Review:Continuous hydrolysis and fermentation for cellulosic ethanol production
Simone Brethauer,Charles E.Wyman *
Center for Environmental Research and Technology and Chemical and Environmental Engineering Department,University of California,Riverside,CA 92507,United States
a r t i c l e i n f o Article history:
Received 31August 2009
Received in revised form 2November 2009Accepted 3November 2009
Available online 14December 2009Keywords:
Continuous fermentation Enzymatic hydrolysis Fuel ethanol
Lignocellulosic biomass
Simultaneous saccharification and fermentation (SSF)
a b s t r a c t
Ethanol made biologically from a variety of cellulosic biomass sources such as agricultural and forestry residues,grasses,and fast growing wood is widely recognized as a unique sustainable liquid transporta-tion fuel with powerful economic,environmental,and strategic attributes,but production costs must be competitive for these benefits to be realized.Continuous hydrolysis and fermentation processes offer important potential advantages in reducing costs,but little has been done on continuous processing of cellulosic biomass to ethanol.As shown in this review,some continuous fermentations are now employed for commercial ethanol production from cane sugar and corn to take advantage of higher vol-umetric productivity,reduced labor costs,and reduced vessel down time for cleaning and filling.On the other hand,these systems are more susceptible to microbial contamination and require more sophisti-cated operations.Despite the latter challenges,continuous processes could be even more important to reducing the costs of overcoming the recalcitrance of cellulosic biomass,the primary obstacle to low cost fuels,through improving the effectiveness of utilizing expensive enzymes.In addition,continuous pro-cessing could be very beneficial in adapting fermentative organisms to the wide range of inhibitors gen-erated during biomass pretreatment or its acid catalyzed hydrolysis.If sugar generation rates can be increased,the high cell densities in a continuous system could enable higher productivities and yields than in batch fermentations.
Ó2009Elsevier Ltd.All rights reserved.
reaction diffusion
1.Introduction
According to the recent report of the Intergovernmental Panel on Climate Change (IPCC)warming of the world’s climate system is unequivocal and is very likely due to the observed increases in anthropogenic greenhouse gas concentrations.Atmospheric con-centrations of carbon dioxide (CO 2),the dominant greenhouse gas,have increased from a pre-industrial value of about 280ppm to 379ppm in 2005,primarily as a result of fossil fuel use (IPCC,2007).Overall,petroleum is the source of about 170quadrillion (1015)BTUs or quads of energy of the total of more than 460quads the world uses,far more than derived from coal,natural gas,hydroelectric power,nuclear energy,geothermal,or other sources.Over half of petroleum in this total is used for transportation,and demand is projected to grow rapidly as vehicle traffic increases throughout the world and even accelerates in Asia.Besides the negative global warming impact of fossil fuels,volatile oil prices and dependency on politically unstable oil exporting countries re-sulted in a significant increase in international interest in alterna-tive fuels and led policy makers in the EU and the US to issue ambitious goals for substitution of alternative for conventional fuels (Galbe and Zacchi,2002;Wyman,2007).
Ethanol made biologically by fermentation from a variety of bio-mass sources is widely recognized as a unique transportation fuel with powerful economic,environmental and strategic attributes.First genera
tion ethanol made from starch-rich materials such as corn and wheat or from sugar feedstock is a mature commodity product with a worldwide annual production of over 13billion US gallons in 2007.However,these raw materials are insufficient to meet the increasing demand for fuels,and concerns have heightened recently that competition between the use of agricultural commod-ities for fuel production is driving up food costs.Furthermore,the use of food crops for fuel production may lead to environmentally detri-mental indirect land use he deforestation of tropical rainforest to gain more farmland.In addition,the reduction of green-house gases resulting from use of starch-based ethanol is not as high as desirable (Farrell et al.,2006;Hahn-Hägerdal et al.,2006).Alter-natively,ethanol can be produced from lignocellulosic materials such as agricultural residues,wood,paper and yard waste in muni-cipal solid waste,and dedicated energy crops,which constitute the most abundant renewable organic component in the biosphere (Cla-assen et al.,1999).
