Comparison of sodium hydroxide and calcium hydroxide pretreatments of giant reed for enhanced enzymatic digestibility and methane production

Accepted Manuscript Comparison of sodium hydroxide and calcium hydroxide pretreatments of giant reed for enhanced enzymatic digestibility and methane production Danping Jiang, Xumeng Ge, Quanguo Zhang, Xuehua Zhou, Zhou Chen, Harold Keener, Yebo Li PII: DOI: Reference:

S0960-8524(17)31372-X http://dx.doi.org/10.1016/j.biortech.2017.08.067 BITE 18676

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

28 June 2017 9 August 2017 10 August 2017

Please cite this article as: Jiang, D., Ge, X., Zhang, Q., Zhou, X., Chen, Z., Keener, H., Li, Y., Comparison of sodium hydroxide and calcium hydroxide pretreatments of giant reed for enhanced enzymatic digestibility and methane production, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.067

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Comparison of sodium hydroxide and calcium hydroxide pretreatments of giant reed for enhanced enzymatic digestibility and methane production

Danping Jiang a,b, Xumeng Ge c,*, Quanguo Zhang b, Xuehua Zhou b, Zhou Chen a, Harold Keener a, Yebo Li c

a Department of Food, Agricultural and Biological Engineering, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH 44691-4096, United States b Key Laboratory of New Materials and Facilities for Rural Renewable Energy of Agricultural Ministry, Collaborative Innovation Center of Biomass Energy, Henan Agricultural University, Zhengzhou 450002, China c Quasar Energy Group, 2705 Selby Road, Wooster, Ohio 44691, United States

* Corresponding author. Tel: +1 330 202 3561; Fax: +1 330 263 3670 E-mail address: [email protected]; [email protected] (X. Ge).

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Abstract NaOH pretreatment with leachate reuse and Ca(OH)2 pretreatment were compared for improved enzymatic digestibility and biogas production from giant reed, a promising energy crop. The NaOH pretreatment with leachate reuse increased glucose yields during enzymatic hydrolysis by 2.6-fold, and methane yields during anaerobic digestion by 1.4- to 1.6-fold, respectively. However, NaOH pretreatment had a negative net benefit (i.e., revenue from increased energy production minus chemical cost). Pretreatment with 7-20% Ca(OH)2 not only improved glucose yield and methane yield by up to 2.3-fold and 1.4-fold, respectively, but also obtained a net benefit of $1.1-5.8 /tonne dry biomass. Thus, Ca(OH)2 pretreatment was shown to be more feasible than NaOH pretreatment for biogas production from giant reed.

Keywords Energy crop; Alkaline pretreatment; Enzymatic digestibility; Biogas production; Benefit-cost analysis

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1. Introduction Giant reed (Arundo donax L.), a perennial rhizomatous grass, has been recognized as a desirable energy crop, because it has a high biomass yield (30-40 tonnes of dry matter per hectare per year) and can adapt to different types of soils and weather conditions (Cavallaro et al., 2014; Ge et al., 2016; Jiang et al., 2016; Liu et al., 2016). Giant reed has been studied for bioenergy production via various approaches, including anaerobic digestion (AD), ethanol fermentation, combustion, and other thermal conversion methods (Ge et al., 2016; Liu et al., 2016; Ragaglini et al., 2014). Among these approaches, AD is one of the most promising options because it can produce biogas (60-70% methane) with a relatively simple reaction system, a robust process, and a low greenhouse gas footprint (Di Girolamo et al., 2013; Petersson et al., 2007) (Corno et al., (2015)). Planting giant reed for biogas production could be more helpful for improving environmental sustainability and saving limited water resources than planting maize as an energy crop (Baldini et al., 2017; Impagliazzo et al., 2017). Giant reed is a lignocellulosic biomass with a complex and rigid structure that is recalcitrant to digestion (Di Girolamo et al., 2013). As a result, pretreatment is generally needed to improve enzymatic digestibility and bioenergy production from giant reed (Di Girolamo et al., 2013). Pretreatment methods can be classified into mechanical, thermal, chemical, and biological (Di Girolamo et al., 2013; Ge et al., 2016). One of the leading chemical pretreatment methods is alkaline pretreatment, which can make the lignocellulosic biomass more accessible for enzymes and bacteria by solvation and saponification reactions (Hendriks and Zeeman, 2009; Xu and Cheng, 2011), and has been widely utilized for improving digestibility of lignocellulosic biomass, especially agricultural residues and herbaceous crops (Xu and Cheng, 2011). Compared to other pretreatment methods, alkaline pretreatment is particularly effective in depolymerization of lignin, the most recalcitrant component of lignocellulose, and does not require 3

