Effects of hydrothermal pretreatment on methane potential of anaerobic digestion sludge cake of cattle manure containing sawdust as bedding materials

Objective The purpose of this study was to analyze the effect of the hydrothermal pretreatment of anaerobic digestion sludge cake (ADSC) of cattle manure on the solubilization of organic matter and the methane yield to improve the anaerobic digestion efficiency of cattle manure collected from the sawdust pens of cattle. Methods Anaerobic digestion sludge cake of cattle manure was thermally pretreated at 160°C, 180°C, 200°C, and 220°C by a hydrothermal pressure reactor, and the biochemical methane potential of ADSC hydrolysate was analyzed. Methane yield recovered by the hydrothermal pretreatment of ADCS was estimated based on mass balance. Results The chemical oxygen demand solubilization degree (CODs) of the hydrothermal hydrolysate increased to 63.56%, 67.13%, 70.07%, and 66.14% at the hydrothermal reaction temperatures of 160°C, 180°C, 200°C, and 220°C, respectively. Considering the volatile solids content obtained after the hydrothermal pretreatment, the methane of 10.2 Nm3/ton-ADSC was recovered from ADSC of 1.0 ton, and methane yields of ADSC hydrolysate increased to 15.6, 18.0, 17.4, and 17.2 Nm3/ton-ADSC. Conclusion Therefore, the optimal hydrothermal reaction temperature that yielded the maximum methane yield was 180°C based on mass balance, and the methane yield from cattle manure containing sawdust was improved by the hydrothermal pretreatment of ADSC.


INTRODUCTION
The total amount of livestock manure generated in Korea in 2019 was about 153,220 ton/d of which the amount of cattle manure was reported to be about 62,608 ton/d. In general, cattle (beef and dairy cattle) breeding facilities mainly adopt sawdust feedlots in which sawdust is adopted as the bedding material. Most of the livestock manure generated in the solid phase at the sawdust feedlot is composted and used as a fertilizer resource for agricultural land. Therefore, in the case of concentrated cattle breeding areas, the existence of nonpoint pollution sources affecting the water system remains a serious concern owing to the outflow of excessive nitrogen and phosphorus resulting from the application of cattle manure as compost to farmland. In particular, in Korea, the 2050 carbonneutral policy, which requires national greenhouse gas emission to be net zero by 2050, has been estab lished, and interest in reducing greenhouse gas emissions by converting livestock manure into bioenergy is increasing. Quantitatively, the energy potential of the biomass that can be converted and used as bioenergy was assessed as 760,032 TOE/yr for cattle manure, 314,493 TOE/yr for sewage sludge, 411,656 TOE/yr for food waste, and 196,320 TOE/yr for pig slurry, respectively. Considering that the bioenergy potential of cattle manure was evaluated to be the highest, a great need exists to promote the conversion of cattle manure into bioenergy as part of future plans to generate bioenergy from livestock manure.
In Korea, cattle manure discharged from cattle breeding facilities consists of a mixture of livestock manure and the sawdust used as bedding material. In particular, sawdust is lignocellulosic biomass and contains a large amount of lignin, which is biologically difficult to decompose and is poorly decomposed during anaerobic digestion. In addition, cattle manure hydrolyzes slowly during the anaerobic digestion process. This decreases the anaerobic decomposition efficiency of organic matter and increases the generation of anaerobic digestion sludge, which impedes the economic feasibility of biogas facilities for processing cattle manure [1]. Especially, anaerobic digestion sludge cake (ADSC) contains the mois ture content above 80%, it is disposed after incineration due to the prohibition of direct landfill and ocean disposal in Korea. Nowadays, the interest in energy conversion of sludge waste is increasing due to high sludge disposal costs and limit ed alternative disposal methods. However, ADSC of cattle manure is characterized by a high solid content that is com posed of lignocellulosic matter. Therefore, it is difficult to be fed into conventional anaerobic digesters. Therefore, recent ly, various technologies to enhance the hydrolysis efficiency, including physical, chemical, and biological pretreatment, have been studied to improve the anaerobic digestion effi ciency of cattle manure [2]. Hydrothermal pretreatment promotes the hydrolysis of difficulttodecompose organic substances based on thermochemical reactions [3,4]. Hy drothermal pretreatment can promote the hydrolysis of organic matter by treating organic material with a moisture content of 70% to 80% with pressurized hot water at 200°C to 300°C. The hydrothermal reaction proceeds via complex mechanisms such as dehydration, carboxylation, decarbox ylation, and condensation to hydrolyze and carbonize organic matter. As a result of these reaction mechanisms, hydrothermal pretreatment has been reported to improve the efficiency of solidliquid separation by increasing the dehydration prop erties of the hydrothermal hydrolysate and the hydrolysis of organic matter [4,5]. Therefore, hydrothermal pretreatment increases the methane production rate by accelerating the hydrolysis of organic matter. This reaction characteristic shortens the hydraulic retention time of the anaerobic di gester, thereby reducing its effective volume [6]. The technical characteristics of this hydrothermal pretreatment can effec tively shorten the operating time of the process by utilizing the byproducts (e.g., anaerobic digestion sludge) of the an aerobic digestion process when applied to conventional anaerobic digestion technology [7,8]. Furthermore, the ap plication of hydrothermal pretreatment technology to the anaerobic digestion of cattle manure containing a large amount of cellulosic material reportedly increases the bioenergy re covery efficiency by 48.2% to 60.0% [911]. However, despite these technical advantages, the application of hydrothermal pretreatment technology to discharged cattle manure con taining sawdust is uncommon in Korea. Therefore, this study aimed to improve the anaerobic digestion efficiency of cattle manure mixed with sawdust by analyzing the effect of hydrothermal pretreatment on the solubilization of organic matter and the potential increase in the amount of methane produced from the ADSC of the cattle manure. Experimen tally, the purpose of this study was to derive the optimal hydrothermal pretreatment temperature to improve the an aerobic digestion efficiency.