Regardless of the feedstock,the final ethanol selling prize must be competitive with that for gasoline,but gasoline benefits from over a century of learning curve improvements and largely paid for capital.Thus,profit margins in ethanol production processes are low,and returns on capital are uncertain due to the tremendous
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*Corresponding author.Tel.:+19517815703;fax:+19517815790.E-mail address:charles.wyman@ucr.edu (C.E.Wyman).Bioresource Technology 101(2010)
4862–4874
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price swings in petroleum prices.In this context,costs must be kept as low as possible,and continuous fermentation of cellulosic bio-mass to ethanol can offer important advantages in terms of greater productivity and lower costs.Unfortunately,although process designs have been conceptualized based on continuous enzymatic hydrolysis and fermentations to take advantage of their low cost potential,limited studies have actually been reported from which to design or advance the technology.Thus,more information is sorely needed on this subject to guide the advancement of lower cost approaches to making ethanol and overcome the significant cost barriers to market entry.
In this paper,we will provide a short introduction to concepts and characteristics of continuous fermentations.Then a summary is presented of experiences and research activities withfirst gener-ation industrial continuous ethanol fermentations as these provide the foundation for second generation cellulose-based processes. Following that,we review current knowledge of continuous fer-mentation of lignocellulosic material,including those based on chemical and enzymatic hydrolysis of cellulose to glucose.
2.Concept of continuous fermentations
In a true continuous fermentation system,substrate is con-stantly fed to the reaction vessel,and a correspondingflow of fer-mented product broth is discharged to keep the reactor volume constant.Furthermore,the balance between feed and discharge is maintained for long enough times to achieve steady state oper-ation with no changes in the conditions within the reactor.Com-pared to a batch reaction,this mode of operation offers reduced vessel down time for cleaning andfilling providing improved vol-umetric productivity that can translate into smaller reactor vol-umes and lower capital investments plus ease of control at steady state.
Two basic types of continuous reactors can be employed:the continuous stirred tank reactor(CSTR)or the plugflow reactor (PFR).In an ideally mixed CSTR,the composition in the reactor is homogenous and identical to that for the outgoingflow.In an ideal PFR,the reactants are pumped through a pipe or tube with a uni-form velocity profile across the radius,and the reaction proceeds as the reagents travel through the PFR with diffusion assumed to be negligible in the axial direction.Consequently,PFR operations imply that inoculum has to be constantly fed to the reactor for fer-mentation processes.Cascading a large number of CSTRs in series will have similar performance to a PFR.
In a system with constant overall reaction stoichiometry that can be described by a single kinetic equation,performing the reac-tion in two or more bioreactors may lead to a higher product con-centrati
on,a higher degree of conversion,a higher volumetric productivity,or a combination of these factors compared to opera-tion of a single CSTR.One approach to optimizing a continuous pro-cess is to determine the reactor configuration that gives the lowest residence time to achieve a certain degree of conversion.If the kinetics are known,a plot of the reciprocal rate against the dimen-sionless substrate concentration S/S feed can be employed to esti-mate the reaction residence time and therefore the reaction volume(de Gooijer et al.,1996).For a CSTR,the area corresponding to a rectangle whose height equals the reciprocal of the rate at the desired conversion will equal the residence time for reaction to this conversion,whereas the residence time for a PFR will corre-spond to the area under the curve(see Fig.1).If the desired conver-sion is higher than the minimum in the curve,a combination of reactors will require less reaction volume.Thus,for the situation depicted in Fig.1,the combination of a CSTR followed by a PFR will be preferred if a conversion of98%is targeted.
