Anda di halaman 1dari 6

Bioresource Technology 175 (2015) 1722

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Pilot scale conversion of wheat straw to ethanol via simultaneous


saccharication and fermentation q
Badal C. Saha , Nancy N. Nichols, Nasib Qureshi, Gregory J. Kennedy, Loren B. Iten, Michael A. Cotta
Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, USA

h i g h l i g h t s
 Conversion of wheat straw to ethanol was scaled up at pilot scale.
 Dilute acid pretreated wheat straw was bioabated by growing a fungus aerobically.
 Recombinant bacterium fermented all sugars to ethanol.
 Maximum ethanol produced from 124 g wheat straw was 36 g in 83 h.
 Ethanol yield was 0.29 g/g wheat straw which is 86% of theoretical ethanol yield.

a r t i c l e

i n f o

Article history:
Received 3 September 2014
Received in revised form 9 October 2014
Accepted 10 October 2014
Available online 18 October 2014
Keywords:
Ethanol
Wheat straw
Simultaneous saccharication and
fermentation
Recombinant ethanologenic Escherichia coli
Ethanol from wheat straw

a b s t r a c t
The production of ethanol from wheat straw (WS) by dilute acid pretreatment, bioabatement of fermentation inhibitors by a fungal strain, and simultaneous saccharication and fermentation (SSF) of the bioabated WS to ethanol using an ethanologenic recombinant bacterium was studied at a pilot scale without
sterilization. WS (124.2 g/L) was pretreated with dilute H2SO4 in two parallel tube reactors at 160 C. The
inhibitors were bio-abated by growing the fungus aerobically. The maximum ethanol produced by SSF of
the bio-abated WS by the recombinant Escherichia coli FBR5 at pH 6.0 and 35 C was 36.0 g/L in 83 h with
a productivity of 0.43 g L1 h1. This value corresponds to an ethanol yield of 0.29 g/g of WS which is 86%
of the theoretical ethanol yield from WS. This is the rst report on the production of ethanol by the
recombinant bacterium from a lignocellulosic biomass at a pilot scale.
Published by Elsevier Ltd.

1. Introduction
Ethanol is the most dominant biofuel. In the USA, nearly 200
operating plants churned out an estimated 13.3 billion gallons of
ethanol from corn starch in 2013 (2014 Ethanol Industry Outlook,
www.ethanolrfa.org). Various agricultural residues [corn stover,
wheat straw (WS), rice straw, barley straw, sugar cane bagasse],
processing byproducts (corn ber, rice hulls), and energy crops
(switchgrass, miscanthus) are available as low cost lignocellulosic
feedstocks for conversion to fuel ethanol (second generation biofuel). WS is one of the most abundant agricultural residues in the
world. The average yield of WS is 1.31.4 kg/kg of wheat grain

q
Mention of trade names or commercial products in this article is solely for the
purpose of providing specic information and does not imply recommendation or
endorsement by the U.S. Department of Agriculture.
Corresponding author at: USDA-ARS-NCAUR, 1815 N. University St., Peoria, IL
61604, USA. Tel.: +1 309 681 6276; fax: +1 309 681 6427.
E-mail address: Badal.Saha@ars.usda.gov (B.C. Saha).

http://dx.doi.org/10.1016/j.biortech.2014.10.060
0960-8524/Published by Elsevier Ltd.

(Montane et al., 1998). The world production of wheat grain in


2013/14 is estimated to be 683 million metric tons (http://
www.igc.int/downloads/gmrsummary/gmrsumme.pdf). WS contains 3545% cellulose, 2030% hemicellulose, and 815% lignin.
This makes WS an attractive feedstock to be converted to ethanol
and other value-added products.
The production of ethanol from WS generally involves four
main steps feedstock pretreatment, enzymatic saccharication,
fermentation, and product recovery. Integration of two or more
process steps is important for simplication of the process and
reduction of production cost. To this effect, simultaneous saccharication and fermentation (SSF) of the pretreated lignocellulosic
feedstock is considered to be an ideal integrated process for ethanol production. It offers distinct advantages over separate hydrolysis and fermentation (SHF) in the production of ethanol from
lignocellulosic feedstock. It can improve the ethanol yield by
eliminating end-product inhibition of cellulose hydrolysis. The
microorganism can utilize the sugars for growth and ethanol

