Bioresource Technology 201 (2016)182-190 Contents lists available at ScienceDirect BIORESOURCE TECHNOLOGY Bioresource Technology ELSEVIER journal homepage:www.elsevier.com/locate/biortech Effect of thermal,acid,alkaline and alkaline-peroxide pretreatments on CrossMark the biochemical methane potential and kinetics of the anaerobic digestion of wheat straw and sugarcane bagasse Silvia Bolado-Rodriguez*,Cristina Toquero,Judit Martin-Juarez,Rodolfo Travaini, Pedro Antonio Garcia-Encina Department of Chemical Engineering and Environmental Technology.University of Valladolid.Calle Doctor Mergelina s/n,47011 Valladolid,Spain HIGHLIGHTS Highest methane productions were obtained from thermally pretreated whole slurries. Furfural and 5-HMF released in acid pretreatment inhibited methane production. High phenolic compounds release required a microorganism's acclimation period. Lignin degradation provided the highest hydrolysis rates when inhibition was defeated. A novel kinetic model is proposed combining hydrolysis and microorganisms inhibition ARTICLE INFO ABSTRACT Article history: The effect of thermal,acid,alkaline and alkaline-peroxide pretreatments on the methane produced by the Received 23 September 2015 anaerobic digestion of wheat straw(WS)and sugarcane bagasse(SCB)was studied,using whole slurry Received in revised form 17 November 2015 and solid fraction.All the pretreatments released formic and acetic acids and phenolic compounds,while Accepted 18 November 2015 Available online 28 November 2015 5-hydroxymetilfurfural(HMF)and furfural were generated only by acid pretreatment.A remarkable inhi- bition was found in most of the whole slurry experiments,except in thermal pretreatment which improved methane production compared to the raw materials(29%for WS and 11%for SCB).The alkaline Keywords: pretreatment increased biodegradability (around 30%)and methane production rate of the solid fraction Biogas Pretreatment of both pretreated substrates.Methane production results were fitted using first order or modified Inhibition Gompertz equations,or a novel model combining both equations.The model parameters provided infor- Lignocellulosic material mation about substrate availability,controlling step and inhibitory effect of compounds generated by Biodegradability each pretreatment. 2015 Elsevier Ltd.All rights reserved. 1.Introduction Among renewable sources,the lignocellulosic biomass is one of the most attractive alternatives for bioenergy production (second Bioenergy production from renewable sources is becoming generation technology)since it is available in high quantities crucial in order to address the growing demand for energy and at a low cost (Badshah et al.,2012).This study focuses on and the need to reduce greenhouse gas emissions,owing to the bioenergy production from two of the major agricultural ligno- unavoidable depletion of fossil fuel reserves and the environmental cellulosic residues:wheat straw (WS)and sugarcane bagasse consequences of global warming (Karagoz et al.,2012). (SCB).Wheat straw represents the largest fraction of agricultural waste in many countries,including Spain.Most of this wheat straw is commonly used for mulching or as fodder and the rest is burnt or left unused.For this reason,its use for biofuel production is grow- Abbreviations:BMP.biochemical methane potential:NP.normalized production ing worldwide (Menon and Rao,2012).Sugarcane bagasse is an of methane:SCB.sugarcane bagasse:TS,total solids:TKN,total Kjeldahl nitrogen: VS.volatile solids:WS,wheat straw. abundant lignocellulosic residue produced in many tropical coun- Corresponding author.Tel.:+34983 423958. tries,such as Brazil,India and Colombia.This bagasse is commonly E-mail address:silvia@iq uvaes (S.Bolado-Rodriguez) used for generating electricity by combustion,as animal feedstock, http://dx.doiorg/10.1016/j.biortech.2015.11.047 0960-8524/2015 Elsevier Ltd.All rights reserved