An important performance criteria is the productivity of the fer-mentation ,the amount of product formed per unit of time and reactor volume,which depends on several factors includ-ing substrate concentration,cell concentration,and dilution rate. We used a simple model based on Monod growth kinetic that in-cludes terms for product and cell inhibition(Lee et al.,1983)to illustrate the influence of some operational parameters.Generally, the productivity in a continuous fermentation syst
em is higher than in a batch reactor.In the model example,a productivity of 3.57g LÀ1hÀ1was calculated for a batch process inoculated with 1g LÀ1yeast cells.In a standard single stage CSTR without cell retention,where the biomass concentration would befixed,a max-imum productivity of4.24g LÀ1hÀ1was calculated for a dilution rate of0.136hÀ1,however,the substrate conversion was only 83%.Generally it is desirable to achieve almost complete substrate conversion at the highest possible productivity to avoid loss of sub-strate or the need for recycle.In a two stage system with properly designed unequal reactor sizes(see Fig.2a),the maximum possible overall productivity would be lower than in a single stage system, but the substrate conversion at identical productivities would be higher.In the two stage system,a maximum overall productivity of4.16g LÀ1hÀ1was calculated at a substrate conversion of92% (Fig.2b).If a substrate conversion of99%were the goal,the pro-ductivity in a single stage CSTR would be 2.77g LÀ1hÀ1but 3.94g LÀ1hÀ1for a two stage system.Generally,a cascade of fer-mentors would be superior to a single vessel for autocatalytic reac-tions such as cell growth which are product-inhibited,but for situations with substrate inhibition,a single stage CSTR is often more favourable to remove as much reactant as possible(de Goo-ijer et al.,1996).
3.Continuous ethanol production from starch and sugar feedstocks
3.1.Industrial continuous ethanol production from sugar cane
Sugar cane is a tropical and subtropical crop that is the primary feedstock for ethanol production in Brazil,India,and Colombia.It contains mainly sucrose,a dimer of glucose and fructose,which is readily assimilated by Saccharomyces cerevisiae(Sanchez and Cardona,2008).Both sugar cane juice and molasses normally con-tain sufficient minerals and nutrients visiae to ferment
1.0
0.8
0.6
0.4
0.2
0.0
CSTR
PFR
S.Brethauer,C.E.Wyman/Bioresource Technology101(2010)4862–48744863
them directly to ethanol(Wheals et al.,1999).In Brazil,70–80%of the distilleries employ fed-batch processes(Melle-Boinot Process) for fermentors with outputs ranging from400to2000m3ethanol per day.Typically,high yeast cell concentrations of between8% and17%achieve fermentation times of only6–10h andfinal etha-nol concentrations of up to11%v/v,corresponding to an average ethanol yield of91%.After each fermentation cycle,the yeast cells are separated,treated with dilute sulphuric acid to kill contaminat-ing bacteria,and then recycled to start a new fermentation.This se-quence can be repeated up to200times and minimizes carbon consumption for yeast growth while providing very high ethanol productivities(Dorfler and Amorim,2007;Godoy et al.,2008; Wheals et al.,1999;Zanin et al.,2000).Thefirst continuous ver-sions of the Melle-Boinot process appeared in the1970s,but sev-eral operational problems were detected,such as a high level of contamination,low productivity,low yields,and problems with solidsflow.Today’s continuous fermentation processes are opti-mized based on kinetic models to achieve high productivities(typ-ically10mL LÀ1hÀ1),high processflexibility and stability,and low consumption of chemicals and are considered to be less expensive for ethanol production than batch processes(Zanin et al.,2000).
An important feature of state-of-the-art continuous processes is the use of multiples stages(typically fo
ur orfive)of variable sizes.The sugar substrate is fed to the top of thefirst reactor together with the recycled yeast cream and leaves through the bottom,flowing then by gravity to the middle of the next stage.Each reac-tor typically uses an external plate-type heat exchanger for cooling of the fermentation broth,with the kinetic energy of the liquid leaving the heat exchanger outlet used to agitate the reactor.The yeast cells produced are separated from the‘‘wine”by disk-bowl centrifuges,forming a yeast cream,which is then sent to acid treat-ment prior to being recycled back to thefirst reactor(Zanin et al., 2000).
