Effect of organic loading, feed-to-inoculum ratio, and pretreatment on the anaerobic digestion of tobacco stalks

Journal Pre-proofs Effect of organic loading, feed-to-inoculum ratio, and pretreatment on the anaerobic digestion of tobacco stalks Hongyan Zhang, Ligong Wang, Zhuangqiang Dai, Ruihong Zhang, Chang Chen, Guangqing Liu PII: DOI: Reference:

S0960-8524(19)31704-3 https://doi.org/10.1016/j.biortech.2019.122474 BITE 122474

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Bioresource Technology

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Please cite this article as: Zhang, H., Wang, L., Dai, Z., Zhang, R., Chen, C., Liu, G., Effect of organic loading, feed-to-inoculum ratio, and pretreatment on the anaerobic digestion of tobacco stalks, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122474

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Effect of organic loading, feed-to-inoculum ratio, and pretreatment on the anaerobic digestion of tobacco stalks Hongyan Zhang a, 1, Ligong Wang a, 1, Zhuangqiang Dai a, Ruihong Zhang b, Chang Chen a, , Guangqing Liu a aBiomass

Energy and Environmental Engineering Research Center, College

of Chemical Engineering, Beijing University of Chemical Technology, Beijing 100029, China 1

Hongyan Zhang and Ligong Wang contributed equally to this work.

bDepartment

of Biological and Agricultural Engineering, University of

California, Davis, CA 95616, United States Corresponding author Chang Chen’s e-mail: [email protected], Phone and Fax: +86-106444-2375 Address: 503-3A Zonghe Building, Beijing University of Chemical Technology, 15 North 3rd Ring East Road, Beijing 100029, China

1

Abstract

This work firstly investigated the suitable organic loading (OL) and feed to inoculum (F/I) ratio of three kinds of tobacco stalks (TS116, TS99, and TS85) during anaerobic digestion (AD) via response surface methodology (RSM). The highest experimental methane yield (EMY) of 148.1 mL/g VS was achieved from TS116 at OL of 20.2 g VS/L and F/I ratio of 1.1. To further increase EMY, various pretreatments including alkaline hydrogen peroxide (AHP), NaOH, KOH, Ca(OH)2, HCl, and oxalic acid (H2C2O4) were implemented on TS116. Results showed that AHP was most effective, and the maximal EMY of 312.8 mL/g VS and biodegradability (Bd) of 75.4% were obtained from 7% AHP pretreated TS116, which respectively increased by 105.6% and 106% than control. XRD, FTIR, and SEM analyses evidenced that the structure of AHP pretreated TS116 was strongly disrupted. This study lays the foundation for applying this waste into AD in future applications. Keywords: tobacco stalks, anaerobic digestion, response surface methodology, pretreatment, methane yield.

2

1 Introduction

Tobacco is one of the most popular planted crops in China with total planted areas of 1.6 million hectares (Sun et al., 2019). The tobacco production of China makes up 40% of the total world’s output and is an important crop to China’s economy (Kazemi et al., 2014; Qin et al., 2018). Tobacco stalks are the major waste associated with tobacco harvest (Cai et al., 2016), and are therefore a highly plentiful material in certain areas with approximately 1.2 million tons produced each year (Cong et al., 2019). Most tobacco stalks are burned or stacked in the field (Qin et al., 2018), a wasteful practice that leads to problematic air pollution. In addition, it has been noted that stacked tobacco stalks may increase the risk of tobacco mosaic virus migrating to the soil and negatively impacting crop health in the following year (Johnson, 1930). Therefore, it is important to explore more environmentally preferred methods for sustainable utilization of tobacco stalks. Anaerobic digestion (AD) is a complex biological process involving degradation of biomass by microorganisms under anoxic conditions to create biogas (Zhao et al., 2017). This method has been successfully applied with a variety of materials including animal manures and agricultural and municipal waste (Paudel et al., 2017a). Tobacco stalks may be a potential feedstock for AD due to its contents of organic compounds such as cellulose, hemicellulose and lignin (Akpinar et al., 2010). However, the 3

efficiency of AD closely depends on many parameters, among which organic loading (OL) and feed to inoculum ratio (F/I) (Zhang et al., 2018b). OL plays a vital role during AD tests with low OLs leading to inefficient performance while excessively high OL can result in the inhibitory accumulation of alcohols, volatile fatty acids (VFAs), and ammonia (NH3) (Rico et al., 2015; Wu et al., 2016). In addition to OL, an appropriate F/I ratio is required to balance the bacteria and archaea associated with the acidification and methanogenic process of AD (Eskicioglu & Ghorbani, 2011; Fagbohungbe et al., 2015). Despite the importance of the sustainable utilization of tobacco stalks, the appropriate levels and combinations of OL and F/I ratio for the efficient digestion is not currently available in the literature. A potential method for determining these parameters is response surface methodology (RSM), a computer-based experimental and statistical technique widely applied for determining the optimal conditions for the operation of AD systems (Lei et al., 2016; Witek-Krowiak et al., 2014; Zhang et al., 2018a; Zhao et al., 2017). The rate of anaerobic degradation of tobacco stalks is limited by the hydrolysis step due to the recalcitrant nature of the tobacco stalks (Paudel et al., 2017a). As a result, the limited availability of complex organic materials such as hemicellulose and cellulose to degradation into monomers can significantly influence the methane yield (Paudel et al., 2017a). Pretreatment has been widely studied as a promising means to 4