Effect of thermal, acid, alkaline and alkaline-peroxide pretreatments on the biochemical methane potential and kinetics of the anaerobic digestion of wheat straw and sugarcane bagasse Silvia Bolado-Rodríguez ⇑ , Cristina Toquero, Judit Martín-Juárez, Rodolfo Travaini, Pedro Antonio García-Encina Department of Chemical Engineering and Environmental Technology, University of Valladolid, Calle Doctor Mergelina s/n, 47011 Valladolid, Spain highlights Highest methane productions were obtained from thermally pretreated whole slurries. Furfural and 5-HMF released in acid pretreatment inhibited methane production. High phenolic compounds release required a microorganism’s acclimation period. Lignin degradation provided the highest hydrolysis rates when inhibition was defeated. A novel kinetic model is proposed combining hydrolysis and microorganisms inhibition. article info Article history: Received 23 September 2015 Received in revised form 17 November 2015 Accepted 18 November 2015 Available online 28 November 2015 Keywords: Biogas Pretreatment Inhibition Lignocellulosic material Biodegradability abstract The effect of thermal, acid, alkaline and alkaline-peroxide pretreatments on the methane produced by the anaerobic digestion of wheat straw (WS) and sugarcane bagasse (SCB) was studied, using whole slurry and solid fraction. All the pretreatments released formic and acetic acids and phenolic compounds, while 5-hydroxymetilfurfural (HMF) and furfural were generated only by acid pretreatment. A remarkable inhibition was found in most of the whole slurry experiments, except in thermal pretreatment which improved methane production compared to the raw materials (29% for WS and 11% for SCB). The alkaline pretreatment increased biodegradability (around 30%) and methane production rate of the solid fraction of both pretreated substrates. Methane production results were fitted using first order or modified Gompertz equations, or a novel model combining both equations. The model parameters provided information about substrate availability, controlling step and inhibitory effect of compounds generated by each pretreatment. 2015 Elsevier Ltd. All rights reserved. 1. Introduction Bioenergy production from renewable sources is becoming crucial in order to address the growing demand for energy and the need to reduce greenhouse gas emissions, owing to the unavoidable depletion of fossil fuel reserves and the environmental consequences of global warming (Karagöz et al., 2012). Among renewable sources, the lignocellulosic biomass is one of the most attractive alternatives for bioenergy production (second generation technology) since it is available in high quantities and at a low cost (Badshah et al., 2012). This study focuses on bioenergy production from two of the major agricultural lignocellulosic residues: wheat straw (WS) and sugarcane bagasse (SCB). Wheat straw represents the largest fraction of agricultural waste in many countries, including Spain. Most of this wheat straw is commonly used for mulching or as fodder and the rest is burnt or left unused. For this reason, its use for biofuel production is growing worldwide (Menon and Rao, 2012). Sugarcane bagasse is an abundant lignocellulosic residue produced in many tropical countries, such as Brazil, India and Colombia. This bagasse is commonly used for generating electricity by combustion, as animal feedstock, http://dx.doi.org/10.1016/j.biortech.2015.11.047 0960-8524/ 2015 Elsevier Ltd. All rights reserved. Abbreviations: BMP, biochemical methane potential; NP, normalized production of methane; SCB, sugarcane bagasse; TS, total solids; TKN, total Kjeldahl nitrogen; VS, volatile solids; WS, wheat straw. ⇑ Corresponding author. Tel.: +34 983 423 958. E-mail address: silvia@iq.uva.es (S. Bolado-Rodríguez). Bioresource Technology 201 (2016) 182–190 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
S.Bolado-Rodriguez et aL/Bioresource Technology 201 (2016)182-190 183 or as fuel in boilers that produce low-pressure steam.However,the pretreatment(Rabelo et al.,2011:Talebnia et al.,2010).The pro- surplus that remains leads to environmental and storage problems cess is usually carried out at mild temperatures.using hydrogen (Costa et al.,2014:Travaini et al.,2013). peroxide(H2O2)and NaOH,leading to a lesser formation of inhibi- Three types of energy can be produced from these lignocellu- tors than in other processes. losic wastes through thermochemical or biochemical processing: The determination of the kinetic of the anaerobic