184 S.Bolado-Rodriguez et aL/Bioresource Technology 201 (2016)182-190 dilute NaOH autoclaving(C),and alkaline peroxide(D).The opera- methane yields are expressed as the volume of methane under tional conditions were the same as those selected by Toquero and standard conditions,i.e.0C and 1 atm for gases,as the Interna- Bolado (2014),according to the optimal experimental settings tional Union of Pure Applied Chemistry(IUPAC)defines,per gram previously reported for each pretreatment (Akhtar et al.,2001: of VS in substrates fed into the assay(N mL CH4/g VS). Cao et al.,2012:Karagoz et al.,2012;Sun and Cheng.2005).In Theoretical methane yields,calculated from the characteriza- autoclave pretreatments A,B,and C,milled and dried WS or SCB tion performed for both substrates,were as follows:449 mL were slurried for 5 min with distilled water,1.5%w/w HCl solution. CH4/g VS for WS;and 420 mL CH4/g VS for SCB.These values are and 1%w/w NaOH solution,respectively,in a 500 mL screw cap consistent with those calculated by Ferreira et al.(2013)for WS bottle with a solid:liquid ratio of 1:10 w/w,and then autoclaved (444 mL CH4/g VS)and Badshah et al.(2012)for SCB(415 mL CH4/ at 121 C for 60 min.In alkaline peroxide pretreatment(D).milled g VS).considering the stoichiometric conversion of the organic and dry WS and SCB were slurried for 5 min with 5%w/w H2O2,in a matter. solid:liquid ratio of 1:20,the pH was then adjusted to 11.5 with 2 M NaOH and the mixture was placed in a rotatory shaker at 2.4.Analytical methods 50C and 120 rpm for 60 min. After pretreatment,and once cooled down to room tempera- Total solids (TS).volatile solids and total Kjeldahl nitrogen ture,the slurry obtained from each pretreatment was recovered. (TKN)were measured following the procedures given in Standard and the residual solid was separated by vacuum filtration till max- Methods for Examination of Water and Wastewater (APHA imum liquid removal and dried in a ventilated oven at 45 C for 2005).Total chemical oxygen demand(TCOD)was determined 48 h.Liquid fractions from every pretreatments were stored in a according to the standard method UNE 77004:2002 based on the refrigeration chamber for biogas production and compositional dichromate method only in the initial raw material (Ferreira analysis.Solid fraction,or whole slurry were used as the substrates et al.,2013). in the subsequent step of anaerobic digestion.All experiments The analytical methods of the National Renewable Energy Lab- were conducted in triplicate and the results were averaged. oratory (NREL)were followed to determine substrate composition in terms of ash,lignin,cellulose (as glucose),and hemicellulose (as 2.3.Anaerobic biodegradability xylose)(Sluiter et al..2012).High performance liquid chromatogra- phy (HPLC)was used to measure glucose,xylose,formic acid,acetic Biochemical methane potential(BMP)tests were carried out to acid,HMF and furfural,using a Bio-Rad HPX-87P column at 80C study the biodegradability of raw and pretreated substrates in with MilliQ water as the mobile phase for sugars and a Bio-Rad duplicate following the protocol of Angelidaki et al.(2009).Batch HPX-87H column at 50C with 0.005 M H2SO4 as the mobile phase mode assays were performed under mesophilic conditions in for acids,both at 0.6 mL/min.A Waters 2414 refractive index was borosilicate glass bottles of 2 L volume (260 mm height,160 mm used as detector (Travaini et al.,2013).The total content of pheno- diameter and a 40 mm bottleneck).The effluent from a pilot- lic compounds in the samples was determined by the Folin-Ciocal- scale mesophilic anaerobic digester processing mixed sludge from teu method (Singleton and Rossi,1965)with gallic acid as the a municipal wastewater treatment plant,with a volatile solids(VS) calibration standard.The biogas composition (CO2.H2S,O2,N2. concentration of 14.0t1.5 g VS/kg was used as inoculum for tests. CH4)was measured by gas chromatography using a varian Two series of experiments (test 1 and test 2)were performed in CP-177 3800 GC-TCD equipped with a CP-Molsieve 5A and a CP- order to determine the influence of the pretreatment and the Pora BOND Q columns,using helium as the carrier gas.All analyses inhibitory effect of compounds present in the liquid phase:(1) were performed in duplicate and all chromatographic standards using the whole slurry (solid and liquid fractions):and (2)using were of analytical grade,and MilliQ Ultrapure water was used. only the solid fraction. Raw substrates,whole slurries or solid fractions from pretreat- ment batches were adjusted to 10%w/w soil content in all the 2.5.Determination of kinetic parameters experiments,using either the pretreatment liquid (assays with whole slurry)or distilled water(assays with raw substrates and The cumulative methane production data from the experiments solid fractions from pretreatments).NaOH or HCI were added,if was fitted either to a first order model (Eq.