An ASABE Meeting Presentation

Paper Number: 12-1338125

Enzymatic Hydrolysis and Fermentation of Whole Sugar Beets

for Ethanol Production
Nurun Nahar, Research Specialist
Agricultural and Biosystems Engineering Dept., North Dakota State University, Fargo, ND
58102, nurun.nahar@ndsu.edu
Scott W. Pryor, Associate Professor
Agricultural and Biosystems Engineering Dept., North Dakota State University, Fargo, ND
58102, scott.pryor@ndsu.edu

Written for presentation at the

2012 ASABE Annual International Meeting
Sponsored by ASABE
Hilton Anatole
Dallas, Texas
July 29 August 1, 2012
Abstract. Sucrose from sugar beets is used for commercial ethanol production in Europe and is
being considered in the United States. Sugar beet biomass also contains cellulose, hemicellulose
and pectin which can be hydrolyzed into monosaccharides and fermented to produce ethanol. Three
different enzymes (pectinase, cellulase and cellobiase) were used for hydrolysis and hydrolyzates
were fermented with Saccharomyces cerevisiae or Escherichia coli KO11 in a simultaneous
saccharification and fermentations (SSF) for ethanol production. This study compared the
effectiveness of Saccharomyces cerevisiae and Escherichia coli KO11 using whole sugar beet as a
substrate. Variations in buffer concentration, solid loading and inoculum loading were also tested to
determine their effects on ethanol yield from whole sugar beets. Maximum ethanol concentrations
obtained were 45 g/L and 41 g/L at 12% solids and 5% inoculum loading for E. coli KO11 and S.
cerevisiae, respectively. However, the volumetric productivity of fermentation yield was higher when
sugar beet was fermented with S. cerevisiae (0.86 g/L/h) than the E. coli (0.21 g/L/h). While solids
loading were increased from 12 to 18% and inoculum loading was adjusted to 3%, ethanol
concentrations of 74 g/L were achieved by S. cerevisiae which corresponds to 92% of theoretical
ethanol yield. S. cerevisiae had higher fermentation yields and rates than E. coli thus making S.
cerevisiae the preferred strain in fermentation of whole sugar beet hydrolyzates.
Keywords. Sugar beet, ethanol, E. coli KO11, Saccharomyces cerevisiae

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Introduction
Biofuels derived from plant biomass can decrease US dependency on petroleum and contribute
to a cleaner environment. The Energy Independence and Security Act (EISA) of 2007 defines
three classes of biofuels (conventional, advanced, and cellulosic) based on potential reduction
of greenhouse gas (GHG) emissions (20, 50, and 60%, respectively). Sugar beets and sugar
cane have been identified as substrates for use in advanced biofuels with expected production
of 4 billion gallons per year by 2022. Commercial biofuel production from sugar beets in Europe,
especially in France and Germany, is twice that from other feedstocks. However, little attention
has been focused on commercialization of biofuel production from sugar beets in the US
(USDA, 2006).
Beets may generally produce twice as much ethanol per acre compared to corn and also
require about 40 percent less water per gallon of ethanol production. Using beets as a feedstock
would also avoid the controversy of using food for fuel as food grade sugar production is limited
by government regulation. Studies also suggested that production costs of biofuel from sugar
beets are lower than the sugar cane (USDA, 2006). Sucrose is the most abundant component
of total dry solids of sugar beet. Traditionally, raw beets are sliced and juice is extracted with hot
water; the juice is then pH-adjusted with lime; sucrose is purified via filtration, drying and
crystallization. After sucrose extraction, beet pulp is dried, pelleted and sold as a relatively lowvalue animal feed. Using whole sugar beets for biofuel production will remove pre-processing,
drying, pelleting, and crystallization steps and will leave a very small amount of waste product
thus reducing both input energy and operational cost.
Most cellulosic feedstocks have high cellulose contents with smaller amounts of hemicellulose
and lignin. Sugar beet is a unique biomass feedstock because it has a high amount of
fermentable sucrose and the remaining pulp has relatively high amounts of hemicellulose and
pectin, moderate cellulose content, and low lignin content. The low lignin and high pectin
content eliminates the need for expensive pretreatment which can make up about 30% of total
industrial costs (Hendriks and Zeeman, 2009). Hemicellulose is primarily a xylose polymer in
most biomass whereas sugar beet hemicellulose is composed of arabinose with lower
concentrations of xylose and galactose. Most other feedstock contain negligible pectin contents,
but sugar beet contains approximately 15% pectin, which can be hydrolyzed to galacturonic acid
(Micard et al., 1996; Spagnuolo et al., 1997). Pectinase and cellulase enzymes are used to
allow release of monomeric sugars by enzymatic hydrolysis. Pectin hydrolysis also improves
cellulose hydrolysis. Therefore, pectinase may be used in addition to cellulase and glucosidase to hydrolyze sugar beet into monosaccharides and galacturonic acid for
fermentation.
Limited research concerning the fermentation of arabinose and galacturonic acid has been
conducted because most biomass feedstock have limited concentrations of these sugars
(Ingram et al., 1987; Sedlak and Ho, 2001). Conventional ethanol-fermenting yeasts such as
Saccharomyces cerevisiae can metabolize glucose, fructose and sucrose (Amutha and
Gunasekaran, 2001) but are typically unable to metabolize either arabinose or galacturonic acid
to produce ethanol. The genetically engineered bacterium Escherichia coli (E. coli KO11) can
ferment arabinose and galacturonic acid with relatively high yields and is tolerant to by-products
(Bothast et al., 1999). However, E. coli KO11 has lower ethanol tolerance (40-60 g/L) compared
to yeasts, which can withstand ethanol concentrations greater than 130 g/L (Doran and Foster,
2000).
Media pH is an important parameter for optimal microbial growth and ethanol production. The
optimal pH range for E. coli KO11 is 6.5 to 7.5. During the fermentation of whole sugar beets or

