A comparison between batch and fed-batch simultaneous. EMT. 2003

Enzyme and Microbial Technology 37 (2005) 195–204

A comparison between batch and fed-batch simultaneoussaccharification and fermentation of steam pretreated spruce

Andreas Rudolf ∗, Malek Alkasrawi, Guido Zacchi, Gunnar LidenDepartment of Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden

Received 20 September 2004; received in revised form 10 January 2005; accepted 10 February 2005

Abstract

In order to improve the process economy it is important to use as high dry matter content as possible in simultaneous saccharification andfermentation (SSF). However, too high dry matter content often gives rise to severe inhibition of the yeast metabolism, due to the increasedlevels of toxic compounds. The aim of the present workwas to increase the fibrous content in SSF of steam pretreated spruce to 10%by adaptingthe yeast to the inhibitory substrate and by using a fed-batch process. Both batch and fed-batch approaches were evaluated. The fed-batchexperiments were started with a batch fermentation containing 6% dry matter. Fibrous slurry from the pretreatment was then added four timesduring the first 24 h giving a final dry matter content corresponding to 10%. The yeast used in the fermentation was produced aerobically onthe hemicellulose hydrolysate obtained from the pretreatment. SSF batch and fed-batch experiments with a cell mass concentration of 2, 3and 5 g/L were carried out. When adapted yeast was used, the available hexoses were completely converted within 72 h and the final ethanolconcentrations reached 40–44 g/L. No major differences in performance between batch and fed-batch were seen, but the ethanol productivityduring the first 24 h was higher in the fed-batch SSF experiments, particularly during the experiments with a cell mass concentration of 2 and3 g/L.© 2005 Elsevier Inc. All rights reserved.

Keywords: Ethanol; Yeast; Fermentation; Enzymatic hydrolysis; Spruce

1. Introduction

Reducing the production costs of ethanol produced fromlignocellulose material is crucial in enabling its commercial-ization. One important factor for the production cost is theethanol concentration in the liquid left after the saccharifica-tion and fermentation. This should be as high as possible [1]in order to minimize the energy costs of evaporation and dis-tillation. By recirculating some of the liquid from the fermen-tation back into the fermentor vessel, the final ethanol con-centration can be increased. However, compounds inhibitoryto the yeast cells and enzymes, formed during the pretreat-ment of lignocellulose material [2–14], are thereby furtherconcentrated, making a high degree of recirculation difficult[15,16]. Raising the dry matter content is another obviousway to reach a higher final ethanol concentration. However,

∗ Corresponding author. Tel.: +46 46 222 82 50; fax: +46 46 14 91 56.E-mail address: [email protected] (A. Rudolf).

the concentrations of compounds inhibitory to yeast and en-zymes are simultaneously increased. In addition, the rheolog-ical properties of a very dense fibrous suspension may causemixing and heat transfer problems.Previously,when using compressedBaker’s yeast inBatch

SSF with a dry matter content of 8% it proved difficult toachieve high ethanol yields (Alkasrawi et al., accepted forpublication). The high concentrations of inhibitors in fermen-tations with a high dry matter content might be overcome byapplying a fed-batch technique [17]. By adding the slurry tothe fermentor vessel continuously or by scheduled additions,the inherent ability of the yeast to detoxify the substrate can bemaintained. This approach has proved successful in fermen-tations of dilute-acid hydrolysates from softwood [18,19].Furthermore, since added fibers are gradually degraded, theinitial viscosity can be kept lower than in a batch process.Provided that it is possible to reduce the inhibition by ap-

plying a fed-batch it could be possible to decrease the amountof yeast used in the SSF. Cultivation of yeast will consume

0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.enzmictec.2005.02.013

196 A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195–204

substrate, which could otherwise be used for ethanol produc-tion thereby decreasing the over-all ethanol yield. In order tominimize the yeast concentration in the SSF, it is importantto have as robust yeast as possible. It has been shown thatthe yeast can be adapted to the inhibiting medium prior tothe fermentations by cultivating the yeast on liquid from thepretreatment (Alkasrawi et al., accepted for publication).The aim of the present work was to enable conversion of

10% dry matter content (the dry matter content only includesthe water insoluble solids) in simultaneous saccharificationand fermentations of steam pretreated spruce by using yeastcultivated on the liquid from the pretreatment and by using afed-batch process. During the fed-batch SSF, the fiber slurry,obtained from the pretreatment, was added five times to thefermentor vessel during the first 25 h. The time between theadditions was varied in order to see how it affected fermen-tation performance. Batch and fed-batch experiments withdifferent cell mass concentrations were carried out in orderto study if it was possible to decrease the amount of yeastadded.The results showed that it was possible to obtain conver-

sion of all available hexose sugars in the fermentation liquidwithin 72 h in SSF using 10% dry matter. Ethanol yields of79–84% of the theoretical yield (based on glucan, glucose,mannose and galactose present in the added substrate) wereachieved.