(1))or to the modified necessary,in order to pre-neutralize the samples up to values of Gompertz equation (Eq.(2))(Lay et al.,1996)or to a combination pH 8 for alkaline samples or pH 5.5 for acid samples.The sludge of both.The first one was applied successfully in many reports on was added in a ratio substrate/inoculum around 0.5 g VS/g VS,in anaerobic biodegradability tests when the hydrolysis reaction was order to obtain a ratio 1 of hydrolysable material in substrate the rate-limiting step of the global process(Ferreira et al..2013). g VS inoculum,considering that sugars (hydrolysable material) The modified Gompertz model described the cumulative methane make up half of the substrate (Ferreira et al.,2014).The pH,after production in batch assays when an inhibitory behavior was adding the activated sludge,was always between 6.5 and 7.The observed,assuming that the methane production was a function working volume was approximately 400 mL in order to have of bacterial growth.Moreover,the model parameters were calcu- enough headspace for gas production.A control test without lated by minimizing the least square difference between observed substrate was also conducted,aiming to check the methanogenic and predicted values. activity of the inoculum. B=Bo·[1-exp(-kH·t)] (1) Before starting the test,the bottles were closed with rubber septa and aluminium crimps.Helium gas was circulated inside the gas chamber for 5 min,and the test started after releasing a-Bexpf-exp+ (2) the pressure.The bottles were placed horizontally in a rotary desk with constant mixing under mesophilic conditions in a thermo- In these equations,B represents the cumulative methane static room (35.10.3C). production (mL CH4/g VS)and t is the length of the assay(d).These Biogas production in the headspace of each bottle was mea- models estimate the methane production potential Bo(mL CH4/ sured periodically by a manual pressure transmitter (PN5007, g VS,related to the substrate biodegradability).the hydrolysis range 0-1 bar,IFM Electronics)over a period of 30 days.Biogas coefficient kH (d-1).the maximum biogas production rate Rm composition was determined by gas chromatography.Specific (mL CH4/g VS-d),and the lag time (d).dilute NaOH autoclaving (C), and alkaline peroxide (D). The opera￾tional conditions were the same as those selected by Toquero and Bolado (2014), according to the optimal experimental settings previously reported for each pretreatment (Akhtar et al., 2001; Cao et al., 2012; Karagöz et al., 2012; Sun and Cheng, 2005). In autoclave pretreatments A, B, and C, milled and dried WS or SCB were slurried for 5 min with distilled water, 1.5% w/w HCl solution, and 1% w/w NaOH solution, respectively, in a 500 mL screw cap bottle with a solid:liquid ratio of 1:10 w/w, and then autoclaved at 121 C for 60 min. In alkaline peroxide pretreatment (D), milled and dry WS and SCB were slurried for 5 min with 5% w/w H2O2, in a solid:liquid ratio of 1:20, the pH was then adjusted to 11.5 with 2 M NaOH and the mixture was placed in a rotatory shaker at 50 C and 120 rpm for 60 min. After pretreatment, and once cooled down to room tempera￾ture, the slurry obtained from each pretreatment was recovered, and the residual solid was separated by vacuum filtration till max￾imum liquid removal and dried in a ventilated oven at 45 C for 48 h. Liquid fractions from every pretreatments were stored in a refrigeration chamber for biogas production and compositional analysis. Solid fraction, or whole slurry were used as the substrates in the subsequent step of anaerobic digestion. All experiments were conducted in triplicate and the results were averaged. 2.3. Anaerobic biodegradability Biochemical methane potential (BMP) tests were carried out to study the biodegradability of raw and pretreated substrates in duplicate following the protocol of Angelidaki et al. (2009). Batch mode assays were performed under mesophilic conditions in borosilicate glass bottles of 2 L volume (260 mm height, 160 mm diameter and a 40 mm bottleneck). The effluent from a pilot￾scale mesophilic anaerobic digester processing mixed sludge from a municipal wastewater treatment plant, with a volatile solids (VS) concentration of 14.0 ± 1.5 g VS/kg was used as inoculum for tests. Two series of experiments (test 1 and test 2) were performed in order to determine the influence of the pretreatment and the inhibitory effect of compounds present in the liquid phase: (1) using the whole slurry (solid and liquid fractions); and (2) using only the solid fraction. Raw substrates, whole slurries or solid fractions from pretreat￾ment batches were adjusted to 10% w/w soil content in all the experiments, using either the pretreatment liquid (assays with whole slurry) or distilled water (assays with raw substrates and solid fractions from pretreatments). NaOH or HCl were added, if necessary, in order to pre-neutralize the samples up to values of pH 8 for alkaline samples or pH 5.5 for acid samples. The sludge was added in a ratio substrate/inoculum around 0.5 g VS/g VS, in order to obtain a ratio 1 of hydrolysable material in substrate/ g VS inoculum, considering that sugars (hydrolysable material) make up half of the substrate (Ferreira et al., 2014). The pH, after adding the activated sludge, was always between 6.5 and 7. The working volume was approximately 400 mL, in order to have enough headspace for gas production. A control test without substrate was also conducted, aiming to check the methanogenic activity of the inoculum. Before starting the test, the bottles were closed with rubber septa and aluminium crimps. Helium gas was circulated inside the gas chamber for 5 min, and the test started after releasing the pressure. The bottles were placed horizontally in a rotary desk with constant mixing under mesophilic conditions in a thermo￾static room (35.1 ± 0.3 C). Biogas production in the headspace of each bottle was mea￾sured periodically by a manual pressure transmitter (PN5007, range 0–1 bar, IFM Electronics) over a period of 30 days. Biogas composition was determined by gas chromatography. Specific methane yields are expressed as the volume of methane under standard conditions, i.e. 0 C and 1 atm for gases, as the Interna￾tional Union of Pure Applied Chemistry (IUPAC) defines, per gram of VS in substrates fed into the assay (N mL CH4/g VS). Theoretical methane yields, calculated from the characteriza￾tion performed for both substrates, were as follows: 449 mL CH4/g VS for WS; and 420 mL CH4/g VS for SCB. These values are consistent with those calculated by Ferreira et al. (2013) for WS (444 mL CH4/g VS) and Badshah et al. (2012) for SCB (415 mL CH4/ g VS), considering the stoichiometric conversion of the organic matter. 2.4. Analytical methods Total solids (TS), volatile solids and total Kjeldahl nitrogen (TKN) were measured following the procedures given in Standard Methods for Examination of Water and Wastewater (APHA, 2005). Total chemical oxygen demand (TCOD) was determined according to the standard method UNE 77004:2002 based on the dichromate method only in the initial raw material (Ferreira et al., 2013). The analytical methods of the National Renewable Energy Lab￾oratory (NREL) were followed to determine substrate composition in terms of ash, lignin, cellulose (as glucose), and hemicellulose (as xylose) (Sluiter et al., 2012). High performance liquid chromatogra￾phy (HPLC) was used to measure glucose, xylose, formic acid, acetic acid, HMF and furfural, using a Bio-Rad HPX-87P column at 80 C with MilliQ water as the mobile phase for sugars and a Bio-Rad HPX-87H column at 50 C with 0.005 M H2SO4 as the mobile phase for acids, both at 0.6 mL/min. A Waters 2414 refractive index was used as detector (Travaini et al., 2013). The total content of pheno￾lic compounds in the samples was determined by the Folin–Ciocal￾teu method (Singleton and Rossi, 1965) with gallic acid as the calibration standard. The biogas composition (CO2, H2S, O2, N2, CH4) was measured by gas chromatography using a Varian CP-177 3800 GC-TCD equipped with a CP-Molsieve 5A and a CP￾Pora BOND Q columns, using helium as the carrier gas. All analyses were performed in duplicate and all chromatographic standards were of analytical grade, and MilliQ Ultrapure water was used. 2.5. Determination of kinetic parameters The cumulative methane production data from the experiments was fitted either to a first order model (Eq. (1)) or to the modified Gompertz equation (Eq. (2)) (Lay et al., 1996) or to a combination of both. The first one was applied successfully in many reports on anaerobic biodegradability tests when the hydrolysis reaction was the rate-limiting step of the global process (Ferreira et al., 2013). The modified Gompertz model described the cumulative methane production in batch assays when an inhibitory behavior was observed, assuming that the methane production was a function of bacterial growth. Moreover, the model parameters were calcu￾lated by minimizing the least square difference between observed and predicted values. B ¼ B0 ½1  expðkH tÞ ð1Þ B ¼ B0 exp  exp Rm e B0 ðk  tÞ þ 1  
ð2Þ In these equations, B represents the cumulative methane production (mL CH4/g VS) and t is the length of the assay (d). These models estimate the methane production potential B0 (mL CH4/ g VS, related to the substrate biodegradability), the hydrolysis coefficient kH (d1 ), the maximum biogas production rate Rm (mL CH4/g VSd), and the lag time k(d). 184 S. Bolado-Rodríguez et al. / Bioresource Technology 201 (2016) 182–190