Accepted Manuscript Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production Badal C. Saha, Nasib Qureshi, Gregory J. Kennedy, Michael A. Cotta PII: DOI: Reference:
S0960-8524(15)00605-7 http://dx.doi.org/10.1016/j.biortech.2015.04.079 BITE 14920
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
5 March 2015 21 April 2015 23 April 2015
Please cite this article as: Saha, B.C., Qureshi, N., Kennedy, G.J., Cotta, M.A., Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.04.079
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BITE-D-15-01301 (Revised) Original research paper
Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production
Badal C. Saha*, Nasib Qureshi, Gregory J. Kennedy, Michael A. Cotta Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture**, Peoria, IL 61604, USA
____________________________________________________________________________________________________________________
* Corresponding author. Address: USDA-ARS-NCAUR, 1815 N. University St., Peoria, IL 61604, USA. Tel: +1-309-681-6276; fax: +1-309-681-6427. E-mail address: [email protected] (B. C. Saha) **Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.
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ABSTRACT
Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.
Keywords: Ethanol, Corn stover, Simultaneous saccharification and fermentation, Recombinant ethanologenic Escherichia coli, Substrate selective inoculum.
1. Introduction
Lignocellulosic biomass has great potential to serve as a widely abundant renewable feedstock for production of second generation fuel ethanol. The production of ethanol from any lignocellulosic biomass generally involves four steps – feedstock pretreatment, enzymatic saccharification, fermentation, and product recovery. Integration of two or more steps is important for lowering the cost of ethanol production from lignocellulosic biomass. Further,
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lignocellulosic biomass, upon pretreatment and enzymatic saccharification, produces a mixture of pentose (xylose, arabinose) and hexose (glucose, galactose) sugars (Saha, 2003). The utilization of all the sugars generated from lignocellulose is essential for the economic production of ethanol (Saha, 2004). The conventional ethanol fermenting yeast (Saccharomyces cerevisiae) or bacterium (Zymomonas mobilis) cannot ferment pentose sugars to ethanol. A major technical hurdle to converting any lignocellulosic feedstock to ethanol is developing an appropriate microorganism for fermentation of both hexose and pentose sugars. A number of recombinant microorganisms such as Escherichia coli, Klebsiella oxytoca, Z. mobilis, and S. cerevisiae have been developed over the last 30 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 can ferment 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 efficiently converting pyruvate into ethanol (Alterthum and Ingram, 1989). The plasmid also contains the genes for ampicillin and tetracycline resistance. The recombinant E. coli strain FBR5 stably maintains the plasmid when grown anaerobically without inclusion of antibiotics in the growth medium and is capable of fermenting both hexose and pentose sugars to ethanol. We studied the long term performance of this recombinant bacterium in a series of continuous culture runs (16-105 days) using wheat straw hydrolyzate as feedstock (Saha and Cotta, 2011). During these studies, no loss of ethanol productivity was observed. Thus the strain showed robustness in performance. However, the bacterium lacked the ability to utilize sugars simultaneously. It preferentially utilized glucose first before utilizing xylose and arabinose during fermentation of lignocellulosic hydrolyzates such as wheat straw,
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barley straw, rice hulls and corn stover hydrolyzates (Saha and Cotta, 2008, 2010; Saha et al., 2011a,b; 2013). This indicates that the xylose and arabinose utilization is catabolite repressed by glucose. Carbon catabolite repression plays an important role for sequential utilization of a mixture of sugars (diauxie phenomenon), where glucose is often the most preferred carbon source (Nichols et al., 2011; Yao and Shimizu, 2012). Desai and Rao (2010) demonstrated that the sugar utilization in E. coli involves multiple layers of regulation, where cells will consume glucose first, then arabinose and finally xylose. The xylose uptake is directly inhibited by the AraC regulator (Desai and Rao, 2010; Jarmander et al., 2014). This indicates that the expression of xylose metabolic genes is repressed and only low levels of xylose transport will occur from the promiscuous pentose transporters in the presence of arabinose. The efficiency of sugar utilization remains a significant bottleneck to economical utilization of the mixed sugars derived from lignocellulosic biomass (Jojima et al., 2010). We are thus interested to investigate the effect of substrate selective inoculum on the production of ethanol by the recombinant E. coli strain FBR5 in order to alleviate the catabolite repression without genetic modification. In this paper, we report that inoculum prepared using glucose, xylose, arabinose, mixed sugars and corn stover hydrolyzate (dilute acid pretreated and enzymatically saccharified, CSH) effected the rate of ethanol production from the CSH and delayed cellulase enzyme addition on simultaneous saccharification and fermentation (SSF) of dilute acid pretreated CS resulted in faster rate of xylose consumption and ethanol production by the recombinant bacterium.
2. Materials and Methods
2.1. Materials
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CS (~ 20%, w/w moisture) was collected from a corn field in the greater Peoria, Illinois area. It was air-dried to ~ 8% (w/w) moisture, chopped and milled in a Hammer mill so as to pass through a 1.27 mm screen. The milled CS was stored at ambient temperature in a tightly closed plastic bag. Celluclast 1.5 L (cellulase) and Novozym 188 (β-glucosidase) were purchased from Brenntag Great Lakes, Milwaukee, WI, USA. Membrane Filter Unit (0.2 mm) was purchased from Nalge Company, Rochester, NY, USA. Aminex HPX 87P column (300 x 7.8 mm), Aminex HPX 87H column (300 x 7.8 mm), De-ashing cartridge (30 x 4.6 mm), Carbo-P micro-guard cartridge (30 x 4.6 mm), and Cation H micro-guard cartridge (30 x 4.6 mm) were purchased from Bio-Rad Laboratories, Inc., Hercules, CA, USA. All other chemicals used were of standard analytical grades.
2.2. Enzyme assays
The cellulase activity was assayed and expressed as filter paper unit (FPU) by the procedure described by Ghose (1987). The β-glucosidase activity was assayed by the procedures described previously (Saha and Bothast, 1996). Both enzyme assays were performed at pH 5.0 and 50 oC. The β-glucosidase activity was expressed in terms of international units (IU, µmole product formed per min). At least triplicate assays were performed for each enzyme.
2.3. Pretreatment of corn stover
Dilute acid (0.75%, w/v) pretreatment of CS (10%, w/w; 7.4% moisture) was carried out at 160 oC with 0 min holding time by the procedure described previously (Avci et al., 2013a). Briefly, it was carried out in a multi-vessel rotating stainless steel reactor system equipped with
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12 stainless steel 200 mL reactors (Labomat BFA-12, Mathis USA Inc. Concord, NC) with 100 mL working volume. The system was heated with infrared heat and water cooled. During pretreatment, the reactors were rotated continuously at 50 rpm (60 sec clockwise and 60 sec counter clockwise) in order to provide efficient mixing and heat transfer. Heating and cooling rates during pretreatment were 3.5oC/min for heating and 6 oC/min for cooling. The holding time of 0 min refers to heating up to the desired temperature and immediately cooling. After pretreatment, the pH was adjusted to 5.0 with Ca(OH)2 for enzymatic saccharification. 2. 4. Enzymatic hydrolysis The enzymatic hydrolysis of dilute acid pretreated CS was carried out at pH 5.0 and 45 oC for 72 h using a cocktail of cellulase (5 FPU) and β-glucosidase (5 U) preparations per g straw. The residual solids were separated from the liquid by centrifugation (12,000 x g, 10 min) before using the liquid portion as enzymatically saccharified CS hydrolyzate (CSH). The commercial enzyme preparations used for enzymatic saccharification contained small quantities of glucose. For simplification purpose, the quantity of glucose present in the enzyme cocktail was subtracted from the measured glucose in each case.