destroy the macroscopic rigidity and complex structure of lignocellulosic materials (Dumas et al., 2015). Chemical pretreatment is a potentially industrially-relevant pretreatment strategy due to its simplicity and effectiveness in accelerating hydrolysis (Paudel et al., 2017b). Acidic and alkaline chemical pretreatments (e.g. alkaline hydrogen peroxide (AHP), NaOH, Ca(OH)2, KOH, and HCl) have been reported to increase methane yield of various biomass materials (Jung et al., 2016; Qin et al., 2011). However, no studies are available detailing the effect of these pretreatment methods on the biomethane production kinetics and yield using tobacco stalks as feedstock. The absence of this data significantly limits the effective industrial utilization of this abundant waste material. Therefore, this study aims to (1) determine the suitable OL and F/I ratio for efficient methane production from tobacco stalks utilizing a RSM experimental design, (2) investigate the effect of six pretreatments (AHP, HCl, NaOH, Ca(OH)2, KOH, H2C2O4) on the tobacco stalk’s structural characteristics and methane production kinetics and yield in AD experiments utilizing the optimal OL and F/I ratio determined previously.

2 Materials and methods

2.1 Substrates and inoculum Three kinds of tobacco stalks common in the Yunnan province of China were selected for analysis including tobacco stalk 116 (TS116), 5

tobacco stalk 99 (TS99), and tobacco stalk 85 (TS85). Chopped tobacco stalks (< 1mm) were ground with a high-speed grinder equipped with an18 mesh screen and then stored in an airtight plastic bag at room temperature (25℃) for subsequent use. The inoculum for AD was collected from Donghuashan biogas plant in Beijing, China. 2.2 Analytical methods Total solids (TS) and volatile solids (VS) of tobacco stalks and inoculum sludge were determined by the APHA method of 23rd edition (Rice et al., 2012). Elemental compositions (C, H, N, S) of tobacco stalks and inoculum sludge were measured by an elemental analyzer (Vario EL cube, Germany). The oxygen content of tobacco stalks was determined by assuming C + H + O + N = 99.5% on a VS basis (Rincón et al., 2012). The contents of cellulose, hemicellulose, and lignin were determined by an A2000 fiber analyzer (ANKOM, USA) according to the Van Soest method (Soest et al., 1991). 2.3 Determination of maximum theoretical methane potential (MMP), experimental methane yield (EMY), and biodegradability (Bd). Based on the elemental composition, the maximum theoretical methane potential (MMP) of tobacco stalks was determined by Eqs.(1) and (2) (Feng et al., 2017) and biodegradability (Bd) was calculated by Eq.(3) by dividing the experimental methane yield (EMY) by the MMP. 6

𝑎 𝑏 3𝑐 𝑛 𝑎 𝑏 3𝑐 𝐶𝑛𝐻𝑎𝑂𝑏𝑁𝑐 + (𝑛 ― ― + )𝐻2𝑂→( + ― ― )𝐶𝐻4 4 2 4 2 8 4 8 𝑛

𝑎

𝑏

3𝑐

+ (2 ― 8 + 4 + 8 )𝐶𝑂2 +𝑐𝑁𝐻3

(1) 𝑛

𝑀𝑀𝑃(𝑚𝐿/𝑔 𝑉𝑆) =

𝑎

𝑏

3𝑐

22.4 × 1000 × (2 + 8 ― 4 ― 8 ) 12𝑛 + 𝑎 + 16𝑏 + 14𝑐

(2) 𝐸𝑀𝑌

𝐵𝑑 = 𝑀𝑀𝑃 × 100%

(3)

Where MMP represents the maximum theoretical methane production resulted by the organic matter in the substrate. n, a, b, and c stand for the number of C, H, O, and N atoms in the molecular formula of the tobacco stalks, respectively. 2.4 Experimental setup by central composite design of RSM A factorial experiment utilizing a central composite design (CCD) was performed using two factors with five levels each to determine the suitable parameters of OL and F/I ratio. The five levels included 0, ±1, and ±α with the value of α were determined by Eq. (4): 𝑘

