food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308

Contents lists available at ScienceDirect

Food and Bioproducts Processing

j ourna l h omepage: www.elsev ier .com/ locate / fbp

Bioconversion of barley straw and corn stover tobutanol (a biofuel) in integrated fermentation andsimultaneous product recovery bioreactors�

N. Qureshi ∗, M.A. Cotta, B.C. SahaUnited States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center forAgricultural Utilization Research (NCAUR), Bioenergy Research Unit, 1815 North University Street, Peoria,IL 61604, USA

a b s t r a c t

In these studies concentrated sugar solutions of barley straw and corn stover hydrolysates were fermented using

Clostridium beijerinckii P260 with simultaneous product recovery and compared with the performance of a control

glucose batch fermentation process. The control glucose batch fermentation resulted in the production of 23.25 g L−1

ABE from 55.7 g L−1 glucose solution resulting in an ABE productivity and yield of 0.33 g L−1 h−1 and 0.42, respectively.

The control reactor (I) was started with 62.5 g L−1 initial glucose and the culture left 6.8 g L−1 unused sugar due to

butanol toxicity resulting in incomplete sugar utilization. Barley straw (BS) hydrolysate sugars (90.3 g L−1) resulted in

the production of 47.20 g L−1 ABE with a productivity of 0.60 g L−1 h−1 and a yield of 0.42. Fermentation of corn stover

(CS) hydrolysate sugars (93.1 g L−1) produced 50.14 g L−1 ABE with a yield of 0.43 and a productivity of 0.70 g L−1 h−1.

These productivities are 182–212% higher than the control run. The culture was able to use 99.4–100% sugars (CS &

BS respectively) present in these hydrolysates and improve productivities which were possible due to simultaneous

product removal. Use of >100 g L−1 hydrolysate sugars was not considered as it would have been toxic to the culture

in the integrated (simultaneous fermentation and recovery) process.

Published by Elsevier B.V. on behalf of The Institution of Chemical Engineers.

Keywords: Barley straw hydrolysate; Corn stover hydrolysate; Butanol/ABE; Simultaneous product recovery; Produc-

tivity

cost substrates or feedstocks compared to corn or molasses;

1. Introduction

As a result of increase in gasoline prices, research interestsin the development of butanol have increased dramaticallyas this biofuel has many superior fuel properties and containhigher energy than ethanol (Anon, 2007, 2008). In fact butanol(acetone butanol ethanol or ABE) production by fermentationranked second to ethanol in importance and history as com-mercial plants during World War I and II produced acetone andbutanol on large scale (Durrie, 1998; Zverlov et al., 2006; Jonesand Woods, 1986). Most of these commercial plants ceased

operation due to development of petrochemically produced

� Mention of trade names or commercial products in this article is sonot imply recommendation or endorsement by the United States Depaemployer.

∗ Corresponding author. Tel.: +1 309 681 6318; fax: +1 309 681 6427.E-mail address: Nasib.Qureshi@ars.usda.gov (N. Qureshi).Received 9 May 2013; Received in revised form 30 October 2013; Accep

0960-3085/$ – see front matter Published by Elsevier B.V. on behalf of Thttp://dx.doi.org/10.1016/j.fbp.2013.11.005

butanol and an increase in the cost of feedstocks such as cornand molasses. For these reasons the fermentation plants couldnot compete with petrochemical route of butanol production.The last plant was shut down in 1982 in South Africa (Jonesand Woods, 1986). In the articles cited below and numerousdiscussions and presentations delivered in clostridia confer-ences around the world, the following recommendations weregiven to revive this fermentation and make butanol a com-mercially viable biofuel (Jones and Woods, 1986; Maddox, 1989;Durrie, 1998; Zverlov et al., 2006; Qureshi, 2009): (i) use of lower

lely for the purpose of providing scientific information and doesrtment of Agriculture. USDA is an equal opportunity provider and

ted 5 November 2013

(ii) increase product concentration above 20 g L−1; (iii) improve

he Institution of Chemical Engineers.

food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308 299

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harvesting, planting season, and baling were provided earlier

ield by utilizing residual acids; and (iv) decrease in prod-ct recovery costs. With the above recommendations Qureshit al. (2013a) focused on each of these and developed a compre-ensive research program with expectations to make butanol

commercially viable biofuel. It should be noted that if thisiofuel (or any other) is to be commercialized, the processhould use agricultural residues and/or new energy crops thatre renewable in nature and economically available. For thisery important reason agricultural biomass including corntover, wheat straw, barley straw, switchgrass, miscanthus,oy molasses, corn fiber, various waste feedstocks containingarbohydrates, and distillers dried grains and soluble (DDGS),nd forest product residues such as saw dust, leaves and barkQureshi et al., 2013a) are being used. The prices of theseesidues are projected to be $60 ton−1 (Lane, 2011) as opposedo corn at $275 ton−1.

In a recent study it was projected that 1.1–1.6 × 109

1.1–1.6 billion tons) dry tons of cellulosic biomass coulde produced in the United States by 2030 (Lane, 2011).ased on calculations 545 × 109 L (141.9 billion gallons) ofcetone–butanol–ethanol (ABE – 3:6:1) of which 326 × 109 L85.1 billion gallons) is butanol, 163.5 × 109 L (42.6 bil-ion gallons) acetone, and 54.5 × 109 L (14.2 billion gallons)thanol could be produced. Currently 514.6 × 109 L (134 bil-ion gallons) of gasoline is used annually in the Unitedtates [(http://www.eia.gov/tools/faq.cfm?id=23&t=10), web-ite accessed December 28, 2012]. This consumption wasbout 6% less than the record high of about 546.7 × 109 L (142.4illion gallons) consumed in 2007. It is assumed that ABE

all three components; the authors are aware that butanolnd ethanol can be used) can be used in cars and automo-ive engines. For these calculations the carbohydrate contentf lignocellulosic biomass was adapted to be 66%, and anBE yield of 0.38. Based on these calculations it is predicted

hat we have potential to be free of foreign oil. Deploy-ent of such a technology will also be expected to generate

tremendous number of jobs in the U.S. The reader isnformed that butanol producing microorganisms can uti-ize all the hexose and pentose sugars as opposed to currentommercial ethanol producing organisms which cannot useentoses.