2.5. Preparation of inoculum
Recombinant E. coli strain FBR5 (provided by Dr. Bruce S. Dien, USDA-ARS, Peoria, IL) was maintained in glycerol vials at -80oC for use as a working stock. It was platted onto Luria Bertani (LB) agar containing 10 g tryptone, 5 g yeast extract, 5 g NaCl and 15 g agar supplemented with 4 g xylose and 20 mg tetracycline per L (pH 6.5). Plates were incubated at 35oC. Cells from a single well-isolated colony were inoculated into a 125 mL Erlenmeyer flask
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containing 100 mL of LB medium with 2 g xylose (unless otherwise specified) and 2 mg tetracycline. Composition of the LB broth was the same as LB agar without the agar. Inoculated liquid culture was incubated semi-anaerobically at 35oC and 100 rpm for 24 h. The cells were centrifuged at 12,000 x g rpm for 10 min, washed with water and resuspended in a volume that gave an OD660nm of 10.0. This cell suspension was used as seed for both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) experiments. Inoculum size was 10%, v/v (final OD 660nm of 1.0). 2.6. Separate hydrolysis and fermentation (SHF) using substrate-selective inoculum
Fermentation experiments were carried out in pH-controlled 250 mL fleakers with a working volume of 200 mL at pH 6.5 and 35 °C. The medium was prepared by dissolving 10 g tryptone and 5 g yeast extract in CSH (per L) and filter-sterilizing using a membrane filter unit. The pH was automatically controlled at 6.5 during fermentation by adding 5 M KOH. Detailed setup of the bioreactors has been reported previously (Saha et al., 2011a). A picture of the pH-controlled fleaker fermentation system was provided in a previous paper (Saha and Nakamura, 2003). Briefly, rubber fleaker caps were drilled to allow the insertion of a pH probe, CO2 vent, sampling needle and a port for addition of base. A magnetic stirrer was located beneath the water bath and maintained at 130 rpm during experiments. Nitrogen gas was bubbled through the fleakers to expel oxygen for 30 min. Samples were withdrawn periodically and the optical densities (A660 nm)
of the samples were monitored immediately after withdrawal. The withdrawn fermentation
broth was centrifuged (25,000 x g, 10 min) to remove cells and kept at -20oC prior to analysis using high pressure liquid chromatography (HPLC). Bioreactor performance was monitored by quantifying unutilized sugars (glucose, xylose, and arabinose) and fermentation product
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(ethanol). Base consumption and pH were recorded. Duplicate parallel fermentation experiments were run for reproducibility and comparison. For simplification purpose, the quantity of ethanol produced from additional sugars (glucose and fructose) present in the enzyme cocktail was subtracted from the measured ethanol yield.
2.7. Simultaneous saccharification and fermentation (SSF) experiments
The pH-controlled SSF experiments were carried out in DasGip Parallel Bioreactor System (Juelich, Germany). The bioreactors are autoclavable glass vessels (350 mL each) with stainless steel head plates with standardized ports for pH-controls, substrate feeding, gassing and sampling and equipped with cooling condensers. They have direct drive head plate mounted stirring with two 6 blade Ruston impellers which allows proper mixing. The fermentation was performed with a working volume of 200 mL at controlled pH 6.5 and 35 oC. The stirring speed was set at 400 rpm initially, then lowered and maintained at 100 rpm after 2 h.
2. 8. Analytical procedures
The composition of corn stover with respect to cellulose, hemicellulose, acid soluble lignin, acid insoluble lignin and ash contents was determined in triplicate using the standard laboratory analytical procedures for biomass analysis provided by National Renewable Energy Laboratory (NREL), Golden, CO, USA (Sluiter et al., 2008a, b). Moisture content was determined using a moisture analyzer (Mark 2, Sartorius Mechatronics Corp., Bohemia, NY, USA). Sugars, ethanol, furfural, and HMF were analyzed by HPLC (Saha and Bothast, 1999). The separation system consisted of a fully integrated solvent delivery system (LC-20AD Prominence, Shimadzu America, Inc., Columbia, MD, USA) equipped with an SIL-20AC autosampler, RID-10A
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refractive index detector, a SDP-20A UV detector, a CTO-10AS VP column heater and a computer software based integration system (LC solutions 1.23 SP1, Shimadzu). 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 oC, and the sugars, furfural and HMF were eluted with Milli-Q filtered water at a flow rate of 0.6 mL/min. The Aminex HPX-87H column was maintained at 65 oC, and the sugars and ethanol were eluted with 10 mM HNO3 prepared using Milli-Q filtered water at a flow rate of 0.6 mL/min. Peaks were detected by refractive index or UV absorption (277 nm) and were identified and quantified by comparison to retention times of authentic standards (glucose, xylose, arabinose, galactose, ethanol, furfural and HMF). The yield of total sugars was calculated by the procedure provided by NREL (Sluiter et al., 2008a). The value is expressed as percentage of maximum theoretical sugar yield. 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) + 2 CO2 (MW 44.01) 3 Xylose (MW 150.13) or 3 Arabinose (MW 150.13) → 5 Ethanol (MW 46.07) + 5 CO2 (MW 44.01) Thus, the theoretical ethanol yield based on stoichiometry is 0.51 g ethanol per g glucose, xylose or arabinose. Cell growth of the bacterium was monitored by measuring the optical density of the appropriately diluted culture broth at 660 nm. The optical density was multiplied by a conversion factor of 0.45 to obtain cell dry weight (cell mass) per L (Saha and Cotta, 2011). The
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cell dry weight was determined from duplicate 10 mL samples. Each sample was centrifuged, washed with distilled water twice, and dried at 105 oC for 24 h.
3. Results and discussion
3. 1. Composition of corn stover and commercial enzyme preparations
CS, collected from a corn field, was used without washing. It contained 37.0±0.1% cellulose, 28.9±0.1% hemicellulose (22.7±0.1 % xylan, 4.5±0.0% arabinan and 1.7±0.0% galactan), 19.4% lignin (17.6±0.0% acid insoluble lignin and 1.8±0.0% acid soluble lignin) and 7.3±0.1% ash (65.9±0.2% total carbohydrates). The composition is similar to the compositions of previously used CS collected from corn fields in greater Peoria area at different times (Avci et al., 2013a,b.c; Saha and Cotta, 2014; Saha et al., 2013). Celluclast contained 30±3 FPU cellulase and Novozym 188 contained 353±11 U β-glucosidase per mL.