beet pulp, E. coli KO11 produces acetate as a galacturonic acid fermentation byproduct; this
decreases the pH and impairs the metabolic activity of the microorganism. In laboratory-scale
fermentations, shake flasks may be used without active pH control. Therefore, high buffer
concentrations are necessary to prevent very fast pH drift. Conversely, high buffer
concentration in the fermentation process might inhibit microbes due to low osmotolerance.
However, no study has been conducted to examine the effect of buffer in fermenting the whole
sugar beet hydrolyzate using E. coli KO11 to determine conversion efficiency and final ethanol
concentrations.
The primary objective of this study was to improve bioconversion of pentose and hexose sugars
in sugar beet hydrolyzates for high ethanol yields and production rates. This study tested
multiple enzymes (pectinase, cellulase and cellobiase) and two microorganisms (S. cerevisiae
and E. coli KO11) to achieve high ethanol concentration and yield from crushed whole sugar
beets. The effect of buffer concentration, solid loading and inoculum loading were also tested to
determine their effects on ethanol yield from sugar beet hydrolyzates.

Materials and Methods

Substrate
Sugar beets were provided by Larry Campbell, USDA-ARS, Fargo, ND. Beets were stored at
-20 C until used. Before using, they were thawed and ground using a food processor and
moisture content was about 77% (w.b.).
Enzymes
The enzymes, NS50013 (cellulase, operating pH: 4.5 - 6.5, operating temperature: 45-50 C),
Novozyme 188 (-glucosidase, operating pH: 2.5 - 6.5, operating temperature: 45-70 C) were
provided by Novozymes North America, Inc. (Franklinton, NC, USA). Pectinex Ultra SPL
(pectinase, operating pH: 4.0 - 6.5, operating temperature 35-65 C) was purchased from
Sigma-Aldrich (St. Louis, MO). The cellulase activity of NS50013 and -glucosidase activity of
Novozyme 188, as determined by Ghose (1987), were 77 filter paper units (FPU)/mL and 500
cellobiase units (CBU)/mL, respectively. Pectinase activity of Pectinex Ultra was 4600 Unit /mL
as determined by Kertesz (1955).