digestion pro- liquid fuels such as bioethanol,gaseous fuels such as biogas,and vides important information about the effect of the inhibitory com- electricity by combustion (Menon and Rao,2012). pounds generated by the pretreatment on the biodegradability. Biogas,composed mainly of methane and carbon dioxide,is and to determine if the hydrolysis is the limiting step.There are considered a clean and renewable form of energy.It has the advan- several models of the kinetic analysis of biogas production process; tage of being easy to implement for consumers,and easy to it all depends on the types of substrate used for anaerobic digestion produce on a local level,such as small-scale farms (Taherdanak and the controlling step. and Zilouei,2014).Biogas can be produced through the anaerobic The Gompertz model is well known among the available models digestion of many types of wastes,and is considered one of the for the kinetic behavior of the anaerobic digestion process consid- most efficient technologies,since high energy recovery and ering inhibition.The Gompertz equation is used to estimate the environmental benefits can be achieved (Ferreira et al.,2013). kinetic parameters;biogas yield potential,duration of the lag Nevertheless,the biodegradability of biomass residues is lim- phase,and maximum biogas production rate (Krishania et al.. ited by its lignocellulosic structure.Therefore,efficient pretreat- 2013).However,when the hydrolysis reaction is the rate limiting ment digestion could accelerate the hydrolysis and improve the step of the overall process,as in the anaerobic degradation of some biogas production(Sambusiti et al.,2013).However,the realization lignocellulosic substrates,the first order model is commonly used of a pretreatment frequently produces degradation compounds to estimate the extent of the reaction,and the hydrolysis constant. that can act as inhibitors:organic acids(acetic,formic and levuli- Both parameters can be used in a global model of the anaerobic nic),furan derivatives [furfural and 5-hydroxymethylfurfural digestion process (such as ADM1)to predict the performance of (5-HMF)]and phenol compounds,affecting overall cell physiology anaerobic digesters (Ferreira et al.,2013). and often decreasing viability and productivity (Chandel et al.. The present study aims to establish the influence of four pre- 2011). treatments (thermal autoclaving.dilute HCI autoclaving.dilute Different pretreatment methods have been studied,depending NaOH autoclaving and alkaline peroxide)in the production of on the characteristics of each lignocellulosic feedstock (Karagoz biogas from sugarcane bagasse and wheat straw,and to study et al.,2012),including biological,chemical,physical processes,or the kinetics of anaerobic digestion in order to determine the a combination of them.Among them,this work focuses on thermal, influence of inhibitory compounds present in both the liquid phase dilute acid,dilute alkaline and oxidative pretreatments. and the solid phase. The thermal pretreatments are considered eco-friendly,green processing technologies.Energy recovery from biomass for fuel is excellent,often with values as high as 80%(Chandra et al., 2.Methods 2012a).Thermal pretreatments have been applied to improve the anaerobic digestibility of different agriculture substrates such as 2.1.Materials wheat straw,sorghum forage and sugarcane bagasse (Costa et al.. 2014;Sambusiti et al.,2013).The non-addition of chemicals avoids Two lignocellulosic substrates were used in this study:WS,pro- the corrosion problems,and decreases the formation of toxic vided by the Castilla Leon Institute of Technological Agriculture compounds.Other advantages include the lower requirement of from Valladolid(Spain).and SCB(surplus after milling in a sugar/ chemicals for the neutralization of the hydrolysates produced, ethanol factory).donated by Usina Vale,City of Onda Verde-SP and the smaller amount of waste produced in comparison to other (Brazil).Wheat straw and sugarcane bagasse were washed for processes(Ferreira et al..2013). particulate material removal,dried in a ventilated oven at 42C Acid pretreatment is widely applied due to its low cost and high and ground in an agricultural crusher