harsh conditions (i.e., high temperature and high pressure) (Di Girolamo et al., 2014). In one study, using the same giant reed biomass, alkaline pretreatment with 20% sodium hydroxide (NaOH) improved the net electrical energy production by 27%, while liquid hot water pretreatment obtained a negative net electrical energy production due to high energy input (Jiang et al., 2016). However, a major drawback of NaOH pretreatment was the relatively high chemical input ($450/tonne), which resulted in a lower net benefit of electricity generation compared to non-pretreatment (Jiang et al., 2016). Reuse of alkaline leachate recovered from the pretreatment system (leachate reuse) was proposed by Park et al. ( 2010) to reduce costs, but it has not been evaluated for AD of giant reed. Another strategy to reduce chemical inputs is to use less costly chemicals, such as calcium hydroxide (Ca(OH)2). Compared to the cost of NaOH ($450/tonne), Ca(OH)2 is less expensive ($116/tonne), and safer to handle (Miller, 2014; Park et al., 2010; Saha and Cotta, 2008; Sierra et al., 2009; Xu et al., 2010). In addition, Ca(OH)2 pretreatment can avoid significant loss of carbohydrates compared to more severe alkaline pretreatment (Park et al., 2010; Sierra et al., 2009). A drawback of Ca(OH)2 is its poor solubility, which limits the reuse of leachate after Ca(OH)2 pretreatment. Ca(OH)2 pretreatments have been studied on rice straw, switchgrass, sugarcane bagasse, corn stover, and wheat straw (Kim et al., 2016; Xu and Cheng, 2011), mainly for enzymatic saccharification and ethanol production (Park et al., 2010; Rabelo et al., 2009; Saha and Cotta, 2008; Xu et al., 2010; Xu and Cheng, 2011). To date, there have been no reports on NaOH pretreatment of giant reed with leachate reuse or on Ca(OH)2 pretreatment of giant reed. As a result, it is unclear whether reuse of NaOH leachate or using Ca(OH)2 can improve the net benefit (i.e., revenue from increased energy production minus chemical cost) of AD of giant reed, and which strategy is more feasible. Furthermore, to the best of authors’ knowledge,

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comparison of NaOH pretreatment with leachate reuse and Ca(OH)2 pretreatment has not been made with any lignocellulosic biomass. The objectives of this study were to 1) evaluate both NaOH pretreatment of giant reed with leachate reuse and Ca(OH)2 pretreatment of giant reed for improved enzymatic hydrolysis and biogas production, and 2) compare these two pretreatment strategies, considering energy/chemical inputs and energy outputs. In order to achieve these objectives, the NaOH pretreatment was conducted in consecutive batches, with the leachate reused for the next batch, and the Ca(OH)2 pretreatment was conducted with different loading rates. All tests used the same source of giant reed biomass. Enzymatic hydrolysis and AD of pretreated giant reed biomass were carried out, respectively. The biomass composition and recovery during pretreatment, sugar yield by enzymatic hydrolysis, and methane yield by AD from pretreated giant reed biomass were measured and compared. The revenue and net benefit from NaOH and Ca(OH)2 pretreatments of giant reed were estimated.

2. Methods and materials 2.1. Raw materials and inoculum for AD Giant reed was collected from the Waterman Farm (Columbus, OH, USA) in August 2016, ground to a ½ inch sieve using a shredder-chipper (Mighty Mac, Mackissic Inc., Parker Ford, PA, USA), and stored in an airtight container at room temperature prior to use. The inoculum for AD was the effluent obtained from a mesophilic liquid anaerobic digester (KB BioEnergy, Akron, OH, USA) that was fed with biosolids. The effluent was stored at 4
and acclimated at 37
for 1 week before use.

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2.2. NaOH pretreatment with leachate reuse For the first batch of NaOH pretreatment, 20 g of giant reed (dry mass) was soaked in a 1-L flask containing 200 mL of 20 g/L NaOH solution to reach a loading ratio of 20% (g NaOH /g initial total solids (TS) of giant reed biomass). The flask was covered with Parafilm and incubated at room temperature (25±1
) for 24h. After collecting the leachate, the pretreated giant reed biomass was washed with tap water using a sieve (325 mesh) until the pH reached around 8, and then 1 N HCl was employed to neutralize the mixture to reach a pH value of about 7.0. The neutralized giant reed was then drained, washed, and dried for further analysis. The second, third, fourth and fifth batches of NaOH pretreatment were conducted in the same manner, except that 100 mL of leachate from the former batch was used to replace half of the 20 g/L NaOH solution, i.e. the NaOH loading was reduced to 10% (g NaOH /g initial TS of giant reed biomass).

2.3. Ca(OH)2 pretreatment Twenty grams of giant reed (dry mass) was added to 200 mL of deionized (DI) water in a 1-L flask with Ca(OH)2 addition of 0.2, 0.6, 1.0, 1.4, 2.4, or 4.0 (g). The loading ratios were 1%, 3%, 5%, 7%, 12%, and 20% (g Ca(OH)2 /g initial TS of giant reed biomass), respectively. The flasks were covered with Parafilm and incubated at room temperature (25±1
) for 24 h. The Ca(OH)2 pretreated giant reed was processed using the same procedure used for the NaOH pretreatment (Section 2.2).