3.2. Effect of substrate-selective inoculum on ethanol production from dilute acid pretreated and enzymatically saccharified corn stover by recombinant E. coli FBR5 CS (10%, w/w, 7.4% moisture) was pretreated with dilute H2SO4 (0.75%, v/v) at 160 oC for 0 min holding time. The pretreated CS contained 2.8±0.2 g glucose, 20.6±0.5 g xylose, 3.3±0.0 g arabinose, and 1.3±0.1 g galactose (total sugars, 28.0±0.8 g) per L. It also contained 3.0±0.1 g acetic acid per L. In addition, it contained 2.2±0.0 mM (211±0.0 mg/L) furfural and 0.08±0.0 mM (10±0.0 mg/L) HMF. Our previous research indicates that CSH with such furfural, HMF and acetic acid concentrations does not need to be detoxified for fermenting to ethanol using the recombinant E. coli FBR5 (Avci et al., 2013). In fact, the pretreatment conditions were optimized in order to release maximum sugars from CS while minimizing the generation of
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common fermentation inhibitors. These data indicate that most of the hemicellulose was solubilized and hydrolyzed to monomers (mainly xylose and arabinose) while the insoluble solid residues contained cellulose. The pretreated CS was then enzymatically saccharified at pH 5.0 and 45 oC for 72 h using a cocktail of cellulase (5 FPU) and β-glucosidase (5 U) preparations per g stover. The enzymatically saccharified hydrolyzate (CSH) contained 33.3±0.3 g glucose, 22.4±0.2 g xylose, 3.4±0.6 g arabinose and 3.4±0.5 g galactose (total sugars, 60.5±1.6 g) per L. In order to investigate the effects of substrate-selective inoculums on the production of ethanol from CSH, seed cultures were prepared by growing the recombinant E. coli FBR5 on 2% (w/v) glucose, xylose, arabinose, mixed sugars (GXA; glucose, xylose, arabinose 1:1:1) and 3 times diluted CSH (0.3xCSH) separately. Each seed preparation was inoculated at a cell density of 1.0 in the medium containing CSH as substrate. The time courses of utilization of xylose, arabinose, total sugars, cell mass and ethanol production for glucose, xylose, arabinose, GXA and 0.3xCSH seed preparations are presented in Fig. 1 (A-E) separately. For all seed preparations, glucose was completely utilized within 16 h (data not shown). During this 16 h period, both furfural and HMF were completely converted to their corresponding alcohols which were considered not toxic (Almeida et al., 2007). Acetic acid was not utilized by the bacterium at all. The xylose utilization at 16 h fermentation was 16.8±0.2, 54.0±3.7, 6.4±0.4, 35.1±1.4 and 12.0±0.6% for glucose, xylose, arabinose, GXA and 0.3xCSH seed, respectively (Fig. 1A). For 20 h fermentation, these values were 40.0±2.0, 99.8±0.0, 23.8±1.2, 84.5±4.9 and 42.1±2.1%, respectively. About 56.9±2.9, 40.1±2.0 and 64.1±3.1% xylose were utilized after 24 h fermentation in the cases of glucose, arabinose and 0.3xCSH seeds, respectively. Xylose was completely utilized for the seed GXA. However, about 5.5±0.4 and17.7±1.0% xylose were left unutilized at 40 h for the seeds grown on glucose and arabinose, respectively. At 47 h of
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fermentation, xylose was completely utilized for all seed inoculums (Fig. 1A). These data indicate that substrate-selective inoculum has profound effect on the rate of xylose utilization from CSH by the recombinant bacterium. The seed prepared growing on arabinose performed the worst followed by glucose and 0.3xCSH. Very similar patterns were observed in the cases of arabinose utilization from CSH for these seed inoculum preparations (Fig. 1B). The time courses of cell growth in terms of cell mass for all the inoculums are shown in Fig. 1D. The initial growth (16 h) was highest for GXA (mixed sugar) inoculum followed by xylose, 0.3xCSH, glucose and arabinose. The time courses of ethanol production by the recombinant bacterium using these substrate-selective inoculums are presented in Fig. 1E. Even though, similar quantities of ethanol were produced using all seeds, the rate (productivity) of ethanol production varied greatly during the initial stages of ethanol production (Table 1). The rates are in the order of the seed inoculum prepared using xylose > GXA > 0.3xCSH > glucose > arabinose. Table 2 summarizes the yield of ethanol as well as ethanol productivity from CSH at the end of fermentation for all substrate-selective seed inoculums. It is clear that the seed inoculum prepared by growing on xylose performed better than the other seed grown on either glucose, arabinose, GXA or 0.3x CSH. Thus xylose is the preferred sugar for growing seed inoculum of the recombinant bacterium for use in ethanol production from CSH.
3.3. Effect of delayed cellulase enzyme addition on simultaneous saccharification and fermentation of pretreated corn stover by recombinant E. coli strain FBR5
The effect of 16 h delayed cellulase cocktail addition on the SSF of pretreated CS by the recombinant bacterium was investigated in order to evaluate whether delayed glucose release from unhydrolyzed cellulose has any effect on the rate of ethanol production or not. The delayed
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cellulase cocktail addition should allow the bacterium to consume xylose and arabinose present in the pretreated CS which is glucose limited. The data of utilization of glucose, xylose, arabinose and total sugars and ethanol production are presented in Fig. 2 (A-E). Fig. 2B shows that xylose was completely utilized by the bacterium within 24 h when enzyme cocktail was added after 16 h. The xylose utilization took much longer time (114 h) to complete when the enzyme cocktail was added simultaneously with the inoculum. Similar patterns were also observed for arabinose utilization (Fig. 2C). The rate of ethanol production for the delayed enzyme addition was faster than adding the inoculum together with the seed preparation (Fig. 2E). The ethanol concentrations were 4.6±0.4 and 9.3±0.7 g/L in 16 h for no delay and delayed cellulase enzyme cocktail addition with corresponding productivities of 0.29±0.02 and 0.58±0.04 g L-1 h-1, respectively. After 24 h fermentation, the ethanol concentrations were 10.3±1.2 and 18.6±0.3 g/L for no delay and delayed cellulase addition with corresponding productivities of 0.43±0.00 and 0.78 g±0.01 L-1 h-1, respectively. These data indicate that the ethanol productivity up to 24 h fermentation was almost doubled for 16 h delayed enzyme addition in comparison to no delayed enzyme addition (Table 3). The delayed enzyme addition allowed the bacterium to consume xylose and arabinose first in the presence of limited glucose from the hemicellulosic hydrolyzates of CS and thus helped to finish the fermentation quicker (Fig. 2 B and 2C). In order to obtain efficient co-fermentation of xylose and glucose in SSF with xylose fermenting recombinant S. cerevisiae, it was necessary to keep the glucose concentration low (Ohgren et al., 2006; Olofsson et al., 2008). The effects of (i) separating the solids from the liquid portion, hydrolyzing it separately at pH 5.0 and 45 oC for 16 h using the cellulase cocktail containing 5 FPU of cellulase and 5 U of βglucosidase per g straw and adding it back to the fermentation after 16 h and (ii) separating the
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solids from the liquid portion and adding the solids and enzyme cocktail to the fermentation of the liquid portion after 16 h were investigated in order to see the effect of fermenting xylose and arabinose first and then adding glucose or non-hydrolyzed cellulose on ethanol production. The data for total sugar utilization and ethanol production are presented in Fig. 3 (A, B). It is clear that separating the solids from the liquid portion helped to complete the fermentation faster in comparison to adding enzyme cocktail along with the inoculum (Fig. 3B and Fig. 2E). The fermentation was completed faster than when the cellulose portion present in the solids was separately hydrolyzed to glucose using enzyme cocktail and then added to the fermentation vessel.
4. Conclusions
Any lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of pentose and hexose sugars. The recombinant E. coli strain FBR5 can convert all these sugars to ethanol. However, the fermentation becomes slower due to catabolite repression of glucose and arabinose on the utilization of xylose. Substrate selective inoculum preparation and fermentation manipulation of SSF is crucial to partially overcome the problem.
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29. Yao, R., Shimizu, K. 2013. Recent progress in metabolic engineering for the production of biofuels and biochemicals from renewable sources with particular emphasis on catabolite regulation and its modulation. Proc. Biochem. 48, 1409-1417.
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Table 1 Comparison of the effects of substrate-selective inoculums on ethanol productivity from dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) and enzymatically saccharified (pH 5.0, 45 oC, 72 h) corn stover (10%, w/w) hydrolyzate by recombinant E. coli FBR5. ______________________________________________________________________________ Inoculum Ethanol productivity (g L-1 h-1) grown on 16 h 20 h 24 h 40 h 47 h 63 h ______________________________________________________________________________ Glucose 1.06±0.05 0.99±0.05 0.97±0.05 0.69±0.03 Xylose
1.43±0.02
1.33±0.02
1.12±0.02
0.72±0.01
Arabinose
0.98±0.05
0.94±0.05
0.88±0.0.4
0.65±0.03
Mixed sugar 1.29±0.00 (glu:xyl:ara, 1:1:1)
1.35±0.03
1.13±0.02
0.70±0.00
0.57±0.02 0.43±0.00
Corn stover 1.14±0.05 1.11±0.00 1.01±0.05 0.72±0.01 hydrolyzate ______________________________________________________________________________ Data presented are averages of two individual experiments. Glu, glucose; xyl, xylose; ara, arabinose.