Materials
Cattle manure was collected from the feedlot of beef cattle farmhouse, and the feedstock for anaerobic digestion was prepared with the mixture of cattle manure and pig slurry for moisture control. Then, the moisture characteristics of cattle manure give difficulty at the mixing of wet type anaer obic digester. Therefore, pig slurry was used as the moisture regulator for the improvement of mixing efficiency of anaer obic digester. Thereafter, the anaerobic digestion sludge of cattle manure was collected from a pilot scale PFR (Plug and flow reactor) type anaerobic digester (effective volume = 100 L) that was operated as the hydraulic retention time of 30 days in the mesophilic condition (38°C). Then, the collected an aerobic digestion sludge was centrifuged at 4,000 rpm for 20 min, thus preparing the ADSC.

Hydrothermal pretreatment
A batchtype hydrothermal reactor was designed and devel oped for the hydrothermal pretreatment of ADSC. The hydrothermal reactor was a closed system with no potential heat loss via vaporization and condensation. The designed hydrothermal reactor had a working volume of 1.5 kg and was equipped with an electric heater (a heating coil), a tem perature sensor, and a pressure gauge. The temperature sensor and pressure gauge were inserted into the reactor to monitor the inner temperature and saturated vapor pressure during the hydrothermal reaction. The sludge cake (1.5 kg) was placed directly in the reactor without additional pro cessing water and the reactor was sealed with an airtight sealant for the hydrothermal reaction test. The temperature settings were 160°C, 180°C, 200°C, and 220°C. When the temperature in the reactor reached each of these settings, isothermic conditions were maintained for 60 min. The in ner vapor pressures corresponding to these temperatures were 0.85 MPa at 160°C, 1.18 MPa at 180°C, 1.78 MPa at 200°C, and 2.51 MPa at 220°C. The hydrothermal reactor was cooled to room temperature at the end of the hydro thermal reaction using a chiller, whereupon the hydrothermal hydrolysates were recovered from the reactor.