However, there are challenges in producing butanol fromellulosic biomass. Butanol production from sugary andtarchy feedstocks has been demonstrated successfully (Jonesnd Woods, 1986). It should be noted that use of lignocellu-osic biomass requires pretreatment and hydrolysis of thiseedstock prior to fermentation to ABE. The pretreatment iserformed using dilute acid (H2SO4), or alkali (NaOH) in ordero provide hydrolytic enzymes access to the cellulosic fibersnd release the sugars contained within (close, if not 100%).uring pretreatment process some of the hemicellulosic sug-rs are converted to furfural, hydroxymethyl (HMF), and otheregradation products that are toxic to the culture. Sincehese inhibitors hinder the fermentation of hydrolysates, theiretoxification becomes essential for successful fermentation

n particular for barley straw and corn stover hydrolysatesQureshi et al., 2010a,b).

Attempts have been made to increase ABE concentrationn the fermentation broth by genetically engineering ABE pro-ucing cultures for improved tolerance to products (Annous,991; Formanek et al., 1997; Shen et al., 2011; Inui et al., 2008;omas et al., 2003; Liu et al., 2010). In some cases butanol con-entration as high as 25 g L−1 has been achieved, however this

s still too low for this fermentation to be commercially viable.

In order to overcome this (to increase butanol or ABE concen-tration per L broth) another approach (process engineering)has been employed where the product was simultaneouslyremoved from the fermentation broth which resulted in ABEproduction of 232.8–461.3 g L−1 broth (Qureshi et al., 2013a)as compared to 25 g L−1 using solely genetic manipulation.This process engineering technique has also improved ABEyield as acids could not leave the system until convertedto ABE thus addressing recommendations number ii and iii.Recommendation number iv was to reduce energy require-ment for product separation. Technologies such as those justmentioned (Qureshi et al., 2013a) to recover ABE simulta-neously from the fermentation broth greatly reduce the energyrequirement for this process.

To further improve process economics of ABE produc-tion from lignocellulosic biomass one of the two processes(separate hydrolysis, fermentation, and recovery: SHFR orsimultaneous saccharification, fermentation, and recovery:SSFR) in combination with use of concentrated sugar (hydrol-ysed cellulosic biomass) solutions should be applied. Use ofconcentrated sugar solutions would reduce capital and oper-ational costs by reducing process stream volumes and reactorsizes. The objective of the current studies was to use SHFRprocess to produce ABE from barley straw and corn stoverhydrolysates. Fermentation of concentrated sugar solutions,in particular cellulosic hydrolysates which are also toxic to themicroorganisms, is a challenge until toxic product (butanol,toxicity level 15–20 g L−1) is simultaneously removed from fer-mentation broth. It should also be noted that some cellulosichydrolysates require detoxification prior to fermentation.

2. Materials and methods

2.1. Culture and inoculum development

Clostridium beijerinckii P260 was a generous gift from Profes-sor David Jones, University of Otago (Dunedin, New Zealand).C. beijerinckii P260 spores were maintained in distilled waterat 4 ◦C. One hundred microlitre spores were heat shockedfor 2 min at 75 ◦C and transferred to cooked meat medium(CMM; Difco Laboratories, Detroit, MI, USA) for germinationas described by Ennis and Maddox (1985) and Qureshi et al.(2007). During germination process the bottles were kept in ananaerobic jar at 35 ◦C. The culture was ready in 16–18 h timeand was termed as stage I inoculum.

The next stage of inoculum preparation (termed as stage II)was developed in P2 medium as described below. One hundredmillilitre of 30 g L−1 glucose (Sigma Chemicals, St. Louis, MO,USA) and 1 g L−1 yeast extract (Bacto-Dickinson & Co., Sparks,MD, USA) were autoclaved at 121 ◦C for 15 min followed bycooling to 35 ◦C and adding 1 mL each of mineral, vitamin, andbuffer solutions (Qureshi et al., 2007). This bottle was inocu-lated with 7–10 mL of stage I inoculum developed above andincubated for approximately 10–12 h at 35 ◦C when the cul-ture was ready for inoculation into 50 mL fermentation bottles(described below) or bioreactor containing barley straw or cornstover hydrolysates.

2.2. Barley straw, pretreatment, and hydrolysis

Barley straw was obtained from Oregon State University, Cor-vallis, OR, USA and its details on Geo-Co-ordinates, verities,

(Qureshi et al., 2010a). The barley straw was milled into fine

300 food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308

Fig. 1 – A schematic diagram of an integrated system ofABE production employing C. beijerinckii P260 andsimultaneous recovery using gas stripping from barleystraw and corn stover hydrolysates.

particles ranging from 1–2 mm using a hammer mill. Eightysix grams per litre of milled barley straw was pretreated withdilute (10 mL L−1) H2SO4 (Sigma Chemicals) at 121 ◦C for 1 hfollowed by cooling to room temperature and adjusting pH to5.0 using 400 g L−1 NaOH (Sigma Chemicals) solution. Follow-ing pH adjustment the pretreated barley straw was hydrolyzedat 45 ◦C for 72 h using 6 mL L−1 each of cellulase (Cellu-clast 1.5 L; Sigma Chemicals; enzyme activity 751 ± 35 U mL−1;carboxymethylcellulose activity), �-glucosidase (Novozyme188; Sigma Chemicals; activity 380 ± 19 U mL−1), and xylanase(Viscostar 150 L; Dyadic Corporation, Jupiter, FL, USA; activ-ity 9837 ± 190 U mL−1) enzymes (Qureshi et al., 2010a). Thehydrolyzed barley straw solution was filter sterilized andstored at 4 ◦C until used for these experiments. The sugar com-position of barley straw hydrolysate has been given in Table 1(total sugar 44.2 g L−1). This sugar concentration was raisedto the desired level by supplementing with 400–500 g L−1 filtersterilized glucose solution.

2.3. Corn stover, pretreatment, and hydrolysis

Corn stover (Pioneer variety) was obtained from a local farmer(Forest, Illinois, USA) and milled to a particle size of 1–2 mmfollowed by suspending in 10 mL L−1 H2SO4 and mixing well.Then the mixture was transferred to a 316 stainless steel reac-tor and heated to 160 ◦C in a fluidized sand bath for 20 min.After cooling to 25 ◦C the pH of the mixture was adjusted to5.0 with 400 g L−1 NaOH solution. Further details have beengiven elsewhere (Qureshi et al., 2010b). Corn stover hydrolysisand further processing was performed as described above forbarley straw. Table 1 shows sugar composition of corn stoverhydrolysate.

2.4. Detoxification of barley straw and corn stoverhydrolysates

Barley straw and corn stover hydrolysates were detoxified byoverliming method as described elsewhere (Qureshi et al.,2010a). The filter sterilized hydrolysates were stored at 4 ◦Cuntil used for fermentation studies.