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Table 2 Summary of the effects of substrate-selective inoculum on maximum ethanol production from dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) and enzymatically saccharified (pH 5.0, 45 oC, 72 h) corn stover (10%, w/w) hydrolyzate by recombinant E. coli FBR5. ______________________________________________________________________________ Inoculum Time Ethanol Ethanol Ethanola Ethanolb Ethanol substrate (h) (g/L) productivity (g/g sugar) (g/g sugar) (g/g straw) (g L-1 h-1) ______________________________________________________________________________ Glucose 40 27.4±1.4 0.69±0.03 0.45±0.02 0.38±0.02 0.27±0.01 Xylose
40
28.7±0.4
0.72±0.01
0.47±0.00
0.40±0.00
0.29±0.00
Arabinose
63
27.2±0.1
0.43±0.00
0.45±0.00
0.38±0.00
0.27±0.00
Mixed sugar 40 (glu:xyl:ara, 1:1:1)
28.1±0.1
0.70±0.00
0.47 ±0.00
0.39±0.00
0.28±0.00
Corn stover 40 28.9±0.2 0.72±0.01 0.48±0.00 0.40±0.00 0.29±0.00 hydrolyzate ______________________________________________________________________________ Data presented are averages of two individual experiments. Glu, glucose; xyl, xylose; ara, arabinose. a
Based on the total sugars present in the corn stover hydrolyzate.
b
Based on the theoretical sugar yield from corn stover.
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Table 3 Ethanol productivities of recombinant E. coli FBR5 for no delay and delayed enzyme cocktail addition in simultaneous saccharification and fermentation (SSF) of dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) corn stover (10%, w/w). ______________________________________________________________________________ Enzyme Ethanol productivity (g L-1 h-1) addition 16 h 24 h 41 h 48 h 68 h 114 h ______________________________________________________________________________ No delayed 0.29±0.02 0.43±0.05 0.43±0.00 0.41±0.03 0.34±0.0 0.23±0.00 Delayed 0.58±0.04 0.78±0.01 0.56±0.02 0.48±0.01 (16 h) ______________________________________________________________________________ Data presented are averages of two individual experiments.
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Legend to Figures:
Fig. 1. Effects of substrate-selective inoculums on the utilization of sugars, cell growth and ethanol production from dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) and enzymatically saccharified (pH 5.0, 45 oC, 72 h) corn stover (10%, w/w) hydrolyzate (CSH) by recombinant E. coli strain FBR5 at pH 6.5 and 35 oC. Data presented are averages of two individual experiments. A, xylose; B, arabinose; C, total sugars; D, cell mass; E, ethanol.
Fig. 2. Effect of delayed enzyme addition on simultaneous saccharification and fermentation (SSF) of dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) corn stover (10%, w/w) to ethanol by recombinant E. coli strain FBR5 at pH 6.5 and 35 oC. Data presented are averages of two individual experiments.
Fig. 3. Effects of (i) separating the solids from dilute acid pretreated (0.75%, w/v H2SO4; 160 o
C, 0 min holding time) corn stover (10%, w/w) and adding them after 16 h with cellulase
cocktail and (ii) separating the solids from the pretreated corn stover, hydrolyzing them for 16 h at pH 5.0 and 45 oC with cellulase cocktail and then adding the hydrolyzate on simultaneous sccahrification and fermentation (SSF) by recombinant E. coli strain FBR5 at pH 6.5 and 35 oC. The cellulase enzyme cocktail contained 5 FPU of cellulase and 5 U of β-glucosidase per g straw. Data presented are averages of two individual experiments. A, utilization of total sugars; B, ethanol production.
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25
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A
B Arabinose (g/L)
Xylose (g/L)
20 15 10 5 0 0
8
16 24 32 40 48 56 64
3 2 1 0 0
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16 24 32 40 48 56 64 Time (h)
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Total sugars (g/L)
60
40 30 20 10
3 2 1 0
0 0
8
16
24
32
40
48
0
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16 24 32 40 48 56 64 Time (h)
Time (h) E
30 Ethanol (g/L)
25 Glucose grown Xylose grown Arabinose grown Mixed sugars grown CSH grown
20 15 10 5 0 0
8
16 24 32 40 48 56 64 Time (h)
Fig. 1
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8
25
6
B 20
0h 16 h
X ylose (g/L)
Glucose (g/L)
A
4
2
0h 16 h
15 10 5 0
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72
96
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35 Total sugars (g/L )
C Arabin ose (g /L)
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Time (h)
3
72
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2
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30
D
25
0h 16 h
20 15 10 5 0
0 0
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96
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72
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Time (h) 35
E
E than ol (g/L )
30 25 20
0h 16 h
15 10 5 0 0
24
48
72
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Fig. 2
24
96
120
96
120
40 A
Total sugars (g/L)
35
Solids added after 16 h Solids hydrolyzed separately for 16 h and added
30 25 20 15 10 5 0 0
8
16
24
32
40
48
56
64
72
80
Time (h) 30
B
Ethanol (g/L)
25 20 15
Solids added after 16 h Solids hydrolyzed separately for 16 h and added
10 5 0 0
8
16
24
32
40
48
56
Time (h)
Fig. 3
25
64
72
80
Highlights: ● Recombinant bacterium fermented all sugars to ethanol. ● Xylose fermentation became slower due to catabolite repression by other sugars. ● Substrate selection inoculum enhanced the rate of xylose utilization by the bacterium. ● Enhanced xylose utilization was achieved by fermentation manipulation.
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S0960-8524(15)00605-7 http://dx.doi.org/10.1016/j.biortech.2015.04.079 BITE 14920
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
5 March 2015 21 April 2015 23 April 2015
Please cite this article as: Saha, B.C., Qureshi, N., Kennedy, G.J., Cotta, M.A., Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.04.079
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BITE-D-15-01301 (Revised) Original research paper
Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production
Badal C. Saha*, Nasib Qureshi, Gregory J. Kennedy, Michael A. Cotta Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture**, Peoria, IL 61604, USA
____________________________________________________________________________________________________________________
* Corresponding author. Address: USDA-ARS-NCAUR, 1815 N. University St., Peoria, IL 61604, USA. Tel: +1-309-681-6276; fax: +1-309-681-6427. E-mail address: [email protected] (B. C. Saha) **Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.
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ABSTRACT
Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.
Keywords: Ethanol, Corn stover, Simultaneous saccharification and fermentation, Recombinant ethanologenic Escherichia coli, Substrate selective inoculum.
