Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors

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Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors夽 N. Qureshi ∗ , M.A. Cotta, B.C. Saha United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center for Agricultural 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; Productivity

1.

Introduction

As a result of increase in gasoline prices, research interests in the development of butanol have increased dramatically as this biofuel has many superior fuel properties and contain higher energy than ethanol (Anon, 2007, 2008). In fact butanol (acetone butanol ethanol or ABE) production by fermentation ranked second to ethanol in importance and history as commercial plants during World War I and II produced acetone and butanol on large scale (Durrie, 1998; Zverlov et al., 2006; Jones and Woods, 1986). Most of these commercial plants ceased operation due to development of petrochemically produced

butanol and an increase in the cost of feedstocks such as corn and molasses. For these reasons the fermentation plants could not compete with petrochemical route of butanol production. The last plant was shut down in 1982 in South Africa (Jones and Woods, 1986). In the articles cited below and numerous discussions and presentations delivered in clostridia conferences around the world, the following recommendations were given to revive this fermentation and make butanol a commercially viable biofuel (Jones and Woods, 1986; Maddox, 1989; Durrie, 1998; Zverlov et al., 2006; Qureshi, 2009): (i) use of lower cost substrates or feedstocks compared to corn or molasses; (ii) increase product concentration above 20 g L−1 ; (iii) improve

夽

Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and employer. ∗ Corresponding author. Tel.: +1 309 681 6318; fax: +1 309 681 6427. E-mail address: [email protected] (N. Qureshi). Received 9 May 2013; Received in revised form 30 October 2013; Accepted 5 November 2013 0960-3085/$ – see front matter Published by Elsevier B.V. on behalf of The Institution of Chemical Engineers. http://dx.doi.org/10.1016/j.fbp.2013.11.005 Please cite this article in press as: Qureshi, N., et al., Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.11.005

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yield by utilizing residual acids; and (iv) decrease in product recovery costs. With the above recommendations Qureshi et al. (2013a) focused on each of these and developed a comprehensive research program with expectations to make butanol a commercially viable biofuel. It should be noted that if this biofuel (or any other) is to be commercialized, the process should use agricultural residues and/or new energy crops that are renewable in nature and economically available. For this very important reason agricultural biomass including corn stover, wheat straw, barley straw, switchgrass, miscanthus, soy molasses, corn fiber, various waste feedstocks containing carbohydrates, and distillers dried grains and soluble (DDGS), and forest product residues such as saw dust, leaves and bark (Qureshi et al., 2013a) are being used. The prices of these residues are projected to be $60 ton−1 (Lane, 2011) as opposed to 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 could be produced in the United States by 2030 (Lane, 2011). Based on calculations 545 × 109 L (141.9 billion gallons) of acetone–butanol–ethanol (ABE – 3:6:1) of which 326 × 109 L (85.1 billion gallons) is butanol, 163.5 × 109 L (42.6 billion gallons) acetone, and 54.5 × 109 L (14.2 billion gallons) ethanol could be produced. Currently 514.6 × 109 L (134 billion gallons) of gasoline is used annually in the United States [(http://www.eia.gov/tools/faq.cfm?id=23&t=10), website accessed December 28, 2012]. This consumption was about 6% less than the record high of about 546.7 × 109 L (142.4 billion gallons) consumed in 2007. It is assumed that ABE (all three components; the authors are aware that butanol and ethanol can be used) can be used in cars and automotive engines. For these calculations the carbohydrate content of lignocellulosic biomass was adapted to be 66%, and an ABE yield of 0.38. Based on these calculations it is predicted that we have potential to be free of foreign oil. Deployment of such a technology will also be expected to generate a tremendous number of jobs in the U.S. The reader is informed that butanol producing microorganisms can utilize all the hexose and pentose sugars as opposed to current commercial ethanol producing organisms which cannot use pentoses. However, there are challenges in producing butanol from cellulosic biomass. Butanol production from sugary and starchy feedstocks has been demonstrated successfully (Jones and Woods, 1986). It should be noted that use of lignocellulosic biomass requires pretreatment and hydrolysis of this feedstock prior to fermentation to ABE. The pretreatment is performed using dilute acid (H2 SO4 ), or alkali (NaOH) in order to provide hydrolytic enzymes access to the cellulosic fibers and release the sugars contained within (close, if not 100%). During pretreatment process some of the hemicellulosic sugars are converted to furfural, hydroxymethyl (HMF), and other degradation products that are toxic to the culture. Since these inhibitors hinder the fermentation of hydrolysates, their detoxification becomes essential for successful fermentation in particular for barley straw and corn stover hydrolysates (Qureshi et al., 2010a,b). Attempts have been made to increase ABE concentration in the fermentation broth by genetically engineering ABE producing cultures for improved tolerance to products (Annous, 1991; Formanek et al., 1997; Shen et al., 2011; Inui et al., 2008; Tomas et al., 2003; Liu et al., 2010). In some cases butanol concentration as high as 25 g L−1 has been achieved, however this is still too low for this fermentation to be commercially viable.

In order to overcome this (to increase butanol or ABE concentration per L broth) another approach (process engineering) has been employed where the product was simultaneously removed from the fermentation broth which resulted in ABE production 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 ABE yield as acids could not leave the system until converted to ABE thus addressing recommendations number ii and iii. Recommendation number iv was to reduce energy requirement for product separation. Technologies such as those just mentioned (Qureshi et al., 2013a) to recover ABE simultaneously from the fermentation broth greatly reduce the energy requirement for this process. To further improve process economics of ABE production from lignocellulosic biomass one of the two processes (separate hydrolysis, fermentation, and recovery: SHFR or simultaneous saccharification, fermentation, and recovery: SSFR) in combination with use of concentrated sugar (hydrolysed cellulosic biomass) solutions should be applied. Use of concentrated sugar solutions would reduce capital and operational costs by reducing process stream volumes and reactor sizes. The objective of the current studies was to use SHFR process to produce ABE from barley straw and corn stover hydrolysates. Fermentation of concentrated sugar solutions, in particular cellulosic hydrolysates which are also toxic to the microorganisms, is a challenge until toxic product (butanol, toxicity level 15–20 g L−1 ) is simultaneously removed from fermentation broth. It should also be noted that some cellulosic hydrolysates require detoxification prior to fermentation.