2.5. Fermentation with or without integrated productrecovery

Fermentation studies without product recovery were per-formed in 50 mL (total volume 67 mL) PyrexTM screw cappedbottles with 35 mL medium. The medium inside the bottleswas not agitated or mixed. To the hydrolysate, 0.35 mL of each

of P2 stock solutions (mineral, vitamin, and buffer; Qureshiet al., 2007) and yeast extract (from presterilized 40 g L−1) to

Table 1 – Sugar concentrations in barley straw and cornstover hydrolysates after hydrolysis at 45 ◦C for 72 husing enzymes.

Sugars Barley strawhydrolysate [g L−1]

Corn stoverhydrolysate [g L−1]

Glucose 23.9 22.1Xylose 16.8 7.2Arabinose 2.2 1.1Galactose 1.3 0.4Mannose 0.0 0.0

Total 44.2 30.8

1 g L−1 level were added. All the transfers were made ascep-tically. The bottles were then placed in an anaerobic jar for48 h for anaerobiosis followed by inoculating with 3 mL of anactively growing stage II culture/inoculum. The bottles wereincubated at 35 ◦C in an anaerobic jar. One millilitre sampleswere taken as needed followed by centrifugation at 15,000 × g.Clear supernatant liquid was stored at −18 ◦C until sugar andABE measurements.

Fermentation with product recovery studies were per-formed in 2.5 L total capacity glass bioreactor (BIOFLO 2000Fermentor; New Brunswick Scientific, New Brunswick, NJ,USA) with 1.25 L working volume. One litre of barley straw orcorn stover hydrolysate was transferred to the reactor followedby adding 12.5 mL of each of P2 medium stock solutions andyeast extract (Bacto-Dickinson & Co.) to 1 g L−1 final level (from40 g L−1 presterilized solution). Additional sugar solution (from400 to 500 g L−1 filter sterilized glucose solution) was added tothis medium to raise sugar level to approximately 100 g L−1 (forproduct recovery experiments). The temperature of the reac-tion mixture was maintained at 35 ◦C and 50 mL min−1 oxygenfree nitrogen gas was sparged/bubbled through the mediumfor 24–48 h prior to inoculation with 100 mL actively growingstage II inoculum. At the time of inoculation, N2 sparging waschanged from sparging to sweeping across the surface of themedium. Gas sweeping continued for 12–24 h or until culturestarted producing its own gases (CO2 and H2). Sparged or sweptgases were cooled in a condenser to capture any volatiles orwater which was returned to the bioreactor.

ABE recovery was started using fermentation gases (CO2

and H2) at a gas recycle rate of 3–4 L min−1 employing atwin-head MasterflexTM (Cole Parmer, Vernon Hills, IL, USA)peristaltic pump and 18 size Norprene (Cole Parmer) tubing.The gases were bubbled through the fermentation mixturefollowed by cooling in a condenser. The condensate contain-ing ABE was removed intermittently and its weight or volumewas recorded followed by measuring ABE concentration. Fig. 1shows a schematic diagram of fermentation and recoveryexperiment. More details of condenser, coolant, and coolingmachine have been given elsewhere (Qureshi et al., 2007).

2.6. Analyses

Fermentation products such as ABE, acetic acid, and butyricacid were measured by gas chromatography (6890 N; Agi-lent Technologies, Wilmington, DE, USA) using a packed glass

column as described elsewhere (Qureshi et al., 2007). Initial

food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308 301

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Fig. 2 – Production of ABE from glucose in a control (I) batchreactor using C. beijerinckii P260. (A) Products and glucoseconcentration vs. Fermentation time; (B) cell concentration

olumn temperature was 100 ◦C which was ramped up at theate of 40 ◦C min−1 to 180 ◦C and held it there for 4 min. Sug-rs were measured using a Surveyor HPLC equipped with anutomatic sampler and injector (Thermo-Electron Corpora-ion, West Palm Beach, FL, USA). The HPLC column (HPX-87P;minex Resin-based) was obtained from Bio-Rad (Hercules,A, USA). Solvent (Milli-Q water) flow rate was maintained at.6 mL min−1 and products were detected using a Refractivendex detector.

ABE productivity was calculated as total ABE produced in L−1 divided by the fermentation time and is expressed as L−1 h−1. Fermentation time was initiated when the bottle oreactor was inoculated. Fermentation time period is defined ashe time difference between inoculation and when fermenta-ion ceased and is expressed in h. Specific productivity (h−1) isefined as productivity in g L−1 h−1 divided by cell concentra-ion in g L−1. ABE yield was calculated as total ABE producedivided by the total sugar utilized (initial sugar minus resid-al sugar; both in g L−1). Cell concentration was measured byn optical density method (� 540 nm) and is presented as dryeight cell concentration (g L−1). To measure optical density,

00 �L fermentation broth was added to 900 �L presterilized.0 g L−1 saline (NaCl) water and mixed well. Dry weight celloncentration was calculated using the following correlation:

= 0.2141x3 − 0.1594x2 + 0.2588x − 0.0009; where y is cell con-entration in the diluted sample in g L−1 and x is opticalensity. Following this, y was multiplied by the dilution factor

10) to calculate cell concentration in the undiluted fermenta-ion broth withdrawn from the bottle or bioreactor.

. Results and discussion

.1. Control fermentation

n order to measure performance of barley straw and corntover fermentation experiments, a control fermentation (I)as run using glucose as substrate. The fermentation was

nitiated with 62.5 g L−1 initial glucose level. Over a period of0 h, the culture accumulated 23.25 g L−1 total ABE (Fig. 2a).t the end of fermentation 6.8 g L−1 glucose was left unusedue to ABE toxicity to the culture, thus using 55.7 g L−1 glu-ose which is 89.1% of that present initially. This experimentesulted in a productivity of 0.33 g L−1 h−1 and an ABE yield of.42. In this fermentation a maximum cell concentration of.60 g L−1 was obtained at 48 h (Fig. 2b). The system resulted in

specific productivity of 0.13 h−1 and a sugar utilization ratef 0.80 g L−1 h−1. A batch system, where product was recoveredimultaneously by gas stripping, resulted in complete utiliza-ion of lactose present in whey permeate medium (Ennis et al.,986). This system indicated that product inhibition can beelieved if toxic product level is kept below toxicity in theeactor by removing it simultaneously.

