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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 low-value 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 β-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

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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 -20º 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 (β-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 β-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

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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 37°C 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 37º 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 121°C. Different solid loadings were used for SSF as described in Table 1, with 100 mL of working volume. Cellulase and β-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 37º 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 µm nylon filter (Pall Corporation; West Chester, PA) and stored at -20ºC 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 (Bio-Rad Laboratories; Hercules, CA) with a mobile phase of 18 mΩ 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 50⁰C and 85⁰C, 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 60⁰C. 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 60⁰C 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.

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Table 1. Simultaneous saccharification and fermentation set-up for different buffer concentration, solid loading, and inoculum loading.

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.

Microorganism Buffer Solid Loading

% (w/v) Inoculum Loading

% (v/v)

E. coli KO11

100 mM Phosphate 12 1

300 mM Phosphate 12 1

500 mM Phosphate 12 1

300 mM Phosphate 12 5

300 mM Citrate 12 5

S. cerevisiae

300 mM Citrate 12 1

300 mM Citrate 12 3

300 mM Citrate 12 5

300 mM Citrate 15 3

300 mM Citrate 18 3

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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

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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

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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

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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%).

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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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