1. Introduction
Lignocellulosic biomass has great potential to serve as a widely abundant renewable feedstock for production of second generation fuel ethanol. The production of ethanol from any lignocellulosic biomass generally involves four steps – feedstock pretreatment, enzymatic saccharification, fermentation, and product recovery. Integration of two or more steps is important for lowering the cost of ethanol production from lignocellulosic biomass. Further,
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lignocellulosic biomass, upon pretreatment and enzymatic saccharification, produces a mixture of pentose (xylose, arabinose) and hexose (glucose, galactose) sugars (Saha, 2003). The utilization of all the sugars generated from lignocellulose is essential for the economic production of ethanol (Saha, 2004). The conventional ethanol fermenting yeast (Saccharomyces cerevisiae) or bacterium (Zymomonas mobilis) cannot ferment pentose sugars to ethanol. A major technical hurdle to converting any lignocellulosic feedstock to ethanol is developing an appropriate microorganism for fermentation of both hexose and pentose sugars. A number of recombinant microorganisms such as Escherichia coli, Klebsiella oxytoca, Z. mobilis, and S. cerevisiae have been developed over the last 30 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 can ferment 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 efficiently converting pyruvate into ethanol (Alterthum and Ingram, 1989). The plasmid also contains the genes for ampicillin and tetracycline resistance. The recombinant E. coli strain FBR5 stably maintains the plasmid when grown anaerobically without inclusion of antibiotics in the growth medium and is capable of fermenting both hexose and pentose sugars to ethanol. We studied the long term performance of this recombinant bacterium in a series of continuous culture runs (16-105 days) using wheat straw hydrolyzate as feedstock (Saha and Cotta, 2011). During these studies, no loss of ethanol productivity was observed. Thus the strain showed robustness in performance. However, the bacterium lacked the ability to utilize sugars simultaneously. It preferentially utilized glucose first before utilizing xylose and arabinose during fermentation of lignocellulosic hydrolyzates such as wheat straw,
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barley straw, rice hulls and corn stover hydrolyzates (Saha and Cotta, 2008, 2010; Saha et al., 2011a,b; 2013). This indicates that the xylose and arabinose utilization is catabolite repressed by glucose. Carbon catabolite repression plays an important role for sequential utilization of a mixture of sugars (diauxie phenomenon), where glucose is often the most preferred carbon source (Nichols et al., 2011; Yao and Shimizu, 2012). Desai and Rao (2010) demonstrated that the sugar utilization in E. coli involves multiple layers of regulation, where cells will consume glucose first, then arabinose and finally xylose. The xylose uptake is directly inhibited by the AraC regulator (Desai and Rao, 2010; Jarmander et al., 2014). This indicates that the expression of xylose metabolic genes is repressed and only low levels of xylose transport will occur from the promiscuous pentose transporters in the presence of arabinose. The efficiency of sugar utilization remains a significant bottleneck to economical utilization of the mixed sugars derived from lignocellulosic biomass (Jojima et al., 2010). We are thus interested to investigate the effect of substrate selective inoculum on the production of ethanol by the recombinant E. coli strain FBR5 in order to alleviate the catabolite repression without genetic modification. In this paper, we report that inoculum prepared using glucose, xylose, arabinose, mixed sugars and corn stover hydrolyzate (dilute acid pretreated and enzymatically saccharified, CSH) effected the rate of ethanol production from the CSH and delayed cellulase enzyme addition on simultaneous saccharification and fermentation (SSF) of dilute acid pretreated CS resulted in faster rate of xylose consumption and ethanol production by the recombinant bacterium.
2. Materials and Methods
2.1. Materials
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CS (~ 20%, w/w moisture) was collected from a corn field in the greater Peoria, Illinois area. It was air-dried to ~ 8% (w/w) moisture, chopped and milled in a Hammer mill so as to pass through a 1.27 mm screen. The milled CS was stored at ambient temperature in a tightly closed plastic bag. Celluclast 1.5 L (cellulase) and Novozym 188 (β-glucosidase) were purchased from Brenntag Great Lakes, Milwaukee, WI, USA. Membrane Filter Unit (0.2 mm) was purchased from Nalge Company, Rochester, NY, USA. Aminex HPX 87P column (300 x 7.8 mm), Aminex HPX 87H column (300 x 7.8 mm), De-ashing cartridge (30 x 4.6 mm), Carbo-P micro-guard cartridge (30 x 4.6 mm), and Cation H micro-guard cartridge (30 x 4.6 mm) were purchased from Bio-Rad Laboratories, Inc., Hercules, CA, USA. All other chemicals used were of standard analytical grades.
2.2. Enzyme assays
The cellulase activity was assayed and expressed as filter paper unit (FPU) by the procedure described by Ghose (1987). The β-glucosidase activity was assayed by the procedures described previously (Saha and Bothast, 1996). Both enzyme assays were performed at pH 5.0 and 50 oC. The β-glucosidase activity was expressed in terms of international units (IU, µmole product formed per min). At least triplicate assays were performed for each enzyme.
2.3. Pretreatment of corn stover
Dilute acid (0.75%, w/v) pretreatment of CS (10%, w/w; 7.4% moisture) was carried out at 160 oC with 0 min holding time by the procedure described previously (Avci et al., 2013a). Briefly, it was carried out in a multi-vessel rotating stainless steel reactor system equipped with
5
12 stainless steel 200 mL reactors (Labomat BFA-12, Mathis USA Inc. Concord, NC) with 100 mL working volume. The system was heated with infrared heat and water cooled. During pretreatment, the reactors were rotated continuously at 50 rpm (60 sec clockwise and 60 sec counter clockwise) in order to provide efficient mixing and heat transfer. Heating and cooling rates during pretreatment were 3.5oC/min for heating and 6 oC/min for cooling. The holding time of 0 min refers to heating up to the desired temperature and immediately cooling. After pretreatment, the pH was adjusted to 5.0 with Ca(OH)2 for enzymatic saccharification. 2. 4. Enzymatic hydrolysis The enzymatic hydrolysis of dilute acid pretreated CS was carried out at pH 5.0 and 45 oC for 72 h using a cocktail of cellulase (5 FPU) and β-glucosidase (5 U) preparations per g straw. The residual solids were separated from the liquid by centrifugation (12,000 x g, 10 min) before using the liquid portion as enzymatically saccharified CS hydrolyzate (CSH). The commercial enzyme preparations used for enzymatic saccharification contained small quantities of glucose. For simplification purpose, the quantity of glucose present in the enzyme cocktail was subtracted from the measured glucose in each case.
2.5. Preparation of inoculum
Recombinant E. coli strain FBR5 (provided by Dr. Bruce S. Dien, USDA-ARS, Peoria, IL) was maintained in glycerol vials at -80oC for use as a working stock. It was platted onto Luria Bertani (LB) agar containing 10 g tryptone, 5 g yeast extract, 5 g NaCl and 15 g agar supplemented with 4 g xylose and 20 mg tetracycline per L (pH 6.5). Plates were incubated at 35oC. Cells from a single well-isolated colony were inoculated into a 125 mL Erlenmeyer flask
6
containing 100 mL of LB medium with 2 g xylose (unless otherwise specified) and 2 mg tetracycline. Composition of the LB broth was the same as LB agar without the agar. Inoculated liquid culture was incubated semi-anaerobically at 35oC and 100 rpm for 24 h. The cells were centrifuged at 12,000 x g rpm for 10 min, washed with water and resuspended in a volume that gave an OD660nm of 10.0. This cell suspension was used as seed for both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) experiments. Inoculum size was 10%, v/v (final OD 660nm of 1.0). 2.6. Separate hydrolysis and fermentation (SHF) using substrate-selective inoculum
Fermentation experiments were carried out in pH-controlled 250 mL fleakers with a working volume of 200 mL at pH 6.5 and 35 °C. The medium was prepared by dissolving 10 g tryptone and 5 g yeast extract in CSH (per L) and filter-sterilizing using a membrane filter unit. The pH was automatically controlled at 6.5 during fermentation by adding 5 M KOH. Detailed setup of the bioreactors has been reported previously (Saha et al., 2011a). A picture of the pH-controlled fleaker fermentation system was provided in a previous paper (Saha and Nakamura, 2003). Briefly, rubber fleaker caps were drilled to allow the insertion of a pH probe, CO2 vent, sampling needle and a port for addition of base. A magnetic stirrer was located beneath the water bath and maintained at 130 rpm during experiments. Nitrogen gas was bubbled through the fleakers to expel oxygen for 30 min. Samples were withdrawn periodically and the optical densities (A660 nm)
of the samples were monitored immediately after withdrawal. The withdrawn fermentation
broth was centrifuged (25,000 x g, 10 min) to remove cells and kept at -20oC prior to analysis using high pressure liquid chromatography (HPLC). Bioreactor performance was monitored by quantifying unutilized sugars (glucose, xylose, and arabinose) and fermentation product
7
(ethanol). Base consumption and pH were recorded. Duplicate parallel fermentation experiments were run for reproducibility and comparison. For simplification purpose, the quantity of ethanol produced from additional sugars (glucose and fructose) present in the enzyme cocktail was subtracted from the measured ethanol yield.