Since sugar concentrations in the barley straw and corntover hydrolysates that were fermented in the integratedxperiments were 90–93 g L−1, another experiment (control II)as conducted with 90.0 g L−1 initial glucose concentration in

he medium. As in the above control, no product recovery waserformed. This study resulted in the production of 23.50 g L−1

otal ABE with a productivity of 0.36 g L−1 h−1 and a yield of.42. At the end of fermentation, the residual sugar concen-ration was 34.0 g L−1. These results suggested that the culture

erformed equally at these two control sugar levels (62.5–90.0)xcept that residual glucose levels were different.

at various fermentation times.

3.2. Barley straw hydrolysate (BSH) fermentation tobutanol and recovery

Following this, experiments were performed where detox-ified barley straw was supplemented with various glucoselevels thus raising total sugar concentration to 63, 80, 100,and 125 g L−1 and fermented. At these sugar levels total ABEconcentrations were 24.06, 21.45, 19.80, and 19.50 g L−1, respec-tively (Table 2). With the increase in sugar concentration,decreased ABE was produced. Cell growth and fermentationsuggested that the culture was capable of tolerating highercellulosic sugar levels (up to 125 g L−1). At 63 g L−1 total sugarconcentration, a productivity of 0.36 g L−1 h−1 and a yield of0.43 were achieved (Table 2). With the increase in sugar con-centration, decreased ABE productivities were achieved. Atan initial sugar concentration of 125 g L−1, a productivity of0.20 g L−1 h−1 was obtained. This productivity (0.20 g L−1 h−1)is 55.6% of 0.36 g L−1 h−1 that was achieved at 63 g L−1 initialsugar level indicating that higher sugar levels were inhibitoryto fermentation. In this fermentation (125 g L−1 initial sugarlevel) ABE yield also decreased from 0.43 (63 g L−1 sugar level)to 0.38 which is a decrease of 11.6%. Decreased productivitiesand yields (with increased sugar concentrations) suggestedthat more sugar was used for maintenance energy and/or pro-duction of reaction intermediates such as acids (acetic andbutyric acids).

Both rate of sugar utilization, and maximum cell concen-tration decreased with increased sugar levels (Table 2). At asugar level of 63 g L−1 a sugar utilization rate of 0.85 g L−1 h−1

was obtained while at 125 g L−1 initial sugar a rate of0.52 g L−1 h−1 was obtained. This decrease is 38.8% based oninitial sugar utilization rate of 0.85 g L−1 h−1. At 80 and 100 g L−1

total initial sugar levels, sugar utilization rates of 0.55 and

0.54 g L−1 h−1 were obtained, respectively. Increased sugar lev-els from 63 to 125 g L−1 resulted in decreased cell growth and

302 food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308

Table 2 – Production of ABE from barley straw and corn stover hydrolysates supplemented with additional sugar tovarious levels.

Initial sugarconc. [g L−1]

ABE[g L−1]

ABEproductivity[g L−1 h−1]

ABEyield

ABEspecificprod. [h−1]

Sugar used[g L−1]

Residualsugar[g L−1]

Sugarused[%]

Sugarutilizationrate [g L−1 h−1]

Final cellconc. [g L−1]

Fermentation parameters: barley straw hydrolysate63 24.06 0.36 0.43 0.42 55.9 7.1 88.70 0.85 0.8580 21.45 0.22 0.40 0.28 53.3 26.7 66.63 0.55 0.80

100 19.80 0.20 0.39 0.25 50.8 49.2 50.80 0.54 0.80125 19.50 0.20 0.38 0.29 51.3 73.7 41.04 0.52 0.70

Fermentation parameters: corn stover hydrolysate60 24.38 0.30 0.44 0.39 55.4 4.6 92.33 0.68 0.7780 10.52 0.11 0.39 0.16 27.0 53.0 33.75 0.28 0.70

100 6.76 0.07 0.36 0.14 18.8 81.2 18.80 0.20 0.50120 6.02 0.14 0.35 0.26 17.2 102.8 14.33 0.39 0.53

0 g L xylose was present, respectively.

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Fig. 3 – Production of ABE from barley straw hydrolysate inan integrated system of production and recovery employing

cell concentration. A sugar concentration of 63 g L−1 resultedin a cell concentration of 0.85 g L−1. This cell concentrationis much lower than the cell concentration achieved in thecontrol fermentation (2.60 g L−1). It should be noted that eventhe detoxified barley straw hydrolysate was inhibitory forcell growth which became even greater as sugar level wasincreased to 125 g L−1. Cell growth decreased from 0.85 to0.70 g L−1 as sugar was increased from 63 to 125 g L−1.

Specific productivity is a measure of productivity per unitcell in the system and is an indication of cell efficiency. In thepresent studies a specific productivity of 0.42 h−1 was achievedat an initial sugar level of 63 g L−1 (Table 2). Further increasein total sugar concentration to 80 g L−1 resulted in a decreasedspecific productivity (0.28 h−1) suggesting that cells were notas efficient, possibly due to inhibition caused by higher sugarconcentration. This was a 33.3% decrease in specific productiv-ity of ABE production. Further increase in sugar concentrationto 100 g L−1, decreased specific productivity to 0.25 h−1. In thefour experiments incomplete sugar utilization was observed(Table 2). The highest sugar utilization was 55.9 g L−1 (88.7%) atan initial sugar level of 63 g L−1. In this experiment a residualsugar concentration of 7.1 g L−1 was left behind. In the subse-quent experiments (80, 100, and 125 g L−1 initial sugar levels),53.3, 50.8, and 51.3 g L−1 sugars were utilized, respectively. Inthese experiments 26.7, 49.2, and 73.7 g L−1 sugar remainedunused. The experiment which contained 125 g L−1 total initialsugar, resulted in the utilization of 41.04% sugar.