2.

Materials and methods

2.1.

Culture and inoculum development

Clostridium beijerinckii P260 was a generous gift from Professor David Jones, University of Otago (Dunedin, New Zealand). C. beijerinckii P260 spores were maintained in distilled water at 4 ◦ C. One hundred microlitre spores were heat shocked for 2 min at 75 ◦ C and transferred to cooked meat medium (CMM; Difco Laboratories, Detroit, MI, USA) for germination as described by Ennis and Maddox (1985) and Qureshi et al. (2007). During germination process the bottles were kept in an anaerobic jar at 35 ◦ C. The culture was ready in 16–18 h time and 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 hundred millilitre 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 by cooling to 35 ◦ C and adding 1 mL each of mineral, vitamin, and buffer solutions (Qureshi et al., 2007). This bottle was inoculated with 7–10 mL of stage I inoculum developed above and incubated for approximately 10–12 h at 35 ◦ C when the culture was ready for inoculation into 50 mL fermentation bottles (described below) or bioreactor containing barley straw or corn stover hydrolysates.

2.2.

Barley straw, pretreatment, and hydrolysis

Barley straw was obtained from Oregon State University, Corvallis, OR, USA and its details on Geo-Co-ordinates, verities, harvesting, planting season, and baling were provided earlier (Qureshi et al., 2010a). The barley straw was milled into fine

Please cite this article in press as: Qureshi, N., et al., Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.11.005

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particles ranging from 1–2 mm using a hammer mill. Eighty six grams per litre of milled barley straw was pretreated with dilute (10 mL L−1 ) H2 SO4 (Sigma Chemicals) at 121 ◦ C for 1 h followed by cooling to room temperature and adjusting pH to 5.0 using 400 g L−1 NaOH (Sigma Chemicals) solution. Following pH adjustment the pretreated barley straw was hydrolyzed at 45 ◦ C for 72 h using 6 mL L−1 each of cellulase (Celluclast 1.5 L; Sigma Chemicals; enzyme activity 751 ± 35 U mL−1 ; carboxymethylcellulose activity), ␤-glucosidase (Novozyme 188; Sigma Chemicals; activity 380 ± 19 U mL−1 ), and xylanase (Viscostar 150 L; Dyadic Corporation, Jupiter, FL, USA; activity 9837 ± 190 U mL−1 ) enzymes (Qureshi et al., 2010a). The hydrolyzed barley straw solution was filter sterilized and stored at 4 ◦ C until used for these experiments. The sugar composition of barley straw hydrolysate has been given in Table 1 (total sugar 44.2 g L−1 ). This sugar concentration was raised to the desired level by supplementing with 400–500 g L−1 filter sterilized 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 mm followed by suspending in 10 mL L−1 H2 SO4 and mixing well. Then the mixture was transferred to a 316 stainless steel reactor 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 to 5.0 with 400 g L−1 NaOH solution. Further details have been given elsewhere (Qureshi et al., 2010b). Corn stover hydrolysis and further processing was performed as described above for barley straw. Table 1 shows sugar composition of corn stover hydrolysate.

2.4. Detoxification of barley straw and corn stover hydrolysates Barley straw and corn stover hydrolysates were detoxified by overliming method as described elsewhere (Qureshi et al., 2010a). The filter sterilized hydrolysates were stored at 4 ◦ C until used for fermentation studies.

2.5. Fermentation with or without integrated product recovery Fermentation studies without product recovery were performed in 50 mL (total volume 67 mL) PyrexTM screw capped bottles with 35 mL medium. The medium inside the bottles was not agitated or mixed. To the hydrolysate, 0.35 mL of each of P2 stock solutions (mineral, vitamin, and buffer; Qureshi et al., 2007) and yeast extract (from presterilized 40 g L−1 ) to

Table 1 – Sugar concentrations in barley straw and corn stover hydrolysates after hydrolysis at 45 ◦ C for 72 h using enzymes. Sugars

Barley straw hydrolysate [g L−1 ]

Corn stover hydrolysate [g L−1 ]