2.7. Simultaneous saccharification and fermentation (SSF) experiments
The pH-controlled SSF experiments were carried out in DasGip Parallel Bioreactor System (Juelich, Germany). The bioreactors are autoclavable glass vessels (350 mL each) with stainless steel head plates with standardized ports for pH-controls, substrate feeding, gassing and sampling and equipped with cooling condensers. They have direct drive head plate mounted stirring with two 6 blade Ruston impellers which allows proper mixing. The fermentation was performed with a working volume of 200 mL at controlled pH 6.5 and 35 oC. The stirring speed was set at 400 rpm initially, then lowered and maintained at 100 rpm after 2 h.
2. 8. Analytical procedures
The composition of corn stover with respect to cellulose, hemicellulose, acid soluble lignin, acid insoluble lignin and ash contents was determined in triplicate using the standard laboratory analytical procedures for biomass analysis provided by National Renewable Energy Laboratory (NREL), Golden, CO, USA (Sluiter et al., 2008a, b). Moisture content was determined using a moisture analyzer (Mark 2, Sartorius Mechatronics Corp., Bohemia, NY, USA). Sugars, ethanol, furfural, and HMF were analyzed by HPLC (Saha and Bothast, 1999). The separation system consisted of a fully integrated solvent delivery system (LC-20AD Prominence, Shimadzu America, Inc., Columbia, MD, USA) equipped with an SIL-20AC autosampler, RID-10A
8
refractive index detector, a SDP-20A UV detector, a CTO-10AS VP column heater and a computer software based integration system (LC solutions 1.23 SP1, Shimadzu). 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 oC, and the sugars, furfural and HMF were eluted with Milli-Q filtered water at a flow rate of 0.6 mL/min. The Aminex HPX-87H column was maintained at 65 oC, and the sugars and ethanol were eluted with 10 mM HNO3 prepared using Milli-Q filtered water at a flow rate of 0.6 mL/min. Peaks were detected by refractive index or UV absorption (277 nm) and were identified and quantified by comparison to retention times of authentic standards (glucose, xylose, arabinose, galactose, ethanol, furfural and HMF). The yield of total sugars was calculated by the procedure provided by NREL (Sluiter et al., 2008a). The value is expressed as percentage of maximum theoretical sugar yield. 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) + 2 CO2 (MW 44.01) 3 Xylose (MW 150.13) or 3 Arabinose (MW 150.13) → 5 Ethanol (MW 46.07) + 5 CO2 (MW 44.01) Thus, the theoretical ethanol yield based on stoichiometry is 0.51 g ethanol per g glucose, xylose or arabinose. Cell growth of the bacterium was monitored by measuring the optical density of the appropriately diluted culture broth at 660 nm. The optical density was multiplied by a conversion factor of 0.45 to obtain cell dry weight (cell mass) per L (Saha and Cotta, 2011). The
9
cell dry weight was determined from duplicate 10 mL samples. Each sample was centrifuged, washed with distilled water twice, and dried at 105 oC for 24 h.
3. Results and discussion
3. 1. Composition of corn stover and commercial enzyme preparations
CS, collected from a corn field, was used without washing. It contained 37.0±0.1% cellulose, 28.9±0.1% hemicellulose (22.7±0.1 % xylan, 4.5±0.0% arabinan and 1.7±0.0% galactan), 19.4% lignin (17.6±0.0% acid insoluble lignin and 1.8±0.0% acid soluble lignin) and 7.3±0.1% ash (65.9±0.2% total carbohydrates). The composition is similar to the compositions of previously used CS collected from corn fields in greater Peoria area at different times (Avci et al., 2013a,b.c; Saha and Cotta, 2014; Saha et al., 2013). Celluclast contained 30±3 FPU cellulase and Novozym 188 contained 353±11 U β-glucosidase per mL.
3.2. Effect of substrate-selective inoculum on ethanol production from dilute acid pretreated and enzymatically saccharified corn stover by recombinant E. coli FBR5 CS (10%, w/w, 7.4% moisture) was pretreated with dilute H2SO4 (0.75%, v/v) at 160 oC for 0 min holding time. The pretreated CS contained 2.8±0.2 g glucose, 20.6±0.5 g xylose, 3.3±0.0 g arabinose, and 1.3±0.1 g galactose (total sugars, 28.0±0.8 g) per L. It also contained 3.0±0.1 g acetic acid per L. In addition, it contained 2.2±0.0 mM (211±0.0 mg/L) furfural and 0.08±0.0 mM (10±0.0 mg/L) HMF. Our previous research indicates that CSH with such furfural, HMF and acetic acid concentrations does not need to be detoxified for fermenting to ethanol using the recombinant E. coli FBR5 (Avci et al., 2013). In fact, the pretreatment conditions were optimized in order to release maximum sugars from CS while minimizing the generation of
10
common fermentation inhibitors. These data indicate that most of the hemicellulose was solubilized and hydrolyzed to monomers (mainly xylose and arabinose) while the insoluble solid residues contained cellulose. The pretreated CS was then enzymatically saccharified at pH 5.0 and 45 oC for 72 h using a cocktail of cellulase (5 FPU) and β-glucosidase (5 U) preparations per g stover. The enzymatically saccharified hydrolyzate (CSH) contained 33.3±0.3 g glucose, 22.4±0.2 g xylose, 3.4±0.6 g arabinose and 3.4±0.5 g galactose (total sugars, 60.5±1.6 g) per L. In order to investigate the effects of substrate-selective inoculums on the production of ethanol from CSH, seed cultures were prepared by growing the recombinant E. coli FBR5 on 2% (w/v) glucose, xylose, arabinose, mixed sugars (GXA; glucose, xylose, arabinose 1:1:1) and 3 times diluted CSH (0.3xCSH) separately. Each seed preparation was inoculated at a cell density of 1.0 in the medium containing CSH as substrate. The time courses of utilization of xylose, arabinose, total sugars, cell mass and ethanol production for glucose, xylose, arabinose, GXA and 0.3xCSH seed preparations are presented in Fig. 1 (A-E) separately. For all seed preparations, glucose was completely utilized within 16 h (data not shown). During this 16 h period, both furfural and HMF were completely converted to their corresponding alcohols which were considered not toxic (Almeida et al., 2007). Acetic acid was not utilized by the bacterium at all. The xylose utilization at 16 h fermentation was 16.8±0.2, 54.0±3.7, 6.4±0.4, 35.1±1.4 and 12.0±0.6% for glucose, xylose, arabinose, GXA and 0.3xCSH seed, respectively (Fig. 1A). For 20 h fermentation, these values were 40.0±2.0, 99.8±0.0, 23.8±1.2, 84.5±4.9 and 42.1±2.1%, respectively. About 56.9±2.9, 40.1±2.0 and 64.1±3.1% xylose were utilized after 24 h fermentation in the cases of glucose, arabinose and 0.3xCSH seeds, respectively. Xylose was completely utilized for the seed GXA. However, about 5.5±0.4 and17.7±1.0% xylose were left unutilized at 40 h for the seeds grown on glucose and arabinose, respectively. At 47 h of
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fermentation, xylose was completely utilized for all seed inoculums (Fig. 1A). These data indicate that substrate-selective inoculum has profound effect on the rate of xylose utilization from CSH by the recombinant bacterium. The seed prepared growing on arabinose performed the worst followed by glucose and 0.3xCSH. Very similar patterns were observed in the cases of arabinose utilization from CSH for these seed inoculum preparations (Fig. 1B). The time courses of cell growth in terms of cell mass for all the inoculums are shown in Fig. 1D. The initial growth (16 h) was highest for GXA (mixed sugar) inoculum followed by xylose, 0.3xCSH, glucose and arabinose. The time courses of ethanol production by the recombinant bacterium using these substrate-selective inoculums are presented in Fig. 1E. Even though, similar quantities of ethanol were produced using all seeds, the rate (productivity) of ethanol production varied greatly during the initial stages of ethanol production (Table 1). The rates are in the order of the seed inoculum prepared using xylose > GXA > 0.3xCSH > glucose > arabinose. Table 2 summarizes the yield of ethanol as well as ethanol productivity from CSH at the end of fermentation for all substrate-selective seed inoculums. It is clear that the seed inoculum prepared by growing on xylose performed better than the other seed grown on either glucose, arabinose, GXA or 0.3x CSH. Thus xylose is the preferred sugar for growing seed inoculum of the recombinant bacterium for use in ethanol production from CSH.