The next experiment was performed in the reactor. Forthis purpose barley straw hydrolysate was supplemented withadditional sugar to raise level to 97 g L−1. Use of over 100 g L−1

sugar solution was not considered as it would have increasedlag time thus resulting in decreased productivity (Table 2).Then cell growth was initiated by inoculation of the reac-tor. As a result of dilution by inoculum addition, total sugarconcentration decreased to 90.3 g L−1. Cell growth was rapidand in 16 h fermentation time 0.80 g L−1 cell mass accumu-lated in the system. Also fermentation was fast and at thattime 4.83 g L−1 total ABE were present in the reactor (Fig. 3a).This was followed by ABE recovery by gas stripping usingfermentation gases. For 16 to 22 h ABE concentration wasnearly constant at about 4.83–4.86 g L−1. Since this ABE con-centration was not toxic to the culture, fermentation becamevigorous and cell growth increased significantly. At 28 h cellconcentration was 2.50 g L−1 and total ABE in the system were12.14 g L−1. This was followed by some oscillations in the total

product concentration in the system which is common inABE fermentations employing C. beijerinckii or C. acetobutylicum

(Huang et al., 2004). After 28 h of fermentation the culturebecame relatively slow and at 39 h a total ABE concentrationof 8.99 g L−1 was recorded. This was followed by vigorous fer-mentation again thus resulting in accumulation of ABE in thereactor. At 51 h, 13.37 g L−1 total ABE were measured. The fer-mentation ceased at 63 h due to complete sugar utilization(Fig. 3b). At the initiation of the reactor 73.89 g L−1 glucose,14.5 g L−1 xylose, 1.4 g L−1 arabinose, and 0.5 g L−1 galactosewere present in the fermentation mixture (Fig. 3b). All thesesugars were utilized simultaneously, though xylose at a muchslower rate than glucose. Both arabinose and galactose werecompletely used during initial 16 h of cell growth and fermen-tation. At 16, 22, 28, 39, 51, and 63 h 12.2, 10.0, 7.7, 4.8, 3.7 and

−1

C. beijerinckii P260. (A) Products vs. fermentation time; (B)sugars in bioreactor vs. fermentation time.

food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308 303

Fig. 4 – Various kinetic parameters of ABE production from barley straw hydrolysate employing C. beijerinckii P260. (A) Sugarutilization rate vs. fermentation time; (B) rate of ABE production (productivity) at various fermentation times; (C) cellconcentration and pH in the reactor at various fermentation times, and (D) specific productivity of ABE production at variousf

1w3BwasswiAwam(wtoccp

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ermentation times.

The rates of sugar utilization are presented in Fig. 4a. At6 h, a rate of sugar utilization of 1.20 g L−1 h−1 was measuredhich increased to 2.26 g L−1 h−1 at 22 h. This rate increased to

.73 g L−1 h−1 at 28 h, and then declined to 0.96 g L−1 h−1 at 39 h.etween 51 and 63 h a rate of sugar utilization of 1.41 g L−1 h−1

as measured. The ABE productivities at various time periodsre shown in Fig. 4b. The productivity was highest at 28 h mea-uring 1.78 g L−1 h−1. The cell concentration and pH values arehown in Fig. 4c. A maximum cell concentration of 2.50 g L−1

as measured at 28 h which was the reason behind achiev-ng a highest productivity employing barley straw hydrolysate.t the initiation of fermentation, pH was adjusted to 6.47hich decreased to 5.35 during initial 16 h of fermentation as

result of production of acetic and butyric acids. At 22 h it waseasured at 5.31 (Fig. 4c). During the course of fermentation

16–63 h) pH remained below 5.6. It should be noted that pHas not controlled by addition of alkali solution, rather cul-

ure controlled it by utilizing acids. The specific productivitiesbtained in this system are shown in Fig. 4d. The highest spe-ific productivity of 0.66 h−1 was obtained at 28 h when the celloncentration was the highest in the system. At 51 h, a specificroductivity was 0.58 h−1.

While concentrations of ABE in the reactor are plotted inig. 3a, their concentrations in the recovered stream are shownn Table 3. At 22 h an ABE concentration of 121.93 g L−1 was

easured in the condensate. This concentration is many foldore than present in the reactor. At 28 h it was 122.34 g L−1. At

9, 51, and 63 h these concentrations were 104.36, 135.15, and00.41 g L−1 respectively. During gas stripping small amountsf acetic and butyric acids were also removed which are pre-ented in Table 3.

In the beginning of the integrated reactor 1250 mL fermen-

ation medium was present with 90.3 g L−1 mixed sugar. Theystem utilized 112.9 g total sugar (in 1250 mL medium; 100%

sugar utilization) and produced 47.20 g total ABE resultingin an ABE yield of 0.42 and an average ABE productivity of0.60 g L−1 h−1 (Table 4). This productivity is 1.82 times (182%)of that achieved in the control experiment. Based on a maxi-mum cell concentration of 2.50 g L−1 that was achieved in thissystem, a specific productivity of 0.24 h−1 was obtained. Thisspecific productivity is 1.85 times than achieved in the controlfermentation. It is pointed out that fermentation of undetox-ified barley straw hydrolysate did not produce more than7.09 g L−1 ABE with a productivity of 0.10 g L−1 h−1 (Qureshiet al., 2010a). Also the non-integrated system where prod-uct was not removed did not produce more than 24.06 g L−1

ABE (Table 2; 63 g L−1 initial sugar) with a productivity of0.36 g L−1 h−1. This demonstrated that an integrated systemusing barley straw hydrolysate containing glucose enrichedsugar solution can be successfully fermented to ABE if thetoxic product is recovered simultaneously. In the control reac-tor (I) the ratio of acids produced per g sugar utilized was0.064 g g−1 while in the integrated system it was 0.026 g g−1

suggesting that in the integrated system less acids wereaccumulated which would result in improved ABE yield andprocess economics. In this reactor system 1 L of barley strawhydrolysate containing 44.2 g L−1 cellulosic sugars was used.To this solution 138 mL of glucose solution containing 500 g L−1

glucose (68.7 g glucose) was added prior to fermentation.

3.3. Corn stover hydrolysate (CSH) fermentation tobutanol and recovery

Since barley straw hydrolysate was fermented to butanol,experiments with corn stover hydrolysate were also per-formed. For this purpose, corn stover hydrolysate was

supplemented with additional sugar to raise total sugar levelto 60–120 g L−1 and fermented without product recovery. This

304 food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308

Table 3 – Concentrations of ABE and acids in recovered stream obtained from barley straw hydrolysate fermentationemploying C. beijerinckii P260.