2. Material and methods

2.1. Raw material

Fresh, chipped spruce was kindly provided by a sawmillin southern Sweden (Harry Nilssons Sagverk, Hastveda,Skane). The wood was chipped and sieved to obtain a chipsize of 2–10mm. The chips were stored in a plastic bag at4 ◦C prior to use. The composition was analyzed using theHagglund method [20] followed by HLPC-analysis.

2.2. Pretreatment

The wood chips were impregnated with SO2 (3%, w/wmoisture) for 20min at room temperature. The impregnationwas performed in tightly closed plastic bags to enable pene-tration of the gas into the wood structure. The amount of SO2absorbedwas determined byweighing the plastic bags beforeand after impregnation. The impregnatedmaterial was steam-pretreated at 215 ◦C for 5min in a steam-pretreatment unitequipped with a 10 L-reactor, which has been described pre-viously [21]. In total about 3.6 kg of impregnated chips werepretreated. Due to the limited size of the reactor, 600 g waspretreated at a time. The slurries obtained from the pretreat-ment runs were mixed together into a single batch and storedat 4 ◦C for future use. The composition of the dry wood andthe dry fibers in the pretreatment slurry was determined bythe Hagglund method [20]. The pretreatment slurry consists

Table 1Composition of the dry wood raw material and the dry washed fibers fromthe two batches of pretreatment slurry

Components Wood (wt.%) Washed pretreatedmaterial (wt.%)Batch 1 Batch 2

Glucan 43 48 48Mannan 12 ∼0 ∼0Xylan 5.4 ∼0 ∼0Galactan 2.3 ∼0 ∼0Lignin 27 47 45The dry matter content of the wood raw material was 56%.

of awater insoluble part mainly glucan and lignin (Table 1) aswell as a liquid fraction containing the sugars released fromthe hydrolysis of the hemicellulose as well as acetic acid andsugar degradation products (Table 2). The water insolublesolids content of both pretreatment batches was 11.5%.

2.3. Cell cultivation

Since the more cell mass had to be produced during someof the initial cultivations, two different reactor set-ups wereused for the cell cultivations. For the first three, SSF-runs(A–C) the cells were cultivated in a 14 L-bioreactor (NLF14, Bioengineering AS, Wald, Switzerland). The cell culti-vations preceding the SSF-experiments D–Lwere performedin a 2.5 L-reactor (BiostatA, B. Braun Biotech International,Melsungen,Germany). The finalworking volume in the 14 L-reactor was 4.3 and 2.0 L in the 2.5 L-reactor. However, allother parameters such as nutrient concentrations, ratio be-tween added pretreatment liquid and batch volume, relativefeed rate increase were identical in the two cultivation set-ups. Thus, the cells produced in the two different reactorsshould be equally resistant to the harsh conditions in theSSF-experiments. In the description of the cultivation proce-dure, the cultivationsmade in the 2.5 L-reactor will bewrittenwithin brackets.

2.3.1. Inoculum culturesFiftymilligramsof pureBaker’s yeast culture (Jastbolaget,

Rotebro, Sweden) from an agar plate was added to a 300mLErlenmeyer-flask,which contained 100mLof sterilemediumwith a pH of 5.5. The medium composition was as follows:glucose, 16.6 g/L; (NH4)2SO4, 7.5 g/L; KH2PO4, 3.5 g/L;

Table 2Composition of the liquid from the two pretreatment batches

Components Concentration (g/L)

Batch 1 Batch 2

Glucose 23.4 22.1Mannose 19.4 18.5Xylose 8.9 8.0Galactose 4.0 4.6Furfural 1.7 1.6HMF 3.0 2.4Acetic acid 3.7 3.4

A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195–204 197

MgSO4, 0.75 g/L; trace metal solution, 10mL/L and vita-min solution, 1mL/L. The trace metal and vitamin solutionswere prepared according to Taherzadeh et al. [22]. The cul-ture was incubated at 30 ◦C for 24 h. The Erlenmeyer-flaskwas closed with a cotton plug.

