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Integrated Two-Stage Alkaline−Oxidative Pretreatment of Hybrid Poplar. Part 2: Impact of Cu-Catalyzed Alkaline Hydrogen Peroxide Pretreatment Conditions on Process Performance and Economics Zhaoyang Yuan,† Sandip Kumar Singh,‡ Bryan Bals,§ David B. Hodge,*,‡,∥ and Eric L. Hegg*,†

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†

Department of Biochemistry & Molecular Biology, Michigan State University, 603 Wilson Road, East Lansing, Michigan 48824, United States ‡ Department of Chemical & Biological Engineering, Montana State University, 306 Cobleigh Hall, Bozeman, Montana 59717, United States § Michigan Biotechnology Institute, 3815 Technology Boulevard, Lansing, Michigan 48910, United States ∥ Division of Sustainable Process Engineering, Luleå University of Technology, 97187 Luleå, Sweden S Supporting Information *

ABSTRACT: Two-stage alkaline/copper 2,2′-bipyridine-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment is an effective strategy for improving the enzymatic digestibility of hybrid poplar. To reduce the chemical inputs and processing costs associated with this process, we investigated the effect of increasing the temperature for both the alkaline pre-extraction and the Cu-AHP pretreatment stages. The results indicate that increasing the alkaline pre-extraction and the Cu-AHP pretreatment temperatures from 30 to 120 and 80 °C, respectively, allowed us to reduce both the pretreatment time of the Cu-AHP stage and the chemical loadings. Incubating alkaline pre-extracted hybrid poplar for 12 h with 10% NaOH (w/w biomass), 8% hydrogen peroxide (w/w biomass), and a Cu2+ and 2,2′-bipyridine (bpy) concentration of 1 mM yielded monomeric sugar yields of approximately 77% glucose and 66% xylose (based on the initial sugar composition) following enzymatic hydrolysis. Technoeconomic analysis (TEA) indicates that these changes to the two-stage alkaline/Cu-AHP pretreatment process could potentially reduce the minimum fuel selling price (MFSP) by more than $1.00 per gallon of biofuel compared to the reference case where both stages were conducted at 30 °C with higher chemical inputs.

1. INTRODUCTION Recently, the biobased economy concept, in which renewable feedstocks such as lignocellulosic biomass are used as the raw material for the production of various commercial products, has received considerable attention. Among lignocellulosic biomass feedstocks, hybrid poplar is an attractive feedstock for large-scale biorefinery applications because of its year-round availability, fast growth rate, and high bulk density relative to that of many other biomass sources.1,2 In addition, compared to softwoods, hardwoods such as hybrid poplar are often less recalcitrant because of their lower lignin content.3 Although the cell wall recalcitrance of hardwoods to deconstruction is greater than that of herbaceous feedstocks,4 the much lower ash content in hardwoods and higher glucan content are advantages for the downstream conversion of polysaccharides to monosaccharides.5 Although various chemical, physical, and biological pretreatment technologies have been configured to improve the release of structural sugars from lignocellulosic © XXXX American Chemical Society

biomass feedstocks, chemical pretreatment is considered to be the most promising technology for commercialization.6−8 Delignification is widely regarded as an important component of many chemical pretreatments.9,10 This is because delignification not only reduces recalcitrance by removing the physical barriers and exposing the crystalline structure of cellulose, it also reduces the presence of ligninderived inhibitors such as phenolics during the subsequent enzymatic hydrolysis and fermentation.9−12 During the chemical delignification process, hemicellulose is often removed as well. Although it has been reported that the removal of hemicellulose can lead to increased exposure of the Special Issue: Biorenewable Energy and Chemicals Received: Revised: Accepted: Published: A

February 15, 2019 July 31, 2019 August 5, 2019 August 5, 2019 DOI: 10.1021/acs.iecr.9b00901 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