𝛼=2

4

(4)

where k reflects the number of parameters (k = 2), resulting in an α of 1.414 for this study. To accurately determine the contour behavior of the model, the experiment was divided into 13 runs including 4 cube points (1, ± 1), (− 1, ± 1), 5 repetitive central points (0, 0), and 4 axial points (0, ± 1.414) (± 1.414, 0). The accuracy of obtained results was evaluated by the lack of fit (LOF) and experimental errors using repetitive central points. Based on previous experience and preliminary investigations of the 7

suitable OL and F/I ratios of various substrates, this experiment investigated an OL range of 5–30 g VS/L and F/I ratio range of 0.8–3. The F/I ratio was determined by the relative contributions of VS from the feed (tobacco stalks) and inoculum (sludge) in the final reactor volume. The coded values of the CCD were converted to real values relating to the OL and F/I ratio parameters. AD test groups of TS116, TS99, and TS85 were determined by CCD measure (see E-supplementary data). A statistical model was fit to the responses according to Eq. (5): 𝑌 = 𝛽0 + 𝛽1𝐴 + 𝛽2𝐵 + 𝛽11𝐴2 + 𝛽22𝐵2 + 𝛽12𝐴𝐵

(5)

where Y denotes the predicted EMY; A and B refer to the F/I ratio and OL; β0 is a constant; and β1, β2, β11, β12, and β22 represent the coefficients of the corresponding terms of the polynomial describing the individual and interactive effects of each factor on the response variable (EMY). The coefficients of the quadratic equation were determined using analysis of variance (ANOVA) by Design-Expert 8.0.6. 2.5 Pretreatment of tobacco stalks The pretreatment reagents consisted of the following: KOH (1.0%, 3.0%, 5.0%, and 7.0%, w/w), NaOH (1.0%, 3.0%, 5.0%, and 7.0%, w/w), Ca(OH)2 (1.0%, 3.0%, 5.0%, and 7.0%, w/w), AHP (1.0%, 3.0%, 5.0%, and 7.0%, w/w), HCl (1.0%, 3.0%, 5.0%, and 7.0%, w/w), and H2C2O4 (1.0%, 3.0%, 5.0%, and 7.0%, w/w). A mass of 20 g of raw TS116 was added to the respective solutions for pretreatment. The moisture content (MC) of 8

each pretreatment vessel was calculated to be 90% according to Eq. (6) (Zheng et al., 2009). All samples were subjected to pretreatment for 24h. The reactors were manually mixed once every four hours and measured for TS and VS. The optimal OL and F/I ratio of the pretreated TS116 obtained from the RSM experiment were used for AD tests. 𝑀𝐶(%) = 1 ―

(

𝑑𝑟𝑦 𝑚𝑎𝑡𝑡𝑒𝑟 𝑤𝑒𝑖𝑔 h𝑡 𝑜𝑓 𝑇𝑆116

)

𝑤𝑒𝑖𝑔 h𝑡 𝑜𝑓 𝑇𝑆 + 𝑤𝑎𝑡𝑒𝑟 𝑎𝑑𝑑𝑒𝑑

× 100%

(6)

2.6 Anaerobic digestion AD was carried out in 500 mL glass bottles with a working volume of 200 mL. Anaerobic conditions were obtained by purging N2 gas to the headspace for 3 min, and then the reactors were tightly closed with rubber stoppers and screw caps to seal the bottles. Each run was performed in triplicate. Control groups containing only inoculum and water for each experimental group were carried out in order to minimize the influence of the background methane production. The headspace pressure of the reactor was used to compute the cumulative biogas yield, which was gauged by a 3151 WAL-BMP-Test system pressure gauge (WAL Messund Regelsysteme GmbH, Oldenburg, Lower Saxony, Germany). The biogas generated in the reactor was discharged under water in order to prevent the intrusion of air into the anaerobic reactors. Afterwards the pressure was remeasured once again. Biogas volume was determined by Eq. (7) (Liu et al., 2015): 9

𝑉𝑏𝑖𝑜𝑔𝑎𝑠 =

𝛥𝑃 × 𝑉ℎ𝑒𝑎𝑑 × 𝐶

(7)

𝑅×𝑇

where Vbiogas is the daily volume of biogas produced (L), ΔP represents the absolute pressure difference inside the reactor before and after discharging the biogas under water (KPa), Vhead is the volume of head space (L), C denotes the molar volume (22.41 L/mol), T indicates the absolute temperature (K), and R reflects the universal gas constant (83.14 L⋅mbar/K/mol). The biogas methane content was determined daily by gas chromatograph (Agilent 7890B, Santa Clara, CA, USA), equipped with an analytical column (Agilent Hayesep Q) and a thermal conductivity detector. The temperatures of column oven and detector were respectively set to 60 and 220 °C for operation and the carrier gas used was helium at a constant pressure of 34.5 kPa. 2.7 Kinetics model. A modified Gompertz equation (8) was utilized to fit the cumulative methane yield (CMY) and describe the kinetics of the AD experiments (Kafle & Sang, 2013) according to Eq. (8):

{

𝐵 = 𝐵0ⅇ𝑥𝑝 ―𝑒𝑥𝑝 [

}

µ𝑚 𝑒 (𝜆 ― 𝑡) + 1] 𝐵0

(8)

where B denotes the simulated cumulative methane production (mL/g VS), B0 is the simulated maximum methane yield (mL/g VS), μm is the maximum methane production rate (mL/g VS/day), e is a constant with the