Methane production potential
The theoretical methane potential (B th ) was calculated stoi chiometrically using Boyle's equation based on the elemental analysis results of the samples (Equation 1 and 2) [12]. 6 corresponding to these temperatures were 0.85 MPa at 160°C, 1.18 MPa at 180°C, 1.78 MPa at 200°C, and 120 2.51 MPa at 220°C. The hydrothermal reactor was cooled to room temperature at the end of the hydrothermal reaction using a chiller, whereupon the hydrothermal hydrolysates were recovered from the 122 reactor.

123
Methane production potential

125
The theoretical methane potential (Bth) was calculated stoichiometrically using Boyle's equation based on 126 the elemental analysis results of the samples (Equation 1 and 2) [12].
The ultimate methane potential (Bu) was assessed by the biochemical methane potential assay [13]. To  Table 1.

138
The inoculum for the biochemical methane potential assay of the hydrothermal hydrolysate was kept 127 132 133 The ultimate methane potential (Bu) was assessed by the biochemical methane potential assay [13]. To  Table 1.

138
The inoculum for the biochemical methane potential assay of the hydrothermal hydrolysate was kept 139 under mesophilic conditions at 38 °C for one week to remove any remaining biodegradable fraction. The 140 substrate to inoculum ratio in all anaerobic batch reactors was equal to 0.5 (g-VSsubstrate/g-VSinoculum  [12]. u) was assessed by the biochemical methane potential assay [13]. To .
The ultimate methane potential (Bu) was assessed by the biochemical methane potential assay [13]. To  Table 1.

138
The inoculum for the biochemical methane potential assay of the hydrothermal hydrolysate was kept . The hydrothermal reactor was cooled to room temperature at the end of the n using a chiller, whereupon the hydrothermal hydrolysates were recovered from the n potential ane potential (Bth) was calculated stoichiometrically using Boyle's equation based on is results of the samples (Equation 1 and 2) [12].
thane potential (Bu) was assessed by the biochemical methane potential assay [13]. To The ultimate methane potential (B u ) was assessed by the biochemical methane potential assay [13]. To assess the biochemical methane potential of the hydrothermal hydro lysate, a batchtype anaerobic reactor was operated under mesophilic conditions (38°C). The anaerobic inoculum was collected from a farmscale anaerobic digester located in Icheon, Korea. The chemical properties of the inoculum are provided in Table 1.
The inoculum for the biochemical methane potential assay of the hydrothermal hydrolysate was kept under mesophilic conditions at 38°C for one week to remove any remaining biodegradable fraction. The substrate to inoculum ratio in all anaerobic batch reactors was equal to 0.5 (gVS substrate /gVS inoculum ). The working volume for anaerobic batch fermentation was 80 mL of a 160 mL serum bottle. The headspace of the serum bottle was filled with N 2 gas and sealed with a butyl rubber stopper. The anaerobic batch reactors for each sample and blank were incubated for up to 90 days in the convection in cubator and manually mixed each day during the fermentation period. Then, the anaerobic batch reactors for each sample and blank were performed in three replicates. The biochemical methane potential was calculated based on the volatile solid (VS) content. The biochemical methane potentials of the samples were corrected using the blank value, and calibrated under standard temperature and pressure (STP) conditions (0°C, 1 atm). The modified Gompertz model (Equation 3) [14] and the parallel firstorder kinetic model (Equation 4) were employed to interpret the progress of cumulative methane production. This enabled the cumulative methane production data to be optimized using these equations [15]. Especially, the modified Gompertz model was applied for the estimation of lag phase time and maximum methane production rate, and the parallel firstorder kinetic model was used the esti mation of organic fractionation composing of ADSC.
where M t is the cumulative methane production (mL), t is the anaerobic fermentation time (days), P is the final methane production (mL), e is the exp (1), R m is the maximum methane production rate (mL/d), and λ represents the lag growth phase time (days).
where B t (mL) is the amount of methane production at time t, B max (mL) is the ultimate amount of methane production, f e (g/g) is the organic distribution constant for the two first order kinetic models, and k 1 and k 2 are the kinetic constants in the parallel firstorder kinetics. The cumulative methane production curves of the hydrothermal hydrolysates were optimized with SigmaPlot (SigmaPlot Version 12.5; Systat Software Inc., Cary, NC, USA) using the modified Gom pertz and parallel firstorder kinetic models, respectively. The degree of optimization by the two mathematical models was evaluated by the root mean square deviation (RMSD) (Equation 5).