2.4. Enzymatic hydrolysis

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Enzymatic hydrolysis was conducted in 20-mL tubes each containing 10 mL of 0.05 M citrate buffer (pH 4.8) based on an NREL Laboratory Analytical Procedure (Selig et al., 2008). Each tube was loaded with untreated or pretreated giant reed (0.25 g dry matter) and the enzyme Cellic CTec2 (Novozymes, Bagsværd, Denmark) to achieve a cellulase concentration of 12 FPU/ g dry matter. Reaction blanks were prepared by loading only giant reed biomass or enzyme to the tube. All tubes were incubated at 50
with shaking at 200 rpm for 72 h, and then the hydrolysate was filtered using Whatman #4 filter paper. After that, the filtrate was collected in a 20-mL tube, which was then boiled for 5 min in order to deactivate the enzyme. The deactivated filtrate was further centrifuged (7826 ×g, 10 min), and then filtered through a 0.2 µm nylon filter before analysis by high performance liquid chromatography (HPLC) (LC-20AB, Shimadzu, Kyoto, Japan). Glucose yield and enzymatic digestibility were calculated according to the following equations:

where Mglucose is the amount of glucose (g) released from pretreated giant reed by enzymatic hydrolysis, Mcellulose is the amount of cellulose (g) in raw giant reed before pretreatment, Ncellulose is the amount of cellulose (g) in pretreated giant reed, and

is the conversion factor

for cellulose to glucose (Jiang et al., 2016; Liu et al., 2015).

2.5. Anaerobic digestion (AD) of giant reed AD was set up by mixing untreated or pretreated giant reed biomass (feedstock), inoculum, and DI water to obtain a feedstock to inoculum (F/I) ratio of 0.5 (w/w, based on 7

volatile solids), and a total solids (TS) content of 5% (w/w). AD test was conducted in 1-L reactors, and each of them was loaded with 600 g of the mixture, sealed with a rubber stopper with an outlet connected to a 5-L Tedlar gas bag (CEL Scientific, Santa Fe Springs, CA, USA), and incubated at 37±1
for 30 days. Biogas volume and composition were measured every 3-5 days during the 30-day AD period. AD with only inoculum was run as a control. Triplicate reactors were run for each condition.

2.6. Analysis methods Total solids (TS), volatile solids (VS), and pH of samples were measured following the Standard Methods for Examination of Water and Wastewater (APHAAmerican Public Health Association. Standard Methods for the Examination of Water and Wastewater, 2006). An elemental analyzer (Elementar Vario Max CNS, Elementar Americas, Mt. Laure, NJ, USA) was employed to determine total carbon (TC) and total nitrogen (TN). The carbon to nitrogen (C/N) ratio was then calculated based on the TC and TN contents. The total ammonia nitrogen (TAN) was determined according to a modified distillation method (ISO, 1984) with a Kjeldahl Distillation Unit B-324 (Buchi, Labortechnik, AG, Switzerland). Dry matter recovery of solids was calculated as the percentage of the water-insoluble mass in the pretreated slurry to the TS in the untreated giant reed. The concentration of NaOH in the pretreatment wastewater was determined by titrating with 0.1N hydrochloric acid using phenolphthalein as the indicator. Cellulose, hemicellulose, and lignin contents were measured following an NREL Laboratory Analytical Procedure (Sluiter et al., 2012). Briefly, samples were extracted with DI water and ethanol using Accelerated Solvent Extraction (ASE 350, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Extractive-free samples were subjected to a two-step acid hydrolysis to release mono-sugars. Sugar analysis was conducted using the HPLC) equipped with an 8

Aminex HPX-87P column (Bio- Rad, Inc., Hercules, CA, USA) and a refractive index detector (RID).The HPLC grade water at a flow rate of 0.3 mL/min was used as the mobile phase. The temperature of the column and detector were set at 60 and 55
, respectively. Cellulose and hemicellulose amounts were calculated by multiplying the amounts of C-6 and C-5 sugars with factors of 0.9 and 0.88, respectively. The acid-soluble lignin and acid-insoluble lignin were determined by ultraviolet-visible (UV-vis) spectroscopy (BioMate 3S spectrophotometer, Waltham, MA, USA) and gravimetric analysis, respectively. Volatile fatty acids (VFAs) in the digestate were measured using a gas chromatograph (GC) (Shimadzu, GC-2010, Columbia, MD, USA) equipped with a 30 m × 0.32 mm × 0.5 µm Stabilwax-DA column and a flame ionization detector (FID). The temperatures for the column and FID were 150 ºC and 250 ºC, respectively. Helium gas was used as the mobile phase at a flow rate of 16.9 mL/min. About 10 g of sample was centrifuged at 7826×g for 10 min. The supernatant was acidified to a pH of around 4.0 using 2 N hydrochloric acid. The acidified solution was filtered through a 0.2 µm nylon syringe filter before GC analysis. Biogas composition (CH4, CO2, N2, and O2) was analyzed by a gas chromatograph (Agilent, HP 6890, Wilmington, DE, USA) equipped with a 30 m× 0.53 mm× 10 µm alumina/KCl deactivation column and a thermal conductivity detector (TCD). Helium gas was used as a carrier gas, and the flow rate was controlled at 5.2 mL/min. The column oven was initially set at 40 ˚C for 4 min and then increased to 60
with an increment of 20
/min and held at 60
for 5 min. The temperature of the column and detector were set at 40
and 200
, respectively. The volume of biogas in the Tedlar gas bag was measured by a drum-type gas meter (Ritter, Bochum, Germany).