3.3. Effect of delayed cellulase enzyme addition on simultaneous saccharification and fermentation of pretreated corn stover by recombinant E. coli strain FBR5
The effect of 16 h delayed cellulase cocktail addition on the SSF of pretreated CS by the recombinant bacterium was investigated in order to evaluate whether delayed glucose release from unhydrolyzed cellulose has any effect on the rate of ethanol production or not. The delayed
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cellulase cocktail addition should allow the bacterium to consume xylose and arabinose present in the pretreated CS which is glucose limited. The data of utilization of glucose, xylose, arabinose and total sugars and ethanol production are presented in Fig. 2 (A-E). Fig. 2B shows that xylose was completely utilized by the bacterium within 24 h when enzyme cocktail was added after 16 h. The xylose utilization took much longer time (114 h) to complete when the enzyme cocktail was added simultaneously with the inoculum. Similar patterns were also observed for arabinose utilization (Fig. 2C). The rate of ethanol production for the delayed enzyme addition was faster than adding the inoculum together with the seed preparation (Fig. 2E). The ethanol concentrations were 4.6±0.4 and 9.3±0.7 g/L in 16 h for no delay and delayed cellulase enzyme cocktail addition with corresponding productivities of 0.29±0.02 and 0.58±0.04 g L-1 h-1, respectively. After 24 h fermentation, the ethanol concentrations were 10.3±1.2 and 18.6±0.3 g/L for no delay and delayed cellulase addition with corresponding productivities of 0.43±0.00 and 0.78 g±0.01 L-1 h-1, respectively. These data indicate that the ethanol productivity up to 24 h fermentation was almost doubled for 16 h delayed enzyme addition in comparison to no delayed enzyme addition (Table 3). The delayed enzyme addition allowed the bacterium to consume xylose and arabinose first in the presence of limited glucose from the hemicellulosic hydrolyzates of CS and thus helped to finish the fermentation quicker (Fig. 2 B and 2C). In order to obtain efficient co-fermentation of xylose and glucose in SSF with xylose fermenting recombinant S. cerevisiae, it was necessary to keep the glucose concentration low (Ohgren et al., 2006; Olofsson et al., 2008). The effects of (i) separating the solids from the liquid portion, hydrolyzing it separately at pH 5.0 and 45 oC for 16 h using the cellulase cocktail containing 5 FPU of cellulase and 5 U of βglucosidase per g straw and adding it back to the fermentation after 16 h and (ii) separating the
13
solids from the liquid portion and adding the solids and enzyme cocktail to the fermentation of the liquid portion after 16 h were investigated in order to see the effect of fermenting xylose and arabinose first and then adding glucose or non-hydrolyzed cellulose on ethanol production. The data for total sugar utilization and ethanol production are presented in Fig. 3 (A, B). It is clear that separating the solids from the liquid portion helped to complete the fermentation faster in comparison to adding enzyme cocktail along with the inoculum (Fig. 3B and Fig. 2E). The fermentation was completed faster than when the cellulose portion present in the solids was separately hydrolyzed to glucose using enzyme cocktail and then added to the fermentation vessel.
4. Conclusions
Any lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of pentose and hexose sugars. The recombinant E. coli strain FBR5 can convert all these sugars to ethanol. However, the fermentation becomes slower due to catabolite repression of glucose and arabinose on the utilization of xylose. Substrate selective inoculum preparation and fermentation manipulation of SSF is crucial to partially overcome the problem.
References
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Alterthum, F., Ingram, L.O., 1989. Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli. Appl. Environ. Microbiol. 55, 1943-1948.
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Avci, A., Saha, B.C., Kennedy, G.J., Cotta, M.A., 2013a. Dilute acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification. Bioresour. Technol. 142, 312-319.
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Avci, A., Saha, B.C., Dien , B.S., Kennedy, G.J., Cotta, M.A., 2013b. Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production. Bioresour. Technol. 130, 603-612.
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Avci, A., Saha, B.C., Kennedy, G.J., Cotta, M.A., 2013c. High temperature dilute phosphoric acid pretreatment of corn stover for furfural and ethanol production. Ind. Crops Products 50, 478-484.
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Desai, T.A., Rao, C.V., 2010. Regulation of arabinose and xylose metabolism in Escherichia coli. Appl. Environ. Microbiol. 76, 1524-1532.
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Dien, B.S., Nichols, N.N., O’Bryan, P.J., Bothast, R.J., 2000. Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass. Appl. Biochem. Biotechnol. 84-86, 181-186.
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Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257-268.
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Jarmander, J., Hallstron, B. M., Larson, G., 2014. Simultaneous uptake of lignocellulosebased monosaccharides by Escherichia coli. Biotechnol. Bioeng. 111, 1108-1115.
10. Jojima, T., Omumasaba, C. A., Inui, M., Yukawa, Y., 2010. Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook. Appl. Microbiol. Biotechnol. 85, 471-480. 11. Nichols, N.N., Dien, B.S., Bothast, R. J., 2001. Use of catabolic repression mutants for fermentation of sugar mixtures to ethanol. Appl. Microbiol. Biotechnol. 56, 120-125.
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12. Ohgren, K., Bengtsson, O., Gorwa-Grauslund, M.F., Galbe, M., Hahn-Hagerdal, B., Zacchi, G., 2006. Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400. J. Biotechnol. 126, 488-498. 13. Olofsson, K., Rudolf, A., Linden, G., 2008. Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. J. Biotechnol. 134, 112-120. 14. Saha, B.C., 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30, 279-291. 15. 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. 2-34. 16. Saha, B.C., Bothast, R.J., 1996. Production, purification, and characterization of a highly glucose-tolerant β-glucosidase from Candida peltata. Appl. Environ. Microbiol. 62, 31653170. 17. Saha, B.C., Bothast, R.J., 1999. Pretreatment and enzymatic saccharification of corn fiber. Appl. Biochem. Biotechnol. 76, 65-77. 18. Saha, B.C., Nakamura, L.K. 2003. Production of mannitol and lactic acid by Lactobacillus intermedius. Biotechnol. Bioeng. 82, 864-871. 19. Saha, B.C., Cotta, M.A., 2008. Lime pretreatment, enzymatic saccharification and fermentation of rice hulls to ethanol. Biomass Bioenergy 22, 971-977. 20. Saha, B.C., Cotta, M.A., 2010. Comparison of pretreatment strategies for enzymatic saccharification and fermentation of barley straw to ethanol. New Biotechnol. 27, 10-16.