to a size of 3-5 mm.Both efficiency to hydrolyze hemicellulose into monomeric sugars with- substrates were kept in an oven at 45C until they reached a out dissolving lignin(Ferreira et al.,2013).However,this pretreat- constant weight prior to compositional analysis and different ment is corrosive and generates high concentrations of toxic pretreatments.The chemical composition of both substrates is compounds,making it necessary to recover the acids in order to presented in Table 1. make the process economically feasible (Talebnia et al.,2010). The main substrates studied for this pretreatment are wheat straw, 2.2.Pretreatments sorghum forage and sugarcane bagasse (Sambusiti et al.,2013). and different acids such as sulphuric,hydrochloric,phosphoric, Four different pretreatments were applied to both substrates in maleic,peracetic or nitric acids have been investigated (Badshah this study:thermal autoclaving(A).dilute HCI autoclaving (B). et al.,2012:Chandel et al.,2011:Costa et al.,2014:Krishania etal,2013). The alkaline pretreatment is typically used in lignocellulosic Table 1 Composition of raw materials. materials with a high lignin content,such as wheat straw and sug- arcane bagasse(Rabelo et al,2011:Taherdanak and Zilouei,2014). Parameter Wheat straw Sugarcane bagasse Alkaline pretreatments performed with bases such as sodium, Total solids(g TS/kg) 916.24±121 91922±084 potassium,calcium and ammonium hydroxides are effective in Volatile solids (g vS/kg) 818.83±1.52 907.96±1.10 modifying the structure and solubilizing the lignin.In addition. N-TKN (g N/kg) 4.85±0.09 2.51±0.02 the alkaline pretreatment reduces the degree of inhibition in TCOD(g Oz/kg) 1150.40±4.99 1188.85±2.43 Cellulose (w/w) 35.19±029 46.21±0.10 methane fermentation and provides a lower cost of production Hemicellulose w/w)" 22.15±021 20.86±0.05 (Ferreira et al.,2013:Krishania et al.,2013). Total lignin w/w)" 22.09±0.80 22.67±0.04 The use of an oxidizing compound in combination with an alka- Acid insoluble lignin w/w) 18.17±021 19.53±0.03 line pretreatment is becoming more common in order to improve Ash w/w) 7.49±029 1.19±0.10 the digestibility of crop residues,compared with an alkaline Dry basis calculated composition
or as fuel in boilers that produce low-pressure steam. However, the surplus that remains leads to environmental and storage problems (Costa et al., 2014; Travaini et al., 2013). Three types of energy can be produced from these lignocellulosic wastes through thermochemical or biochemical processing: liquid fuels such as bioethanol, gaseous fuels such as biogas, and electricity by combustion (Menon and Rao, 2012). Biogas, composed mainly of methane and carbon dioxide, is considered a clean and renewable form of energy. It has the advantage of being easy to implement for consumers, and easy to produce on a local level, such as small-scale farms (Taherdanak and Zilouei, 2014). Biogas can be produced through the anaerobic digestion of many types of wastes, and is considered one of the most efficient technologies, since high energy recovery and environmental benefits can be achieved (Ferreira et al., 2013). Nevertheless, the biodegradability of biomass residues is limited by its lignocellulosic structure. Therefore, efficient pretreatment digestion could accelerate the hydrolysis and improve the biogas production (Sambusiti et al., 2013). However, the realization of a pretreatment frequently produces degradation compounds that can act as inhibitors: organic acids (acetic, formic and levulinic), furan derivatives [furfural and 5-hydroxymethylfurfural (5-HMF)] and phenol compounds, affecting overall cell physiology and often decreasing viability and productivity (Chandel et al., 2011). Different pretreatment methods have been studied, depending on the characteristics of each lignocellulosic feedstock (Karagöz et al., 2012), including biological, chemical, physical processes, or a combination of them. Among them, this work focuses on thermal, dilute acid, dilute alkaline and oxidative pretreatments. The thermal pretreatments are considered eco-friendly, green processing technologies. Energy recovery from biomass for fuel is excellent, often with values as high as 80% (Chandra et al., 