2. MATERIALS AND METHODS 2.1. Materials. Hybrid poplar (Populus nigra var. charkoviensis × caudina cv. NE-19) was grown at the University of Wisconsin Arlington Agricultural Research Station. The airdried wood logs (moisture content ∼5.5% w/w) were chipped and hammer milled (HammerHead, Muson Co., Inc., USA) to pass through a 5 mm screen. The milled biomass was stored in airtight bags prior to use. Enzyme cocktails Cellic CTec3 (197.3 mg protein/g) and HTec3 (164 mg protein/g) were kindly provided by Novozymes A/S (Bagsværd, Denmark). All other chemicals were reagent grade and purchased from Fisher Scientific (USA) unless otherwise noted. 2.2. Two-Stage Pretreatment. 2.2.1. Alkaline Preextraction. Alkaline pre-extraction of biomass was performed using a 100 mL stainless steel Parr reactor (Parr Instruments Company, Moline, IL, USA), model 4560 mini benchtop reactor. The reactor was fitted with two six-blade turbine impellers, heated by an electric heater, and the temperature was controlled with a fully proportional integral and derivative controller, model 4848. The reactor was cooled with cold water circulating through a serpentine coil. A series of alkaline pre-extraction experiments were conducted over temperatures of 30−155 °C at 10% NaOH loading (based on the weight of biomass). The liquid-to-wood ratio was fixed at 10 L/kg (10% w/v). Alkaline pre-extraction reactions were performed by mixing 5.3 g of air-dried (≤5 mm) biomass (equivalent to 5 g of oven-dried biomass), 47.2 mL of deionized water, and 2.5 mL of 5 M NaOH into the Parr reactor at the appropriate temperature for 1 h. The solid and liquid fractions were separated by filtration, and the solid fraction was thoroughly washed with approximately 1 L of deionized water and stored at 4 °C. All experiments were performed at least in duplicate. 2.2.2. Cu-AHP Pretreatment. The moisture content of the wet alkaline pre-extracted poplar biomass was determined to calculate the amount of biomass required for Cu-AHP pretreatment. Cu-AHP pretreatment was conducted using the same Parr reactor described above at a biomass loading of 10% (w/v) (based on the original dry biomass). Having demonstrated that increasing the reaction temperature could reduce processing costs,26,28 we investigated the possibility of reducing the reaction time, H 2 O 2 loading, and bpy concentration sequentially. The Cu-AHP pretreatment conditions were therefore varied to explore the effects of temperature (30−95 °C), reaction time (3−24 h), H2O2 loading (0−10% w/w), and bpy concentration (0−2 mM). The H2O2 was added in a fed-batch manner in 10 equal aliquots during the first 40% of the reaction time. Following Cu-AHP pretreatment, the reactor was cooled in an ice/water bath, the solid fraction was separated from the liquor via filtration, thoroughly washed with deionized water (approximately 1 L), and stored at 4 °C prior to compositional analysis and enzymatic hydrolysis. In one set of experiments, following Cu-AHP pretreatment, the whole reaction mixture was collected and stored at 4 °C for enzymatic hydrolysis. 2.3. Enzymatic Hydrolysis. Enzymatic hydrolysis was performed in 15 mL Falcon tubes using a 1:1 ratio of CTec3/ HTec3 with a total enzyme loading of 15 mg of protein/g of glucan (based on the initial glucan content). Reactions were incubated at 5% (w/v) solid loading (5 g of biomass per 100 mL of liquid added) and 50 °C for 72 h with 50 mM sodium citrate buffer (pH 5) in a shaking incubator (C24KC refrigerated incubator shaker, New Brunswick Scientific, NJ,