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2.7. Energy and cost analysis The methane (CH4) yield based on the initial TS of giant reed biomass was calculated using: (3) Biomass energy production from giant reed biomass was calculated as the product of the initial TS- based methane yield, the lower heating value of methane (33 kЈ/L at 25
and 101 kPa), and an assumed effective electric efficiency of 0.6 for a combined heat and power (CHP) system (USEPA, 2013). The benefit of electricity generation ($/tonne initial TS) was calculated by multiplying the biomass energy production by 1000 (kg/tonne) and the average electricity price ($ 2.91×10-5/kЈ or 10.48 cents/kW h) (USEIA, 2016). The revenue due to pretreatment was calculated by subtracting the value of electricity generation for untreated giant reed from that of pretreated giant reed. The cost for chemical inputs ($/tonne initial TS) was estimated based on the loading ratio and price of Ca(OH)2 ($116/tonne) (Miller, 2014) or NaOH ($450/tonne) (Kumar and Murthy, 2011). The revenue due to pretreatment minus the cost for chemical inputs was determined and reported as the net benefit from pretreatment.

2.8. Statistical analysis Statistical significance was assessed by analysis of variance (ANOVA) (α = 0.05) using Minitab (Version 17, Minitab, Inc., State College, PA, USA).

3. Results and discussion 3.1. Effect of NaOH and Ca(OH)2 pretreatments on biomass composition and recovery The leachate obtained during each batch of NaOH pretreatment was about 120 mL with a pH of about 12.5 (Table 1). As shown in Table 1, leachate generation was stable during 10

consecutive batches of NaOH pretreatment, with no significant differences in volume and pH value of leachate among 5 batches of NaOH pretreatment. After the 1st batch of NaOH pretreatment (20% NaOH), the cellulose content of giant reed increased from 34.9% to 40.5%, the xylan content remained nearly at the same level, and the lignin content decreased from 20.1% to 15.1% (Table 1). Compared to the 1st batch of NaOH pretreatment, the 2nd batch (10% NaOH + leachate from 1st batch) obtained a similar change in biomass composition, while the 3rd-5th batches (each with 10% NaOH + leachate from the previous batch) showed less of an increase in cellulose content and less of a decrease in lignin content (Table 1). The last three batches of NaOH pretreatments resulted in a very consistent biomass composition, with 36.7-37.5% cellulose, 16.7-17.5% xylan, and 17.0-17.7% lignin (Table 1). For the Ca(OH)2 pretreatment of giant reed, higher loading rates resulted in a smaller volume of leachate after pretreatment, which indicated that the water holding capacity of pretreated biomass increased with Ca(OH)2 loading (Table 1). During alkaline pretreatment, ester bonds between hemicellulose and lignin are broken, leading to a swelling of pretreated biomass, which increases the porosity and internal surface area (Di Girolamo et al., 2014). Therefore, more links between lignin and structural carbohydrates could be loosened as more Ca(OH)2 is added, resulting in a pretreated biomass that can hold more water. After pretreatment with 1% Ca(OH)2, the pH in the leachate was similar to that of untreated giant reed, which implies that the 1% loading was too low for pretreatment (Table 1). The pH in leachate increased with the loading of Ca(OH)2, and 7-20% Ca(OH)2 obtained comparable pH values (12.0-12.4) to those of the 10-20% NaOH pretreatment (Table 1). Ca(OH)2 pretreatment with lower loadings (1%, 3%, and 5%) gave lower cellulose contents (31.1%, 33.3%, and 34.7%, respectively) compared to that of untreated giant reed (34.9%). Ca(OH)2 pretreatment with further increased

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loadings (7-20%) achieved higher cellulose contents (36.0-36.9%) compared to untreated giant reed (Table 1). Both xylan contents and lignin contents obtained from Ca(OH)2 pretreatment were lower than those in the untreated giant reed, except for the lignin content with Ca(OH)2 loading of 1%, which obtained 21.1%. The xylan contents increased from 13.7% to 17.1% when Ca(OH)2 loading increased from 1% to 7%, while higher Ca(OH)2 loadings (12-20%) led to a decrease of xylan contents from 16.9% to 16.7%. Lignin contents decreased gradually from 21.1% to 17.6% as Ca(OH)2 loading increased from 1% to 20% (Table 1). At the same dosage of 20%, Ca(OH)2 pretreatment of giant reed resulted in lower cellulose content and xylan content, and higher lignin content, compared to NaOH pretreatment. The reason could be that NaOH is more effective than Ca(OH)2 for alkaline pretreatment of giant reed at the same dosage, which is supported by the results of biomass recovery as described below. Figure 1 shows a comparison of biomass recovery between NaOH pretreatment with leachate reuse and Ca(OH)2 pretreatment with different loadings. Both the NaOH pretreatment and Ca(OH)2 pretreatment recovered more than 77% of the cellulose and more than 73% of the xylan from giant reed biomass (Figure 1). The NaOH pretreatment with leachate reuse recovered 74-78% of the dry matter, while Ca(OH)2 pretreatment with different loadings recovered 92-95% of the dry matter (Figure 1). Ca(OH)2 is less severe than NaOH, thus Ca(OH)2 pretreatment can recover more cellulose, xylan, and dry matter, but will have lower lignin removal than NaOH pretreatment at the same loading ratio (Kim et al., 2016). Lignin removal efficiencies of 36-48% and 2-19% were obtained during NaOH pretreatment and Ca(OH)2 pretreatment, respectively (Figure 1). Similar lignin removal (4.4-24.3%) was reported in a study on Ca(OH)2 pretreatment of rice straw with 4-12% Ca(OH)2 (Song et al., 2013). Besides, the lower lignin removal by Ca(OH)2 pretreatment than by NaOH pretreatment could also be attributed to the formation of a

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calcium-lignin complex, which decreases lignin solubility during pretreatment processes (Song et al., 2013).