Estimation of fraction of organic matter
The parallel firstorder kinetic model (Equation 4) considers that the degradation of organic matter sequentially occurs in two stages. In addition, f e distributes the characteristics of the two types of substrates with different reaction rates under anaerobic conditions, and k 1 and k 2 indicate the firstorder   kinetics constants for the first and second organic degradation stages, respectively. In this study, based on the characteristics of the parallel firstorder kinetic model, the characteristics of the organic material composition of the substrate were estimat ed from the analysis results of the rate of the decomposition reaction of the organic material. The total volatile solids (VS T ) of the substrate were assumed to consist of a biode gradable volatile solid fraction (VS B ) and a nonbiodegradable volatile solid fraction (VS NB ), as in Equation 6. The biode gradable organic fraction was defined as consisting of easily biodegradable volatile solids (VS e ) that were readily decom posed in the early stage of anaerobic digestion and persistently biodegradable volatile solids (VS p ) that were slowly decom posed owing to their resistance to decomposition as in Equation 7 [7]. Then, the composition fraction of VS e and VS p can be estimated by the f e (the organic distribution constant for the two firstorder kinetics, g/g) as in Equation 8.

191
( 6) where VS T is the total volatile solid content (g), VS B is the bio degradable volatile solids content (g), and VS NB is the non biodegradable volatile solids content (g). In Equation 6, VS B may be considered to be represented by B u /B th . Then, Equation 7 is induced.
where B u is the ultimate methane potential (Nm 3 CH 4 /kg VS added ) and B th is the theoretical methane potential (Nm 3 CH 4 /kgVS added ).
where VS e is the easily biodegradable volatile solid content (%, w/w), VS p is the persistently biodegradable volatile solid content (%, w/w), and f e is the organic distribution constant for the two firstorder kinetics models (VS e /VS B , g/g).

Analysis
The total solids (TS), VS, pH, chemical oxygen demand (COD Cr ), soluble chemical oxygen demand (SCOD Cr ), total kjeldahl nitrogen (TKN), ammonium nitrogen (NH 4 + N), and alkalinity were determined based on standard methods [16]. The total volatile fatty acids (TVFAs) were measured using a gas chromatograph (GC2010; Shimadzu Scientific Instruments, Inc., Columbia, MD, USA) equipped with a flame ionization detector with an automatic sampler. This chemical analysis was performed in three replicates. The elemental composition (C, H, N, O, S) was determined using an element analyzer (EA1108; Thermo Finnigan LLC, San Jose, CA, USA) and the COD Cr solubilization degree (CODs) of the hydrothermal hydrolysate was calculated by Equation 9, where COD s represents the COD Cr solubilization degree of the hydrothermal hydrolysate, SCOD Crhydrolysate represents the SCOD Cr of the hydrothermal hydrolysate, and SCOD CrADSC represents the SCOD Cr of ADSC.  In the anaerobic batch reactor experiment, the total gas production was measured daily for the first five days and then every two or three days. The gas that was produced dis placed an acidified brine solution in a burette and the volume of displaced solution was recorded after correcting for atmo spheric pressure [17]. The CH 4 and CO 2 concentrations in the gas samples were determined using a gas chromatograph (Clarus 680; PerkinElmer, Inc., Waltham, MA, USA) equipped with a thermal conductivity detector and a HayeSepQ packed column (CRS Inc., Louisville, KY, USA). The column was operated with helium carrier gas at a flow rate of 5 mL/min. The temperatures of the injector, oven, and detector were set to 150°C, 90°C, and 150°C, respectively [14].