remaining lignin, thereby causing increased adsorption and reduced productivity of the cellulases during hydrolysis,13−15 it has also been observed that the removal of hemicellulose can facilitate enzymatic hydrolysis efficacy by increasing the accessibility of cellulose.6−8 Therefore, it is critical to ascertain the ideal level of pretreatment severity to achieve maximal monomeric sugar yields following enzymatic hydrolysis. Alkaline hydrogen peroxide (AHP) pretreatment is a chemical pretreatment technology that is highly effective for the delignification of several lignocellulosic feedstocks, including wood, agricultural residues, and grasses.16−19 In addition, mild AHP pretreatment enables lignin to be isolated with its original macromolecular structure intact,20 allowing for subsequent valorization to high-value products. A challenge of traditional AHP pretreatment, however, is that the loading of H2O2 is relatively high (25 to 100% of the biomass weight),21−23 leading to excessive processing costs.24 Various strategies have been employed to improve the economic viability of the AHP process. Previously, we demonstrated that adding copper 2,2′-bipyridine complexes [Cu(bpy)] during AHP pretreatment (Cu-AHP) improved the efficiency of H2O2 utilization and led to significantly increased sugar yields following enzymatic hydrolysis compared to uncatalyzed AHP.25−29 A second strategy that we21,30,31 and others32 have employed is the addition of an alkaline preextraction step prior to AHP that can remove acetyl ester groups and partially solubilize lignin oligomers and hemicellulose, thus allowing the severity of the AHP pretreatment to be reduced. For example, Bhalla et al. reported that the sequential two-stage pretreatment comprising alkaline preextraction followed by Cu-AHP pretreatment substantially improved the enzymatic digestibility of poplar.26 Following enzymatic hydrolysis, a monomeric sugar yield of around 80% was achieved when conducting Cu-AHP pretreatment at a H2O2 loading of 10% (w/w biomass).26 Importantly, the dissolved lignin and hemicellulose in alkaline pre-extraction liquors can be recovered,33,34 allowing it be used in a variety of applications.35−37 Earlier work demonstrated that increasing the reaction temperature of the alkaline pre-extraction step allowed the chemical inputs during the Cu-AHP pretreatment stage to be reduced without significantly lowering the sugar yields following enzymatic hydrolysis, thereby reducing the processing cost.26,28 In the current work, we sought to build on those results and gain more insight into the effects of the alkaline pre-extraction temperature as well as evaluate the impact that the second-stage Cu-AHP pretreatment temperature has on sugar yields and processing costs. Alkaline pre-extraction temperatures of 30−155 °C were screened, and the amounts of lignin and hemicellulose removed were determined. Subsequent Cu-AHP pretreatment of the pre-extracted poplar was performed under a variety of conditions, including temperatures of 30−95 °C, reaction times of 3−24 h, H2O2 loadings of 2−10% w/w biomass, and 2,2′-bipyridine (bpy) concentrations of 0−2 mM. The two-stage pretreated substrate was then subjected to enzymatic hydrolysis at a modest enzyme loading to assess the digestibility of the two-stage pretreated biomass. A technoeconomic analysis (TEA) indicated that increasing the pretreatment temperature allowed a substantial reduction in processing cost by lowering chemical inputs while maintaining high sugar yields. B

DOI: 10.1021/acs.iecr.9b00901 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research USA) at 210 rpm. Following enzymatic hydrolysis, the pH of the reaction mixture was measured again to verify the reaction conditions. The Falcon tubes were placed in the autoclave empty at 121 °C for 15 min prior to being used. After enzymatic hydrolysis, the hydrolysate was centrifuged at 5000 rpm for 10 min to separate the liquid from the solid residues. Then, the sugar concentrations were measured using a highperformance liquid chromatography (HPLC) system (Agilent 1260 Series) following National Renewable Energy Laboratory (NREL) protocol.38 The sugar yields (glucose and xylose) were expressed on the basis of the total sugar content of the original biomass (dry weight basis). 2.4. Analytical Methods. The moisture content of solid samples was measured by drying at 105 ± 2 °C for 6 h to constant weight. The chemical compositions of original and pretreated solid samples were determined using NREL standard protocols.38 In brief, the solid samples were airdried and ground with a Wiley mill to pass through a 20 mesh screen. The ground sample (0.1 g) was digested by a two-step H2SO4 hydrolysis protocol. After hydrolysis, Klason lignin (acid insoluble ligning) was separated by filtration through a Whatman no. 1 filter paper (Fisher Scientific Co., USA) and weighed after drying at 105 ± 2 °C. Acid-soluble lignin in the hydrolysate was measured at 205 nm using a UV−vis spectrophotometer (8453 UV−visible spectrophotometer, Hewlett-Packard, USA). To determine carbohydrates, the acid hydrolysate was refiltered using a 0.22 μm syringe filter (Millipore Sigma, MA, USA) and then analyzed using an HPLC system (Agilent 1260 series equipped with an infinity refractive index detector) fitted with a Biorad Aminex HPX87H column (Bio-Rad Laboratories, USA), operating at 65 °C with a mobile phase of 5.0 mM sulfuric acid at a flow rate of 0.6 mL/min as described by Sluiter et al.38 Because the HPX87H column cannot separate xylose, mannose, and galactose, the measured value of xylose was a combination of these three sugars. Sugars were quantified using a standard curve prepared by using pure glucose and xylose. The ash content of solid samples was determined by completely ashing about 0.5 g of 20-mesh milled dry biomass at 550 °C for 12 h to a constant weight. 2.5. Technoeconomic Analysis. To estimate the impact of changes in the reaction conditions on the cost of producing biofuel, TEA was performed using a Microsoft Excel model. An NREL model for producing hydrocarbons via the catalytic conversion of sugars was used as the basis of this model.39 All pieces of equipment, material streams, and major energy flows were accounted for in this model and replicated in Microsoft Excel. The biomass preprocessing and pretreatment segments were removed and replaced with a customized model of CuAHP pretreatment as well as standard preprocessing for poplar.28 Downstream processes from the NREL model were then adjusted on the basic of the input and output streams from pretreatment and hydrolysis as obtained from the experimental data. For example, wastewater treatment capital equipment was resized based on either water use or total solubles (depending on which was more suitable for each major piece of equipment), and major heat and power inputs were adjusted on the basis of the changes in material streams. For the boiler and turbogenerator, both pieces of equipment were resized on the basis of the amount of combustable biomass remaining and the breakdown of steam and electricity requirements for the biorefinery. Ash was assumed to be disposed of in the manner outlined in Davis et al.39