3.2. Effect of NaOH and Ca(OH)2 pretreatments on glucose yield from giant reed biomass During NaOH pretreatment with leachate reuse, the 1st batch obtained a glucose yield of 51% which was 3 times that of untreated giant reed biomass (16%), while glucose yields for the 2nd-5th batches were 42-43%, which was about 2.5 times that of untreated giant reed biomass (Figure 2). The glucose yield was slightly improved by pretreatment with 1% Ca(OH)2, and increased gradually up to 35% with the increase of Ca(OH)2 loading. Glucose yields obtained by pretreatment with 3-20% Ca(OH)2 were 1.5- to 2.3-fold that of untreated giant reed (Figure 2). As presented in Figure 3, the enzymatic digestibility of both Ca(OH)2 pretreatment and NaOH pretreatment were correlated with lignin removal. This result was consistent with our previous study on alkaline pretreatment of giant reed with different NaOH loadings, in which higher lignin removal led to higher enzymatic digestibility (Jiang et al., 2016). Ca(OH)2 pretreatment was less effective than NaOH pretreatment in improving glucose yield from giant reed, which could be explained by the lower lignin removal efficiency of Ca(OH)2 pretreatment (Figure 1).

3.3. Effect of NaOH and Ca(OH)2 pretreatments on methane production from AD of giant reed The daily methane yields of NaOH pretreated giant reed peaked at day 6, and showed higher maximum daily methane yields than untreated giant reed (Figure 4a). The 1st batch of NaOH pretreatment obtained the highest maximum daily methane yield (27.4 L/kg VS/d), and the 2nd-5th batches showed a stable methane production with similar maximum daily methane yields (about 20 L/kg VS/d) (Figure 4a). Ca(OH)2 pretreatment at loading rates of 1-5% did not improve the daily methane yield (Figure 4b). Giant reed pretreated with 7-20% Ca(OH)2 showed 13

higher daily methane yields than untreated biomass after day 6, although the improvement was lower than those from the NaOH pretreatment (Figure 4a and 4b). Generally, methane contents from untreated giant reed and treated giant reed increased to about 50% in 9 days, which indicated healthy AD processes (Figure 4c and 4d). The effect of pretreatment on cumulative methane yield from giant reed is further shown in Figure 5. The 1st batch of NaOH pretreatment achieved the highest methane yield (353 L/kg VS), which was 61% higher than that of untreated giant reed (213 L/kg VS) (Figure 5). The 2nd5th batches of pretreatment obtained cumulative methane yield of 341, 310, 304, and 306 L/kg VS, respectively, with a 40-56% increase (Figure 5). These methane yields were close to those (316-355 L/kg VS) reported in a previous study using 12- 20% NaOH for pretreatment of giant reed (Jiang et al., 2016). Pretreatment with 1-5% Ca(OH)2 did not improve cumulative methane yields, while pretreatment with higher Ca(OH)2 loadings (7%-20%) significantly (p<0.05) improved cumulative methane yield with a 7-34% increase (Figure 5). Based on the results of cumulative methane yields from this study, NaOH pretreatment was more effective than Ca(OH)2 pretreatment in increasing methane yield from giant reed, which is also consistent with its performance in reducing lignin (Figure 1) and improving glucose yield (Figure 2). These results were further demonstrated by final VFAs as shown in Table 2. VFAs were detected in the AD digestate of untreated and Ca(OH)2 pretreated giant reed, while no VFAs were found in the digestate of NaOH pretreated giant reed (Table 2). Besides, the amount of VFAs decreased with Ca(OH)2 loading (Table 2). It is notable that the accumulation of VFAs during the AD process indicates inadequate degradation of biomass (Cui et al., 2011). The pH of untreated giant reed after AD was lower than those of giant reed pretreated with Ca(OH)2 and NaOH leachate reuse, which supported the results of VFAs (Table 2). Besides,

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more CO2 could be dissolved in digestate at higher pH, resulting in lower CO2 content in the gas phase as shown in Figure 4c and 4d. This result was also consistent with that observed in another study of lime treatment of smooth cordgrass for biogas production (Liang et al., 2011).

3.4. Energy production and cost-efficiency analysis Based on its methane yield, untreated giant reed could generate 3853 kЈ/kg initial TS of electrical energy via AD with CHP (Table 3), which was comparable to that obtained in a previous study using the same giant reed biomass (Jiang et al., 2016). As described in the previous study, NaOH pretreatment increased biomass energy production to 4918 kЈ/kg initial TS with 20% NaOH, and a similar result was obtained in this study with the same condition (the 1st batch of NaOH pretreatment) (Ge et al., 2015; Jiang et al., 2016; Monlau et al., 2012; Zhao et al., 2014; Zheng et al., 2009; Zhu et al., 2010). However, the additional revenue due to pretreatment was lower than the cost of chemical inputs, leading to a negative net benefit from pretreatment (Table 3). During consecutive batches with leachate reuse, biomass energy production decreased from 4886 to 4433 kЈ/kg initial TS, which was still higher than that of untreated giant reed. Since half of the NaOH solution was replaced with leachate from the former batch, the chemical input cost was also reduced by half (from $90 to $45/tonne initial TS). However, the revenue due to consecutive batches of NaOH pretreatment with leachate reuse did not offset the cost for NaOH input, which still resulted in negative net benefits from pretreatment (Table 3). As shown in Table 3, the biomass energy produced for 1-5% Ca(OH)2 pretreated giant reed was 3383, 3620, and 3992 kЈ/kg initial TS, respectively, all of which were lower than or close to that of untreated giant reed (3853 kЈ/kg initial TS) due to the dry matter loss during pretreatment (Figure 1) and the unimproved methane yield during AD (Figure 4). Therefore, 15