Glucose Xylose Arabinose Galactose Mannose

23.9 16.8 2.2 1.3 0.0

22.1 7.2 1.1 0.4 0.0

Total

44.2

30.8

3

Fig. 1 – A schematic diagram of an integrated system of ABE production employing C. beijerinckii P260 and simultaneous recovery using gas stripping from barley straw and corn stover hydrolysates. 1 g L−1 level were added. All the transfers were made asceptically. The bottles were then placed in an anaerobic jar for 48 h for anaerobiosis followed by inoculating with 3 mL of an actively growing stage II culture/inoculum. The bottles were incubated at 35 ◦ C in an anaerobic jar. One millilitre samples were taken as needed followed by centrifugation at 15,000 × g. Clear supernatant liquid was stored at −18 ◦ C until sugar and ABE measurements. Fermentation with product recovery studies were performed in 2.5 L total capacity glass bioreactor (BIOFLO 2000 Fermentor; New Brunswick Scientific, New Brunswick, NJ, USA) with 1.25 L working volume. One litre of barley straw or corn stover hydrolysate was transferred to the reactor followed by adding 12.5 mL of each of P2 medium stock solutions and yeast extract (Bacto-Dickinson & Co.) to 1 g L−1 final level (from 40 g L−1 presterilized solution). Additional sugar solution (from 400 to 500 g L−1 filter sterilized glucose solution) was added to this medium to raise sugar level to approximately 100 g L−1 (for product recovery experiments). The temperature of the reaction mixture was maintained at 35 ◦ C and 50 mL min−1 oxygen free nitrogen gas was sparged/bubbled through the medium for 24–48 h prior to inoculation with 100 mL actively growing stage II inoculum. At the time of inoculation, N2 sparging was changed from sparging to sweeping across the surface of the medium. Gas sweeping continued for 12–24 h or until culture started producing its own gases (CO2 and H2 ). Sparged or swept gases were cooled in a condenser to capture any volatiles or water 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 a twin-head MasterflexTM (Cole Parmer, Vernon Hills, IL, USA) peristaltic pump and 18 size Norprene (Cole Parmer) tubing. The gases were bubbled through the fermentation mixture followed by cooling in a condenser. The condensate containing ABE was removed intermittently and its weight or volume was recorded followed by measuring ABE concentration. Fig. 1 shows a schematic diagram of fermentation and recovery experiment. More details of condenser, coolant, and cooling machine have been given elsewhere (Qureshi et al., 2007).

2.6.

Analyses

Fermentation products such as ABE, acetic acid, and butyric acid were measured by gas chromatography (6890 N; Agilent Technologies, Wilmington, DE, USA) using a packed glass column as described elsewhere (Qureshi et al., 2007). Initial

Please cite this article in press as: Qureshi, N., et al., Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.11.005

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3.

Results and discussion

3.1.

Control fermentation

In order to measure performance of barley straw and corn stover fermentation experiments, a control fermentation (I) was run using glucose as substrate. The fermentation was initiated with 62.5 g L−1 initial glucose level. Over a period of 70 h, the culture accumulated 23.25 g L−1 total ABE (Fig. 2a). At the end of fermentation 6.8 g L−1 glucose was left unused due to ABE toxicity to the culture, thus using 55.7 g L−1 glucose which is 89.1% of that present initially. This experiment resulted in a productivity of 0.33 g L−1 h−1 and an ABE yield of 0.42. In this fermentation a maximum cell concentration of 2.60 g L−1 was obtained at 48 h (Fig. 2b). The system resulted in a specific productivity of 0.13 h−1 and a sugar utilization rate of 0.80 g L−1 h−1 . A batch system, where product was recovered simultaneously by gas stripping, resulted in complete utilization of lactose present in whey permeate medium (Ennis et al., 1986). This system indicated that product inhibition can be relieved if toxic product level is kept below toxicity in the reactor by removing it simultaneously. Since sugar concentrations in the barley straw and corn stover hydrolysates that were fermented in the integrated experiments were 90–93 g L−1 , another experiment (control II) was conducted with 90.0 g L−1 initial glucose concentration in the medium. As in the above control, no product recovery was performed. This study resulted in the production of 23.50 g L−1 total ABE with a productivity of 0.36 g L−1 h−1 and a yield of 0.42. At the end of fermentation, the residual sugar concentration was 34.0 g L−1 . These results suggested that the culture performed equally at these two control sugar levels (62.5–90.0) except that residual glucose levels were different.

25

70 60

20

Products [gL-1]

50 15

40

10

30 20

5

10

0

0 0

16

25

36

48

64

Glucose [gL-1]

a

Acetone Butanol Ethanol HAc HBu ABE Glucose

70

Fermentation Time [h]

b

3 2.5

Cell Conc [gL-1]

column temperature was 100 ◦ C which was ramped up at the rate of 40 ◦ C min−1 to 180 ◦ C and held it there for 4 min. Sugars were measured using a Surveyor HPLC equipped with an automatic sampler and injector (Thermo-Electron Corporation, West Palm Beach, FL, USA). The HPLC column (HPX-87P; Aminex Resin-based) was obtained from Bio-Rad (Hercules, CA, USA). Solvent (Milli-Q water) flow rate was maintained at 0.6 mL min−1 and products were detected using a Refractive Index detector. ABE productivity was calculated as total ABE produced in g L−1 divided by the fermentation time and is expressed as g L−1 h−1 . Fermentation time was initiated when the bottle or reactor was inoculated. Fermentation time period is defined as the time difference between inoculation and when fermentation ceased and is expressed in h. Specific productivity (h−1 ) is defined as productivity in g L−1 h−1 divided by cell concentration in g L−1 . ABE yield was calculated as total ABE produced divided by the total sugar utilized (initial sugar minus residual sugar; both in g L−1 ). Cell concentration was measured by an optical density method ( 540 nm) and is presented as dry weight cell concentration (g L−1 ). To measure optical density, 100 ␮L fermentation broth was added to 900 ␮L presterilized 9.0 g L−1 saline (NaCl) water and mixed well. Dry weight cell concentration was calculated using the following correlation: y = 0.2141x3 − 0.1594x2 + 0.2588x − 0.0009; where y is cell concentration in the diluted sample in g L−1 and x is optical density. Following this, y was multiplied by the dilution factor (10) to calculate cell concentration in the undiluted fermentation broth withdrawn from the bottle or bioreactor.

2 1.5 1 0.5 0 0

16

25

36

48

64

70

Fermentation Time [h]

Fig. 2 – Production of ABE from glucose in a control (I) batch reactor using C. beijerinckii P260. (A) Products and glucose concentration vs. Fermentation time; (B) cell concentration at various fermentation times.