2.3.2. Aerobic batch cultivationIn order to produce cell mass before the fed-batch cul-

tivation phase, batch cultivation, with a working volume of2.5 L [1.2 L],1 was carried out at 30 ◦C under sterile condi-tions. Themedium contained the following components: glu-cose, 17 g/L; (NH4)2SO4, 20 g/L; KH2PO4, 9.4 g/L;MgSO4,2.0 g/L; trace metal solution, 26mL/L and vitamin solution,3mL/L. The pH was maintained at 5.0 by automatic additionof 2M NaOH. The stirrer speed was kept at 700 rpm and theaeration was maintained at 2.5 L/min [1.3 L/min]1. The cul-tivation was started by adding 80mL [40mL]1 of inocolum.

2.3.3. Aerobic fed-batch cultivationSubsequent to depletionof the ethanol producedduring the

batch phase, feeding of the pretreatment liquid was started.The liquid was enriched with 28 g glucose/L. 1.8 L [0.85 L]1of glucose enriched pretreatment liquid was added during16 h. The feed rate was initially set to 0.07 L/h [0.035 L/h]1and was increased linearly to 0.15 L/h [0.07 L/h]1 after 16 h.The pH was maintained at 5.0 by automatic addition of 2MNaOH. The stirrer speedwas kept at 700 rpm and the aerationwas maintained at 2.5 L/min [1.3 L/min]1.

2.3.4. Cell harvestThe cultivation liquid was centrifuged (1000× g) in

700mL containers using a HERMLE Z 513K centrifuge(HERMLE Labortechnik, Wehingen, Germany). The pelletswere resuspended in 0.9% NaCl-solution in order to obtaina cell suspension with a cell mass concentration of about75 g dw/L. The time lapse between the end of the fed-batchphase and the addition of the harvested cells to the SSF-fermentations was always kept below 3 h.

2.4. SSF-experiments

2.4.1. Fed-batchSSF-experiments with a final working weight of 1.6 kg

(ca. 1.5 L) were performed in a 2.5 L-reactor (Biostat A,B. Braun Biotech International, Melsungen, Germany). Thepretreatment slurry was diluted with fresh water in order toget an initial dry matter content of 6%. The reactor contain-ing the diluted slurry was autoclaved at 120 ◦C for 20min.The fermentation medium was supplemented with nutrients:NH4H2PO4, 0.87 g/L;MgSO4·7H2O, 0.025 g/L andyeast ex-tract, 1.0 g/L (based on final volume). The experiments werestarted by adding cell suspension, giving a final cell concen-tration, after all slurry additions, of 2, 3 or 5 g/L (Table 3).

1 Refers to the cultivations performed in the 2.5 L-reactor.

Table 3The complete experimental series

SSF-index Pretreatmentbatch

Time betweenslurryadditions (h)

N2-flushing Cell massconcentration(g/L)

A 1 Batch Yes 5B 1 1.5 Yes 5C 1 3 Yes 5D 1 5 Yes 5E 2 Batch Yes 5F 2 1.5 Yes 5G 2 Batch Yes 3H 2 1.5 Yes 3I 2 Batch Yes 2J 2 1.5 Yes 2K 2 Batch No 5L 2 1.5 No 5

The enzyme used preparations were Novozyme 188 with a!-glucosidase activity of 362 IU/g and Celluclast 1.5 L witha cellulase activity of 75 FPU/g and a !-glucosidase activityof 12 IU/g. The enzyme preparations were kindly providedby Novozymes A/S (Bagsvaerd, Denmark). The amount ofNovozyme 188 added corresponded to 4% (w/w) on the drymatter, giving a total !-glucosidase activity of 36.2 IU/g glu-can (the combined activity of Celluclast 1.5 L and Novozyme188) and the amount of Celluclast 1.5 L added correspondedto 24% (w/w) on the dry matter (a cellulase activity of37.5 FPU/g of glucan). After the initial batch phase with 6%dry matter, pretreatment slurry, which had been pH-adjustedto 4.9 with 6M NaOH, was added four times giving a totaldry matter content of 10%. Experiments with a time lapsebetween the slurry additions of 1.5, 3 and 5 h were carriedout (Table 3). The fermentation pH was maintained at 5.0 bythe addition of 3M NaOH. All SSF experiments were per-formed at 37 ◦C for 72 h. The CO2 evolution rate (CER) wasmonitored by flushing N2 through the reactor and analyzingthe CO2 amount in the exhaust gas with a gas analyzer (Tan-Dem, Adaptive Biosystems, Luton, United Kingdom). TheN2-flow was 400mL/min during the first 24 h and was thendecreased to 200mL/min.