10

value of 2.72, λ is the lag phase time (day), and t is the digestion time (day). 2.8 Structural Analysis XRD analysis. The crystallinity index (CrI) of the cellulose component was obtained by X-ray diffraction (XRD) utilizing a Bruker D8-Advance (Germany) device at 40 kV and 40 mA with Cu Kα radiation. All samples were scanned from 5 to 60° at a rate of 5°/min. Eq. (9) was used to determine the CrI of pretreated TS116 (Li et al., 2014). 𝐶𝑟𝐼 = [(𝐼002 ―𝐼𝑎𝑚𝑜𝑟𝑝

h𝑜𝑢𝑠)/𝐼002

] × 100%

(9)

where I002 is the intensity of the [002] peak at 2θ = 21.8° and Iamorphous is the intensity of the amorphous zone diffraction at 2θ = 18°. FTIR analysis. A Nicolet 6700 Fourier transform infrared (FTIR) spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a 400–4000 cm-1 DLATGS detector was used to detect the structural features of pretreated samples. Raw and pretreated TS116 were analyzed by grinding with KBr (1:100, w/w) and pressing into transparent pellets SEM analysis The changes of surface structure of TS116 before and after pretreatment were observed and analyzed by scanning electron microscopy (SEM) (S11

4700, Hitachi, Japan) at a magnification of 500×. 2.9 Statistical analysis. Origin Pro 8.0 (Origin Lab, USA) was used to deal with all the data and graphs. The data was statistically analyzed by analysis of variance (ANOVA) with a statistical significance threshold of 0.05.

3 Results and Discussion

3.1 Characteristics of TS116, TS99, and TS85 The physicochemical characteristics of the three kinds of tobacco stalks and inoculum could be found in Table.1. The TS of three kinds tobacco stalks were similar, between 91.3% and 92.5% with VS between 88 and 89%. The VS made up the vast majority (95.7% to 96.6%) of the TS. This suggested that the tobacco stalks had extremely high organic contents, a desirable trait for AD. In addition, cellulose, and hemicellulose made up 45.7%–52.3% and 18.3%-19.5% of the biomass’ dry weight, respectively, indicating that structural carbohydrates accounted for the majority (63.0%-71.8%) of the dry biomass. The tobacco stalks had lower lignin contents (14.9%-15.3% ) than many straws previously investigated for AD such as wheat straw, cotton straw, and rice straw (Ahmad et al., 2018). Relatively lower lignin contents in tobacco stalks were advantageous for increasing the efficiency of AD because it would make a 12

more access for enzymes to cellulose in tobacco stalks. According to organic element analysis, the C/N ratio of TS116, TS99, and TS85 were 27.2, 17.6, and 14.8, respectively, which were all within the preferred range for AD of 15-30 (Feng et al., 2013). The MMP of TS116, TS99, and TS85 were calculated to be 430.7 mL/g VS, 405.8 mL/g VS, and 404.3 mL/g VS, respectively. Taken together, the physicochemical characteristics indicated promise for using the tobacco stalks for biomethane production. 3.2 Effects of OL and F/I ratio on AD performance of TS116, TS99, and TS85 The effect of OL and F/I ratio on the EMY could be visualized in the 3D response surface graph and its contour plot in Fig.1. As seen in Fig.1A and B, the EMY of TS116, for a constant OL, generally decreased as the F/I ratio increased. Meanwhile, for a constant F/I ratio, the EMY generally increased with increasing OL although plateaus and slight decreases in EMY were observed at high OL levels. The EMY of TS116 were obtained and there were favorable EMY from a lower F/I ratio and properly larger OL (runs 9,11, and12) (see E-supplementary data). The general behavior of the effect of OL and F/I ratio on the EMY was similar for the TS99 and TS85 substrates compared to the TS116 material (Fig.1C-F). However, some important differences should be noted. For instance, both TS99 and TS85 displayed generally lower EMY values compared to TS116 for similar OL and F/I ratio conditions. Additionally, the decrease in EMY observed for 13

high OL and low F/I condition in TS116 was less pronounced or not present in TS99 or TS85. These results demonstrated that the methane yield of TS116, TS99, and TS85 suffered from both F/I ratio and OL, which was coherent with previous report (Zhao et al., 2017). Generally, a high OL meant more methane yield (Duan et al., 2019), which could be explained why EMY increased as OL increased for a set F/I ratio well. The reason that EMY decreased as F/I increased for a set OL might be relative insufficient microbes to convert feedstock or the VFAs accumulation as a result of the depletion rate of VFAs could not keep up with its production rate (Zhao et al., 2017; Zhu et al., 2014). It was a useful finding that high OL could be utilized in digesters as long as the F/I ratio is low enough. That is, there were enough microbes present to efficiently handle the large amount of biomass VS loaded to the reactor. This had practical implications for the industry as the ability to increase the organic OL could allow reductions in reactor volumes, among other things. The slight difference of EMY within these three kinds of tobacco stalks might be due to different varieties. 3.3 Optimization of OL and F/I ratio for methane production The experimental data was analyzed using Design-Expert 8.0.6 software and the following second-order polynomials were obtained for TS116, TS99, and TS85, respectively:

14

𝑌1 = 99.38391 ― 48.00066𝐴 + 7.85208𝐵 + 2.00784𝐴𝐵 ― 1.10902𝐴2 ― 0.24709𝐵2 (10) 𝑌2 = 78.54652 ― 63.84467𝐴 + 6.71133𝐵 ― 0.00064377 + 16.01461 𝐴2 ― 0.11543𝐵2 (11) 𝑌3 = 30.21085 ― 21.45394𝐴 + 8.42091𝐵 + 0.38956𝐴𝐵 + 1.68547𝐴2 ― 0.17933𝐵2 (12) Where Y1, Y2, and Y3 are the predicted EMY of TS116, TS99, and TS85, respectively while A and B refer to F/I ratio and OL, respectively. The model F-values of TS116, TS99, and TS85 were 88.11, 16.44, and 26.23, respectively, suggesting that there was some validity to fitting a model to this data aside from the null hypothesis. There was only less than 1% chance that the large F-value had been aroused by the noise. The ‘‘Lack of Fit F-value” of 2.57, 0.97, and 12.43 implied the ‘‘Lack of Fit” had a bad relationship with the pure error. The R2 of the three equations (0.9844, 0.9215, and 0.9493) were greater than 0.75 that exhibited the fitting degree of the quadratic polynomial was good (Naik, 2014). According to Design Expert Software, the optimal EMY of TS116 was calculated to be 148.1 mL/g VS at an OL of 20.2 g VS/L and F/I ratio of 1.1. For TS99, the optimal EMY was 129.1 mL/g VS at OL of 23.7 g VS/L and F/I ratio of 0.9. Meanwhile, F/I ratio of 1.1 and OL of 26.3 g VS/L were the optimal conditions for TS85 where the EMY reached 117.2 mL/g VS. Among the three kinds of tobacco stalks, the highest EMY was obtained from TS116, showing highest methanogenesis potential. Therefore, TS116 was selected for further pretreatment research.

15

3.4 Effluent characteristics and system stability The characteristics of the final AD vessels including pH and VFAs after 60 days of AD were determined (see E-supplementary data). It could be observed that the pH values of all vessels were in an acceptable region for AD (6.5-7) (Cun-Fang et al., 2008), indicating the high buffering capacity and stability of the AD system. Excessive accumulation of VFAs resulted in the reduction of pH, thereby limiting the activity of methanogens (Yuan & Zhu, 2016). However, the concentration of VFAs in this study were all below 38.17 mg/L, indicating that excessive VFA accumulation did not occur during the AD trials. 3.5 Effect of various pretreatments on methane yield of TS116 The pretreatment of TS116 with increasing concentrations of NaOH, KOH, and Ca(OH)2 resulted in progressively delayed peaks in daily methane yield (see E-supplementary data). For example, the 1% and 3% NaOH treatments reached an initial daily methane yield peak within 3 or 4 days while higher concentration NaOH treatments (5%, 7%) required 8 or 11 days before a similar peak was observed. This result might be owed to increased acclimation times required for the microorganisms in response to the increasingly harsh biomass pretreatment conditions. The CMY of the TS116 residues pretreated with NaOH, KOH, and Ca(OH)2 were shown in Fig.2A, B, and C. The TS116 pretreated with NaOH 16

and KOH showed an improvement in CMY compared to the control (170.5mL/g VS) at all of the pretreatment concentrations tested (Fig.2A, B). For the NaOH pretreatment, the maximum CMY of 321.7 mL/g VS (88.6% improvement compared to untreated) was found at the 5% NaOH treatment while 3% and 7% NaOH pretreatments also displayed high CMY (Fig.2A). The CMY of the NaOH pretreated biomass at all concentrations tested was significantly higher than the untreated control (P<0.05) and the CMY of the 3%, 5%, and 7% NaOH treatments were significantly higher than the 1% NaOH treatment (P<0.05). A reduction in the CMY was found with increased NaOH concentration from 5% to 7%, implying that an optimum concentration for NaOH pretreatment exists, above or below which nonoptimal results are obtained. This finding was in agreement with the reported results of Dai et al who noted a similar optimal concentration of chemical for the pretreatment of rice straw (Dai et al., 2017). The maximum CMY of TS116 pretreated by 7% KOH was 309.8 mL/g VS (Fig.2B), a significant increase of 79.3% compared to the control (P<0.05). The CMY of TS116 pretreated at different concentrations of Ca(OH)2 was shown in Fig.2C. The CMYs were 227.2 mL/g VS, 236.2 mL/g VS, and 195.4 mL/g VS after pretreatment with 1%, 3%, and 5% Ca(OH)2, which were slightly higher than that of the untreated group. Moreover, a significant difference (P<0.05) existed among the CMY of Ca(OH)2 experimental groups. The highest concentration of Ca(OH)2 (7%) resulted in a notable decrease in 17