3.2. Barley straw hydrolysate (BSH) fermentation to butanol and recovery Following this, experiments were performed where detoxified barley straw was supplemented with various glucose levels thus raising total sugar concentration to 63, 80, 100, and 125 g L−1 and fermented. At these sugar levels total ABE concentrations were 24.06, 21.45, 19.80, and 19.50 g L−1 , respectively (Table 2). With the increase in sugar concentration, decreased ABE was produced. Cell growth and fermentation suggested that the culture was capable of tolerating higher cellulosic sugar levels (up to 125 g L−1 ). At 63 g L−1 total sugar concentration, a productivity of 0.36 g L−1 h−1 and a yield of 0.43 were achieved (Table 2). With the increase in sugar concentration, decreased ABE productivities were achieved. At an initial sugar concentration of 125 g L−1 , a productivity of 0.20 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 initial sugar level indicating that higher sugar levels were inhibitory to fermentation. In this fermentation (125 g L−1 initial sugar level) ABE yield also decreased from 0.43 (63 g L−1 sugar level) to 0.38 which is a decrease of 11.6%. Decreased productivities and yields (with increased sugar concentrations) suggested that more sugar was used for maintenance energy and/or production of reaction intermediates such as acids (acetic and butyric acids). Both rate of sugar utilization, and maximum cell concentration decreased with increased sugar levels (Table 2). At a sugar 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 of 0.52 g L−1 h−1 was obtained. This decrease is 38.8% based on initial 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 levels from 63 to 125 g L−1 resulted in decreased cell growth and

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Table 2 – Production of ABE from barley straw and corn stover hydrolysates supplemented with additional sugar to various levels. Initial sugar conc. [g L−1 ]

ABE [g L−1 ]

ABE ABE productivity yield [g L−1 h−1 ]

ABE specific prod. [h−1 ]

Sugar used [g L−1 ]

Residual sugar [g L−1 ]

Sugar used [%]

Final cell conc. [g L−1 ]

Sugar utilization rate [g L−1 h−1 ]

Fermentation parameters: barley straw hydrolysate 24.06 0.36 0.43 63 21.45 0.22 0.40 80 19.80 0.20 0.39 100 19.50 0.20 0.38 125

0.42 0.28 0.25 0.29

55.9 53.3 50.8 51.3

7.1 26.7 49.2 73.7

88.70 66.63 50.80 41.04

0.85 0.55 0.54 0.52

0.85 0.80 0.80 0.70

Fermentation parameters: corn stover hydrolysate 24.38 0.30 0.44 60 10.52 0.11 0.39 80 6.76 0.07 0.36 100 6.02 0.14 0.35 120

0.39 0.16 0.14 0.26

55.4 27.0 18.8 17.2

4.6 53.0 81.2 102.8

92.33 33.75 18.80 14.33

0.68 0.28 0.20 0.39

0.77 0.70 0.50 0.53

(Huang et al., 2004). After 28 h of fermentation the culture became relatively slow and at 39 h a total ABE concentration of 8.99 g L−1 was recorded. This was followed by vigorous fermentation again thus resulting in accumulation of ABE in the reactor. At 51 h, 13.37 g L−1 total ABE were measured. The fermentation 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 galactose were present in the fermentation mixture (Fig. 3b). All these sugars were utilized simultaneously, though xylose at a much slower rate than glucose. Both arabinose and galactose were completely used during initial 16 h of cell growth and fermentation. At 16, 22, 28, 39, 51, and 63 h 12.2, 10.0, 7.7, 4.8, 3.7 and 0 g L−1 xylose was present, respectively.

a

16 14

Products [gL-1]

12

Acetone

10

Butanol

8

Ethanol

6

HAc

4

HBu

2

ABE

0 0

16

22

28

39

51

63

Fermentation Time [h]

b

Sugars [gL-1]

cell concentration. A sugar concentration of 63 g L−1 resulted in a cell concentration of 0.85 g L−1 . This cell concentration is much lower than the cell concentration achieved in the control fermentation (2.60 g L−1 ). It should be noted that even the detoxified barley straw hydrolysate was inhibitory for cell growth which became even greater as sugar level was increased to 125 g L−1 . Cell growth decreased from 0.85 to 0.70 g L−1 as sugar was increased from 63 to 125 g L−1 . Specific productivity is a measure of productivity per unit cell in the system and is an indication of cell efficiency. In the present studies a specific productivity of 0.42 h−1 was achieved at an initial sugar level of 63 g L−1 (Table 2). Further increase in total sugar concentration to 80 g L−1 resulted in a decreased specific productivity (0.28 h−1 ) suggesting that cells were not as efficient, possibly due to inhibition caused by higher sugar concentration. This was a 33.3% decrease in specific productivity of ABE production. Further increase in sugar concentration to 100 g L−1 , decreased specific productivity to 0.25 h−1 . In the four experiments incomplete sugar utilization was observed (Table 2). The highest sugar utilization was 55.9 g L−1 (88.7%) at an initial sugar level of 63 g L−1 . In this experiment a residual sugar concentration of 7.1 g L−1 was left behind. In the subsequent 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. In these experiments 26.7, 49.2, and 73.7 g L−1 sugar remained unused. The experiment which contained 125 g L−1 total initial sugar, resulted in the utilization of 41.04% sugar. The next experiment was performed in the reactor. For this purpose barley straw hydrolysate was supplemented with additional 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 increased lag time thus resulting in decreased productivity (Table 2). Then cell growth was initiated by inoculation of the reactor. As a result of dilution by inoculum addition, total sugar concentration decreased to 90.3 g L−1 . Cell growth was rapid and in 16 h fermentation time 0.80 g L−1 cell mass accumulated in the system. Also fermentation was fast and at that time 4.83 g L−1 total ABE were present in the reactor (Fig. 3a). This was followed by ABE recovery by gas stripping using fermentation gases. For 16 to 22 h ABE concentration was nearly constant at about 4.83–4.86 g L−1 . Since this ABE concentration was not toxic to the culture, fermentation became vigorous and cell growth increased significantly. At 28 h cell concentration was 2.50 g L−1 and total ABE in the system were 12.14 g L−1 . This was followed by some oscillations in the total product concentration in the system which is common in ABE fermentations employing C. beijerinckii or C. acetobutylicum

