydrolysis［38］．Saha et al［39］improved the yield of total sugars from wheat straw to 84%using a microwave for heating to 160℃ with a sulfuric acid concentration of 0．5%(w/v)for 10 minutes．

5．2 Woody Biomass

The main sources of woody biomass can broadly be grouped as forest residuals and wood chips．In general，woody biomass tends to respond better to the combination of dilute acid pretreatment with steam explosion than just dilute acid alone［23］．In an example of this dilute acid pretreatment coupled with steam explosion，Emmel et al［40］was able to recover 70%of hemicellulose sugars using 0．175%acid at 210℃ for a two－minute pretreatment time．In the same study，a 90% cellulose conversion was achieved at 200℃under similar pretreatment conditions．Examining only xylose recovery，Esteghlalian et al［41］were able to achieve close to 90% of the theoretical yield from poplar under pretreatment conditions between 170～180℃ using in excess of 0．9%sulfuric acid and residence times of minutes．

5．3 Herbaceous Biomass

Generally，switchgrass stands out as a popular choice of research because of a high yield of biomass per hectare(5．2 ～11．1 dry－Mg/ha)，the option to cultivate it on marginal land，and a potentially favorable net energy gain［40］．Young switchgrass pretreated with 1．5%acid at 120℃ for 60 minutes was shown to yield close to 80%glucose and above 90%nonglucose(xylose and arabinose)sugars．At 1．5%acid and 150℃ for 20 minutes(with 10 minute preheat)young switchgrass yielded about 85%glucose and 80%non－glucose sugars［42］．Switchgrass was also pretrated at 180℃ and 1．2%(mass fraction)sulfuric acid to yield 90%glucose from cellulose after enzymatic hydrolysis．

However，switchgrass is not the only possibility of an herbaceous energy crop and therefore other options more specific to certain locales have been investigated as well．Vegetative stage reed canary grass was pretreated with 1．5%acid at 120 ℃ for 60 minutes to yield above 80%glucose and 90%non－glucose su－gars［42］．Sun and Cheng［43］were able to show the enzymatic glucose conversion of about 82%for coastal bermudagrass，a grass grown for hog waste nutrient management in the southeastern of USA，after pretreatments at 1．5% (mass fraction)sulfuric acid at 121℃ for 60 and 90 minutes．Corresponding xylose conversion，however，was only about 60%．

6 Degradation Products

Dilute acid，especially when coupled with high temperatures，unfortunately has a propensity to degrade biomass compounds into products which are inhibitory to downstream processes like enzymatic hydrolysis and fermentation．Inhibitory levels of these compounds，which will be discussed further below，are generally not very high and therefore restrict the severity of pretreatment conditions feasible for successful downstream process．A balance between severity and yield can be difficult to reach and some researchers have generated higher yields for monomeric sugars from hemicellulose and cellulose，only to find that the yeast are inhibited and those pretreatment conditions cannot be used to make bioethanol［44］．The inhibitory levels of each of these compounds and the corresponding degrees of inhibition are not as clear because of interaction effects between inhibitors and other factors like yeast concentration［45］．

6．1 Sugar Degradation Products

During high severity pretreatment，as pentose polymers are hydrolyzed into monomers，those monomers can be further degraded into furfural．After continued exposure to severe conditions，furfural can be degraded into levulinic acid．The same process occurs with hexoses as well，generating hydroxymethylfurfural(HMF)and formic acid respective of exposure to pretreatment conditions．Reactions 1 and 2 below outline the path of sugar degradation．

Hemicellulose →Pentose→Furfural→

Cellulose or Hemicellulose→Hexose→

Generally，furfural and HMF have been reported as causing a lag phase in yeast growth before sugar consumption begins because the yeast take up the two degradation compounds first(furfual faster than HMF)before moving on to converting sugars to ethanol．Levulinic and formic acids are generally shown as helpful at levels up to 100mmol and inhibitory after levels from 100 ～ 200 mmol．This has been confirmed by experiments done by Larsson et al［46］which showed furfural and HMF as not inhibitory to ethanol yield，while weak acids(formic，levulinic，and acetic)at a combined concentration greater than 100 mmol did inhibit ethanol yield．Navarro［47］showed the increased inhibition of yeast productivity and growth rate corresponding to higher levels of furfural．He also reported on how increasing the initial yeast concentration could decrease the inhibitory effects of furfural．In a review of furfural and HMF inhibition studies，Almeida et al［48］showed that the pooled research on this topic confirms some level of inhibition to yeast and ethanol production at a variety of furfural，HMF，and initial yeast concentrations．

6．2 Lignin Degradation Products

Just as lignin molecules can include a variety of different chemical compounds，the resulting degradation compounds possible are just as varied．In general，lignin degrades into phenolic compounds with a variety of molecular weights［49］．Details regarding phenolic inhibition are limited due to a lack of accurate investigation，but it is suspected that low molecular weight phenolic compounds are more inhibitory to fermentation and that there are interaction effects with furfural and HMF which increase overall inhibitory effects of both compounds［45］．

7 Conclusion

Bioethanol appears to be a good gasoline replacement to start with until a better liquid alternative becomes more feasible．Research using dilute acid pretreatment，especially when coupled with steam explosion，across all types of lignocellulosic feedstocks should continue in order to refine optimal pretreatment conditions that maximize sugars and minimize inhibitory compounds．Commercial scale adoptions of dilute acid pretreatment should also be used to further refine process conditions for the benefit of process economics．

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