Guerreiro et al.(1997)described an expert system for the de-sign of such an industrial continuous fermentation plant which combines expert knowledge and industrial practices with kinetic modelling.As parameters are taken from industrial fermentations, differences between theoretical calculations and practical results are claimed to be minimal.In the example presented,an input medium containing170–190g LÀ1sugar was fed at a rate of 143m3hÀ1to a four stage reactor train with volumes of215, 274,324,and213m3to maximize performance.In this case,the steady state concentrations in thefirst and last stages were54 and1g LÀ1of sugar,42and66g LÀ1of ethanol,and29and 31g LÀ1of yeast biomass,respectively,and the process productiv-ity was given as7.7g LÀ1hÀ1.Generally,the volumes of the tanks influence the productivity which varied from6.1to7.9g LÀ1hÀ1. Through optimization,it was p
ossible to replace a previous fed-batch plant that consisted of24fermenters of200m3volume each (total4800m3)producing400m3of96%ethanol per day with a continuous plant with a total volume of2500m3producing about 440m3of96%ethanol per day(Guerreiro et al.,1997).However, larger continuous plants exist with capability to produce up to 600m3ethanol per day(Zanin et al.,2000).
In some Brazilian distilleries,processes based onflocculent yeast strains are employed,with cell separation in settlers to avoid costly centrifuges.Yeastflocculation is a reversible,asexual,cal-cium dependent process of self-aggregation in which cells adhere to formflocs consisting of thousands of cells.Because of their macroscospic size and mass,the yeastflocs rapidly settle out of the fermenting medium,thus providing natural cell immobiliza-tion(Verbelen et al.,2006).Compared to the classical Melle-Boinot process,it is claimed that up to1.5%higher fermentation efficiency is obtained,ethanol production costs are ca.$7/m3lower,and con-sumption of chemicals such as antifoam is reduced(Zanin et al., 2000).
Despite such desirable attributes,there are also critical opinions about replacing batch fermentations with continuous processes.In one study of the advantages and disadvantages of continuous and batch fermentation processes for62distilleries over a time span of 9years(1998–2007),batch processes with yeast recycle were shown to be less susceptible to bacterial contamination and the corresponding loss
in productivity(Godoy et al.,2008).Lactobacil-lus contaminations,in particular,are regarded as the major factor that can reduce ethanol yield and also impair yeast centrifugation, and greater quantities of antibiotics are needed to address this is-sue for continuous processes.Also,slightly more sulphuric acid was consumed in continuous processes.Yet,continuous processes have the advantages of lower installation costs due to smaller fer-mentor volumes and less heat exchanger demands as well as lower costs due to greater automation(Godoy et al.,2008).
3.2.Continuous ethanol production from corn
Up to now,corn is the major feedstock for ethanol production in the US,which surpasses Brazil as the largest ethanol producer. Corn kernels contain about70%by weight starch on a dry weight basis.Starch is a D-glucose polymer,consisting of about30%amy-lose,a linear chain of a-1,4linked glucose units with a
helical 4864S.Brethauer,C.E.Wyman/Bioresource Technology101(2010)4862–4874
structure and70%amylopectin,a highly branched polymer with additional a-1,6glycolytic bonds.Ethanol from corn can be pro-duced by either a dry grind(67%of the fuel ethanol)or wet mill (33%)process with recent growth in the industry mostly with dry grind plants due to their lower capital costs(Bothast and Schli-cher,2005).
In the wet mill process,the grain is separated into its four basic components of starch,germ,fiber,and protein to recover higher value co-products including corn oil,corn gluten meal,corn gluten feed,and germ.On the other hand,the dry grind process is much simpler in that the entire corn kernel is ground and mixed with water to form a mash.
The isolated starch from wet-milling and the mash from the dry grind process are treated identically to produce ethanol.First,a thermostable alpha-amylase,which breaks down the starch poly-mer to soluble dextrins by hydrolyzing a1–4bonds,is added. The mixture is heated to over100°C to liquefy the mash over a holding time of at least30min.Then,glucoamylase is added, which converts liquefied starch to glucose at an optimal tempera-ture of65°C.In thefinal fermentation step,which is performed either coupled(simultaneous saccharification and fermentation, SSF)or subsequent to glucoamylase treatme
nt(separate hydrolysis and fermentation,SHF),the mash is cooled to32°C,and yeast is added as well as ammonium sulphate or urea as a nitrogen source. Alternatively,proteases are added to break down corn protein to free amino acids for use as a nitrogen source.Fermentation is com-pleted in48–72h to afinal ethanol concentration of10–12%v/v and higher.Over the course of fermentation,the pH drops to4.0 or lower,which helps to prevent bacterial contamination.Many plants use simultaneous saccharification and fermentation,be-cause it lowers the risk of contamination,lowers the initial osmotic stress on the yeast,and is generally more energy-efficient(Bothast and Schlicher,2005).