18

B.C. Saha et al. / Bioresource Technology 175 (2015) 1722

production as they are formed. Moreover, SSF does not require separate reactors for enzymatic saccharication and fermentation of
generated sugars to ethanol. There are a number of studies available related to SSF of pretreated lignocellulosic biomass using Saccharomyces cerevisiae at 3035 C (Alfani et al., 2000; Ohgren et al.,
2006, Olofsson et al., 2008; Tomas-Pejo et al., 2008; Saha et al.,
2013).
WS, upon pretreatment and enzymatic saccharication, produces a mixture of pentose (xylose and arabinose) and hexose sugars (glucose and galactose) (Saha, 2003). The utilization of all
sugars generated from WS is essential for economical production
of ethanol (Saha, 2004). The conventional ethanol fermenting yeast
(S. cerevisiae) or bacterium (Zymomonas mobilis) cannot ferment
xylose and arabinose to ethanol. A number of recombinant microorganisms such as Escherichia coli, Klebsiella oxytoca, Z. mobilis, and
S. cerevisiae have been developed over the last 25 years with a goal
of fermenting both hexose and pentose sugars to ethanol (Saha,
2003). Our research unit has developed a recombinant E. coli
(strain FBR5) that ferments mixed multiple sugars to ethanol
(Dien et al., 2000). The strain carries the plasmid pLOI297, which
contains the genes for pyruvate decarboxylase (pdc) and alcohol
dehydrogenase (adh) from Z. mobilis necessary for efciently converting pyruvate into ethanol (Alterthum and Ingram, 1989). The
plasmid also contains the genes for ampicillin and tetracycline
resistance. It selectively maintains the plasmid when grown anaerobically and is capable of fermenting both hexose and pentose sugars to ethanol. In our previous papers, we reported about the
production of ethanol from WS by dilute acid, lime, alkaline peroxide and microwave pretreatments, enzymatic saccharication, and
fermentations of the hydrolyzates by both SHF and SSF using this
recombinant E. coli strain FBR5 (Saha et al., 2005, 2008, 2011a,b,
2013; Saha and Cotta, 2006, 2007, 2011) at laboratory scale
(350 ml in a 500 ml eaker). The minimum and maximum ethanol
produced in these studies were 13.0 2.0 and 41.8 0.0 g/L from
pretreated WS (86150 g/L) which are equivalent to 0.17 and
0.28 g ethanol per g straw, respectively. The yields varied between
0.37 and 0.50 g per g of available sugars depending on the type of
pretreatment used. The fermentation time also varied greatly from
17 to 136 h which was also highly dependent on the type of pretreatment and the inhibitory compounds present in the pretreated
hydrolyzate. We also studied the long term performance of this
recombinant bacterium in a series of continuous culture runs
(16105 days) using alkaline peroxide pretreated and enzymatically saccharied wheat straw hydrolyzate (WSH) as feedstock
(Saha and Cotta, 2011). During these studies, no loss of ethanol
productivity was observed which indicates that the strain showed
stability and robustness in performance. We were thus interested
to study the ethanol production from WS at a pilot scale by SSF.
In this paper, we report the production of ethanol from WS by
the recombinant bacterium at pilot scale.

USA. Membrane Filter Unit (0.2 lm) was purchased from Nalge
Nunc Int., Rochester, NY, USA. Lactoside V (Virginiamycin) was
supplied by Lallemand Biofuels and Distilled Spirits, Milwaukee,
WI, USA. Yeast extract and casein peptone type M were obtained
from Marcor Development Corp., Carlstadt, NJ, USA. Biospumex
153K antifoam was from Cognis Corp., Tucson, AZ, USA. Hydrated
lime was obtained from Mississippi Lime Co., St. Louis, MO. All other
chemicals used were of standard analytical grades.
2.2. Enzyme assays
The cellulase activity in terms of lter paper activity was
assayed and expressed as lter paper unit (FPU) by the procedure
described by Ghose (1987). Carboxymethyl cellulase (CMCase),
b-glucosidase, xylanase, b-xylosidase, a-L-arabinofuranosidase,
and ferulic acid esterase activities were assayed by the procedures
described previously (Saha et al., 2005). All enzyme assays were
performed at pH 5.0 and 45 C and the activities were expressed
in terms of international units (IU, lmole product formed per min).
2.3. Dilute acid pretreatment of wheat straw
Two steam heated jacketed parallel tube reactors (each 10 L
working volume) were used. Milled WS (124.2 g/L, dry basis) was
slurried in 0.75% (v/v) H2SO4 and pretreated in the tube reactors
at 160 C for 20 min holding time. The heating and cooling times
of the reactors were around 15 min each. The reactors were cooled
using chilled tap water. One set of pretreatment generated 20 L of
pretreated material.
The severity factor (SF) for a gradually heating, holding and
gradually cooling process was determined by the following equation (Rubio et al., 1998):

SF LogR0  log10

Z
0

exp


Tt  100
dt
14:75

where t is the residence time in min and T is the temperature of pretreatment in C at one min residence intervals during heating, holding and cooling. This is necessary due to long heating and cooling
times. R0 originally designated by Overend and Chornet (1987) as
reaction ordinate or severity parameter is commonly used to represent SF.
The combined severity factor (CSF) for dilute acid pretreatment
takes into account of temperature, time and acid concentration
(pH) and is calculated using the following equation (Nguyen
et al., 2000):

CSF LogR0   pH
where pH of the reaction mixture for use in CSF was measured after
pretreatment.
The pH of the pretreated WS was adjusted to 6.5 using commercial grade hydrated lime.