100 90 80 70 60 50 40 30 20 10 0

Glu Xyl Arab Man gal Tot Sug

0

16

22

28

39

51

63

Fermentation Time [h] Fig. 3 – Production of ABE from barley straw hydrolysate in an integrated system of production and recovery employing C. beijerinckii P260. (A) Products vs. fermentation time; (B) sugars in bioreactor vs. fermentation time.

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Fig. 4 – Various kinetic parameters of ABE production from barley straw hydrolysate employing C. beijerinckii P260. (A) Sugar utilization rate vs. fermentation time; (B) rate of ABE production (productivity) at various fermentation times; (C) cell concentration and pH in the reactor at various fermentation times, and (D) specific productivity of ABE production at various fermentation times. The rates of sugar utilization are presented in Fig. 4a. At 16 h, a rate of sugar utilization of 1.20 g L−1 h−1 was measured which increased to 2.26 g L−1 h−1 at 22 h. This rate increased to 3.73 g L−1 h−1 at 28 h, and then declined to 0.96 g L−1 h−1 at 39 h. Between 51 and 63 h a rate of sugar utilization of 1.41 g L−1 h−1 was measured. The ABE productivities at various time periods are shown in Fig. 4b. The productivity was highest at 28 h measuring 1.78 g L−1 h−1 . The cell concentration and pH values are shown in Fig. 4c. A maximum cell concentration of 2.50 g L−1 was measured at 28 h which was the reason behind achieving a highest productivity employing barley straw hydrolysate. At the initiation of fermentation, pH was adjusted to 6.47 which decreased to 5.35 during initial 16 h of fermentation as a result of production of acetic and butyric acids. At 22 h it was measured at 5.31 (Fig. 4c). During the course of fermentation (16–63 h) pH remained below 5.6. It should be noted that pH was not controlled by addition of alkali solution, rather culture controlled it by utilizing acids. The specific productivities obtained in this system are shown in Fig. 4d. The highest specific productivity of 0.66 h−1 was obtained at 28 h when the cell concentration was the highest in the system. At 51 h, a specific productivity was 0.58 h−1 . While concentrations of ABE in the reactor are plotted in Fig. 3a, their concentrations in the recovered stream are shown in Table 3. At 22 h an ABE concentration of 121.93 g L−1 was measured in the condensate. This concentration is many fold more than present in the reactor. At 28 h it was 122.34 g L−1 . At 39, 51, and 63 h these concentrations were 104.36, 135.15, and 100.41 g L−1 respectively. During gas stripping small amounts of acetic and butyric acids were also removed which are presented in Table 3. In the beginning of the integrated reactor 1250 mL fermentation medium was present with 90.3 g L−1 mixed sugar. The system utilized 112.9 g total sugar (in 1250 mL medium; 100%

sugar utilization) and produced 47.20 g total ABE resulting in an ABE yield of 0.42 and an average ABE productivity of 0.60 g L−1 h−1 (Table 4). This productivity is 1.82 times (182%) of that achieved in the control experiment. Based on a maximum cell concentration of 2.50 g L−1 that was achieved in this system, a specific productivity of 0.24 h−1 was obtained. This specific productivity is 1.85 times than achieved in the control fermentation. It is pointed out that fermentation of undetoxified barley straw hydrolysate did not produce more than 7.09 g L−1 ABE with a productivity of 0.10 g L−1 h−1 (Qureshi et al., 2010a). Also the non-integrated system where product 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 of 0.36 g L−1 h−1 . This demonstrated that an integrated system using barley straw hydrolysate containing glucose enriched sugar solution can be successfully fermented to ABE if the toxic product is recovered simultaneously. In the control reactor (I) the ratio of acids produced per g sugar utilized was 0.064 g g−1 while in the integrated system it was 0.026 g g−1 suggesting that in the integrated system less acids were accumulated which would result in improved ABE yield and process economics. In this reactor system 1 L of barley straw hydrolysate 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 to butanol and recovery Since barley straw hydrolysate was fermented to butanol, experiments with corn stover hydrolysate were also performed. For this purpose, corn stover hydrolysate was supplemented with additional sugar to raise total sugar level to 60–120 g L−1 and fermented without product recovery. This

Please cite this article in press as: Qureshi, N., et al., Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.11.005

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Table 3 – Concentrations of ABE and acids in recovered stream obtained from barley straw hydrolysate fermentation employing C. beijerinckii P260. Products [g L−1 ]

Recovery time [h]