According to a United States Department of Agriculture(USDA) survey in2002,27%of the dry grind distilleries in the US employ continuous fermentation processes which are more common in large plants producing more than400m3ethanol per day(Shapo-uri and Gallagher,2005).To the best of our knowledge,no perfor-mance data for industrial continuous corn ethanol fermentations are published.However,Bai et al.(2008)described in their review a commercial plant employing a self-flocculating yeast with a pro-duction capacity of680m3per day which started operation in 2005in China.In this system,six fermentors with volumes of 1000m3each were arranged in a cascade,and corn meal hydroly-zate,with a sugar concentration of200–220g LÀ1,was fed to the fermentation system at a dilution rate of0.05hÀ1.Thefinal ethanol concentration was reported to be11–12%v/v.Yeast
flocs were re-tained within the fermentor by baffles to effectively immobilize them,and the yeast concentration within the fermentors was maintained at40–60g DCW LÀ1.
4.Continuous production of second generation ethanol from lignocellulosic materials
Although composition of lignocellulosic materials varies in dif-ferent plants,the three main components are cellulose(36–61%), hemicellulose(13–39%),and lignin(6–29%)(Olsson and Hahn-Hägerdal,1996).Cellulose is a D-glucose polymer,where the sub-units are linearly linked by b-1,4glycosidic bonds and exists in crystalline and amorphous forms.Hemicellulose is composed of linear and branched heteropolymers of ,xylose and arabinose)and ,mannose,glucose,and galactose).Lig-nin is a polymer that can consist of three different phenylpropane units(p-coumaryl,coniferyl and sinapyl alcohol)that bind the plant together.In order to release fermentable sugar monomers,cellulose and hemicellulose are hydrolyzed chemically,enzymati-cally,or by their combination(Gray et al.,2006;Hendriks and Zee-man,2009;Wyman et al.,2004).
Lignocellulosics can be hydrolyzed chemically by addition of acids,with sulphuric acid most often preferred based on price and toxicity,and acid hydrolysis can be divided in two categories: concentrated acid hydrolysis and dilute acid hydrolysis.Concen-trated acid processes operate at low te
,40°C,and give high sugar ,90%of theoretical glucose yield.How-ever,acid consumption is high,a lot of energy is consumed for acid recovery and recycle,the equipment can suffer from corrosion,and reaction times of2–6h are required.The dilute acid process is characterized by a low acid consumption and very short reaction times at high temperatures.Hemicellulose is generally much more susceptible to acid hydrolysis than cellulose,and yields of more than85%can be obtained at relatively mild conditions,with only a small part of the cellulose converted to glucose.More severe con-ditions required to achieve high glucose yields from cellulose, however,lead to degradation of hemicellulose sugars,resulting in low yields and unwanted side-products that are also strong fer-mentation inhibitors.Potential inhibitors that can be formed or re-leased from hemicellulose,cellulose,and lignin during such thermochemical routes include furfural,5-hydroxymethylfurfural (HMF),levulinic acid,acetic acid,formic acid,uronic acid,4-hydroxybenzoic acid,vanillic acid,vanillin,phenol,cinnamalde-hyde,and formaldehyde.To reduce degradation of monosaccha-rides at high temperature,dilute acid hydrolysis is typically carried out in two stages,with hemicellulose solubilized in thefirst under relatively mild conditions and the residual solids hydrolyzed in the second under the more severe conditions needed to break-down cellulose.With this procedure,hemicellulose derived sugar yields are in the range of90%,while glucose yields are only about 40–60%at realistic residence times.However,it has been reported that alternative reactor configu
rations to classic batch reactors, such as a shrinking-bed reactor give glucose yields of up to90% (Taherzadeh and Karimi,2007).