2. Methods
2.4. Bioabatement of dilute acid pretreated wheat straw hydrolyzate
2.1. Materials
WS, supplied by Dr. Matthew Digman, U.S. Dairy Forage Research
Center, Madison, WI, was dried in a forced-air oven at 55 C for 24 h
and milled in a hammer mill to pass through a 1.27 mm screen. The
milled WS was stored at room temperature. Celluclast 1.5 L (cellulase) and Novozym 188 (b-glucosidase) were purchased from
Brenntag Great Lakes, Milwaukee, WI, USA. Aminex HPX 87P column (300  7.8 mm), Aminex HPX 87H column (300  7.8 mm),
De-ashing cartridge (30  4.6 mm), Carbo-P micro-guard cartridge
(30  4.6 mm), and Cation H micro-guard cartridge (30  4.6 mm)
were purchased from Bio-Rad Laboratories, Inc., Hercules, CA,

The fungus Coniochaeta ligniaria NRRL 30616 was used to bioabate the dilute acid pretreated WS (Nichols et al., 2005). The
detailed procedure for biobatement of pretreated WS by the fungal
strain was described previously (Saha et al., 2011a). For bioabatement at 2 L scale, the seed culture was grown aerobically in a
500 ml bafed ask containing 125 ml of the liquid portion of pretreated WS, 0.1% (NH4)2SO4 and 2 ppm Virginiamycin at pH 6.5,
30 C and 225 rpm. For bioabatement at pilot scale, the seed culture was grown in a 10 L fermentor (Biostat B, B. Braun Biotech.,
Inc., Sartorius Stedium North America, Inc., Behima, NY) with 6 L
of the liquid portion of pretreated WS, 0.1% (NH4)2SO4 and 2 ppm

19

B.C. Saha et al. / Bioresource Technology 175 (2015) 1722

Virginiamycin at pH 6.5, 30 C, 225 rpm and aeration at 0.5 vvm.


The time courses of abatements of furfural and HMF were monitored 34 times during growth using high pressure liquid chromatography (HPLC) to determine when to terminate the process. The
bioabatement of the pretreated WS was performed at pH 6.5, 30 C,
350 rpm and aeration at 0.5 vvm without removing the solid
residues.
2.5. Ethanologenic recombinant E. coli strain and preparation of
inoculums
Recombinant E. coli strain FBR5 (provided by Dr. Bruce S. Dien of
our Research Unit) was maintained in glycerol vials at 80 C for
use as a working stock. It was platted onto Luria broth (LB, 10 g
tryptone, 5 g yeast extract, and 5 g NaCl) containing 4 g xylose
and 20 mg tetracycline solidied with 15 g agar per L (pH 6.5).
Plates were incubated at 35 C. Cells from a single well-isolated
colony were inoculated into a 125 ml Erlenmeyer ask containing
100 ml of LB with 20 g xylose and 20 mg tetracycline per L. Cultures were incubated at 35 C and 100 rpm for 24 h. This grown
culture was used as seed culture for 2 L fermentation experiments.
The inoculum size was 5% (v/v). For pilot scale fermentor runs, the
inoculum (5 L) was prepared in 5 L fermentor with LB medium
without NaCl containing 20 g xylose per L at controlled pH 6.5
and 35 C for 24 h.

software based integration system (Chromquest 4.0, Spectra-Physics). Two ion moderated partition chromatography columns
(Aminex HPX-87P with De-ashing and Carbo-P micro-guard cartridges, Aminex HPX 87H with Cation H micro-guard cartridge)
were used. The Aminex HPX-87P column was maintained at
85 C, and the sugars were eluted with Milli-Q (Millipore Corp.,
Bedford, MA) ltered deionized water at a ow rate of 0.6 ml/
min. The Aminex HPX-87H column was maintained at 65 C, and
the sugars, organic acids, ethanol, furfural, and HMF were eluted
with 10 mM HNO3 prepared using Milli-Q ltered water at a ow
rate of 0.6 ml/min. Peaks were detected by refractive index or UV
absorption (277 and 215 nm) and were identied and quantied
by comparison to retention times of authentic standards (glucose,
xylose, arabinose, galactose, ethanol, succinic acid, acetic acid, furfural, furfuryl alcohol and HMF). The theoretical yield of each sugar
was calculated by the procedure provided by NREL (Sluiter et al.,
2008a). The equations for calculation of theoretical ethanol yield
from glucose, xylose and arabinose are as follows:

Glucose MW 180:16 ! 2 Ethanol MW 46:07


2CO2 MW 44:01
3 Xylose MW 150:13 or 3 Arabinose MW 150:13
! 5 Ethanol MW 46:07 5 CO2 MW 44:01

2.6. Simultaneous saccharication and fermentation (SSF)


Thus, the theoretical ethanol yield based on stoichiometry is
0.51 g ethanol per g glucose, xylose or arabinose.

The SSF experiments were carried out semi-anaerobically in 2 L


(Biostat B) and 100 L (Biostat D) fermentors (B. Braun) with pH and
temperature controls and working volumes of 1.5 L and 70 L,
respectively at pH 6.0 and 35 C. The medium contained 10 g
casein peptone and 5 g yeast extract per L in addition to bioabated
WS. It also contained Biospumex 153 K antifoam (0.25 ml/L). The
medium was then inoculated with E. coli FBR5 at 35 C, pH 6.5
and 250 rpm and the fermentation was continued for 6 h before
adding a lter sterilized enzyme cocktail of 100 lL cellulase and
10 lL b-glucosidase per g WS. The SSF experiment was continued
until completion at 180 rpm. The pH was controlled at 6.0 using
4 M NaOH. Samples were withdrawn periodically, centrifuged to
remove cells and residual solids, and kept at 20 C prior to HPLC
analysis. Reactor performance was monitored by quantifying
unutilized sugars (glucose, xylose, arabinose and galactose) and
fermentation products (ethanol, succinic acid). Base consumption
and pH were also recorded. For simplication purpose, the quantity of ethanol produced from the additional sugars (glucose, fructose) present in the enzyme cocktail was subtracted from the
measured ethanol yield in each case. A simple schematic process
diagram showing the process steps used for pilot scale run is
shown in Fig. 1.

The WS used in this study contained 34.4 1.4% cellulose,


24.7 1.4% hemicellulose (total carbohydrate content, 59.1
2.8%), 18.4 1.4% lignin, and 7.4 0.2% ash on dry basis (Table 1).
These values are, somewhat, different from the WS (obtained several times from a local farmer in greater Peoria area) used in our
previous research (Saha et al., 2005, 2008, 2011a). Our previous
research also showed that the composition of WS obtained from
the same farmer varied somewhat from season to season.
WS (124.2 g/L, dry basis) was pretreated with 0.75% (v/v) H2SO4
at 160 C for 20 min holding time. Two commercial enzyme preparations (cellulase and b-glucosidase) were used for SSF of pretreated WS. The activity levels of cellulase (lter paper), CMCase,
b-glucosidase, xylanase, b-xylosidase, a-L-arabinofuranosidase,
and ferulic acid esterase in these two enzyme preparations are
given in Table 2.

2.7. Analytical methods

3.2. SSF of pretreated wheat straw at 2 L scale

The composition of WS with respect to cellulose, hemicellulose,


lignin and ash contents was determined using the standard laboratory analytical procedures for biomass analysis reported by
National Renewable Energy Laboratory (NREL) (Sluiter et al.,
2008a,b). Moisture content was determined using a moisture analyzer (Mark 2, Sartorius Mechatronics Corp., Bohemia, NY, USA).
Sugars, ethanol, succinic acid, acetic acid, furfural, and HMF were
analyzed by HPLC (Saha and Bothast, 1999). The separation system
consisted of a solvent delivery system (P2000 Pump, Spectra-Physics, San Jose, CA, USA) equipped with an autosampler (717, Waters
Chromatography Division, Millipore Corp., Milford, MA, USA), a
refractive index detector (410 Differential Refractometer, Waters),
a dual k absorbance detector (2487, Waters), and a computer

The liquid portion of pretreated WS (124.2 g/L on dry basis)


contained 4.9 g glucose, 19.5 g xylose, 3.2 g arabinose and 1.3 g
galactose (total sugars, 28.9 g) per L. It also contained 1958 mg furfural, 214 mg HMF and 2.9 g acetic acid per L. The SF and CSF values for the pretreatment were calculated as 3.2 and 2.3,
respectively. Both bioabatement of fermentation inhibitors and
SSF of the bioabated WS to ethanol were performed in the same
fermentor. About 1.5 L of pretreated WS was used and 75 ml of
the seed culture (C. ligniaria) was inoculated into the fermentor.
The disappearance of furfural and HMF was monitored to determine when to terminate the bioabatement (data not shown).
The time courses of unutilized total sugars and ethanol production for two separate SSF runs are shown in Fig. 2. Both SSF runs