16 22 28 39 51 63

Acetone

Butanol

Ethanol

0.00 34.41 33.81 35.64 45.55 36.06

0.00 86.52 87.60 67.10 88.10 62.90

0.00 1.00 0.93 1.60 1.50 1.45

HAc 0.00 – – 1.30 – –

HBu

Acids

0.00 0.66 0.69 0.53 – –

0.00 0.66 0.69 1.83 – –

ABE 0.00 121.93 122.34 104.36 135.15 100.41

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 tolerance to the culture. At 60 g L−1 initial sugar level, the culture produced 24.38 g L−1 total ABE (Table 2). Further increase in sugar concentration to 80 g L−1 resulted in the production of 10.52 g L−1 ABE which further declined to 6.76 and 6.02 g L−1 at sugar levels of 100 and 120 g L−1 , respectively. At these initial sugar levels, ABE productivities and yields were also evaluated which are presented in Table 2. At a sugar concentration of 60 g L−1 an ABE productivity of 0.30 g L−1 h−1 was obtained which decreased to 0.07 g L−1 h−1 at a sugar level of 100 g L−1 demonstrating that CSH in combination with supplemented sugar was more toxic to the culture than BSH. Even at a sugar concentration of 60 g L−1 , ABE productivity in CSH medium was reduced by approximately 17% as compared to productivity in BSH medium. In this case (CSH), as sugar concentration was increased, productivity and cell growth decreased, further reinforcing cell growth and fermentation toxicity due to CSH. It should be noted that glucose alone at 100–120 g L−1 is not toxic to the cells of C. beijerinckii P260 (Qureshi et al., 2007). In CSH experiments ABE yields were 0.44, 0.39, 0.36, and 0.35 for the four fermentations with initial sugar levels of 60, 80, 100, and 120 g L−1 , respectively. When compared to BSH, cell concentrations in CSH fermentations were lower (Table 2). At an initial sugar level of 60 g L−1 , a cell concentration of 0.77 g L−1 was obtained which decreased to 0.50 g L−1 as initial sugar level was increased to 100 g L−1 . At this sugar level cell concentration was reduced by 35.1%. It should be noted that sugar utilization rates were also low in case of CSH fermentation. At 60 g L−1 sugar, a sugar utilization rate of 0.68 g L−1 h−1 was observed which was reduced to 0.20 g L−1 h−1 as sugar level was increased to 100 g L−1 . In these experiments with CSH as feedstock, specific productivities 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 of 60 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

utilization decreased to 27 g L−1 which further decreased to 17.2 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 recovery. Fermentation was initiated with an initial sugar level of 93.1 g L−1 . At 19 h, 1.66 g L−1 acetone 3.32 g L−1 butanol and 0.68 g L−1 ethanol were measured thus totaling to 5.66 g L−1 ABE (Fig. 5a). Concentrations of acetic and butyric acids were 6.32 g L−1 and 2.11 g L−1 , respectively, thus totaling to 8.43 g L−1 . At this time recovery of products (ABE) was initiated and as a result of simultaneous product removal total ABE decreased to 4.62 g L−1 at 24 h. As a result of efficient recovery of fermentation products total product concentration decreased to 2.09 g L−1 thus reducing inhibitory effect significantly and for that reason the culture became highly active and initiated producing ABE vigorously. Both fermentation and recovery were continued and at 47, 55, and 72 h, total ABE were 6.7, 12.87, and 9.49 g L−1 , respectively. At 72 h fermentation ceased as nearly all the sugars fed to the reactor were utilized 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 were present in the reactor (Fig. 5b). During the first 24 h of fermentation glucose was reduced from 84.4 to 58.1 g L−1 while xylose was decreased from 7.20 to 3.97 g L−1 . At this time arabinose and galactose were completely used. At 47 h, 29.2 g L−1 glucose and 2.2 g L−1 xylose were present in the system. Fermentation stopped at 72 h and at that time 0.52 g L−1 xylose was present in the reactor; all other sugars were completely utilized. In this integrated experiment, rates of sugar utilization at various times are presented in Fig. 6a. At 19 h, a rate of sugar utilization 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 was 2.16 g L−1 h−1 which reduced to 0.79 g L−1 h−1 at 72 h. ABE productivities at various times are presented in Fig. 6b. The highest productivity was observed at 55 h with a value of

Table 4 – A comparison of ABE production from barley straw and corn stover hydrolysates in integrated fermentation product removal system. Feed stock Cont-Glu Int-BSH Int-CSH

AC2 O [g L−1 ] 7.70 13.56 14.04

BuOH [g L−1 ] 13.89 30.86 34.77

EtOH [g L−1 ] 1.66 2.78 1.33

ABE [g L−1 ] 23.25 47.20 50.14

Sugar used [g L−1 ] 55.7 90.3 93.1

ABE Prod [g L−1 h−1 ] 0.33 0.60 0.70

Yield 0.42 0.42 0.43

Cont-Glu: control (I) – glucose concentration 62.5 g L−1 in the medium. Int-BSH, integrated barley straw hydrolysate; Int-CSH, integrated corn stover hydrolysate; AC2 O, BuOH, and EtOH – acetone, butanol, and ethanol, respectively.

Please cite this article in press as: Qureshi, N., et al., Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.11.005

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a

14

Products [gL-1]

12 10

Acetone

8

Butanol

6

Ethanol HAc

4

HBu

2

ABE

0 0

19

24

31

47

55

72

Fermentation Time [h]

Sugars [gL-1]

b

100 90 80 70 60 50 40 30 20 10 0

Glu Xyl Arab Man Gal Tot Sug

0

19

24

31

47

55

72

Fermentation Time [h]