Cellulase enzymes from the fungus Trichoderma reesei can hydrolyze biomass to sugars at near ambient temperatures,result-ing in little degradation.However,because sugar yields from raw biomass are very low,the biomass is subjected to a pretreatment step.Numerous pretreatment methods have been developed including pretreatment with steam,liquid hot water,dilute acid, lime,ammonia,and wet oxidation and are discussed in more detail elsewhere(Hendriks and Zeeman,2009;Mosier et al.,2005;Wy-man et al.,2005,2009).Effective pretreatments are thought to en-hance enzymatic digestibility of biomass due to several effects: disruption of the lignocellulosic structure by loosening the hemi-cellulose lignin entanglement,hemicellulose hydrolysis,lignin sol-ubilisation and disruption,decrystallization of cellulose,and increased accessible surface area(Lynd et al.,2002;Zhang and Lynd,2004,2006).During many pretreatments,fermentation inhibitors such as acetic acid,lignin breakdown products,and fur-fural are released.After pretreatment,several process configura-tions are possible,as recently reviewed in detail(Cardona and Sanchez,2007).In the separate hydrolysis and fermentation (SHF)approach,the liquid and solid phases are separated after pre-treatment,and the solid phase may be subjected to additional washing steps.The solids,in case of dilute acid and steam p
retreat-ment,contain most of the lignin and cellulose from the raw bio-mass,with the latter hydrolyzed to glucose by addition of cellulolytic enzymes that are comprised of endo-and exoglucanase and b-glucosidase activities,often supplemented with additional b-glucosidase derived from Aspergillus niger.The resulting hexose solution is then fermented to ethanol using conventional yeast or other suitable microorganisms.A suitable pentose fermenting strain can convert the liquid stream from pretreatment containing
S.Brethauer,C.E.Wyman/Bioresource Technology101(2010)4862–48744865
solubilized hemicellulose to ethanol in a separate unit usually after a detoxification step such as overliming to reduce fermentation inhibitors and make the hydrolyzate fermentable.Hydrolysis and fermentation were initially separated to better match the pH and temperatures to those that are optimal for each step,with about 50°C preferred for enzymatic hydrolysis and about32°C often best for fermentations.Alternatively,enzymes could be added to the whole pretreatment slurry without separation of the liquid from the solids,followed by fermentation of pentoses and hexoses to ethanol,a process which we call separate hydrolysis and co-fer-mentation(SHcF).This more integrated approach is economically very attractive,but the fermentation step is much more challeng-ing than in SHF and complicates hydrolyzate conditioning to re-move inhibitors due to the presence of the solids(Cardona a
nd Sanchez,2007).
Because cellulases are inhibited by their hydrolysis products cellobiose and glucose,a favoured processing mode is to combine hydrolysis and the fermentation,a process termed simultaneous saccharification and fermentation(SSF),thereby keeping sugar concentrations low(Gauss et al.,1976;Spindler et al.,1987;Takagi et al.,1977;Wright et al.,1987;Wyman et al.,1986).Although ini-tial applications subjected only the washed solids fraction from pretreatment to SSF with the liquid pentose stream processed sep-arately,these two steps can be combined in what is termed simul-taneous saccharification and co-fermentation(SScF)(Wooley et al., 1999).Despite the need to reduce the temperature for SSF from the optimal levels for enzymes to accommodate fermentative organ-isms available to date,SSF was shown to achieve higher rates, yields,and concentrations than SHF by overcoming the major ef-fects of end-product inhibition(Spindler et al.,1987;Wright et al.,1987).In addition,SSF,and even more so SScF,reduces fer-mentation equipment demands,and the presence of ethanol im-pedes invasion by unwanted organisms.Thus,until enzymes are found that can overcome end-product inhibition,SSF or SScF are likely to be preferred in terms of productivity,yields,and ethanol concentrations.In the following sections,results for continuous fermentations with hydrolzates from acid and enzymatic hydroly-sis and for SSF applications are summarized.
4.1.Fermentation of hexoses in enzymatic hydrolyzates
Fermentation of hexose sugars derived from enzymatic hydro-lysis of washed pretreated lignocellulosic material generally does not pose special difficulties(see Table1),as the inhibitor concen-tration should be very low.However,compared to starch and sug-arcane fermentations,the sugar concentration after hydrolysis are often low with values approaching typically not more than70g LÀ1 due to challenges in feeding solids concentrations higher than about10%by weight to the fermentors and end-product inhibition of cellulase enzymes by the sugars released.Thus,a concentration ,vacuum evaporation,might be needed to achieve higher concentrations,with additional extra costs possibly counterbal-anced by savings in thefinal distillation step(Maiorella et al., 1984).