both the revenue and the net benefit for pretreatment with 1-5% Ca(OH)2 was negative. When the Ca(OH)2 loading rate was increased to 7%, the biomass energy production was significantly improved to 4334 kЈ/kg initial TS, and the benefit of electricity generation was increased to $125.2/tonne TS. Pretreatment with 7% Ca(OH)2 achieved a revenue of $13.9/tonne TS with a chemical input cost of $8.1/tonne TS, resulting in a net benefit of $5.8/tonne TS. Increasing the Ca(OH)2 loading to 12-20% further improved the biomass energy production, benefit of electricity generation and revenue of pretreatment to 4485-4693 kЈ/kg initial TS, $129.6-135.6, and $18.3-24.3/tonne TS, respectively. Pretreatment with 12% and 20% Ca(OH)2 obtained net benefits of $4.4/tonne TS and $1.1/tonne TS, respectively. Compared to 7% Ca(OH)2, the 1220% Ca(OH)2 did not further increase the net benefit due to the increased cost of chemical inputs (Table 3). In summary, pretreatment of giant reed with 7-20% Ca(OH)2 can have positive net benefits, and thus is more feasible than NaOH pretreatment for biogas production from giant reed via AD with CHP.

4. Conclusions NaOH pretreatment with/without leachate reuse was more effective than Ca(OH)2 pretreatment for removing lignin, improving glucose yield from giant reed biomass by enzymatic hydrolysis and increasing methane yield by AD. However, the improvement in methane yield did not offset the high cost of NaOH, resulting in a negative net benefit from NaOH pretreatment. In contrast, Ca(OH)2 pretreatment at a loading rate of 7-20% not only increased the glucose yield and methane yield from giant reed, but also had positive net benefits of $1.1-5.8/tonne TS due to the low cost of Ca(OH)2.

Acknowledgement 16

This study is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2012-10008-20302 (U.S.); by state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research and Development Center (U.S.); and by the Collaborative Innovation Center of Biomass Energy, Henan Province (China). The authors thank Mrs. Mary Wicks for her comprehensive review and thoughtful comments.

References 1. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. 21st ed. Washington, DC. 2. Baldini, M., da Borso, F., Ferfuia, C., Zuliani, F., Danuso, F., 2017. Ensilage suitability and bio-methane yield of Arundo donax and Miscanthus × giganteus. Ind. Crops Prod. 95, 264–275. 3. Cavallaro, V., Patanè, C., Cosentino, S.L., Di Silvestro, I., Copani, V., 2014. Optimizing invitro large scale production of giant reed (Arundo donax L.) by liquid medium culture. Biomass Bioenergy 69, 21–27. 4. Corno, L., Pilu, R., Tambone, F., Scaglia, B., Adani, F., 2015. New energy crop giant cane (Arundo donax L.) can substitute traditional energy crops increasing biogas yield and reducing costs. Bioresour. Technol. 191, 197–204. 5. Cui, Z., Shi, J., Li, Y., 2011. Solid-state anaerobic digestion of spent wheat straw from horse stall. Bioresour. Technol. 102, 9432–9437. 6. Di Girolamo, G., Bertin, L., Capecchi, L., Ciavatta, C., Barbanti, L., 2014. Mild alkaline pre-treatments loosen fibre structure enhancing methane production from biomass crops and residues. Biomass Bioenergy 71, 318–329. 7. Di Girolamo, G., Grigatti, M., Barbanti, L., Angelidaki, I., 2013. Effects of hydrothermal pre-treatments on Giant reed (Arundo donax) methane yield. Bioresour. Technol. 147, 152–159. 8. Ge, X., Matsumoto, T., Keith, L., Li, Y., 2015. Fungal pretreatment of Albizia chips for enhanced biogas production by solid-state anaerobic digestion. Energy Fuels 29, 200–204. 9. Ge, X., Xu, F., Vasco-correa, J., Li, Y., 2016. Giant reed : A competitive energy crop in comparison with miscanthus. Renew. Sustain. Energy Rev. 54, 350–362. 10. Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10–18. 11. Impagliazzo, A., Mori, M., Fiorentino, N., Di Mola, I., Ottaiano, L., De Gianni, D., 17