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21. 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, 477-487. 22. Saha, B.C., Cotta, M.A., 2012. Ethanol production from lignocellulosic biomass by recombinant Escherichia coli FBR5. Bioengineered 3, 197-202. 23. Saha, B.C., Cotta, M.A., 2014. Alkaline peroxide pretreatment of corn stover for enzymatic saccharification and ethanol production. Ind. Biotechnol. 10, 34-41. 24. Saha, B.C., Nichols, N. N., Qureshi, N., Cotta, M.A., 2011a. Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant Escherichia coli strain FBR5. Appl. Microbiol. Biotechnol. 92, 865-874. 25. 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, 10892-10897. 26. Saha, B.C., Yoshida, T., Cotta, M.A., Sonomoto, K., 2013. Hydrothermal pretreatment and enzymatic saccharification of corn stover for efficient ethanol production. Ind. Crops Products 44, 367-372. 27. 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 28. 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
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29. Yao, R., Shimizu, K. 2013. Recent progress in metabolic engineering for the production of biofuels and biochemicals from renewable sources with particular emphasis on catabolite regulation and its modulation. Proc. Biochem. 48, 1409-1417.
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Table 1 Comparison of the effects of substrate-selective inoculums on ethanol productivity from dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) and enzymatically saccharified (pH 5.0, 45 oC, 72 h) corn stover (10%, w/w) hydrolyzate by recombinant E. coli FBR5. ______________________________________________________________________________ Inoculum Ethanol productivity (g L-1 h-1) grown on 16 h 20 h 24 h 40 h 47 h 63 h ______________________________________________________________________________ Glucose 1.06±0.05 0.99±0.05 0.97±0.05 0.69±0.03 Xylose
1.43±0.02
1.33±0.02
1.12±0.02
0.72±0.01
Arabinose
0.98±0.05
0.94±0.05
0.88±0.0.4
0.65±0.03
Mixed sugar 1.29±0.00 (glu:xyl:ara, 1:1:1)
1.35±0.03
1.13±0.02
0.70±0.00
0.57±0.02 0.43±0.00
Corn stover 1.14±0.05 1.11±0.00 1.01±0.05 0.72±0.01 hydrolyzate ______________________________________________________________________________ Data presented are averages of two individual experiments. Glu, glucose; xyl, xylose; ara, arabinose.
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Table 2 Summary of the effects of substrate-selective inoculum on maximum ethanol production from dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) and enzymatically saccharified (pH 5.0, 45 oC, 72 h) corn stover (10%, w/w) hydrolyzate by recombinant E. coli FBR5. ______________________________________________________________________________ Inoculum Time Ethanol Ethanol Ethanola Ethanolb Ethanol substrate (h) (g/L) productivity (g/g sugar) (g/g sugar) (g/g straw) (g L-1 h-1) ______________________________________________________________________________ Glucose 40 27.4±1.4 0.69±0.03 0.45±0.02 0.38±0.02 0.27±0.01 Xylose
40
28.7±0.4
0.72±0.01
0.47±0.00
0.40±0.00
0.29±0.00
Arabinose
63
27.2±0.1
0.43±0.00
0.45±0.00
0.38±0.00
0.27±0.00
Mixed sugar 40 (glu:xyl:ara, 1:1:1)
28.1±0.1
0.70±0.00
0.47 ±0.00
0.39±0.00
0.28±0.00
Corn stover 40 28.9±0.2 0.72±0.01 0.48±0.00 0.40±0.00 0.29±0.00 hydrolyzate ______________________________________________________________________________ Data presented are averages of two individual experiments. Glu, glucose; xyl, xylose; ara, arabinose. a
Based on the total sugars present in the corn stover hydrolyzate.
b
Based on the theoretical sugar yield from corn stover.
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Table 3 Ethanol productivities of recombinant E. coli FBR5 for no delay and delayed enzyme cocktail addition in simultaneous saccharification and fermentation (SSF) of dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) corn stover (10%, w/w). ______________________________________________________________________________ Enzyme Ethanol productivity (g L-1 h-1) addition 16 h 24 h 41 h 48 h 68 h 114 h ______________________________________________________________________________ No delayed 0.29±0.02 0.43±0.05 0.43±0.00 0.41±0.03 0.34±0.0 0.23±0.00 Delayed 0.58±0.04 0.78±0.01 0.56±0.02 0.48±0.01 (16 h) ______________________________________________________________________________ Data presented are averages of two individual experiments.
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Legend to Figures:
Fig. 1. Effects of substrate-selective inoculums on the utilization of sugars, cell growth and ethanol production from dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) and enzymatically saccharified (pH 5.0, 45 oC, 72 h) corn stover (10%, w/w) hydrolyzate (CSH) by recombinant E. coli strain FBR5 at pH 6.5 and 35 oC. Data presented are averages of two individual experiments. A, xylose; B, arabinose; C, total sugars; D, cell mass; E, ethanol.
Fig. 2. Effect of delayed enzyme addition on simultaneous saccharification and fermentation (SSF) of dilute acid pretreated (0.75%, w/v H2SO4; 160 oC, 0 min holding time) corn stover (10%, w/w) to ethanol by recombinant E. coli strain FBR5 at pH 6.5 and 35 oC. Data presented are averages of two individual experiments.
Fig. 3. Effects of (i) separating the solids from dilute acid pretreated (0.75%, w/v H2SO4; 160 o
C, 0 min holding time) corn stover (10%, w/w) and adding them after 16 h with cellulase
cocktail and (ii) separating the solids from the pretreated corn stover, hydrolyzing them for 16 h at pH 5.0 and 45 oC with cellulase cocktail and then adding the hydrolyzate on simultaneous sccahrification and fermentation (SSF) by recombinant E. coli strain FBR5 at pH 6.5 and 35 oC. The cellulase enzyme cocktail contained 5 FPU of cellulase and 5 U of β-glucosidase per g straw. Data presented are averages of two individual experiments. A, utilization of total sugars; B, ethanol production.
22
25
4
A
B Arabinose (g/L)
Xylose (g/L)
20 15 10 5 0 0
8
16 24 32 40 48 56 64
3 2 1 0 0
8
Time (h)
16 24 32 40 48 56 64 Time (h)
5
D
C 4
50
Cell mass (g/L)
Total sugars (g/L)
60
40 30 20 10
3 2 1 0
0 0
8
16
24
32
40
48
0
8
16 24 32 40 48 56 64 Time (h)
Time (h) E
30 Ethanol (g/L)
25 Glucose grown Xylose grown Arabinose grown Mixed sugars grown CSH grown
20 15 10 5 0 0
8
16 24 32 40 48 56 64 Time (h)
Fig. 1
23
8
25
6
B 20
0h 16 h
X ylose (g/L)
Glucose (g/L)
A
4
2
0h 16 h
15 10 5 0
0 0
24
48
72
96
0
120
24
48
4
120
35 Total sugars (g/L )
C Arabin ose (g /L)
96
Time (h)
Time (h)
3
72
0h 16 h
2
1
30
D
25
0h 16 h
20 15 10 5 0
0 0
24
48
72
96
0
120
24
48
72
Time (h)
Time (h) 35
E
E than ol (g/L )
30 25 20
0h 16 h
15 10 5 0 0
24
48
72
Time (h)
Fig. 2
24
96
120
96
120
40 A
Total sugars (g/L)
35
Solids added after 16 h Solids hydrolyzed separately for 16 h and added
30 25 20 15 10 5 0 0
8
16
24
32
40
48
56
64
72
80
Time (h) 30
B
Ethanol (g/L)
25 20 15
Solids added after 16 h Solids hydrolyzed separately for 16 h and added
10 5 0 0
8
16
24
32
40
48
56
Time (h)
Fig. 3
25
64
72
80
Highlights: ● Recombinant bacterium fermented all sugars to ethanol. ● Xylose fermentation became slower due to catabolite repression by other sugars. ● Substrate selection inoculum enhanced the rate of xylose utilization by the bacterium. ● Enhanced xylose utilization was achieved by fermentation manipulation.
26