The Cu-AHP pretreatment reactor was modeled as a series of vertical stainless steel reactors with screws to transport biomass into and out of reactors. Sodium hydroxide, water, copper sulfate, and bpy were all modeled to be added at the first reactor stage, while H2O2 was modeled to be added in a fed-batch manner as described above. The pre-extraction stage was modeled as a single vertical reactor. Lignin recovery was modeled as acid precipitation of the soluble material remaining after the alkaline pre-extraction and the Cu-AHP pretreatment stages followed by filtration. The remaining liquid stream can either be used for hydrolysis or sent to wastewater treatment, and both options were modeled. No attempt was made to recover the pretreatment chemicals; the sodium and copper were assumed to be recovered and disposed of as ash. The cost and design assumptions for these reactors were based on a previous model.28 Total capital expenditures were determined by summing the installed capital costs and applying multipliers as described in Davis et al.39 Total material expenditures were determined by multiplying the hourly inputs needed by 8400 h per year. All of the extracted lignin from both the pre-extraction stage and the Cu-AHP pretreatment stage was assumed to be sold as a coproduct at a constant price of $0.50/kg with no further processing performed. The final analysis was performed by determining the minimum fuel selling price (MFSP), as determined by targeting the net present value of the biorefinery to be $0. The net present value was determined via the same method as in Davis et al.39 Major assumptions used in the economic model are shown in Table 1. Table 1. Major Assumptions Used for the Technoeconomic Model

a

item

value

units

biorefinery size operating hours plant lifetime interest rate internal rate of return poplar cost enzyme cost H2O2 cost CuSO4 cost bipyridine cost

2000 8400 30 8% 10% $50 $5 $1000 $1500 $30

dry Mga/day h/y years

per per per per per

dry Mg g protein Mg Mg kg

Mg: metric ton.

2.6. Statistical Analysis. All experiments were singlefactor multilevel comparisons. Data collected from triplicate measurements were analyzed by one-way analysis of variance (one-way ANOVA) using Unscrambler v10.5 software (CAMO software, Japan). Multiple comparisons among treatments were performed using the Tukey post hoc test. Significant differences between treatment conditions were evaluated at p ≤ 0.05. Error bars in all graphs refer to 95% confidence intervals.

3. RESULTS AND DISCUSSION 3.1. Impact of Alkaline Pre-extraction on the Properties of Poplar. We previously demonstrated that the twostage pretreatment process comprising an alkaline preextraction step followed by a Cu-AHP alkaline oxidative step is effective at pretreating hardwoods under relatively mild conditions.26−28 In addition, Bhalla et al. reported that C

DOI: 10.1021/acs.iecr.9b00901 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research increasing the alkaline pre-extraction temperature to 120 °C could theoretically reduce the ethanol unit production cost by up to 25%.28 To evaluate the possibility of further reducing chemical inputs and processing costs, the effect of increasing the temperature of both the alkaline pre-extraction process and the Cu-AHP pretreatment process on the properties of the hybrid poplar was investigated. Moreover, it should be noted that rather than using 20 mesh (≤0.85 mm) biomass, as in our previous studies,26−28 larger particle size (≤5 mm) hybrid poplar was used in this work. The objectives of the alkaline pre-extraction stage are to solubilize some of the lignin and hemicellulose, remove acetyl groups, increase the surface area of the biomass, and reduce the generation of lignin-derived inhibitors, thereby increasing the accessibility of both the remaining lignin to pretreatment chemicals in the subsequent Cu-AHP stage and the polysaccharides to hydrolytic enzymes. Figure 1 shows the