Nocerino, S., Fagnano, M., 2017. Crop growth analysis and yield of a lignocellulosic biomass crop (Arundo donax L.) in three marginal areas of Campania region. Ital. J. Agron. 12, 1–7. 12. ISO, 1984. Water Quality – Determination of Ammonium – Distillation and Titration Method. ISO 5664, International Standard Organization, Geneva. 13. Jiang, D., Ge, X., Zhang, Q., Li, Y., 2016. Comparison of liquid hot water and alkaline pretreatments of giant reed for improved enzymatic digestibility and biogas energy production. Bioresour. Technol. 216, 60–68. 14. Kim, J.S., Lee, Y.Y., Kim, T.H., 2016. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour. Technol. 199, 42–48. 15. Kumar, D., Murthy, G.S., 2011. Impact of pretreatment and downstream processing technologies on economics and energy in cellulosic ethanol production. Biotechnol. Biofuels 4, 2–19. 16. Liang, Y., Zheng, Z., Hua, R., Luo, X., 2011. A preliminary study of simultaneous lime treatment and dry digestion of smooth cordgrass for biogas production. Chem. Eng. J. 174, 175–181. 17. Liu, S., Ge, X., Liu, Z., Li, Y., 2016. Effect of harvest date on Arundo donax L. (giant reed) composition, ensilage performance, and enzymatic digestibility. Bioresour. Technol. 205, 97–103. 18. Liu, S., Ge, X., Niee, L., Liu, Z., Li, Y., 2015. Effect of urea addition on giant reed ensilage and subsequent methane production by anaerobic digestion. Bioresour. Technol. 192, 682–688. 19. Miller, M., 2014. Lime. Available at: . 20. Monlau, F., Barakat, A., Steyer, J.P., Carrere, H., 2012. Comparison of seven types of thermo-chemical pretreatments on the structural features and anaerobic digestion of sunflower stalks. Bioresour. Technol. 120, 241–247. 21. Park, J.Y., Shiroma, R., Al-Haq, M.I., Zhang, Y., Ike, M., Arai-Sanoh, Y., Ida, A., Kondo, M., Tokuyasu, K., 2010. A novel lime pretreatment for subsequent bioethanol production from rice straw - Calcium capturing by carbonation (CaCCO) process. Bioresour. Technol. 101, 6805–6811. 22. Petersson, A., Thomsen, M.H., Hauggaard-Nielsen, H., Thomsen, A.B., 2007. Potential bioethanol and biogas production using lignocellulosic biomass from winter rye, oilseed rape and faba bean. Biomass Bioenergy 31, 812–819. 23. Rabelo, S.C., Filho, R.M., Costa, A.C., 2009. Lime pretreatment of sugarcane bagasse for bioethanol production. Appl. Biochem. Biotechnol. 153, 139–150. 24. Ragaglini, G., Dragoni, F., Simone, M., Bonari, E., 2014. Suitability of giant reed (Arundo donax L.) for anaerobic digestion: Effect of harvest time and frequency on the biomethane yield potential. Bioresour. Technol. 152, 107–115.

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25. Saha, B.C., Cotta, M.A., 2008. Lime pretreatment, enzymatic saccharification and fermentation of rice hulls to ethanol. Biomass Bioenergy 32, 971–977. 26. Selig, M.J., Weiss, N., Ji, Y., 2008. Enzymatic Saccharification of Lignocellulosic Biomass, Technical Report NREL/TP-510-42629. 27. Sierra, R., Granda, C., Holtzapple, M.T., 2009. Short-term lime pretreatment of poplar wood. Biotechnol. Prog. 25, 323–332. 28. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2012. Determination of structural carbohydrates and lignin in Biomass, Technical Report NREL/TP-510-42618. 29. Song, Z. lin, Yag, G. he, Feng, Y. zhong, Ren, G. xin, Han, X. hui, 2013. Pretreatment of rice straw by hydrogen peroxide for enhanced methane yield. J. Integr. Agric. 12, 1258– 1266. 30. USEIA, 2016. Annual Energy Outlook 2016 with projections to 2040, Available at :. 31. USEPA, 2013. Methods for Calculating Efficiency, Available at: . 32. Xu, J., Cheng, J.J., 2011. Pretreatment of switchgrass for sugar production with the combination of sodium hydroxide and lime. Bioresour. Technol. 102, 3861–3868. 33. Xu, J., Cheng, J.J., Sharma-shivappa, R.R., Burns, J.C., 2010. Lime pretreatment of switchgrass at mild temperatures for ethanol production. Bioresour. Technol. 101, 2900– 2903. 34. Zhao, J., Ge, X., Vasco-correa, J., Li, Y., 2014. Fungal pretreatment of unsterilized yard trimmings for enhanced methane production by solid-state anaerobic digestion. Bioresour. Technol. 158, 248–252. 35. Zheng, M., Li, X., Li, L., Yang, X., He, Y., 2009. Enhancing anaerobic biogasification of corn stover through wet state NaOH pretreatment. Bioresour. Technol. 100, 5140–5145. 36. Zhu, J., Wan, C., Li, Y., 2010. Enhanced solid-state anaerobic digestion of corn stover by alkaline pretreatment. Bioresour. Technol. 101, 7523–7528.