Recovery time [h] Products [g L−1]

Acetone Butanol Ethanol HAc HBu Acids ABE

16 0.00 0.00 0.00 0.00 0.00 0.00 0.0022 34.41 86.52 1.00 – 0.66 0.66 121.9328 33.81 87.60 0.93 – 0.69 0.69 122.3439 35.64 67.10 1.60 1.30 0.53 1.83 104.3651 45.55 88.10 1.50 – – – 135.1563 36.06 62.90 1.45 – – – 100.41

Recovery was initiated at 16 h and first condensate was collected at 22 h.HAc and HBu – acetic and butyric acids.

was performed to evaluate corn stover hydrolysate sugar tol-erance to the culture. At 60 g L−1 initial sugar level, the cultureproduced 24.38 g L−1 total ABE (Table 2). Further increase insugar concentration to 80 g L−1 resulted in the production of10.52 g L−1 ABE which further declined to 6.76 and 6.02 g L−1 atsugar levels of 100 and 120 g L−1, respectively. At these initialsugar levels, ABE productivities and yields were also evalu-ated which are presented in Table 2. At a sugar concentrationof 60 g L−1 an ABE productivity of 0.30 g L−1 h−1 was obtainedwhich decreased to 0.07 g L−1 h−1 at a sugar level of 100 g L−1

demonstrating that CSH in combination with supplementedsugar was more toxic to the culture than BSH. Even at a sugarconcentration of 60 g L−1, ABE productivity in CSH mediumwas reduced by approximately 17% as compared to productiv-ity in BSH medium. In this case (CSH), as sugar concentrationwas increased, productivity and cell growth decreased, furtherreinforcing cell growth and fermentation toxicity due to CSH.It should be noted that glucose alone at 100–120 g L−1 is nottoxic to the cells of C. beijerinckii P260 (Qureshi et al., 2007). InCSH experiments ABE yields were 0.44, 0.39, 0.36, and 0.35 forthe four fermentations with initial sugar levels of 60, 80, 100,and 120 g L−1, respectively.

When compared to BSH, cell concentrations in CSH fer-mentations were lower (Table 2). At an initial sugar level of60 g L−1, a cell concentration of 0.77 g L−1 was obtained whichdecreased to 0.50 g L−1 as initial sugar level was increased to100 g L−1. At this sugar level cell concentration was reducedby 35.1%. It should be noted that sugar utilization rates werealso low in case of CSH fermentation. At 60 g L−1 sugar, asugar utilization rate of 0.68 g L−1 h−1 was observed whichwas reduced to 0.20 g L−1 h−1 as sugar level was increased to100 g L−1. In these experiments with CSH as feedstock, specificproductivities ranged from 0.39 to 0.14 h−1 (Table 2). Further,sugar utilization was 55.4 g L−1 at an initial sugar level of60 g L−1 thus leaving behind 4.6 g L−1 unused sugar (Table 2).In this experiment 92.33% of sugar was used of the available

in feed. As initial sugar was increased to 80 g L−1, total sugar

Table 4 – A comparison of ABE production from barley straw anproduct removal system.

Feed stock AC2O [g L−1] BuOH [g L−1] EtOH [g L−1] ABE

Cont-Glu 7.70 13.89 1.66 23.Int-BSH 13.56 30.86 2.78 47.Int-CSH 14.04 34.77 1.33 50.

Cont-Glu: control (I) – glucose concentration 62.5 g L−1 in the medium.Int-BSH, integrated barley straw hydrolysate; Int-CSH, integrated corn stoverespectively.

utilization decreased to 27 g L−1 which further decreased to17.2 g L−1 at an initial sugar of 120 g L−1 in feed. At 120 g L−1

initial sugar only 14.33% sugar was utilized by the culture.Next, an experiment (in reactor) was conducted where CSH

was used in combination with simultaneous product recov-ery. Fermentation was initiated with an initial sugar level of93.1 g L−1. At 19 h, 1.66 g L−1 acetone 3.32 g L−1 butanol and0.68 g L−1 ethanol were measured thus totaling to 5.66 g L−1

ABE (Fig. 5a). Concentrations of acetic and butyric acidswere 6.32 g L−1 and 2.11 g L−1, respectively, thus totaling to8.43 g L−1. At this time recovery of products (ABE) was initi-ated and as a result of simultaneous product removal totalABE decreased to 4.62 g L−1 at 24 h. As a result of efficientrecovery of fermentation products total product concentrationdecreased to 2.09 g L−1 thus reducing inhibitory effect signifi-cantly and for that reason the culture became highly activeand initiated producing ABE vigorously. Both fermentationand recovery were continued and at 47, 55, and 72 h, total ABEwere 6.7, 12.87, and 9.49 g L−1, respectively. At 72 h fermenta-tion ceased as nearly all the sugars fed to the reactor wereutilized by the culture.

In the beginning of fermentation, 84.4 g L−1 glucose,7.2 g L−1 xylose, 1.1 g L−1 arabinose and 0.4 g L−1 galactose werepresent in the reactor (Fig. 5b). During the first 24 h of fermen-tation glucose was reduced from 84.4 to 58.1 g L−1 while xylosewas decreased from 7.20 to 3.97 g L−1. At this time arabinoseand galactose were completely used. At 47 h, 29.2 g L−1 glucoseand 2.2 g L−1 xylose were present in the system. Fermentationstopped at 72 h and at that time 0.52 g L−1 xylose was presentin the reactor; all other sugars were completely utilized.

In this integrated experiment, rates of sugar utilization atvarious times are presented in Fig. 6a. At 19 h, a rate of sugarutilization was 0.71 g L−1 h−1 which increased to 3.47 g L−1 h−1

at 24 h. Between 47 and 55 h, a rate of sugar utilization was2.16 g L−1 h−1 which reduced to 0.79 g L−1 h−1 at 72 h. ABE pro-ductivities at various times are presented in Fig. 6b. The

highest productivity was observed at 55 h with a value of

d corn stover hydrolysates in integrated fermentation

[g L−1] Sugar used [g L−1] ABE Prod [g L−1 h−1] Yield

25 55.7 0.33 0.4220 90.3 0.60 0.4214 93.1 0.70 0.43

r hydrolysate; AC2O, BuOH, and EtOH – acetone, butanol, and ethanol,

food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308 305

0

2

4

6

8

10

12

14

0 19 24 31 47 55 72

Pro

du

cts

[gL

-1]

Fer menta tio n Time [h]

a

b

Acetone

Butanol

Ethanol

HAc

HBu

ABE

0

10

20

30

40

50

60

70

80

90

100

0 19 24 31 47 55 72

Su

gars

[g

L-1

]

Fer menta tio n Time [h]

Glu

Xyl

Arab

Man

Gal

Tot Sug

Fig. 5 – Production of ABE from corn stover hydrolysate inan integrated bioreactor. Simultaneous ABE production andrecovery using C. beijerinckii P260. (A) Products vs.fermentation time; (B) sugars in bioreactor vs. fermentationtime.