3. Results and discussion


3.1. Composition of wheat straw and commercial enzyme preparations

20

B.C. Saha et al. / Bioresource Technology 175 (2015) 1722

Wheat Straw

Milling

+ 0.75% (w/v)
H2SO4

Bioabatement seed
culture,
10 L fermentor

2. Bioabatement
3. SSF with E. coli
1. Dilute acid
pretreatment
160oC, 20min

E. coli FBR5 seed


culture, 5 L
fermentor

Steam
Heated
Jacketed
Tube
Reactors

+ Enzymes
2, 3
100 L
Fermentor

+ Ca(OH)2

Residual Solids

Distillation

Fig. 1. A Schematic diagram showing the process steps used for conversion of wheat straw to ethanol at pilot scale.

Table 1
Composition of wheat straw on dry basis before pretreatment and after simultaneous
saccharication and fermentation.
Component

Glucan
Xylan
Arabinan
Galactan
Total carbohydrates
Lignin
Ash

3.3. SSF of pretreated wheat straw at pilot scale

g/100 g of original material on dry basis


a

Original material

Solid residues after SSF

34.4 1.4
19.3 0.9
3.8 0.2
1.6 0.3
59.1 2.8
18.4 1.4
7.4 0.2

6.2 0.1
1.2 0.1
00
00
7.4 0.2
16.3 0.3
8.6 0.2

Data presented are averages standard deviations of triplicate estimations.


a
61% of the original material was solubilized.

Table 2
Activity levels of two commercial enzyme preparations used in simultaneous
saccharication and fermentation of dilute acid pretreated and bioabated wheat
straw.
Enzyme

Filter paper activity (FPU)


Carboxymethyl cellulase
b-Glucosidase
Xylanase
a-L-Arabinofuranosidase
b-Xylosidase
Ferulic acid esterase

anol per L. In 96 h, the ethanol concentration was only increased by


0.1 g/L to 33.2 0.1 g/L.

Activity (U/ml)a
Celluclast 1.5 L
(cellulase)

Novozym 188
(b-glucosidase)

52 4
833 42
21 3
1006 21
32 2
28 2
00

00
51 2
785 53
85 15
20
12 1
00

The data presented are means standard deviation for triplicate assays.
a
At pH 5.0 and 45 C.

showed similar pattern of sugar utilization and ethanol production


and were essentially complete in 76 h, producing 33.1 0.1 g eth-

Similar to the 2 L SSF runs, both bioabatement of fermentation


inhibitors and SSF of the bioabated WS to ethanol were performed
in the same fermentor. Seventy L of pretreated WS was used. The
seed culture of C. ligniaria NRRL 30616 was prepared using 6 L of
the liquid portion of the pretreated WS in a 10 L fermentor for
20 h. This seed culture was then inoculated in the 100 L fermentor.
The bioabatement was carried out with aeration rate at 0.5 vvm.
The disappearance of furfural, HMF and total sugars during bioabatement for both fermentor runs is shown separately in Fig. 3.
For run 1, the furfural (1.56 g/L) and HMF (0.18 g/L) levels
decreased to 0.31 and 0.11 g/L, respectively, in 22 h and there
was no sugar loss during this period. For run 2, the furfural
(1.92 g/L) and HMF (0.22 g/L) levels decreased to 0.39 and 0.15 g/
L, respectively, in 41 h and there was no sugar loss during this period also. Thus, the quantity of furfural and HMF produced varied
from batch to batch pretreatment. This variation may be due to
unequal heat transfer during pretreatment as the tube reactors
did not have a mixing option. The recombinant E. coli strain FBR5
cannot grow at all at these levels of furfural and HMF concentrations (Saha and Cotta, 2012). Treatment with C. ligniaria for 22 h
reduced the furfural level by 79.9% to 0.31 g and HMF level by
40.2% to 0.11 g per L for run 1. On the other hand, treatment with
the fungal strain under the same conditions reduced the furfural
level by 79.7% to 0.39 g and HMF level by only 27.9% to 0.15 g/L
in 41 h. These data indicate that utilization of furfural is much
more rapid than that of HMF by the fungal strain under the conditions used. Similar ndings were also observed in our previous
studies on bioabatement of dilute acid pretreated WS with this
fungal strain (Saha et al., 2011a,b). Our previous research shows
that the recombinant E. coli FBR5 can tolerate and grow at these