Fig. 5 – Production of ABE from corn stover hydrolysate in an integrated bioreactor. Simultaneous ABE production and recovery using C. beijerinckii P260. (A) Products vs. fermentation time; (B) sugars in bioreactor vs. fermentation time. 1.25 g L−1 h−1 . In this fermentation the reactor was started with an initial cell concentration of 0.001 g L−1 . During the first 19 h period, cell concentration increased to 0.55 g L−1 which kept increasing until 55 h when it was 2.56 g L−1 (Fig. 6c). In

the beginning of fermentation, pH was 6.43 which decreased to 5.57 during 19 h of cell growth and fermentation period. During the fermentation, the culture maintained pH between 5.38 and 5.57. At the end of fermentation a pH of 5.50 was recorded (Fig. 6c). In this fermentation, specific productivities ranged from 0.37 to 0.71 h−1 . The highest specific productivity was measured at 24 h with a value of 0.72 h−1 (Fig. 6d). In the condensate, concentrations of various products including reaction intermediates (acetic and butyric acid) were measured and they are presented in Table 5. Recovery was started at 19 h and first condensate was collected at 24 h. At this 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 in the recovered product. At 31 h concentration of ABE decreased to 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 their concentrations in the condensate rose to 121.4 g L−1 (55 h) and 127.57 g L−1 (72 h). As in the case of barley straw hydrolysate fermentations, small amounts of acetic and butyric acids were measured in the condensate. In the integrated bioreactor system 93.1 g L−1 total sugars were present in the beginning of the experiment with a total 116.3 g sugar (in 1250 mL medium). During the experiment 99.4% sugar utilization occurred thus producing 50.14 g total ABE representing a yield of 0.43. This fermentation product recovery system resulted in an average productivity of 0.70 g L−1 h−1 which is 212.1% of that achieved in the control fermentation process. A total of 2.76 g of acids were recovered in the product stream. It should be noted that unlike barley straw hydrolysate fermentation and recovery experiment, corn stover hydrolysate fermentation and recovery system resulted in accumulation of 4.16 and 1.80 g L−1 acetic and butyric acids in the reactor. At this point reasons for accumulation of more acids are not known.

Fig. 6 – Various kinetic parameters of ABE production from corn stover hydrolysate employing C. beijerinckii P260. (A) Sugar utilization rate vs. fermentation time; (B) rate of ABE production (productivity) at various fermentation times; (C) cell concentration and pH in the reactor at various fermentation times, and (D) specific productivity of ABE production at various fermentation times. Please cite this article in press as: Qureshi, N., et al., Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.11.005

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Table 5 – Concentrations of ABE and acids in recovered stream obtained from corn stover hydrolysate fermentation employing C. beijerinckii P260. Products [g L−1 ]

Recovery time [h]

19 24 31 47 55 72

Acetone

Butanol

0.00 32.59 23.80 30.34 43.31 45.90

0.00 88.01 39.46 57.09 76.41 79.91

Ethanol 0.00 1.26 0.86 1.07 1.69 1.76

HAc 0.00 – 0.50 0.63 – –

HBu

Acids

0.00 0.54 0.66 0.43 – –

0.00 0.54 1.16 1.06 – –

ABE 0.00 121.86 64.12 88.50 121.41 127.57

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

In the above reactor 1 L of corn stover hydrolysate containing 30.8 g cellulosic sugar was used. To this solution 171 mL of 500 g L−1 glucose (85.5 g glucose) solution was added. It should be noted that 86 g L−1 corn stover resulted in 30.80 g L−1 total sugars upon hydrolysis. For this cellulosic deconstruction, hydrolytic conditions were not optimized and for that reason sugar concentration in the hydrolysate was low which could be improved upon process optimization. Currently, most of the ethanol production industries use batch process to produce this fuel or chemical and it would be beneficial if those companies can be adapted for butanol production without making any significant changes or modifications to their infrastructure. Making no significant changes in ethanol producing plants would benefit the economics of this process. Butanol can be produced from dilute sugar solutions (60 g L−1 ) without simultaneous product recovery and concentrated sugar solutions (90–200 g L−1 ) with simultaneous product recovery. Use of concentrated sugar solutions in combination with simultaneous product recovery is expected to be economical for butanol production from concentrated sugar solutions. However, the culture should be able to grow in concentrated sugar medium. Previous studies suggested that C. beijerinckii P260 is able to grow and produce ABE in sugar solutions as high as 200 g L−1 (Qureshi et al., 2007) while C. saccharobutylicum P262 (previously known as C. acetobutylicum P262) 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 lignocellulosic biomass, rather than using glucose and xylose

mixture, for the production of butanol. For the present studies glucose was used to supplement the cellulosic hydrolysate and study the fermentation characteristics of concentrated fermentable sugar mixture. This sugar mixture, though toxic to some extent, was fermented completely. Furthermore, we were able to develop a process of simultaneous saccharification, fermentation, and recovery (SSFR) using agricultural residues as the sole substrate and carbon source (Qureshi et al., 2013b) without a need to supplement with additional pure sugar mixture such as glucose and xylose. Lignocellulosic biomass substrates require pretreatment using dilute acid or alkali solutions at high temperature. During pretreatment process sugar degradation products such as furfural, hydroxymethyl furfural, acids (acetic, ferulic, glucuronic and ␳-coumaric), and phenolic compounds (Ezeji et al., 2007) are generated that inhibit fermentation. To avoid this inhibition, such solutions are detoxified by treating with lime (Qureshi et al., 2010a,b). In our various studies a number of agricultural residues were used and it was discovered that the cellulosic hydrolysates were toxic to the culture’s cell growth and fermentation in the following order: corn fiber > corn stover > barley straw > wheat straw > glucose (non-integrated systems; Table 6). Although lower sugar concentrations were used in the integrated systems of this study (Table 6), higher ABE productivities were obtained which improves process economics. However, overliming or other detoxification processes add to the cost of butanol production and hence this problem should be circumvented using one of the two approaches:

Table 6 – Toxicity due to various lignocellulosic feedstock hydrolysates to C. beijerinckii P260. Feedstock

Sugar concentration culture tolerated [g L−1 ]

ABE productivity [g L−1 h−1 ]

Reference

Non-integrated systems Glucose Corn fiber hydrolysateb Wheat straw hydrolysate Barley straw hydrolysate Corn stover hydrolysate

200a 46.3–54.3 170.2 125.0 120.0

0.15 0.10 0.11 0.20 0.14

Qureshi et al. (2007) Qureshi et al. (2008) Qureshi et al. (2007) This work This work

Integrated systems Glucose Corn fiber hydrolysatec Wheat straw hydrolysate Barley straw hydrolysate Corn stover hydrolysate

161.7b 60.0 + 5.0 128.3 90.3 93.1

0.59 0.47 0.36 0.60 0.70

Ezeji et al. (2003) Qureshi et al. (2006) Qureshi et al. (2007) This work This work

a b c

At 250 g L−1 glucose level no cell growth and fermentation was observed. C. beijerinckii BA101. Corn fiber arabino xylan (60 g L−1 xylan + 5 g L−1 xylose) fermented by C. beijerinckii P260.