1w1k

butyric acids in the reactor. At this point reasons for accu-

Fucf

.25 g L−1 h−1. In this fermentation the reactor was startedith an initial cell concentration of 0.001 g L−1. During the first

−1

9 h period, cell concentration increased to 0.55 g L whichept increasing until 55 h when it was 2.56 g L−1 (Fig. 6c). In

ig. 6 – Various kinetic parameters of ABE production from corn

tilization rate vs. fermentation time; (B) rate of ABE production

oncentration and pH in the reactor at various fermentation timeermentation times.

the beginning of fermentation, pH was 6.43 which decreasedto 5.57 during 19 h of cell growth and fermentation period. Dur-ing the fermentation, the culture maintained pH between 5.38and 5.57. At the end of fermentation a pH of 5.50 was recorded(Fig. 6c). In this fermentation, specific productivities rangedfrom 0.37 to 0.71 h−1. The highest specific productivity wasmeasured at 24 h with a value of 0.72 h−1 (Fig. 6d).

In the condensate, concentrations of various productsincluding reaction intermediates (acetic and butyric acid) weremeasured and they are presented in Table 5. Recovery wasstarted at 19 h and first condensate was collected at 24 h. Atthis time 32.59 g L−1 acetone, 88.01 g L−1 butanol and 1.26 g L−1

ethanol thus totaling to 121.89 g L−1 ABE were measured.Traces of acetic acid and 0.54 g L−1 butyric acid were present inthe recovered product. At 31 h concentration of ABE decreasedto 64.12 g L−1 followed by an increase to 88.50 g L−1 at 47 h.As concentration of ABE in the reactor was high at 55 h theirconcentrations in the condensate rose to 121.4 g L−1 (55 h) and127.57 g L−1 (72 h). As in the case of barley straw hydrolysatefermentations, small amounts of acetic and butyric acids weremeasured in the condensate.

In the integrated bioreactor system 93.1 g L−1 total sug-ars were present in the beginning of the experiment with atotal 116.3 g sugar (in 1250 mL medium). During the experi-ment 99.4% sugar utilization occurred thus producing 50.14 gtotal ABE representing a yield of 0.43. This fermentation prod-uct recovery system resulted in an average productivity of0.70 g L−1 h−1 which is 212.1% of that achieved in the controlfermentation process. A total of 2.76 g of acids were recoveredin the product stream. It should be noted that unlike bar-ley straw hydrolysate fermentation and recovery experiment,corn stover hydrolysate fermentation and recovery systemresulted in accumulation of 4.16 and 1.80 g L−1 acetic and

mulation of more acids are not known.

stover hydrolysate employing C. beijerinckii P260. (A) Sugar(productivity) at various fermentation times; (C) cells, and (D) specific productivity of ABE production at various

306 food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308

Table 5 – Concentrations of ABE and acids in recovered stream obtained from corn stover hydrolysate fermentationemploying C. beijerinckii P260.

Recovery time [h] Products [g L−1]

Acetone Butanol Ethanol HAc HBu Acids ABE

19 0.00 0.00 0.00 0.00 0.00 0.00 0.0024 32.59 88.01 1.26 – 0.54 0.54 121.8631 23.80 39.46 0.86 0.50 0.66 1.16 64.1247 30.34 57.09 1.07 0.63 0.43 1.06 88.5055 43.31 76.41 1.69 – – – 121.4172 45.90 79.91 1.76 – – – 127.57

Recovery was initiated at 19 h and first condensate was collected at 24 h.

In the above reactor 1 L of corn stover hydrolysate contain-ing 30.8 g cellulosic sugar was used. To this solution 171 mLof 500 g L−1 glucose (85.5 g glucose) solution was added. Itshould be noted that 86 g L−1 corn stover resulted in 30.80 g L−1

total sugars upon hydrolysis. For this cellulosic deconstruc-tion, hydrolytic conditions were not optimized and for thatreason sugar concentration in the hydrolysate was low whichcould be improved upon process optimization.

Currently, most of the ethanol production industries usebatch process to produce this fuel or chemical and it wouldbe beneficial if those companies can be adapted for butanolproduction without making any significant changes or modifi-cations to their infrastructure. Making no significant changesin ethanol producing plants would benefit the economics ofthis process. Butanol can be produced from dilute sugar solu-tions (60 g L−1) without simultaneous product recovery andconcentrated sugar solutions (90–200 g L−1) with simultaneousproduct recovery. Use of concentrated sugar solutions in com-bination with simultaneous product recovery is expected to beeconomical for butanol production from concentrated sugarsolutions. However, the culture should be able to grow in con-centrated sugar medium. Previous studies suggested that C.beijerinckii P260 is able to grow and produce ABE in sugarsolutions as high as 200 g L−1 (Qureshi et al., 2007) while C.saccharobutylicum P262 (previously known as C. acetobutylicumP262) can tolerate 200–227 g L−1 sugars (Maddox et al., 1985;Qureshi and Maddox, 2005).

The ultimate goal of our research program is to use lig-

nocellulosic biomass, rather than using glucose and xylose

Table 6 – Toxicity due to various lignocellulosic feedstock hydro

Feedstock Sugar concentrationculture tolerated [g L−1]

Non-integrated systemsGlucose 200a

Corn fiber hydrolysateb 46.3–54.3

Wheat straw hydrolysate 170.2

Barley straw hydrolysate 125.0

Corn stover hydrolysate 120.0

Integrated systemsGlucose 161.7b

Corn fiber hydrolysatec 60.0 + 5.0

Wheat straw hydrolysate 128.3

Barley straw hydrolysate 90.3

Corn stover hydrolysate 93.1

a At 250 g L−1 glucose level no cell growth and fermentation was observedb C. beijerinckii BA101.c Corn fiber arabino xylan (60 g L−1 xylan + 5 g L−1 xylose) fermented by C. b

mixture, for the production of butanol. For the present stud-ies glucose was used to supplement the cellulosic hydrolysateand study the fermentation characteristics of concentratedfermentable sugar mixture. This sugar mixture, though toxicto some extent, was fermented completely. Furthermore, wewere able to develop a process of simultaneous saccharifi-cation, fermentation, and recovery (SSFR) using agriculturalresidues as the sole substrate and carbon source (Qureshiet al., 2013b) without a need to supplement with additionalpure sugar mixture such as glucose and xylose.

Lignocellulosic biomass substrates require pretreatmentusing dilute acid or alkali solutions at high temperature. Dur-ing pretreatment process sugar degradation products suchas furfural, hydroxymethyl furfural, acids (acetic, ferulic, glu-curonic and �-coumaric), and phenolic compounds (Ezeji et al.,2007) are generated that inhibit fermentation. To avoid thisinhibition, such solutions are detoxified by treating with lime(Qureshi et al., 2010a,b). In our various studies a number ofagricultural residues were used and it was discovered that thecellulosic hydrolysates were toxic to the culture’s cell growthand fermentation in the following order: corn fiber > cornstover > barley straw > wheat straw > glucose (non-integratedsystems; Table 6). Although lower sugar concentrations wereused in the integrated systems of this study (Table 6), higherABE productivities were obtained which improves process eco-nomics.