2.4.2. BatchBatch experiments with a working weight of 1.6 kg (ca.

1.5 L) and a drymatter content of 10%were also run (Table 3).For the batch fermentations a 2.5 L-bioreactor was used(BIOFLO III, New Brunswick Scientific, Edison, NJ, USA).The fermentation conditions, nutrient concentrations and en-zyme loadingswere the same as in the fed-batch experiments.

2.5. Fermentation of sugar-containing solutions withoutfibers

Fermentations with pulse addition of pure glucose solu-tions or pretreatment liquidwere carried out in order to exam-ine what caused the decrease in CER response to the slurryadditions during the SSF fed-batch experiments. The glucose


solution used had a concentration of 36 g/L, which equalledthe sum of glucose and mannose in the pretreatment liquid.Glucose solution or pretreatment liquid were added every 3 hduring the first 12 h. After 24 h glucose solution (36 g/L) wasadded a last time in both experiments. During the experi-ments, a glucose solution (188 g/L) was continuously fed at9mL/h in order to simulate the slow release of glucose duringa SSF experiment. The final volume was 1.5 L and the cellmass concentration 5 g/L. The yeast was cultivated exactly inthe same way as prior to the SSF experiments.

2.6. Analysis

2.6.1. Cell mass concentrationThe cell mass concentration during the aerobic cell culti-

vationwasmeasured by taking duplicate 10mL samples. Thesampleswere centrifuged (1000× g) for 4min at 3000 rpm (Z200 A, HERMLE Labortechnik, Wehingen, Germany). Thesupernatantswere discarded and the pelletswerewashedwithdeionised water and recentrifuged. The pellets were dried at105 ◦C for 24 h and subsequently weighed.

2.6.2. Cell viabilitySamples for cell viability testswere takenfive times during

the batch and fed-batch experiments (E–J). One milliliter offermentation liquid was diluted 104–105 times and 75"L ofdiluted sample was applied to an agar plate. Five to six plateswere prepared for every sample. The plates were incubatedat 30 ◦C for 24 h. The cell viability was expressed as the totalnumber of colony forming units.

2.6.3. HPLC-analysisSamples of the fermentation liquid were centrifuged

(16,000× g) in 2mL eppendorf tubes at 14,000 rpm for5min. (Z 160M, HERMLE Labortechnik, Wehingen, Ger-many). The supernatant was filtered with 0.2"m sterile fil-ters and the filtered samples were stored at−20 ◦C. Analysisof the most common metabolites and substrates was madeusing HPLC. The concentrations of glucose, mannose xy-lose and galactose were determined using a polymer column(Aminex HPX-87P, Bio-Rad Laboratories, Munchen, Ger-many) at 85 ◦C. The eluent used was MilliQ-water with aflow rate of 0.6mL/min. Ethanol, glycerol, acetate, lactate,HMF and furfural were analyzed using an Aminex HPX-87H column (Bio-Rad Laboratories, Munchen, Germany) at60 ◦C. The eluent used was 5mM H2SO4 with a flow rate of0.6mL/min. The compounds of interest were detected with arefractive index detector (Waters 2410,Waters,Milford,MA,USA).

2.6.4. Yield calculationsThe ethanol yields, YSE, were calculated based on the total

amount of fermentable sugars added to the fermentations, i.e.the sum of glucose, mannose and galactose present in theliquid part of the added pretreatment slurry plus the glucosein the form of glucan.

The theoretical amounts of glucose released during thehydrolysis are 1.11 times the amount of glucan (due to theaddition of water when the glycosidic bonds are hydrolysed).The relative ethanol yield, Y∗

SE, i.e. the actual ethanol yield,YSE, divided by the theoretical ethanol yield is described byEq. (1).