2012a). Thermal pretreatments have been applied to improve the anaerobic digestibility of different agriculture substrates such as wheat straw, sorghum forage and sugarcane bagasse (Costa et al., 2014; Sambusiti et al., 2013). The non-addition of chemicals avoids the corrosion problems, and decreases the formation of toxic compounds. Other advantages include the lower requirement of chemicals for the neutralization of the hydrolysates produced, and the smaller amount of waste produced in comparison to other processes (Ferreira et al., 2013). Acid pretreatment is widely applied due to its low cost and high efficiency to hydrolyze hemicellulose into monomeric sugars without dissolving lignin (Ferreira et al., 2013). However, this pretreatment is corrosive and generates high concentrations of toxic compounds, making it necessary to recover the acids in order to make the process economically feasible (Talebnia et al., 2010). The main substrates studied for this pretreatment are wheat straw, sorghum forage and sugarcane bagasse (Sambusiti et al., 2013), and different acids such as sulphuric, hydrochloric, phosphoric, maleic, peracetic or nitric acids have been investigated (Badshah et al., 2012; Chandel et al., 2011; Costa et al., 2014; Krishania et al., 2013). The alkaline pretreatment is typically used in lignocellulosic materials with a high lignin content, such as wheat straw and sugarcane bagasse (Rabelo et al., 2011; Taherdanak and Zilouei, 2014). Alkaline pretreatments performed with bases such as sodium, potassium, calcium and ammonium hydroxides are effective in modifying the structure and solubilizing the lignin. In addition, the alkaline pretreatment reduces the degree of inhibition in methane fermentation and provides a lower cost of production (Ferreira et al., 2013; Krishania et al., 2013). The use of an oxidizing compound in combination with an alkaline pretreatment is becoming more common in order to improve the digestibility of crop residues, compared with an alkaline pretreatment (Rabelo et al., 2011; Talebnia et al., 2010). The process is usually carried out at mild temperatures, using hydrogen peroxide (H2O2) and NaOH, leading to a lesser formation of inhibitors than in other processes. The determination of the kinetic of the anaerobic digestion provides important information about the effect of the inhibitory compounds generated by the pretreatment on the biodegradability, and to determine if the hydrolysis is the limiting step. There are several models of the kinetic analysis of biogas production process; it all depends on the types of substrate used for anaerobic digestion and the controlling step. The Gompertz model is well known among the available models for the kinetic behavior of the anaerobic digestion process considering inhibition. The Gompertz equation is used to estimate the kinetic parameters; biogas yield potential, duration of the lag phase, and maximum biogas production rate (Krishania et al., 2013). However, when the hydrolysis reaction is the rate limiting step of the overall process, as in the anaerobic degradation of some lignocellulosic substrates, the first order model is commonly used to estimate the extent of the reaction, and the hydrolysis constant. Both parameters can be used in a global model of the anaerobic digestion process (such as ADM1) to predict the performance of anaerobic digesters (Ferreira et al., 2013). The present study aims to establish the influence of four pretreatments (thermal autoclaving, dilute HCl autoclaving, dilute NaOH autoclaving and alkaline peroxide) in the production of biogas from sugarcane bagasse and wheat straw, and to study the kinetics of anaerobic digestion in order to determine the influence of inhibitory compounds present in both the liquid phase and the solid phase. 2. Methods 2.1. Materials Two lignocellulosic substrates were used in this study: WS, provided by the Castilla & León Institute of Technological Agriculture from Valladolid (Spain), and SCB (surplus after milling in a sugar/ ethanol factory), donated by Usina Vale, City of Onda Verde-SP (Brazil). Wheat straw and sugarcane bagasse were washed for particulate material removal, dried in a ventilated oven at 42 C and ground in an agricultural crusher to a size of 3–5 mm. Both substrates were kept in an oven at 45 C until they reached a constant weight prior to compositional analysis and different pretreatments. The chemical composition of both substrates is presented in Table 1. 