subsequent Cu-AHP pretreatment. It was expected that the solubilization of approximately 25% xylan and 27% lignin will considerably increase the porosity of the biomass and increase the accessibility of the remaining lignin to the copper catalyst, H2O2, and NaOH used in the subsequent Cu-AHP pretreatment stage.45 3.2. Impact of Cu-AHP Pretreatment Conditions on the Properties of Pretreated Poplar. During Cu-AHP pretreatment, Cu2+ (added as CuSO4) in combination with bpy is the catalyst. Although the specific role of the bpy ligand in the reaction is not well understood, both the N heteroatoms and the aromaticity appear to be very important on the basis of our previous survey of potential ligands (unpublished results). We speculate that bpy may help the copper ions penetrate the plant cell wall and localize to the lignin, presumably via π−π interactions. It may also be important to modulate copper’s redox potential and the reactivity of the active oxidant. Because the cost of bpy is very high (∼$30 per kg), decreasing the bpy concentration is an appropriate strategy for reducing the biofuel production cost. Other potential targets based on sensitivity analysis of the process include H2O2 loading and the biomass resident time. Thus, we next investigated the effect of temperature, time, H2O2 loading, and bpy concentration during Cu-AHP pretreatment on the overall sugar yields following enzymatic hydrolysis (Figure 2). We first explored the effect of reaction temperature on the properties of the two-stage pretreated poplar biomass (≤5 mm). Washed alkaline pre-extracted biomass was subjected to Cu-AHP pretreatment at different temperatures (30−95 °C) while keeping all the other parameters fixed [10% NaOH loading (100 mg per gram of initial biomass), 10% H2O2 loading (100 mg per gram of initial biomass), 10% (w/v based on initial biomass) solid loading, 1 mM Cu2+, 2 mM bpy, and a reaction time of 23 h]. Figure 2a illustrates the sugar yields following both Cu-AHP pretreatment and enzymatic hydrolysis of the two-stage pretreated substrates. With increasing CuAHP pretreatment temperature, the glucose yields (based on the initial glucan content) also steadily increased until approximately 80 °C, at which point they began to plateau at ∼82% glucose release. The relatively small increase in glucose yields from 80 to 95 °C, which mirrors the minor changes in lignin and xylan removal between those temperatures (additional file in Table S2) is consistent with the number of accessible substrate sites being similar between the two temperatures.46 To further improve glucose release, more severe Cu-AHP pretreatment conditions or additional pretreatment strategies (e.g., mechanical, chemical) might be required to increase the surface area and the number of accessible substrate sites. The xylose yields (based on initial xylan content) also increased with increasing Cu-AHP pretreatment temperature until reaching a plateau at approximately 65 °C (Figure 2a). For example, when conducting Cu-AHP pretreatment at 65 °C, the overall xylose yield was 72.4% (based on the initial xylan content). By increasing the Cu-AHP pretreatment temperature to 80 or even 95 °C, the xylose yields were 72.6 and 70.7% (based on the initial xylan content), respectively. Overall, the data in Figure 2a indicate that Cu-AHP pretreatment at approximately 80 °C improves the overall monomeric sugar yields from poplar biomass. We further explored the effect of Cu-AHP pretreatment time on sugar yields. The Cu-AHP reaction time was varied from 3 to 24 h while the other parameters remained fixed [80 °C, 1 mM Cu2+, 2 mM bpy, 10% (w/w) H2O2 loading (100 mg per

Figure 1. Impact of the alkaline pre-extraction temperature on the solubilization of glucan, xylan, and acid-insoluble lignin from poplar.