Y∗SE = YSE

0.51(%) (1)

2.6.5. Estimation of ethanol evaporationN2-flushing will strip substantial amounts of the produced

ethanol, even if the exhaust gas passes a condenser. In thepresent work, the rate of ethanol evaporation was studied inexperiments performedwith the fermentors used for the SSF-experiments. After a SSF-experiment, the residual cells werekilled by a short elevation of temperature (55 ◦C for 20min).Additional ethanol was added in order to get an initial ethanolconcentration of about 30 g/L. The decrease in ethanol con-centration was then measured during 48 h. The experimentswere run with a N2-flow of 400 or 200mL/min. A relation-ship between the molar ethanol fraction in the fermentationliquid and the molar ethanol fraction in the exhaust gas wasestimated based on the ethanol evaporation data for the twoN2-flows (Eq. (2)):

y = 0.57x (2)

where y is the mole fraction of ethanol in exhaust gas and xis the mole fraction of ethanol in the fermentation liquid.

3. Results

3.1. Cell cultivation

The cell yield on glucose was about 0.35 g/g for the aero-bic batch growth (after consumption of ethanol formed) andabout 0.48 g/g for aerobic fed-batch growth. The lower yieldsduring the batch phases are due to the well-known less ef-ficient diauxic growth on glucose and subsequently on theethanol formed as a result of over-flow metabolism [23]. Ad-ditionally, ethanol evaporation will lower the yield further.The final cell mass concentration after the fed-batch phasereached 16–17 g/L.

3.2. SSF-experiments

Batch and fed-batch fermentations with a dry matter con-tent of 10% were carried out. In the experiments A–D, theinfluence of the feed profile on the fermentation performanceand process dynamics was studied. In experiments E–J, theeffect of cell mass concentration on the fermentation perfor-mance in batch and fed-batch SSF were evaluated. Addition-ally, one fed-batch (experiment L) and one batch-experiment(experiment K) were run without N2-flushing in order to ex-clude that the flushing affected the fermentation performance,


Table 4Final ethanol concentrations and ethanol yields on available fermentablesugars

Experiment Final ethanolconcentration (g/L)

YSE (72 h) (g/g) Y∗SE (72 h) % oftheoretical yield

A 42.6 0.42 82B 44.0 0.43 84C 40.0 0.40 78D 43.5 0.43 84E 44.5 0.43 84F 44.5 0.43 84G 41.9 0.42 82H 43.7 0.43 84I 41.6 0.41 80J 41.0 0.41 80K 42.2 0.43 84L 42.2 0.43 84The final ethanol concentrations have been compensated for evaporation(except for experiments K and L).

and also to confirm the estimated ethanol evaporation in ex-periments A–J. The model used to estimate ethanol evapo-ration gave final ethanol yields close to the expected values(Table 4).Over-all, the fermentation performance was satisfying.

The final ethanol concentrations were above 40 g/L (aftercompensation for ethanol evaporation) in all experiments andthe ethanol yields, YSE, on the total amount of fermentablesugars were above 0.40 g/g in all experiments (Table 4). Thefinal ethanol yields did not differ significantly between batch

and fed-batch. The cell mass concentration could be de-creased to 2 g/L without any significant deterioration in finalethanol yield.The responses in CER subsequent to the slurry additions

were fast and sharp (Figs. 1 and 2). However, the responsesdecreased with time, particularly, when the time between theslurry additions was 3 or 5 h (Fig. 1). At 24 h, 3 g of glucosewas added to the fermentation medium (marked by arrowsin Figs. 1 and 2) in order to examine the fermentative ca-pacity of the yeast cells after extended time in the inhibitingmedium. The responses in CER to the pulse additions of glu-cose strongly suggest that the enzymatic hydrolysis is therate limiting process. However, the responses were small inexperiments A and G (Figs. 1 and 2).The glucose and mannose concentrations during the fed-

batch fermentations were in general below 5 g/L and did notexceed 1.5 g/L after 72 h (Figs. 3–5). The measured hex-ose concentrations during the first 24 h were higher in thebatch fermentations compared to the fed-batch fermentations.These differences in hexose concentration between batch andfed-batchweremore significant in the experiments with a cellmass concentration of 2 and 3 g/L (Fig. 4, experiments G–J).The total number of colony forming units (CFU)wasmea-

sured during some of the fermentations (Table 5). The CFUdecrease during the fermentations suggests that the yeast cellssuffered from inhibition and/or starvation. It is thus likely thatthe sugar consumption for cell growthwas very low. TheCFUdecreased somewhat slower during the fed-batch fermenta-

Fig. 1. CER during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch 1) was used as substrate. The yeast was cultivated onthe liquid from the pretreatment. The cell mass concentration was 5 g/L. Pulse additions of 3 g of glucose are marked with arrows. (A) SSF batch, (B) SSFfed-batch with substrate additions every 1.5 h, (C) SSF fed-batch with substrate additions every 3 h and (D) SSF fed-batch with substrate additions every 5 h.