CMY from the control group. This phenomenon might be explained by a decline in the accessibility of cellulose caused by the formation of insoluble white particles that might have adhered to the surface of the material. Thus, 3% was routinely regarded as the most suitable concentration for Ca(OH)2. Two peaks in daily methane yield were commonly observed after pretreatment with HCl and H2C2O4 (see E-supplementary data). The first daily methane yield peak occurred within 2-3 days. The next summit appeared in HCl-treated TS116 on 8th or 9th day while in H2C2O4-treated TS116, it emerged on 7th or 8th day. There was no obvious distinction in the lag phase of the HCl and H2C2O4 treatments similar maximum daily methane yields obtained in both, implying that the mechanism of these two acids on the pretreatment of the TS116 material was probably similar. For the HCl treatment, a concentration of 5% produced the maximum CMY of 203.3 mL/g VS, which was significantly higher (P < 0.05) compared to the 1%, 3%, 7% HCl, and control groups. The highest CMY obtained with the H2C2O4 treatment occurred at a concentration of 7% and was 206.5 mL/g VS (Fig.1F). However, the CMY of TS116 treated with 7% H2C2O4 did not reveal a significant difference (P < 0.05) compared with that treated with 1%, 3%, 5% H2C2O4, and the control group. The maximum CMY (350.7 mL/g VS) of TS116 following treatment with AHP was obtained with an AHP concentration of 7%, an increase in CMY of 105.6% compared with control group. Significant differences (P < 18

0.05) were observed between all AHP treatments with increasing CMYs obtained with increasing AHP concentrations. The maximum CMY obtained from the six pretreatment strategies were presented in Fig.3. The highest CMY and peak of daily methane yield (see E-supplementary data) were obtained after treatment with 7% AHP. A significant difference (P < 0.05) was found between 5% NaOH-treated TS116 and 7% KOH-treated TS116 and this appearance was also happening between 7% H2C2O4-treated TS116 and 5% HCl-treated TS116. The CMY of TS116 after H2C2O4 and HCl pretreatment were lower compared to the NaOH and KOH treatments. The CMY of 3% Ca(OH)2-treated TS116 was 14.4% higher than 7% H2C2O4-treated TS116 and 16.2% higher than 5% HCl-treated TS116. Based on these results, AHP and alkaline pretreatments were more effective than acidic pretreatment in maximizing the methane yield of TS116. Apart from the promising results obtained, considering the sustainability aspects of AD process of tobacco stalks, the advanced sustainability assessment tools such as LCA, exergy, and their combinations could be used for well-scrutinizing these aspects in the future industrial application (Rosen, 2018). 3.6 Kinetic analysis The kinetic parameters obtained from applying the modified Gompertz model could be found in Table.2. The model successfully

19

described the variation in the data as indicated by the R2 values, which were between 0.954 and 0.999. The difference between the B0 values predicted from the model and the EMY was relatively subtle, further indicating that this model was well suited to predict the CMY within the set range of selected values. The λ parameter is a reflection of the length of start-up time and the λ values for all pretreatments were higher than untreated group. This observation is consistent with the lag period noticed with these treatments (Fig.3). For all conditions investigated, an increase in B0 values was observed compared with that of control group, especially with the AHP pretreatment. The um parameter corresponds to the maximum methane yield rate predicted by the model. For all pretreatments except 7% H2C2O4 and 5% HCl, a higher um was observed compared to the control group. Additionally, each pretreated group had higher biodegradability (Bd) than the untreated group with the maximum Bd value (75.4%) being obtained from 7% AHPtreated TS116, which indicated that it had the most favorable digestion efficiency among the pretreatment methods. 3.7 Analysis of modifications in the microcosmic and surface structure, as well as cellulose, hemicellulose, and lignin of TS116 after different pretreatments FTIR analysis The FTIR spectra of untreated TS116 and pretreated TS116 were

20

illustrated (see E-supplementary data). The peak at 1374.6 cm−1 could be attributed to C-H deformation in cellulose and hemicellulose, indicating the existence of deformed cellulose and hemicellulose (Faulon et al., 1994; Pandey & Pitman, 2004). The absorbance at 1738.6 cm−1 was responsible for unconjugated C=O in xylans, which also indicated the existence of hemicellulose (Ding et al., 2012; Pandey & Pitman, 2004). The absorption bands of 1374.6 cm−1 and 1738.6 cm−1 had weakened or distorted after pretreatment, which was consistent with the decline of cellulose and hemicellulose as shown in Table.2 for the pretreated samples. The absorbance of 1510.5 cm−1 was aromatic skeletal that was the characteristic of lignin (Correia et al., 2013; Pandey & Pitman, 2004). This absorbance band was greatly reduced in the pretreated biomass materials, indicating that lignin was removed to different degrees by pretreatment. XRD analysis To further characterize the structure of cellulose, the XRD patterns of untreated TS116 and pretreated TS116 were depicted (see E-supplementary data). The range of 2θ from 22-24° was generally recognized as the cellulose crystallographic plane (Mulinari et al., 2009). A reduction in intensity over this range was observed in TS116 treated with 7% KOH and could be attributed to a decrease in crystalline structure of cellulose. No significant differences in cellulose structure of TS116 subjected to all of the