21

2.1

30

1.8

20

Total sugars (Run 1)


Ethanol (Run 1)
Total sugars (Run 2)
Ethanol (Run 2)

15
10
5
0

1.5
1.2

12

24

36

48

60

72

84

12
8

0.3

4
0

12

18

24

Time (h)

Time (h)

32

2.1

28

Furfural or HMF (g/L)

1.8

Furfural
HMF
Total sugars

1.5
1.2

20
12

0.6

0.3
0.0

24
16

0.9

4
0

12

18

24

30

36

0
42

Time (h)
Fig. 3. Patterns of utilization of furfural, HMF and total sugars from the pretreated
wheat straw hydrolyzate by Coniochaeta ligniaria NRRL 30616 at pH 6.5 and 30 C.
Virginiamycin (2 ppm) was used to suppress the contamination without sterilizing
the fermentation medium. The data for both pilot scale runs are shown separately.
Above, for Run 1; below, for Run 2.

35
Total sugars or Ethanol (g/L)

levels of inhibitors (Avci et al., 2013). The strain was able to convert furfural to furfuryl alcohol and HMF to hydroxymethyl furfuryl
alcohol which are considered less toxic (Almeida et al., 2007).
Unlike our previous studies mentioned above where there was
about 57% sugar loss during bioabatement, the fungal strain did
not utilize any sugar at all in both cases of run 1 and run 2.
The data on sugar utilization and ethanol productions for both
fermentor runs are presented separately in Fig. 4. The two fermentation runs showed very similar patterns of ethanol production for
up to 48 h. The run 1 was complete in 83 h producing 36.0 g ethanol per L while in run 2 the ethanol concentration was 32.8 g per L
in 115 h and at 144 h, the ethanol concentration was 34.3 g per L.
The residue left (39% of original material) after SSF of the bioabated
WS contained 6.2 0.1 g glucan, 1.2 0.1 g xylan, 16.3 0.3 g
lignin per 100 g original WS (Table 1). The summary of each SSF
experiment including ethanol yield per g WS is given in Table 3.
Both 2 L and pilot scale fermentations were run without sterilizing the fermentation medium. Instead we used Virginiamycin, a
commonly used antibiotic used in the fuel ethanol industry, in
low dose (2 ppm) to suppress bacterial contamination. This helped
the fermentation runs not only avoid sugar loss due to sterilization,
but also prevent contamination by lactic acid bacteria. Without the
addition of Virginiamycin, lactic acid bacteria prevailed over the
recombinant E. coli and produced signicant quantities of lactic
acid in addition to ethanol during SSF (data not shown). No lactic
acid was detected in the fermentation broth after bioabatement
with the fungal strain and also SSF of the bioabated WS with
2 ppm Virginiamycin. The source of lactic acid bacterial contamination was not investigated further. In pilot scale runs, the recombinant E. coli FBR5 utilized all sugars and produced ethanol even
though run 2 took much longer time (144 h) to complete in comparison to run 1 (83 h). In pilot scale run 1, the ethanol yield was
0.44 g/g of theoretically available sugars from WS and 0.50 g/g of
available sugars (Table 3) based on solid residue composition after
SSF (Table 1). This corresponds to an ethanol yield of 86% of the
theoretical ethanol yield from WS. The ethanol yield was 0.29 g/g
of WS on dry basis. This means that 8.69 kg of WS (used to prepare
70 L of pretreated WS) was converted to 2.52 kg of ethanol. Maas
et al. (2008) studied pilot scale (100 L) conversion of lime pretreated WS with high solid content (35%, w/w) to ethanol via
SSF using commercial hydrolytic enzymes and bakers yeast. They
obtained an ethanol concentration of 21.4 g/L corresponding to a

20

0.6

96

Fig. 2. Time courses of utilization of total available sugars and ethanol production
by mixed sugar utilizing ethanologenic recombinant Escherichia coli FBR5 at 2 L
scale by simultaneous saccharication and fermentation of dilute acid pretreated
(160 C, 20 min holding time) and bioabated (pH 6.5, 30 C, 20 h) wheat straw
(124.2 g/L, dry basis) hydrolyzate at pH 6.0 and 35 C. The enzyme cocktail
contained 100 lL of cellulase and 10 lL of b-glucosidase per g straw. Two separate
experiments were performed. The data are presented separately for each run.