2.2. Pretreatments Four different pretreatments were applied to both substrates in this study: thermal autoclaving (A), dilute HCl autoclaving (B), Table 1 Composition of raw materials. Parameter Wheat straw Sugarcane bagasse Total solids (g TS/kg) 916.24 ± 1.21 919.22 ± 0.84 Volatile solids (g VS/kg) 818.83 ± 1.52 907.96 ± 1.10 N-TKN (g N/kg)* 4.85 ± 0.09 2.51 ± 0.02 TCOD (g O2/kg)* 1150.40 ± 4.99 1188.85 ± 2.43 Cellulose (% w/w)* 35.19 ± 0.29 46.21 ± 0.10 Hemicellulose (% w/w)* 22.15 ± 0.21 20.86 ± 0.05 Total lignin (% w/w)* 22.09 ± 0.80 22.67 ± 0.04 Acid insoluble lignin (% w/w)* 18.17 ± 0.21 19.53 ± 0.03 Ash (% w/w)* 7.49 ± 0.29 1.19 ± 0.10 * Dry basis calculated composition. S. Bolado-Rodríguez et al. / Bioresource Technology 201 (2016) 182–190 183
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 operational 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 temperature, the slurry obtained from each pretreatment was recovered, and the residual solid was separated by vacuum filtration till maximum 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 pilotscale 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 pretreatment 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 thermostatic room (35.1 ± 0.3 C). Biogas production in the headspace of each bottle was measured 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 International 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 characterization 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 Laboratory (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 chromatography (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 phenolic compounds in the samples was determined by the Folin–Ciocalteu 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 CPPora 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 calculated 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. 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S.Bolado-Rodriguez et aL/Bioresource Technology 201(2016)182-190 185 3.Results and discussion et al..2011).However,Sambusiti et al.(2013)reported the release of furfural (0.2-0.4 g/100g VS raw material)in the thermal pre- 3.1.Composition of pretreatment liquids treatment of WS at 100C and 160C.Costa et al.(2014)obtained considerably higher sugar release (1.94 g/100g raw material)and The presence and concentration of sugars,volatile solids and some HMF production from the thermal pretreatment of SCB degradation compounds released into the liquid during pretreat- working with a higher temperature (150C)but lower reaction ments can significantly affect the anaerobic digestion of pretreated time(30 min).Their experiments of SCB acid pretreatment,work- biomass.Table 2 shows these values for percentages of sugars ing at 120C and 40 min with 0.63 M HCI.provided appreciably (glucose plus xylose),volatile solids and degradation compounds lower degradation compounds (0.001 g of 5-HMF and 0.8 g of formed and released into the liquid during the four pretreatments furfural by 100 g of raw material). for both substrates Rabelo et al.(2011)reported high sugar concentrations The pretreatment liquids of both substrates presented low sug- (1.504-2.904 g/L)with lower solid concentrations of sugarcane ars release,with values below 4.04 g/100g raw material in all bagasse(4-8%)in a pretreatment with Ca(OH)2 at 90C over a long cases except for the liquids from the diluted acid pretreatment period (90 h).They also obtained higher sugar concentrations (B).which reached a higher content due to solubilization of hemi- (7.565-21.759 g/L)in an alkaline hydrogen peroxide pretreatment celluloses produced by this type of pretreatment.The experimental at 25C with solids concentrations of between 4%and 15%over pretreatment conditions in this work are identical to those applied 1 h.These high sugar release results may be due to a higher by Toquero and Bolado(2014)for ethanol production from wheat concentration of H202(7.36%(v/v). straw,so