Buffer
Three different phosphate buffer concentrations (100, 300 and 500 mM) were selected to
conduct the fermentation of whole sugar beet hydrolyzates using E. coli KO11. Citrate buffer
(300 mM) was also used with E. coli KO11 for ethanol yield comparison. Citrate buffer (300 mM)
was used for all Saccharomyces cerevisiae fermentations.

Microorganisms
E. coli KO11 (ATCC 55124) was provided by American Crystal Sugar Company (Moorhead,
MN). The inoculation seed was prepared in a solution of 50 g/L glucose, 10 g/L tryptone, 5 g/L
yeast extract, 5/L g NaCl, and 40 mg/L chloramphenicol at 37oC and 100 rpm for 24 h.
Chloramphenicol was added after autoclaving. The resulting cell culture was mixed with sterile
80% glycerol to produce a 40% glycerol solution. Aliquots (1 mL) were dispensed into sterile

cryovials and stored until use at -20oC. For each experiment, one cryovial was added to 100 mL
of inoculum medium containing 50 g/L glucose, 10 g/L tryptone, and 5 g/L yeast extract. The
inoculum was incubated at 37C and 100 rpm for 18 h.
Saccharomyces cerevisiae (industrial strain obtained from POET, LLC; Sioux Falls, SD) was
prepared by inoculating 0.15 g of yeast granules in sterile 50 mM citrate buffer media containing
2 g/L yeast extract and 10 g/L glucose. The pH was adjusted to 4.0 with 0.1 N HCl. After
inoculation, the media was incubated in a water bath rotary shaker (MaxQ 7000, Thermo
Scientific; Dubuque, IA) at 37 C and 100 rpm for 24 h.
Simultaneous saccharification and fermentation (SSF)
Simultaneous saccharification and fermentation (SSF) was carried out with E. coli KO11 and
Saccharomyces cerevisiae separately along with Pectinex, NS50013, and Novozyme 188 to
convert all sugar components into ethanol. Ground sugar beet was added into 500-mL
Erlenmeyer flasks and autoclaved for 20 min at 121C. Different solid loadings were used for
SSF as described in Table 1, with 100 mL of working volume. Cellulase and -glucosidase were
added at 45 FPU/g cellulose and 30 CBU/g cellulose, respectively. Pectinase was loaded at
2760 Units/dry g. Airlocks were used to maintain anaerobic conditions and release carbon
dioxide. The biomass samples were mixed with different buffer concentration (Table 1) and
agitated in a water bath shaker (MaxQ 7000, Thermo Scientific; Dubuque, IA, USA) at 37 C
and 100 rpm. Samples (1 mL) were taken every 24 h and centrifuged at 13,000 rpm for 5 min
(Galaxy 16 micro-centrifuge, VWR International; Bristol, CT, USA). After centrifugation, the
supernatant was filtered through a 0.2 m nylon filter (Pall Corporation; West Chester, PA) and
stored at -20C until analysis via HPLC.
Chloramphenicol (40 mg/L) was added to prevent contamination for SSF with E. coli KO11.
Fermentation was carried out for 192 h and pH was adjusted as needed throughout the
experiment. The pH of the E. coli KO11 and S. cerevisiae fermentations were adjusted to 6.5
and 4.8, respectively, with 6N NaOH or 0.1N HCL.
High performance liquid chromatography (HPLC) analysis
SSF samples were analyzed for individual sugars, ethanol and organic acids by two separate
HPLC systems (Waters Corporation; Milford, MA). Samples were analyzed for neutral sugars
(sucrose, glucose, arabinose, and galactose) using a Bio-Rad Aminex HPX-87P column (BioRad Laboratories; Hercules, CA) with a mobile phase of 18 m water at a flow rate of 0.6
mL/min and quantified using a refractive index (RI) detector (model 2414, Waters Corporation)
with column and detector temperatures of 50C and 85C, respectively. Ethanol was separated
using a Bio-Rad Aminex HPX-87H column (Bio-Rad Laboratories; Hercules, CA) with a mobile
phase of 5 mM sulfuric acid at a constant flow of 0.6 mL/min at 60C. Galacturonic acid was
also separated with a Bio-Rad Aminex 87H column with a mobile phase of 5 mM sulfuric acid at
a constant flow of 0.6 mL/min at 60C but detection was carried out using a photodiode array
detector (model 2996, Waters Corporation) at 210 nm wavelength. All components were
quantified using 4-point external standard curves.