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Table 1 Effect of NaOH and Ca(OH)2 pretreatments on composition of giant reed biomass

Chemical loading rate (% DM of biomass)

Leachate

Composition (% DM of biomass)

Volume (mL)

pH

Cellulose

Xylan

Lignin

34.9±2.6

17.4±1.6

20.1±1.1

No pretreatment N/A

N/A

5.5

NaOH pretreatment (Consecutive reuse of leachate) 20 (1st batch)

120

12.4

40.5

17.1

15.1

10 (2nd batch)

122

12.6

40.7

18.2

16.3

10 (3rd batch)

123

12.5

36.7

17.1

17.4

10 (4th batch)

120

12.4

37.2

16.7

17.7

10 (5th batch)

120

12.4

37.5

17.5

17.0

Ca(OH)2 pretreatment (Single-Batch) 1

134

5.7

31.1

13.7

21.1

3

132

9.3

33.3

15.6

20.4

5

130

10.7

34.7

16.8

19.9

7

129

12.0

36.5

17.1

19.5

12

128

12.3

36.9

16.9

18.0

20

120

12.4

36.0

16.7

17.6

DM: dry matter N/A: not applicable.

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Table 2 Effect of NaOH and Ca(OH)2 pretreatments on final pH, alkalinity and VFAs after AD Pretreatment conditions

Final pH

Alkalinity (g/kg)

VFAs (g/kg)

Untreated

7.66±0.08

7.08±0.02

0.48±0.20

1st batch, NaOH

7.72±0.05

6.79±0.20

ND

2nd batch, NaOH

7.89±0.02

6.57±0.10

ND

3rd batch, NaOH

8.07±0.13

7.03±0.41

ND

4th batch, NaOH

8.03±0.02

6.70±0.03

ND

5th batch, NaOH

7.91±0.10

6.81±0.17

ND

1%, Ca(OH)2

7.73±0.27

6.92±0.05

0.16±0.14

3%, Ca(OH)2

7.75±0.28

6.93±0.04

0.09±0.08

5%, Ca(OH)2

8.06±0.08

6.94±0.09

0.07±0.06

7%, Ca(OH)2

8.07±0.03

6.92±0.09

0.06±0.05

12 %, Ca(OH)2

8.02±0.02

6.95±0.04

0.05±0.04

20 %, Ca(OH)2

8.08±0.01

6.78±0.17

0.02±0.04

VFAs=Acetic acid + propionic acid + isobutyric acid + butyric acid + isovaleric acid + valeric acid ND: not detectable

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Table 3 Effect of NaOH and Ca(OH)2 pretreatments on net benefit of electricity generation from using biogas from AD of giant reed biomass.

Chemical loading rate (% DM of biomass)

Gross biomass energy production (kJ/kg TS)

Benefit of electricity generation ($/tonne TS)

Revenue due to pretreatment ($/tonne TS)

Cost for chemical input ($/tonne TS)

Net benefit from pretreatment ($/tonne TS)

0

0

0

Untreated None

3853

*

111.3

NaOH pretreatment (Consecutive batch with leachate reuse) 20 (1st batch)

4886

141.2

29.9

90.0

Negative

10 (2nd batch)

4831

139.6

28.3

45.0

Negative

10 (3rd batch)

4512

130.4

19.1

45.0

Negative

10 (4th batch)

4415

128.6

16.3

45.0

Negative

10 (5th batch)

4433

128.1

16.8

45.0

Negative

Ca(OH)2 pretreatment (Single-Batch) 1

3383

97.8

Negative

1.2

Negative

3

3620

104.6

Negative

3.5

Negative

5

3992

115.4

4.1

5.8

Negative

7

4334

125.2

13.9

8.1

5.8

12

4485

129.6

18.3

13.9

4.4

20

4693

135.6

24.3

23.2

1.1

Note: All values are based on initial TS of giant reed biomass. *: Gross biomass energy production = methane yield × VS%-dry × solid recovery × lower heating value of methane × effective electric efficiency = 213 L/kg VS × 91.36% kg VS/ kg TS × 100% × 33 kЈ/L × 0.6 = 3853 kJ/kg TS.

22

32 33

34 35

36 37

38 39 40 41 42

Figure 1. Effect of NaOH and Ca(OH)2 pretreatments on (a) cellulose recovery, (b) xylan recovery, (c) lignin removal, and (d) dry matter recovery of giant reed biomass

23

43 44 45

Figure 2. Effect of NaOH and Ca(OH)2 pretreatments on glucose yield from enzymatic hydrolysis of giant reed biomass

46 47 48 49 50 51 52 53 54 55 56 57 58 59

24

60 61 62

Figure 3. Correlation between lignin removal and enzymatic digestibility of giant reed pretreated by NaOH or Ca(OH)2

63 64 65 66 67 68 69 70 71 72 73 74

25

(a)

(b)

(c)

(d)

Figure 4. Daily methane yield and methane content during AD of giant reed biomass pretreated with NaOH and Ca(OH)2

26

Figure 5. Cumulative methane yield during AD of giant reed biomass pretreated with NaOH or Ca(OH)2

27

Highlights

•

Sodium hydroxide and calcium hydroxide pretreatments of giant reed were compared.

•

Revenue from sodium hydroxide (NaOH) pretreatment can’t offset the chemical input.

•

Calcium hydroxide (Ca(OH)2) pretreatment improved glucose yield and methane yield.

•

Pretreatment with 7% Ca(OH)2 had a positive net benefit of $5.8/tonne dry biomass.

•

Ca(OH)2 is more feasible than NaOH for improving biogas production from giant reed.

28