However, overliming or other detoxification processes addto the cost of butanol production and hence this problem

should be circumvented using one of the two approaches:

lysates to C. beijerinckii P260.

ABE productivity[g L−1 h−1]

Reference

0.15 Qureshi et al. (2007)0.10 Qureshi et al. (2008)0.11 Qureshi et al. (2007)0.20 This work0.14 This work

0.59 Ezeji et al. (2003)0.47 Qureshi et al. (2006)0.36 Qureshi et al. (2007)0.60 This work0.70 This work

.

eijerinckii P260.

food and bioproducts processing 9 2 ( 2 0 1 4 ) 298–308 307

Table 7 – A comparison of fermentation parameters for the production of ABE from barley straw and corn stoverhydrolysates employing various microbial cultures.

Substrate Microbial culture Total ABE [g L−1] Yield Productivity [g L−1 h−1] Reference

Non-integrated systemsBarley straw C. beijerinckii P260 26.64 0.43 0.39 Qureshi et al. (2010a)Corn stover C. acetobutylicum P262 25.70 0.34 – Parekh et al. (1988)Corn stover C. beijerinckii P260 26.27 0.44 0.31 Qureshi et al. (2010b)Corn cobs C. acetobutylicum IFP913 13.55–17.90 0.30–0.33 0.23–0.47 Marchal et al. (1992)Corn fiber C. beijerinckii BA101 9.30 0.39 0.10 Qureshi et al. (2008)

Integrated systemsBarley straw C. beijerinckii P260 47.20 0.42 0.60 This work

0

(aaottae2whliNgp

nitr1l(urhmsubbfHt

fnansftbt

4

If

Corn stover C. beijerinckii P260 50.14

i) development of a tolerant culture that can efficiently grownd produce ABE using lignocellulosic hydrolysates thus toler-ting hydrolysis inhibitors; and/or, (ii) investigate applicationf other pretreatment techniques that generate low concen-rations of toxic chemicals. There are a number of cultureshat have been developed including C. beijerinckii BA101, C.cetobutylicum, and Escherichia coli (Annous, 1991; Formanekt al., 1997; Shen et al., 2011; Inui et al., 2008; Tomas et al.,003; Liu et al., 2010) and they should be used in combinationith hydrolysates. Furthermore, a number of other techniquesave been used for lignocellulosic biomass pretreatment fol-

owed by hydrolysis and ethanol production. These techniquesnclude hot water, ammonia fiber expansion (AFEX), and diluteaOH treatment of lignocellulosics (Saha, 2003). Hydrolysatesenerated using these approaches should be evaluated for ABEroduction using efficient strains such as C. beijerinckii P260.

There are several simultaneous product removal tech-iques that can be applied to remove butanol (or ABE)

ncluding adsorption, gas stripping, liquid-liquid extrac-ion, perstraction, vacuum fermentation, pervaporation, andeverse osmosis (Maddox, 1989; Qureshi, 2009; Groot et al.,992; Roffler et al., 1984, 1988). Gas stripping, pervaporation,iquid-liquid extraction (Groot et al., 1992), and adsorptionQureshi et al., 2005) are viewed to be cost effective prod-ct removal techniques. In situ removal of ABE by adsorptionesults in adsorption of nutrients (Ennis et al., 1987) andence adsorbents should not be brought in contact with fer-entation broth. For this reason, ABE should be removed by

team stripping or gas stripping before further concentrationsing adsorption. Gas stripping is a novel technique for in situutanol removal which does not require any chemical or mem-rane, can be applied at fermentation temperature, and evenermentation gases that are produced in the system (CO2 and

2) can be used for recovery thus reducing operation cost. Forhese reasons, gas stripping was applied to these systems.

The purpose of these experiments was to produce ABErom concentrated lignocellulosic sugar solutions in combi-ation with simultaneous product recovery which has beenchieved successfully. Initially barley straw hydrolysate didot produce >7.1 g L−1 ABE (Qureshi et al., 2010a) while corntover hydrolysate was not able to support cell growth andermentation at all (Qureshi et al., 2010b). By using detoxifica-ion and simultaneous product recovery techniques we haveeen able to utilize concentrated sugar solutions derived fromhese feedstocks.

. Conclusions

n a control batch experiment (I), 23.25 g L−1 ABE was producedrom 55.7 g L−1 glucose (feed glucose concentration 62.5 g L−1)

.43 0.70 This work

resulting in an ABE productivity and yield of 0.33 g L−1 h−1 and0.42, respectively. At the end of fermentation 6.8 g L−1 glucosewas left unused due to product toxicity. Use of concentratedbarley straw and corn stover hydrolysates in combinationwith simultaneous product recovery was also investigated.From 90.3 g L−1 barley straw hydrolysate sugars 47.20 g L−1

total ABE was produced with a productivity of 0.60 g L−1 h−1.This productivity is 182% of that achieved in the control(glucose) fermentation process. In this process an ABE yieldof 0.42 was obtained and the process resulted in completesugar utilization. Use of greater than 100 g L−1 cellulosic sugarwas not considered in the integrated system as it wouldhave resulted in substrate inhibition. Fermentation of cornstover hydrolysate was also possible with 93.1 g L−1 initialcellulosic sugar concentration. In this process 99.4% sugarswere used and the integrated reactor resulted in the produc-tion of 50.14 g L−1 ABE resulting in a reactor productivity of0.70 g L−1 h−1 which is 212.1% of that achieved in the controlexperiment. This system resulted in an ABE yield of 0.43. Inboth cases (barley straw and corn stover hydrolysates) higherspecific productivities were obtained when compared to thecontrol (glucose alone) experiment. Use of more concentratedsugar solutions was toxic to the culture. It is recommendedthat use of other pretreatment techniques and newly devel-oped strains should be explored. The productivities, ABEconcentrations, and yields obtained in these studies are supe-rior to the results reported by other investigators (Table 7).

Acknowledgments

The author (NQ) would like to thank Professor David Jones(Otago University, Dunedin, New Zealand) for his generous giftof C. beijerinckii P260. N. Qureshi would also like to thank Pro-fessor Patrick Hayes (Oregon State University, Corvallis, OR,USA) for his kind gift of barley straw, and Loren Iten, AdamWallenfang, and Greg Kennedy for helping with preparationof barley straw and corn stover hydrolysates.

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