fraction of the components removed from poplar following alkaline pre-extraction at various temperatures. As shown in Figure 1, only 5−10% of glucan (based on initial glucan) was solubilized during alkaline pre-extraction under elevated temperatures (95−155 °C), presumably because the high crystallinity of glucan makes it very recalcitrant toward degradation by alkali.40,41 Figure 1 also demonstrates that, as expected for high-temperature-facilitated delignification,42 the fraction of solubilized acid-insoluble lignin (Klason lignin) increased from 14 to 34% (based on initial lignin) as the reaction temperature increased from 95 to 155 °C. Finally, increasing the severity of alkaline pre-extraction also increased xylan removal from 20 to 40% (based on initial xylan), consistent with previous studies on the alkaline extraction of xylan from hardwood or herbaceous crops.43,44 Technoeconomic analysis of the effect of the alkaline preextraction temperature on the processing cost was performed (Figure S1). By including the solubilized glucan and xylan in the alkaline pre-extraction liquor, the cost of biofuel initially decreased with increasing alkaline pre-extraction temperature until approximately 115−125 °C, but it increased thereafter because of the increased heat input and the degradation of xylan that occurred in the pretreatment liquor. On the basis of these results, hybrid poplar biomass pre-extracted with 10% NaOH (w/w biomass) at 120 °C for 1 h was selected for D

DOI: 10.1021/acs.iecr.9b00901 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 2. Impact of Cu-AHP pretreatment conditions on overall sugar yields following the enzymatic hydrolysis of hybrid poplar: (a) Cu-AHP temperature, (b) Cu-AHP reaction time, (c) H2O2 loading, and (d) 2,2′-bipyridine concentration. Enzymatic hydrolysis was performed at 50 °C and pH 5 using 15 mg of total protein per g of glucan (based on the initial glucan content) for 72 h. The points are the averages of triplicate experiments, and error bars indicate standard deviations from the means.

obtained with 10% H2O2 loading (81%). Our ability to decrease the H2O2 loading while maintaining high sugar yields can be attributed to the higher temperatures used during CuAHP pretreatment in this study, which facilitates delignification as confirmed by compositional analysis (additional file in Table S4). Improved delignification increases the porosity of the biomass, thereby enhancing the surface area of glucan accessible to the enzyme cocktail.10,17,47 The overall xylose yields following enzymatic hydrolysis only slightly varied with varying H2O2 loadings (Figure 2c). This is likely because the fraction of residual xylan following the two-stage pretreated substrate could be easily digested after solubilizing 11−20% more xylan during the Cu-AHP pretreatment stage (additional file in Table S4). In total, Figure 2c indicates that 8% H2O2 loading (based on the initial biomass) can be used during CuAHP to efficiently improve the enzymatic digestibility of hybrid poplar. In a fourth set of experiments, we explored the possibility of reducing the concentration of the costly ligand (bpy) during Cu-AHP pretreatment. We varied the concentration of bpy from 0 to 2 mM while keeping the remaining variables fixed [8% H2O2 loading (on initial biomass), 1 mM Cu2+, 80 °C, 10% solid loading, 12 h] (Figure 2d; additional file in Table S5). Similar to the above results, the overall glucose yields gradually increased with the increase in bpy concentration until reaching a plateau at a bpy concentration of approximately 1 mM. For example, further increasing the bpy concentration from 1 to 2 mM resulted only in the increase in the overall glucose yields from 77 to 80% (based on the initial glucan

gram of initial biomass), 10% (w/v) solid loading (based on initial biomass)] (Figure 2b; additional file in Table S3). As expected, the sugar yields increased with increasing reaction times. Under the investigated conditions, however, the increases in sugar yields after 12 h were relatively minimal. For example, although the overall yields of glucose and xylose at 12 h reached approximately 81 and 65% (based on the initial composition), respectively, increasing the reaction time to 24 h resulted only in an increase in the glucose and xylose yields by additional 3.7 and 2.1% (based on the initial composition). This is consistent with the compositional analysis of the poplar biomass pretreated at different times (additional file in Table S3), which revealed that increasing the reaction time initially solubilized substantial amounts of lignin and xylan but that the amount solubilized began to level off at 12 h. Thus, performing Cu-AHP pretreatment at 80 °C for 12 h appears to be a promising combination for achieving high sugar yields. According to our earlier work,28 H2O2 loading represents an important contribution to the processing cost for cellulosic biofuel production using the Cu-AHP pretreatment technology. In this set of experiments, H2O2 loading was varied during Cu-AHP pretreatment while keeping other variables constant [80 °C, 12 h, 1 mM Cu2+, 2 mM bpy, 10% (w/v) solid loading (based on initial biomass)], and the impact on sugar yields and chemical composition was determined (Figure 2c; additional file in Table S4). As shown in Figure 2c, the H2O2 loading could be reduced to 6−8% (based on the initial biomass) while maintaining high overall glucose yields (75−79% of the initial glucan content), which were only slightly lower than that E