Fig. 2. CER during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch 2) was used as substrate. The yeast was cultivated onthe liquid from the pretreatment. The time lapse between the substrate additions during the fed-batch experiments was 1.5 h. Pulse additions of 3 g of glucoseare marked with arrows. (E) SSF batch with a cell mass concentration of 5 g/L, (F) SSF fed-batch with a cell mass concentration of 5 g/L, (G) SSF batch with acell mass concentration of 3 g/L, (H) SSF fed-batch with a cell mass concentration of 3 g/L, (I) SSF batch with a cell mass concentration of 2 g/L and (J) SSFfed-batch with a cell mass concentration of 2 g/L.

tions, suggesting that the pulse wise addition decreased theinhibition and cell death rate.The extent of yeast inhibitionwas also studied by perform-

ing fermentations with pulse addition (every 3 h) of glucose

solution (36 g/L) and liquid from the pretreatment. When apure glucose solution was fed the responses in CER did notdecrease significantly during 24 h (Fig. 6), suggesting thatneither the elevated temperature (37 ◦C) nor the periods of

Table 5Total number of colony forming units (CFU) in experiments E–J

Experiment 1 h (CFU× 10−9) 7 h (CFU× 10−9) 24 h (CFU× 10−9) 48 h (CFU× 10−9) 72 h (CFU× 10−9)

E 124 85 73 34 31F 173 117 91 79 55G 94 59 44 12 6H 107 133 110 45 37I 74 52 41 17 8J 86 70 50 22 13The degree of error is ±10%.


Fig. 3. Ethanol (!), glucose (!) and mannose (⋆) concentrations during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch 1)was used as substrate. The yeast was cultivated on the liquid from the pretreatment. The cell mass concentration was 5 g/L. (A) SSF batch, (B) SSF fed-batchwith substrate additions every 1.5 h, (C) SSF fed-batch with substrate additions every 3 h and (D) SSF fed-batch with substrate additions every 5 h.

carbon limitation had a significant effect on cell viability.However, in the experiment with pulse feeding of pretreat-ment liquid, the responses in CER decreased steadily (Fig. 6),indicating cell inhibition most likely caused by certain com-pounds formed during the pretreatment. The decrease in CERresponse to the substrate additions was in agreement withwhat has previously been observed during SSF-experimentsC and D.

4. Discussion

Increasing the fibrous content to 10% worked satisfactoryusing the yeast cultivated on the pretreatment liquid. Theethanol yields on available sugars were in all cases above0.40 g/g and final ethanol concentrations above 40 g/L wereattained (Table 4). To reach a high ethanol concentration wasone of the main targets of the present work since the finalethanol concentration in the fermentation liquid is criticalwhen trying to reduce ethanol production costs [1]. In shakeflask fermentations of pretreatment liquid or glucose – usingthe same yeast preparation and medium composition as inthe SSF-experiments – ethanol yields on fermentable sugarsreached 0.45–0.46 g/g. The slightly lower ethanol yields inthe SSF-experiments could be due to the fact that a small frac-tion of the glucanwas not completely hydrolyzedwithin 72 h.The small difference in fermentation performance be-

tween batch and fed-batchwas somewhat surprising. In previ-

ous fermentation experiments using dilute-acid hydrolysateas substrate, fed-batch fermentation proved to be superior tobatch fermentation [19,24]. However, the pretreatment priorto the SSF is less severe than the hydrolysis used to producedilute-acid hydrolysates. Furthermore, the cell cultivation onthe pretreatment liquid probably adapted the yeast to theinhibitory compounds present in the fermentation medium.Adaptation by cultivating the yeast on the pretreatment liq-uid have previously proved to be successful (Alkasrawi et al.,accepted for publication).Even when the cell mass concentration was decreased to