24
16

0.9

0.0

28
Furfural
HMF
Total sugars

Total sugars (g/L)

25

32

Total sugars (g/L)

35

Furfural or HMF (g/L)

Total sugars or Ethanol (g/L)

B.C. Saha et al. / Bioresource Technology 175 (2015) 1722

30
25
20
Total sugars (Run 1)
Ethanol (Run 1)
Total sugars (Run 2)
Ethanol (Run 2)

15
10
5
0

24

48

72

96

120

144

Time (h)
Fig. 4. Time courses of utilization of total available sugars and ethanol production
by mixed sugar utilizing ethanologenic recombinant Escherichia coli FBR5 at pilot
scale by simultaneous saccharication and fermentation of dilute acid pretreated
(160 C, 20 min holding time) and bioabated (pH 6.5, 30 C) wheat straw (124.2 g/L,
dry basis) hydrolyzate at pH 6.0 and 30 C. The enzyme cocktail contained 100 lL of
cellulase and 10 lL of b-glucosidase per g wheat straw. Two separate experiments
were performed. The data are presented separately for each run.

22

B.C. Saha et al. / Bioresource Technology 175 (2015) 1722

Table 3
Summary of simultaneous saccharication and fermentation (SSF) of dilute acid pretreated (0.75%, w/v H2SO4; 160 C, 20 min) and bioabated (30 C, pH 6.5) wheat straw
(124.2 g/L, dry basis).

a
b

Fermentor

Time (h)

Ethanol (g/L)

Ethanol productivity (g L1 h1)

Ethanol yielda (g/g sugar)

Ethanol yieldb (g/g sugar)

Ethanol yield (g/g straw)

2 L run 1
2 L run 2
Pilot run 1
Pilot run 2

76
76
83
144

33.0
33.2
36.0
34.3

0.43
0.44
0.43
0.24

0.46
0.46
0.50
0.48

0.40
0.40
0.44
0.42

0.27
0.27
0.29
0.28

Based on theoretical total sugar yield from original wheat straw minus theoretical total sugar yield from residual wheat straw after SSF.
Based on theoretical total sugar yield from original wheat straw.

48% glucan to ethanol conversion of the theoretical maximum.


Thus 16.7 kg of pretreated WS could be converted to 1.7 kg of
ethanol.
4. Conclusions
Scale up of the conversion of wheat straw to ethanol from bench
to a pilot scale is a critical step leading to commercialization. In
this paper, we have successfully demonstrated the conversion of
wheat straw to ethanol by dilute acid pretreatment, detoxication
of fermentation inhibitors using a novel fungal strain and SSF of
detoxied WS from 2 L to pilot scale without sterilization. To our
knowledge, this is the rst report on the conversion of WS to ethanol at pilot scale using the mixed sugar utilizing recombinant ethanologenic E. coli FBR5.
References
Alfani, A., Gallifuoco, A., Saporosi, A., Spera, A., Cantarella, M., 2000. Comparison of
SHF and SSF processes for the bioconversion of steam-exploded wheat straw. J.
Ind. Microbiol. Biotechnol. 25, 184192.
Almeida, J.R., Modig, T., Petersson, A., Hahn-Hagerdal, B., Linden, G., GorwaGrauslund, M.F., 2007. Increased tolerance and conversion of inhibitors in
lignocellulosic hydrolyzates by Saccharomyces cerevisiae. J. Chem. Technol.
Biotechnol. 83, 340349.
Alterthum, F., Ingram, L.O., 1989. Efcient ethanol production from glucose, lactose,
and xylose by recombinant Escherichia coli. Appl. Environ. Microbiol. 55, 1943
1948.
Avci, A., Saha, B.C., Kennedy, G.J., Cotta, M.A., 2013. Dilute acid pretreatment of corn
stover for enzymatic hydrolysis and efcient ethanol production by
recombinant Escherichia coli FBR5 without detoxication. Bioresour. Technol.
142, 312319.
Dien, B.S., Nichols, N.N., OBryan, P.J., Bothast, R.J., 2000. Development of new
ethanologenic Escherichia coli strains for fermentation of lignocellulosic
biomass. Appl. Biochem. Biotechnol. 8486, 181186.
Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257
268.
Maas, R.H.W., Bakker, R.R., Boersma, A.R., Bisschop, I., Pels, J.R., de Jong, E.,
Weusthuis, R., Reith, H., 2008. Pilot-scale conversion of lime-treated wheat
straw into bioethanol: quality assessment of bioethanol and valorization of side
streams by anaerobic digestion and combustion. Biotechnol. Biofuels 1, 14.
Montane, D., Farriol, X., Salvado, J., Jollez, P., Chernet, E., 1998. Application of steam
explosion to the fractionation and rapid vapour-phase alkaline pulping of wheat
straw. Biomass Bioenergy 14, 261276.
Nguyen, Q., Tucker, M.P., Keller, F.A., Eddy, F.P., 2000. Two-stage dilute acid
pretreatment of softwoods. Appl. Biochem. Biotechnol. 8486, 561576.
Nichols, N.N., Dien, B.S., Guisado, G.M., Lpez, M.J., 2005. Bioabatement to remove
inhibitors from biomass-derived sugar hydrolysates. Appl. Biochem. Biotechnol.
121124, 379390.
Ohgren, K., Bengtsson, O., Gorwa-Grauslund, M.F., Galbe, M., Hahn-Hagerdal, B.,
Zacchi, G., 2006. Simultaneous saccharication and co-fermentation of glucose