DOI: 10.1021/acs.iecr.9b00901 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 3. Correlation between (a) lignin removal or (b) xylan solubilization and overall glucose hydrolysis yields. Note that the overall glucose yields include only the glucan solubilized during the Cu-AHP pretreatment stage and the subsequent enzymatic hydrolysis.

idea that improving delignification and xylan solubilization during the two-stage alkaline/Cu-AHP pretreatment is a critical factor in facilitating the downstream enzymatic hydrolysis of glucan and that lignin and xylan removal can be used to predict subsequent glucose enzymatic hydrolysis yields (Figure 3). 3.4. Technoeconomic Analysis. In general, the conditions with the lowest MFSP were those with the highest or nearly the highest sugar yields, as expected. Revenue from lignin was much lower than sugar yields, indicating that increasing sugar yields or finding high-value lignin products to increase its value has the largest impact on the revenue stream. The value of the residual solids is very low because of the high volumes of sugar and lignin recovered, and most of the scenarios tested were net consumers of electricity rather than net exporters as in the baseline scenario in the NREL model (Davis et al.).39 Increasing the temperature of the Cu-AHP pretreatment decreased the MFSP until 65 °C was reached, after which the MFSP was relatively stable (Figure S2). While the 95 °C experiment produced the lowest MFSP, the difference was negligible compared to the chosen optimum of 80 °C. Likewise, decreasing the pretreatment residence time from 24 to 12 h had little effect on the MFSP, but the MSFP increased if reduced beyond 12 h (Figure S3). The decrease in capital expenditure caused by the reduction of pretreatment reactors was offset by reductions in sugar and lignin yields. Reducing the hydrogen peroxide loading to 8% substantially reduced the MFSP (Figure S4), driven by the high cost of peroxide. Surprisingly, eliminating the bpy entirely resulted in the lowest MFSP (Figure S5), simply because of the high cost of the bpy. The relatively small reduction in sugar yields is completely offset by the high price of bpy. On the basis of these results, the MFSP can be reduced by over 40% relative to the base case, as shown in Figure 4. The largest reductions in MFSP occur as a result of the change in temperature, the reduction of bpy, and the recovery of the liquid medium from Cu-AHP pretreatment. Decreasing the residence time and H2O2 loading had little impact on the MFSP, and in this analysis, the decreased residence time actually slightly increased the selling price. However, given the uncertainties surrounding the capital cost of these reactors as well as the relatively small changes in the sugar yield, this difference may not be significant. In contrast, increasing the

content). The overall xylose yields changed only slightly (from 63 to 69% of the initial glucan content) with varying bpy concentration (Figure 2d); this is presumably due to the solubilization of about 25 and 20% of the xylan during the alkaline pre-extraction and Cu-AHP pretreatment stages, respectively (Table S1; additional file in Table S5). On the basis of the results shown in Figure 2d, when the Cu-AHP reaction is performed at 80 °C following alkaline pre-extraction (at 120 °C), the bpy concentration can be lowered to 1 mM (from 2 mM in the base case)28 while maintaining high sugar yields. To provide an overview of the two-stage alkaline/CuAHP pretreatment process, a summary of the overall mass balance on 100 g of oven-dried poplar biomass is reported in the Supporting Information (additional file in Scheme S1). Briefly, following enzymatic hydrolysis of the whole quantity of Cu-AHP pretreated material, 38.2 g of glucose and 11.9 g of xylose were released, which corresponded to the yields of 76.9 and 66.3% of the initial glucan and xylan in raw poplar, respectively. Taken together, the results presented in Figure 2 demonstrate that increasing the Cu-AHP pretreatment temperature enables a reduction in both the reaction time and chemical inputs without extensively decreasing the overall sugar yields. 3.3. Correlation between Glucose Yields and the Removal of Lignin and Xylan. The alteration in the bulk composition of the biomass by the two-stage pretreatment (alkaline pre-extraction followed by oxidative Cu-AHP pretreatment) is one of the key outcomes of pretreatment. To provide further insight into how the compositional changes impact the enzymatic digestibility of the biomass, the glucose yields following enzymatic hydrolysis were plotted against the removal of lignin and xylan during pretreatment (Figure 3). Strong positive correlations can be observed between the overall glucose yields and the removal of lignin and xylan, although this correlation weakens slightly at high levels of lignin and xylan removal. The overall correlation is not surprising because these two classes of cell wall biopolymers play a key role in limiting access to cellulose. In addition, these results display a trend similar to the one observed in Figure 2, in which the glucose yields begin to reach a plateau with increasing pretreatment severity. Moreover, in agreement with the literature,48−50 these results also collectively support the F

DOI: 10.1021/acs.iecr.9b00901 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.9b00901.