2 and 3 g/L the differences in over-all fermentation perfor-mance between batch and fed-batch remained small. How-ever, the ethanol productivity during the first 24 h – and es-pecially during the first 7 h – was considerably higher duringthe fed-batch experiments (H and J) compared to the batchexperiments (G and I). This suggests that the fed-batch ap-proach actually reduces the cell inhibition, but since the rateof hydrolysis during the entire SSF is low compared to the po-tential sugar uptake by the cells, all hexoses are metabolizedeven though the cells initially are severely inhibited and thecell viability decreases throughout the fermentation. The factthat the potential sugar uptake rate by the cells exceeds thehydrolysis rate, suggests that there could be a potential for fur-ther decreasing the amount of yeast addedwhile still maintaingood fermentation performance. When using cell mass con-centrations below 2 g/L, a fed-batch approach is probably themost feasible option. The glucose concentrations were lower


Fig. 4. Ethanol (!), glucose (!) and mannose (⋆) concentrations during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch2) was used as substrate. The yeast was cultivated on the liquid from the pretreatment. The time lapse between the substrate additions during the fed-batchexperiments was 1.5 h. (E) SSF batch with a cell mass concentration of 5 g/L, (F) SSF fed-batch with a cell mass concentration of 5 g/L, (G) SSF batch with acell mass concentration of 3 g/L, (H) SSF fed-batch with a cell mass concentration of 3 g/L, (I) SSF batch with a cell mass concentration of 2 g/L and (J) SSFfed-batch with a cell mass concentration of 2 g/L.

Fig. 5. Ethanol (!), glucose (!-) and mannose (⋆) concentrations during SSF-experiments with a dry matter content of 10%. The SSF experiments were carriedout without N2-flushing. Steam-pretreated spruce (batch 2) was used as substrate. The yeast was cultivated on the liquid from the pretreatment. The cell massconcentration was 5 g/L. (K) SSF batch and (L) SSF fed-batch with substrate additions every 1.5 h.


Fig. 6. CER during pulse feeding experiments with glucose solution andliquid from the pretreatment. The yeast was cultivated on the liquid from thepretreatment. The cell mass concentration was 5 g/L, ( ) pulse feeding ofpretreatment liquid and (· · ·) pulse feeding of glucose solution (36 g/L).

during the first 24 h in the fed-batch experiments comparedto the batch experiments. Thus, a fed-batch approach couldreduce the end-product inhibition of the cellulases. However,since there were no major differences in ethanol yield be-tween the two methods, end-product inhibition did not seemto affect the hydrolysis rate considerably.In order to increase the ethanol productivity further, clearly

the rate of enzymatic hydrolysis has to be increased. Due tothe high prize of currently available commercial cellulasepreparations, addition of more enzymes is not an attractiveoption.Alternatively, the rate of hydrolysis can be acceleratedby raising the temperature; an increase from 37 to 50 ◦C canresult in a 29% increase in enzymatic activity [25]. At suchtemperatures, however, SSF is not a feasible process, sinceSaccharomyces cerevisiae cannot tolerate such high temper-atures. The enzymatic hydrolysis would in such a case beperformed in a separate step, after which fermentation of thesugar solution can take place (separate hydrolysis and fer-mentation, SHF). The higher productivity is thus paid forby an increased investment cost [1]. Speaking against SHFis furthermore the fact that in previous experiments usingsteam-pretreated spruce as a substrate, the ethanol yield ontotal hexoses has been higher using a SSF configuration com-pared to a SHF configuration [26]. A way forward whichcombines the advantages of SSF and SHF could be to run ashort hydrolysis at elevated temperature andwhen the hydrol-ysis becomes end product inhibited switch to SSF by addingyeast and lower the temperature. Another option would be todevelop a more thermostable yeast strain. This would haveseveral benefits, not only could the enzymatic hydrolysis beperformed closer to the optimum temperature, but the risk ofcontamination would be reduced.Due to the current high prices of commercial cellulases a

reduction of the amount of cellulases added could improvethe process economy even more than an increase in ethanolproductivity. There is a potential for decreasing the enzymeloading while still maintaining a high ethanol yield sinceapproximately 95% of the ethanol is produced during the first

48 h of the fermentations (Fig. 5). Thus, even if the enzymatichydrolysis is slower high ethanol yields could still be reachedwithin 72 h.

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Documents

A comparison between batch and fed-batch simultaneous. EMT. 2003