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Table 7 – A comparison of fermentation parameters for the production of ABE from barley straw and corn stover hydrolysates employing various microbial cultures. Total ABE [g L−1 ]

Yield

Productivity [g L−1 h−1 ]

Non-integrated systems C. beijerinckii P260 Barley straw Corn stover C. acetobutylicum P262 Corn stover C. beijerinckii P260 Corn cobs C. acetobutylicum IFP913 Corn fiber C. beijerinckii BA101

26.64 25.70 26.27 13.55–17.90 9.30

0.43 0.34 0.44 0.30–0.33 0.39

0.39 – 0.31 0.23–0.47 0.10

Qureshi et al. (2010a) Parekh et al. (1988) Qureshi et al. (2010b) Marchal et al. (1992) Qureshi et al. (2008)

Integrated systems C. beijerinckii P260 Barley straw C. beijerinckii P260 Corn stover

47.20 50.14

0.42 0.43

0.60 0.70

This work This work

Substrate

Microbial culture

(i) development of a tolerant culture that can efficiently grow and produce ABE using lignocellulosic hydrolysates thus tolerating hydrolysis inhibitors; and/or, (ii) investigate application of other pretreatment techniques that generate low concentrations of toxic chemicals. There are a number of cultures that have been developed including C. beijerinckii BA101, C. acetobutylicum, and Escherichia coli (Annous, 1991; Formanek et al., 1997; Shen et al., 2011; Inui et al., 2008; Tomas et al., 2003; Liu et al., 2010) and they should be used in combination with hydrolysates. Furthermore, a number of other techniques have been used for lignocellulosic biomass pretreatment followed by hydrolysis and ethanol production. These techniques include hot water, ammonia fiber expansion (AFEX), and dilute NaOH treatment of lignocellulosics (Saha, 2003). Hydrolysates generated using these approaches should be evaluated for ABE production using efficient strains such as C. beijerinckii P260. There are several simultaneous product removal techniques that can be applied to remove butanol (or ABE) including adsorption, gas stripping, liquid-liquid extraction, perstraction, vacuum fermentation, pervaporation, and reverse osmosis (Maddox, 1989; Qureshi, 2009; Groot et al., 1992; Roffler et al., 1984, 1988). Gas stripping, pervaporation, liquid-liquid extraction (Groot et al., 1992), and adsorption (Qureshi et al., 2005) are viewed to be cost effective product removal techniques. In situ removal of ABE by adsorption results in adsorption of nutrients (Ennis et al., 1987) and hence adsorbents should not be brought in contact with fermentation broth. For this reason, ABE should be removed by steam stripping or gas stripping before further concentration using adsorption. Gas stripping is a novel technique for in situ butanol removal which does not require any chemical or membrane, can be applied at fermentation temperature, and even fermentation gases that are produced in the system (CO2 and H2 ) can be used for recovery thus reducing operation cost. For these reasons, gas stripping was applied to these systems. The purpose of these experiments was to produce ABE from concentrated lignocellulosic sugar solutions in combination with simultaneous product recovery which has been achieved successfully. Initially barley straw hydrolysate did not produce >7.1 g L−1 ABE (Qureshi et al., 2010a) while corn stover hydrolysate was not able to support cell growth and fermentation at all (Qureshi et al., 2010b). By using detoxification and simultaneous product recovery techniques we have been able to utilize concentrated sugar solutions derived from these feedstocks.

4.

Conclusions

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

Reference

resulting in an ABE productivity and yield of 0.33 g L−1 h−1 and 0.42, respectively. At the end of fermentation 6.8 g L−1 glucose was left unused due to product toxicity. Use of concentrated barley straw and corn stover hydrolysates in combination with 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 yield of 0.42 was obtained and the process resulted in complete sugar utilization. Use of greater than 100 g L−1 cellulosic sugar was not considered in the integrated system as it would have resulted in substrate inhibition. Fermentation of corn stover hydrolysate was also possible with 93.1 g L−1 initial cellulosic sugar concentration. In this process 99.4% sugars were used and the integrated reactor resulted in the production of 50.14 g L−1 ABE resulting in a reactor productivity of 0.70 g L−1 h−1 which is 212.1% of that achieved in the control experiment. This system resulted in an ABE yield of 0.43. In both cases (barley straw and corn stover hydrolysates) higher specific productivities were obtained when compared to the control (glucose alone) experiment. Use of more concentrated sugar solutions was toxic to the culture. It is recommended that use of other pretreatment techniques and newly developed strains should be explored. The productivities, ABE concentrations, and yields obtained in these studies are superior 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 gift of C. beijerinckii P260. N. Qureshi would also like to thank Professor Patrick Hayes (Oregon State University, Corvallis, OR, USA) for his kind gift of barley straw, and Loren Iten, Adam Wallenfang, and Greg Kennedy for helping with preparation of barley straw and corn stover hydrolysates.

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Please cite this article in press as: Qureshi, N., et al., Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.11.005