■

Chemical composition of alkaline pre-extracted biomass under different temperatures; chemical composition of alkaline pre-extracted biomass following Cu-AHP pretreatment under different temperatures, times, H2O2 loadings, and 2,2′-bipyridine concentrations; example of the mass balance of the two-stage alkaline/ Cu-AHP pretreatment process; technoeconomic analysis of the alkaline pre-extraction stage; effects of the secondstage Cu-AHP pretreatment temperature, time, H2O2 loading, and 2,2′-bipyridine concentration on the minimum fuel selling price (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

Figure 4. Reduction in minimum fuel selling price as a percentage from the initial baseline conditions. Alkaline pre-extraction: 10% NaOH loading (100 mg per gram of initial biomass), 10% (w/v, based on initial biomass) solid loading, 30 °C, and a reaction time of 1 h. Cu-AHP pretreatment: 10% NaOH loading (100 mg per gram of initial biomass), 10% H2O2 loading (100 mg per gram of initial biomass), 10% (w/v, based on the initial biomass) solid loading, 1 mM Cu2+, 2 mM bpy, 30 °C, and a reaction time of 23 h. The changes in conditions are (1) increasing the temperatures of the first-stage alkaline pre-extraction and the second Cu-AHP pretreatment to 120 and 80 °C, respectively, (2) decreasing the residence time of the CuAHP pretreatment stage to 12 h, (3) reducing hydrogen peroxide loading to 8% (w/w biomass), (4) decreasing the bpy concentration to 1 mM, and (5) using the Cu-AHP pretreatment medium as the hydrolysate media.

ORCID

Zhaoyang Yuan: 0000-0003-2445-9505 Sandip Kumar Singh: 0000-0003-0094-6253 David B. Hodge: 0000-0002-9313-941X Eric L. Hegg: 0000-0003-2055-5495 Notes

The authors declare the following competing financial interest(s): E.L.H. and D.B.H. are listed as inventors on a related patent (Multi-Ligand Metal Complexes and Methods of Using Same to Perform Oxidative Catalytic Pretreatment of Lignocellulosic Biomass 2015/0352540 A1). As holders of this patent, we may benefit financially from advances in the technology discussed in this article.

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temperature and decreasing the bpy content had a substantial impact on the MFSP because of increasing sugar yields for the former and decreasing the raw material costs for the latter. The single largest impact, however, was due to using the liquid phase of the Cu-AHP pretreatment as the hydrolysis medium. It is imperative that these sugars are not lost during the process, given the large impact this has on the cost of the biofuel. More details on precisely how these sugars, as well as the sugars removed during the pre-extraction phase, can be incorporated into hydrolysis while maintaining a high solids loading require more careful investigation in the future.

ACKNOWLEDGMENTS This work was funded by DOE EERE (DE-EE0008148). The CTec3 and HTec3 enzyme cocktails were kindly provided by Novozymes.

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LIST OF ABBREVIATIONS bpy = 2,2′-bipyridine Cu-AHP = copper-catalyzed alkaline hydrogen peroxide HPLC = high-performance liquid chromatography MFSP = minimum fuel selling price Mg = metric ton TEA = technoeconomic analysis

4. CONCLUSIONS

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Overall, the results in this study demonstrated that increasing the reaction temperature in both the alkaline pre-extraction stage and the Cu-AHP pretreatment stage to 120 and 80 °C, respectively, allow for reductions in the reaction time, H2O2 loading, and bpy concentration while still achieving high overall yields of glucose (∼77% of the initial glucan) and xylose (∼67% of the initial xylan). Technoeconomic analysis revealed that the cost of biofuels produced could be reduced considerably using these modified Cu-AHP pretreatment conditions. As expected, the extents of delignification and xylan solubilization were the two main factors governing the overall glucose hydrolysis yields.

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