Statistical analysis
The tables in this article present the mean values and stan dard deviations of the data obtained from the experiments. The statistical analysis of the results of this experiment was analyzed using the general linear model procedure of the SAS program package (SAS ver. 9.4; SAS instrument Inc., Cary, NC, USA), and the significant difference (p<0.05) of the mean between treatments was tested through Duncan's multiple range test. Table 2 presents the elemental analysis results and theoreti cal methane potentials of the hydrothermal hydrolysate obtained by the hydrothermal pretreatment of ADSC at 160°C, 180°C, 200°C, and 220°C and the anaerobically di gested sludge cake (ADSC) of the cattle feedlot manure. The carbon (C) content of the ADSC was 34.0%, and that of the hydrothermal hydrolysate at the reaction temperatures of 160°C, 180°C, 200°C, and 220°C was 37.5%, 37.2%, 36.4%, and 35.2%, respectively. Based on the results of the elemental analysis, the theoretical methane potential was stoichiomet rically calculated according to Boyle's equations (Equation 1 and 2), and the theoretical methane potential (B th ) of ADSC was 0.425 Nm 3 /kgVS added . The B th of the hydrothermal hydro lysates were 0.478, 0.462, 0.496, and 0.466 Nm 3 /kgVS added at the hydrothermal reaction temperatures of 160°C, 180°C, 200°C, and 220°C, respectively. Therefore, the hydrothermal pretreatment of ADSC increased the carbon content and theoretical methane potential of the hydrolysate, with the highest theoretical methane potential corresponding to the reaction temperature of 200°C for hydrothermal pretreat ment. Table 3 presents the physicochemical analysis of ADSC and hydrothermal hydrolysate. The TS and VS con tents of the hydrothermal hydrolysate have the lowest values of 203,799 and 140,056 mg/kg, respectively, at the reaction temperature of 200°C. The value of SCOD Cr was 39,595, 43,900, 48,215, and 42,620 mg/L for the 160°C, 180°C, 200°C, and 220°C hydrothermal hydrolysates, respectively. The sol ubilization degree of COD (COD s ) increased to 63.56%, 67.13%, 70.07%, and 66.14% for the hydrothermal hydroly sates at 160°C, 180°C, 200°C, and 220°C compared to ADSC, respectively. Generally, hydrothermal pretreatment using pig manure, cattle manure, chicken manure, etc. reportedly dif fers in terms of the solubilization degree of organic matter and the effect of temperature, depending on the characteristics of the raw material [1820]. In addition, as the hydrother mal pretreatment entails the hydrolysis and carbonization reactions of the organic matter, the amount of elemental carbon and the theoretical methane potential increased, as reported previously [21]. Therefore, the hydrothermal hy drolysate can be easily converted to methane in an anaerobic digester [22]. Figure 1 shows the cumulative methane production curve of the ADSC hydrothermal hydrolysate optimized with the modified Gompertz model (Equation 3). The parameters obtained by the modified Gompertz model are listed in Table  4. The methane potential (B u G) of the ADSC hydrother mal hydrolysate, estimated with the modified Gompertz model, was 0.075, 0.092, 0.112, and 0.104 Nm 3 /kgVS added at the hydrothermal pretreatment reaction temperatures of 160°C, 180°C, 200°C, and 220°C, respectively. Moreover, the methane yields increased by 59.57%, 95.74%, 138.30%, and 121.28% relative to the B u G of ADSC (0.047 Nm 3 /kg VS added ), respectively, and the hydrothermal pretreatment reaction temperature of 200°C yielded the highest amount of methane. The maximum methane production rates (R m ) of the ADSC hydrothermal hydrolysate were 3.9, 4.8, 5.8, All data means the average value from three replicates (n = 3). ADSC, anaerobic digestion sludge cake. 1) Dry basis.