Table 1. Simultaneous saccharification and fermentation set-up for different buffer

concentration, solid loading, and inoculum loading.

Microorganism

E. coli KO11

S. cerevisiae

Solid Loading
% (w/v)

Inoculum Loading
% (v/v)

100 mM Phosphate

12

300 mM Phosphate

12

500 mM Phosphate

12

300 mM Phosphate

12

300 mM Citrate

12

300 mM Citrate

12

300 mM Citrate

12

300 mM Citrate

12

300 mM Citrate

15

300 mM Citrate

18

Buffer

Results and Discussion

SSF with E. coli KO11
Results of SSF with E. coli KO11 are shown in Figure 1. Phosphate buffer concentrations of
100, 300 and 500 mM at 12% solid loading with 1% inoculum loading produced 30.6, 30.5 and
29.4 g/L ethanol, respectively, and their differences were not statistically significant (Fig. 1). At
the end of SSF, glucose concentrations were around 1 g/L for all treatments; E. coli KO11 can
ferment glucose, arabinose and galacturonic acid to ethanol (Grohmann et al., 1994; Rorick et
al., 2011). Although variations in ethanol concentration using three different concentrations were
not significant, higher buffer concentration reduced ethanol yields because of high osmolarity in
500 mM buffer. Volumetric productivity (1.2 g/L/h) and ethanol yield (54.6%) was also lower in
500 mM phosphate buffer compared to 100 and 300 mM buffers. Theoretical ethanol yields
(56.7%) and volumetric productivity (1.3 g/L/h) for 100 and 300 mM phosphate buffer were not
statistically different.
Although ethanol concentrations were very similar in the 100 and 300 mM treatments, pH drift
was greater with the 100 mM buffer and it required more frequent pH adjustment than did the
300 mM phosphate buffer (Fig. 2). The pH of the 100 mM treatment dropped from 6.5 to 5.8 in 4
h and decreased again to 5.6 by 24 h while the pH of 300 and 500 mM treatments were 6.0 to
6.3, respectively, at 24 h. Within 72 h there was no need of pH adjustment for 300 and 500 mM
phosphate buffer, whereas pH adjustment was needed for 100 mM phosphate buffer until 192 h.
Therefore, 300 mM buffer was selected for further SSF studies.

Figure 1. Effect of buffer concentration on ethanol yield from E. coli KO11

Figure 2. Impact of buffer concentration on pH of fermentation with E. coli KO1. The pH was
adjusted to 6.5 at each time point.
Relatively low ethanol yields for E. coli KO11 could have been due to low pH during the
fermentation. pH 6 has been considered an optimum pH for an effective E. coli fermentation
which minimizes CO2 solubilization (Moniruzzaman et al., 1998). The exposure of E. coli to low
pH (<6.0) reduces the ethanol yield (Takahashi et al., 1999; Moniruzzaman et al., 1998). Active
control of pH during the fermentation should allow more complete and rapid sugar utilization for
ethanol production.
Arabinose concentration was high (11 g/L at 192 h) at the end of SSF with E. coli (data not
shown). It was not clear why the residual arabinose generated by hydrolysis was not utilized by

E. coli KO11 for ethanol production. Rorick et al. (2011) showed concurrent utilization of
glucose, arabinose and galacturonic acid when using sugar beet pulp in SSF with E. coli KO11.
Others have also reported that arabinose utilization is significantly higher in the presence of
other sugars (Dien et al., 2003). One possible explanation could be preferential consumption of
the readily available sucrose and glucose at the beginning of the whole sugar beet fermentation
producing 30 g/L of ethanol. Arabinose utilization in Z. mobilis which is an E. coli KO11 gene
donor strain, has also been shown to decline at ethanol concentrations higher than 30 g/L
(Lawford and Rousseau, 2002; Mohagheghi et al., 2002).
Inoculum volume also had impact on ethanol yield obtained in E. coli fermentation. Increasing
inoculum volume from 1 to 5% increased ethanol yield from 56.6 % to 73%, respectively. Higher
inoculum levels (5%) produced 9 g/L more ethanol than the 1% inoculum loading (Fig. 3). The
impact of E. coli inoculum level was also reported by Okuda et al. (2008). Increasing E. coli
loading from 0.2 to 0.8 g(DCW)/L when fermenting wood hydrolyzates showed higher ethanol
yield in shorter time.