In one study,sugar cane bagasse was delignified by autoclaving in1%NaOH for1h,and the solids were washed several times prior to hydrolysis sei cellulases.In a single stage continuous fer-mentation visiae with a16%glucose feed,several dilution rates were tested tofind a maximum ethanol productivity of 4.1g LÀ1hÀ1at a dilution rate of0.13hÀ1.At this point,steady state concentrations of90g LÀ1glucose,31g LÀ1ethanol,and 3.8g LÀ1biomass were measured.To avoid washout of large amounts of unfermented glucose,a continuous single stage cell re-cycle fermentation system was set up,and the maximal productiv-ity reached18.3g LÀ1hÀ1at a dilution rate of0.3hÀ1with
a steady state glucose concentration of22g LÀ1(Ghose and Tyagi,1979).
Lee et al.(2000)employed an enzymatic hydrolyzate derived from washed steam exploded oak chips(3min at215°C).To re-duce fermentation inhibitors,the hydrolyzate was sterilized for 120min at60°C,rather than at a typical temperature of121°C. Continuous cultures were performed in a reactor equipped with an internal membranefiltration module to retain cells inside the reactor.At a dilution rate of0.22hÀ1and a feed glucose concentra-tion of180g LÀ1,77g LÀ1ethanol was produced,corresponding to a productivity of16.9g LÀ1hÀ1and a yield of0.43g gÀ1.In a batch fermentation in a similar medium containing170g LÀ1glucose, only57g LÀ1ethanol was produced in210h,with35g LÀ1glucose not utilized,leading to a very low productivity of0.3g LÀ1hÀ1.No problems were experienced with bacterial contamination despite the low sterilization temperature.
When the solid–liquid separation and solids washing steps are omitted and the whole slurry is enzymatically hydrolyzed,fermen-tations are much more difficult,as exemplified by the work of Palmqvist et al.(1998)who employed a hydrolyzate of spruce pre-treated by steam explosion for5min at215°C after sulphur diox-ide impregnation.Two different batches of hydrolyzate were used, containing25–50g LÀ1glucose and approximately10g LÀ1man-nose,that were both supplemented with mineral vi-siae ATCC96581,a strain isolated from a spent sulphite liquor(SSL) fermentatio
n plant running since1940and showing a7-fold high-er maximum growth rate on SSL than bakers yeast,was employed for the study.At a pH of4.6in a batch system,cells metabolized
Table1
Fermentation of hexoses in enzymatic hydrolyzates.
Medium Sugar concentration
(g LÀ1)Reactor type Dilution
rate
(hÀ1)
Ethanol
(g LÀ1)
cell dry
weight
(g LÀ1)
Ethanol
yield
(g gÀ1)
Ethanol
productivity
(g LÀ1hÀ1)
References
Sugar cane bagasse pretreated with NaOH;washed solids enzymatically hydrolyzed,concentration by vacuum evaporation;addition of‘‘cheap
nitrogen source”,CaCl2,MgSO4Reducing sugars:160Single stage CSTR0.1331 3.80.19 4.1Ghose and
Tyagi
(1979)
Single stage CSTR
with cell recycle
0.358300.3618.3
Steam exploded oak chips,washed solids enzymatically hydrolyzed,concentrated by vacuum evaporation,sterilization
for120min at60°C Glucose:180Single stage CSTR
with cell retention by
membrane module
0.2277n.d.0.4316.9Lee et al.
(2000) Glucose:170Batch–570.340.3
(fermentation
time of
210h)
Steam exploded spruce,whole slurry enzymatically hydrolyzed,addition of complete mineral
medium salts Glucose:25–50,
mannose:10
Single stage CSTR0.05200.90.320.5Palmqvist
et al.
(1998)
0.1Washout
Single stage CSTR
with cell recycle
0.123Maximum
26
0.51 2.3
4866S.Brethauer,C.E.Wyman/Bioresource Technology101(2010)4862–4874
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