Biochemical methane potential assay
2) Theoretical methane potential. All data means the average value from three replicates (n = 3). ADSC, anaerobic digestion sludge cake; SEM, standard error of the mean; TS, total solid; VS, volatile solid; TKN, total kjeldahl nitrogen; NH 4 + -N, ammonium nitrogen; SCOD Cr , soluble chemical oxygen demand; COD s , chemical oxygen demand solubilization degree. 1) COD solubilization degree of ADSC by the hydrothermal pretreatment. 2) Wet basis. a-d Mean with different letter differs significantly between treatment (DMRT; Duncan's multiple range test, p < 0.05). and 5.3 mL/d, respectively. In comparison with the R m (2.6 mL/d) of ADSC, the R m of the ADSC hydrothermal hydro lysate increased to 50.00%, 88.46%, 123.08%, and 103.85% at hydrothermal pretreatment reaction temperatures of 160°C, 180°C, 200°C, 220°C, respectively. The lag growth phase time (λ) of ADSC was 2.7 days and the lag growth phase times (λ) of the ADSC hydrothermal hydrolysate were 1.7, 1.6, 1.1, and 1.6 days at the hydrothermal pretreat ment reaction temperatures of 160°C, 180°C, 200°C, and 220°C, respectively. Thus, the hydrothermal pretreatment time of ADSC was shortened by 37.04%, 40.74%, 59.26%, and 40.74% compared to λ of ADSC, respectively. Figure 2 shows the cumulative methane production curve of the ADSC hydrothermal hydrolysate optimized with the parallel firstorder kinetic model (Equation 4). The parameters de termined with this model are listed in Table 5. The methane potential (B u P) of the ADSC hydrothermal hydrolysate es timated with the parallel firstorder kinetic model, was 0.086, 0.105, 0.124, and 0.118 Nm 3 /kgVS added at the hydro thermal pretreatment reaction temperatures of 160°C, 180°C, 200°C, and 220°C, respectively. Thus, the hydro thermal pretreatment reaction temperature of 200°C yielded the largest amount of methane. A comparison of the per formance of the modified Gompertz model and the parallel   firstorder kinetic model with respect to optimizing the cumulative methane production curve of the ADCS hydro thermal hydrolysate, respectively, revealed that the RMSD of the former model was in the range of 0.006 to 0.010, and   that of the latter model ranged from 0.001 to 0.002. There fore, the parallel firstorder kinetic model was more suitable for the analysis of the cumulative methane production curve of the ADSC hydrothermal hydrolysate containing persis tently biodegradable VS.

Changes of VS fractionation and methane production
The organic distribution constant (f e ), which indicates the distribution of the easily biodegradable volatile solids (VS e ) and persistently biodegradable volatile solids (VS p ), was esti mated to be 0.066 for the ADSC.  Figure 3). Therefore, the optimal hydrothermal reaction temperature that yielded the maximum methane yield was 180°C based on mass balance. Hydrothermal pretreatment was shown to be an efficient method for hydrolyzing cattle manure containing difficult todecompose organic matter. However, hydrothermal pretreatment has been reported to lead to different degrees of solubilization of organic substances depending on the constituents of the raw materials, reaction temperature, and reaction time [21,23,24]. In this study, a hydrothermal reac tion temperature of 200°C was determined to be the optimal temperature at which the methane yield is maximized. How ever, as the hydrothermal reaction temperature increased, the VS e and VS p fractions increased simultaneously with the VS p content (persistently biodegradable) changing most sig nificantly at the hydrothermal reaction temperature of 200°C. MarinBatista et al [10] reported a methane yield of 0.111 Nm 3 /kgVS added from the anaerobic digestion of cattle ma nure and reported yields of 0.294, 0.235, and 0.080 Nm 3 /kg VS added from the hydrothermal hydrolysates at hydrothermal reaction temperatures of 170°C, 200°C, and 230°C, respec tively. In addition, Kim et al [25] reported methane potentials Figure 3. Methane yield and VS fractionation in the hydrothermal pretreatment of ADSC (Vertical bar means standard error, n = 3). The ADSC indicates anaerobic digestion sludge cake, and H160°C, H180°C, H200°C, and H220°C indicate hydrothermal pre-treatment reaction temperature. The VS BN means non-biodegradable volatile solid fraction, the VS e means easily biodegradable volatile solid fraction, the VS p means persistently biodegradable volatile solid fraction, and the filled cycle (•) shape represents CH 4 production.