Figure 3. Effect of E. coli KO11 inoculum volume on ethanol concentration

In this study, we also tested two different buffers with similar concentration (300 mM citrate and
phosphate) to see which buffer had better pH stabilization capacity throughout fermentation
process. Citrate buffer showed better result than phosphate buffer. SSFs in citrate buffer (300
mM) produced 6 g/L more ethanol than the same concentration of phosphate buffer (Fig. 4).
Overall, citrate buffer increased ethanol yield by 73 to 83.5% at 12% solid loadings and 5%
inoculums loading, respectively.
SSF with Saccharomyces cerevisiae
The fermentations with S. cerevisiae were completed within 48 h. Glucose was consumed within
24 h and concentrations stabilized at less than 1 g/L. Arabinose was present but was not utilized
during the SSF with yeast as S. cerevisiae does not have the ability to utilize arabinose (Barnett

et al., 1990; Grohmann et al., 1994). Three different inoculum loadings (1, 3 and 5%) were
tested using 12% solids for ethanol conversion efficiency. The effect of inoculum on SSF with
yeast at 12% solid loading is presented in Figure 5. Increasing the inoculum loading from 1 to
3% resulted in increasing ethanol production (at 48 h) from 37 to 48 g/L. Increasing inoculum
loading from 3 to 5% decreased ethanol production by 8 g/L. Therefore, 3% inoculum was used
for further studies. Other studies also found that higher yeast inoculums (10 to 20%) did not
provide any benefit of ethanol yield (Gibbons, 1996).

Figure 4. Effect of buffer on ethanol concentration from fermentation with E. coli KO11

Figure 5. Effect of yeast inoculum volume on ethanol concentration

8

Ethanol concentrations were 48.2, to 61.6 and 74.4 g/L for sugar beet solid loadings of 12, to 15
and 18%, respectively (Fig. 6). Higher solid loading rates increased higher sugar concentrations
and thus ethanol concentrations. For all solid loading levels, fermentations were nearly
complete by 48 h with yeast. Therefore, 18% solid loading was chosen for further fermentation
studies. High solid loading during enzymatic hydrolysis or fermentation increases ethanol
concentration and lowers product recovery and equipment costs.

Figure 6. Effect of solid loading on yeast fermentation

Results showed that whole sugar beet can be effectively fermented into ethanol using E. coli
KO11 or S. cerevisiae. SSF with S. cerevisiae produced higher ethanol yields (92%) than SSF
with E. coli KO11 (83%). With regards to the fermentation time, S. cerevisiae was found to
consume sugar more rapidly than E. coli increasing reactor productivity. Maintaining optimal pH
(4.8) for S. cerevisiae throughout fermentation may have contributed to the higher yield.
Comparison of the performance of different microorganisms is often hampered by the variations
in experimental conditions such as sugar type and concentration, media nutrient levels, initial
cell density (Saha et al., 2005; Lau et al., 2010).
Dien et al. (2003) acknowledged that an economically attractive cellulosic technology requires
microorganisms to achieve ethanol yield, titer and rate higher than 90%, 40 g/L and 1.0 g/L/h. In
comparison, S. cerevisiae is the most relevant for industrial production for its ability to ferment
whole sugar beet due to overall ethanol yield, titer and rate achieved by this microorganism.

Conclusions
Whole sugar beets can be effectively fermented into ethanol using E. coli KO11 or S. cerevisiae.
SSF with S. cerevisiae produced higher ethanol yields (92%) than SSF with E. coli KO11 (83%).

Ethanol titers and yields of more than 70 g/L and 90% are feasible for fermentations of whole
sugar beet hydrolyzates without the need for sucrose extraction. Ethanol yields can be improved
by optimizing fermentation parameters such as buffer concentration, pH, and inoculum loading.

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