and xylose in steam-pretreated corn stover at high ber content with


Saccharomyces cerevisiae TMB3400. J. Biotechnol. 126, 488498.
Olofsson, K., Rudolf, A., Linden, G., 2008. Designing simultaneous saccharication
and fermentation for improved xylose conversion by a recombinant strain of
Saccharomyces cerevisiae. J. Biotechnol. 134, 112120.
Overend, R.P., Chornet, E., 1987. Fractionation of lignocellulosics by steam-aqueous
pretreatments. Philos. Trans. R. Soc. Lond. A321, 523536.
Rubio, M., Tortosa, J.P., Quesada, J., Gomez, D., 1998. Fractionation of
lignocellulosics. Solubilization of corn stock hemicelluloses by autohydrolysis
in aqueous medium. Biomass Bioenergy 15, 483491.
Saha, B.C., Bothast, R.J., 1999. Pretreatment and enzymatic saccharication of corn
ber. Appl. Biochem. Biotechnol. 76, 6577.
Saha, B.C., 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30, 279
291.
Saha, B.C., 2004. Lignocellulose biodegradation and applications in biotechnology.
In: Saha, B.C., Hayashi, K. (Eds.), Lignocellulose Biodegradation. American
Chemical Society, Washington, DC, pp. 234.
Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y.V., 2005. Dilute acid pretreatment, enzymatic
saccharication, and fermentation of wheat straw to ethanol. Process Biochem.
40, 36933700.
Saha, B.C., Cotta, M.A., 2006. Ethanol production from alkaline peroxide pretreated
enzymatically saccharied wheat straw. Biotechnol. Prog. 22, 449453.
Saha, B.C., Cotta, M.A., 2007. Enzymatic hydrolysis and fermentation of lime
pretreated wheat straw to ethanol. J. Chem. Technol. Biotechnol. 82, 913919.
Saha, B.C., Cotta, M.A., 2012. Ethanol production from lignocellulosic biomass by
recombinant Escherichia coli FBR5. Bioengineered 3, 197202.
Saha, B.C., Biswas, A., Cotta, M.A., 2008. Microwave pretreatment, enzymatic
saccharication and fermentation of wheat straw to ethanol. J. Biobased Mater.
Bioenergy 2, 210217.
Saha, B.C., Cotta, M.A., 2011. Continuous ethanol production from wheat straw
hydrolysate by recombinant ethanologenic Escherichia coli strain FBR5. Appl.
Microbiol. Biotechnol. 90, 477487.
Saha, B.C., Nichols, N.N., Qureshi, N., Cotta, M.A., 2011a. Comparison of separate
hydrolysis and fermentation and simultaneous saccharication and
fermentation processes for ethanol production from wheat straw by
recombinant Escherichia coli strain FBR5. Appl. Microbiol. Biotechnol. 92, 856
874.
Saha, B.C., Nichols, N.N., Cotta, M.A., 2011b. Ethanol production from wheat straw
by recombinant Escherichia coli strain FBR5 at high solid loading. Bioresour.
Technol. 102, 1089210897.
Saha, B.C., Nichols, N.N., Cotta, M.A., 2013. Comparison of separate hydrolysis and
fermentation versus simultaneous saccharication and fermentation of
pretreated wheat straw to ethanol by Saccharomyces cerevisiae. J. Biobased
Mater. Bioenergy 7, 409414.
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D.,
2008a. Determination of structural carbohydrates and lignin in biomass.
Technical
Report
NREL/TP-510-42618.
http://www.nrel.gov/biomass/
analytical_procedures.html.
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008b.
Determination of ash in biomass. Technical Report NREL/TP-510-42622. http://
www.nrel.gov/biomass/analytical_procedures.html.
Tomas-Pejo, E., Oliva, J.M., Ballesteros, M., Olsson, L., 2008. Comparison of SHF and
SSF processes from steam-exploded wheat straw for ethanol production by
xylose-fermenting and robust glucose-fermenting Saccharomyces cerevisiae
strains. Biotechnol. Bioeng. 100, 11221131.