21

other pretreatments were found compared to the untreated group. The CrI of untreated TS116 was 27.87% and it increased to 43.51%, 40.04%, 31.47%, 37.59%, 33.26%, and 36.83% after 7% AHP, 5% NaOH, 7% KOH, 3% Ca(OH)2, 7% H2C2O4, and 5% HCl pretreatment (Table.2), respectively, probably owing to the loss of non-crystalline components including lignin and hemicellulose. SEM analysis The SEM analysis of TS116 biomass subjected to 7% AHP, 5% NaOH, 7% KOH, 3% Ca(OH)2, 7% Ca(OH)2, 1% H2C2O4, 7% H2C2O4, and 5% HCl pretreatment were done compact structure was observed in untreated TS116 and various damages to this structure were observed regardless of the pretreatment methods (see E-supplementary data). Pretreatment with 7% AHP, 5% NaOH, 7% KOH, 3% Ca(OH)2, and 7% Ca(OH)2 resulted in extremely destructive behavior with the development of rectangular holes in the biomass structure, which might be due to the degradation of lignin−carbohydrate linkages and removal of lignin. The surface of TS116 subjected to 3% Ca(OH)2 and 7% Ca(OH)2 pretreatment did observe some white substances and the area covered by these white substances followed the increase in Ca(OH)2 concentration. Although Ca(OH)2 pretreatment resulted in the improvement of delignification and the exposure of cellulose, the adsorbed particles interfered with the utilization of cellulose, partially

22

negating the positive influence given by the Ca(OH)2 pretreatment. In this sense, excessive concentrations of Ca(OH)2 might have a negative effect on methane yield, as observed in this study. Also, there was only a subtle transformation on the surface of TS116 treated with 1% H2C2O4 while a more dramatic effect was observed when the concentration was increased to 7%. The surface structure was clearly not as disrupted from the acidic pretreatments compared to the AHP and alkaline pretreatment methods, which was consistent with the lower CMY and biodegradability values observed for the acidic pretreatments in this study. Effect of various pretreatments on cellulose, hemicellulose and lignin of TS116 The changes in cellulose, hemicellulose, and lignin present in the pretreated TS116 were shown in Table.2. All pretreated samples had variations in the cellulose, hemicellulose, and lignin contents compared to the control. Significant loss in cellulose and hemicellulose was observed in the samples treated with 5% NaOH, 7% KOH, and 3% Ca(OH)2. Alkaline reagents were effective in terms of reducing cellulose crystallinity and dissolving, partially or totally, the hemicellulose (Chandra et al., 2012). The most valuable result of the pretreatment process was the removal of lignin because lignin protected the cellulose and hemicellulose from hydrolyzing enzymes (Karagöz et al., 2012). The rates of delignification were 60.3%, 39.7%, 45.9%, 41.7%, 41.0%, and 13.0% from TS116 treated with 7% AHP,

23

5% NaOH, 7% KOH, 3% Ca(OH)2, 7% H2C2O4, and 5% HCl, respectively. These results revealed the subsequent enzymatic hydrolysis during AD would be facilitated by all the pretreated methods, as was observed in the study. These results revealed the subsequent enzymatic hydrolysis during AD would be facilitated by all the pretreated methods, as was observed in the study.

4 Conclusions

The determination of the optimal AD condition and pretreatment for tobacco stalk was implemented in this study. TS116 achieved the maximum EMY at OL of 20.2 g VS/L and F/I ratio of 1.1. Among all the pretreatments, AHP pretreated TS116 showed a significant increase of 105.6% in methane yield (312.8 mL/g VS) compared to the untreated with a maximum Bd of 75.4%, indicating a favorable digestion efficiency. In summary, this study lends insight into a possible method for the full utilization of tobacco stalks in a more ecologically friendly manner that has the added benefit of producing renewable energy.

Acknowledgements

This work was financially supported by the National Key Research and

Development

Program

of

China

(2018YFD0800103,

2017YFD0800801). Special thanks to Dr. Tyler Barzee for his suggestions 24

on this paper. We also would like to acknowledge the support from International Clean Energy Talent Project (iCET) provided by the China Scholarship Council (CSC).

Appendix A. Supplementary data

E-supplementary data of this work can be found in online version of the paper.

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Table.1 Characteristics of three kinds of tobacco stalks and inoculum Paraments

TS116

TS99

TS85

Inoculum

TS (%)a

91.32 ± 0.37

92.51 ± 0.15

92.07 ± 0.11

9.58 ± 0.03

VS (%)a

88.19 ± 0.91

89.76 ± 0.89

89.02 ± 0.80

2.68 ± 0.01

3.13 ± 0.02

3.75 ± 0.41

3.67 ± 0.05

6.89 ± 0.02

96.59 ± 0.03

95.95 ± 0.12

95.68 ± 0.08

27.99 ± 0.06

45.72 ± 0.87

52.34 ± 0.51

50.91 ± 0.81

ND

18.34 ± 0.48

19.50 ± 0.93

19.01 ± 0.31

ND

lignin (%)b

14.85 ± 0.67

15.27 ± 0.64

14.88 ± 0.10

ND

C (%)b

Ash

(%)a

VS/TS (%) cellulose

(%)b

hemicellulose

(%)b

43.99 ± 0.64

41.93 ± 0.12

41.78 ± 0.37

13.77 ± 0.53

H

(%)b

6.03 ± 0.10

6.01 ± 0.06

6.00 ± 0.04

2.57 ± 0.07

O

(%)b

44.47 ± 0.41

45.15 ± 0.09

44.59 ± 0.27

9.83 ± 0.36

N

(%)b

1.62 ± 0.39

2.38 ± 0.04

2.83 ± 0.07

1.68 ± 0.04

S (%)b

0.07 ± 0.03

0.14 ± 0.01

0.18 ± 0.03

0.44 ± 0.02

C/N

27.15 ± 0.65

17.59 ± 0.21

14.76 ± 0.12

8.20 ± 0.23

ND: Not detectable; a As the total weight of samples; b As the TS of the samples.

29

Table.2 Analysis of cellulose, hemicellulose, and lignin of TS116 after different pretreatments, as well as kinetic parameters of the modified Gompertz model

Cellulose (%)

Hemicellulose (%)

Lignin (%)

CrI (%)

EMY (mL/g VS)

Bd (%)

Improved compared to the control (%)

untreated

45.72 ± 0.87

18.34 ± 0.48

14.85 ± 0.67

27.87

170.6

36.6

7% AHP

45.80 ± 0.04

8.23 ± 0.12

5.90 ± 0.01

43.51

350.7

5% NaOH

34.46 ± 0.01

6.33 ± 0.34

8.96 ± 0.04

40.04

7% KOH

37.99 ± 0.05

7.27 ± 0.02

8.04 ± 0.02

3% Ca(OH)2

37.61 ± 0.01

9.18 ± 0.04

7% H2C2O4

39.89 ± 0.06

5% HCl

41.60 ± 0.05

Parameters

Modified Gompertz model B0

μm

λ

(mL/g VS)

(mL/g VS/day)

(day)

-

141.6

6.2

1

0.954

75.4

105.6

326.3

25.9

4.7

0.997

321.7

69.2

88.6

286

13.2

8.2

0.998

31.47

309.7

66.6

81.6

298.6

15

9.9

0.999

8.66 ± 0.02

37.59

236.2

50.8

38.5

206.1

13

5.9

0.984

12.69 ± 0.02

8.76 ± 0.08

33.26

206.5

44.4

21.1

192.5

6.6

6.2

0.985

11.00 ± 0.04

12.92 ± 0.02

36.83

203.3

43.7

19.2

179.4

7.04

2.7

0.965

30

R2

Figure.1 Methane production of tobacco stalk at different OLs and F/I ratios. (A) The response surface graph of TS116; (B) the contour plots of TS116; (C) the response surface graph of TS99; (D) the contour plots of TS99; (E) the response surface graph of TS85; (F) the contour plots of TS85. Figure.2 Cumulative methane yield of TS116, (A NaOH-treated TS116; (B) KOH-treated TS116; (C) Ca(OH)2-treated TS116; (D) AHP-treated TS116; (E) HCl-treated TS116; (F) H2C2O4-treated TS116 Figure.3 Cumulative methane yield at optimal conditions of different pretreatments for TS116

Author Contributions H. Zhang and L. Wang carried out the experiments, collected the data, and performed the statistical analyses. All authors discussed the experiments results. L. Wang wrote the paper. C. Chen and G. Liu conceived and designed the experiments.

31

Figure.1 Methane production of tobacco stalk at different OLs and F/I ratios. (A) The response surface graph of TS116; (B) the contour plots of TS116; (C) the response surface graph of TS99; (D) the contour plots of TS99; (E) the response surface graph of TS85; (F) the contour plots of TS85.

32

Figure.2 Cumulative methane yield of TS116, (A NaOH-treated TS116; (B) KOH-treated TS116; (C) Ca(OH)2-treated TS116; (D) AHP-treated TS116; (E) HCl-treated TS116; (F) H2C2O4-treated TS116

33

Figure.3 Cumulative methane yield at optimal conditions of different pretreatments for TS116

34

Highlights：

The OL and F/I ratio of tobacco stalks for AD were optimized via RSM

Various pretreatments for improving methane yield from TS116 were examined.

AHP pretreated TS116 achieved the highest methane yield.

Graphical Abstract (for review)

35