the sugar release results are also the same for this material.For most of the pretreatments tested,the sugar release 3.2.Methane production was higher for WS than for SCB.In contrast,the concentration of volatile solids in the pretreatment liquids was greater,in all cases 3.2.1.Test 1:BMP of untreated materials and of pretreated whole for SCB that for WS.The highest volatile solids concentration was slurry found in the liquid phases from the basic pretreatment(C),despite The influence of pretreatments applied to both substrates was the low sugar release in this pretreatment.This high volatile solids studied in terms of methane production,considering three param- release may be related to the remarkable effect of the dilute eters:methane yield,defined as the volume of methane gas alkaline pretreatment disrupting the lignin structure.Liu et al. produced per gram of volatile solid fed;biodegradability,defined (2015)observed a lignin reduction of up to 54.7%in wheat straw as the percentage of the theoretical methane yield determined pretreatment with 50%KOH solution.Costa et al.(2014)extracted for raw substrates;and normalized production of methane (NP). approximately 80%of the lignin content of sugarcane bagasse defined as the ratio between the production of methane per gram using alkaline pretreatment (130C,20 min,NaOH 1 M). of VS from treated and untreated substrates. The main degradation compounds found in the pretreatment Fig.I presents the cumulative methane production curves from liquids were organic acids and phenolics from lignin.Acetic acid test 1,working with whole slurry;the results of WS (a)and SCB(b) and phenolic compounds appear in the liquid phase of all the are shown together for comparison.Results from untreated pretreated samples.HMF and furfural were only detected in hydro- substrates are also presented in this figure. lysates from acid pretreatment samples(B).where the formic acid By comparing the experimental values obtained in the BMP concentration was very low or undetectable.Comparing both tests of raw WS with the theoretical methane yield(449 mL CH4/ substrates,except for in the thermal pretreatment,which pro- g VS).it was determined that 48%of the VS were converted into duced very low concentrations of inhibitors,the production of methane.This result is very close to the 51%obtained with WS degradation compounds was higher for SCB than for WS.The high- by Ferreira et al.(2013).For untreated SCB,the experimental est concentrations of degradation compounds were detected in biodegradability was slightly higher,reaching 52%of the theoreti- samples from the basic pretreatment (C).with 5.80 g/100g of cal value (420 mL CH4/g VS). raw material for WS,and with 8.36 g/100 g of raw material for Concerning the assays with whole slurry of pretreated materi- SCB,respectively.This pretreatment provided the highest releases als,the highest methane yields were obtained for substrates of acetic acid and phenolic compounds,related to the previously pretreated by thermal autoclaving (A).both substrates followed noted high lignin removal effect. comparable behaviors during the digestion process.The anaerobic Sugars and volatile solids release,and degradation compounds biodegradability of WS_A1 increased up to 62%and the variable concentrations obtained in this study are within the range of most NP reached a value of 1.29.Thermal pretreatment also caused an of the results found in the literature (Bustos et al.,2003;Chandel increase in methane production from sample SCB_A1,which Table 2 Sugars (glucose plus xylose).volatile solids and degradation compounds(g/100 g of raw material)released to the liquid during pretreatments of wheat straw and sugarcane bagasse. Sample Degradation compounds Sugars Volatile solids Formic acid Acetic acid HMF Furfural Total phenolics WS_A 1.48±0.01 9.88 0.08±0.01 0.13±0.03 ND ND 0.42±0.01 WS_B 1774±300 27.8 0.06±0.00 0.72±000 0.04±0.00 1.10±0.07 0.80±0.00 WS_C 1.99±0.12 33.42 0.25±0.03 2.60±0.14 ND ND 2.95±0.00 WS_D 4.04±0.07 12.74 0.79±0.01 1.23±0.06 ND ND 0.48±0.01 SCB_A ND 10.43 0.01±0.00 0.08±0.00 ND ND 0.50±0.01 SCB_B 21.42±2.09 30.52 ND 4.11±0.13 0.02±0.00 122±0.11 1.32±0.00 SCB_C 0.98±0.00 37.41 0.12±0.01 4.30±0.24 ND ND 3.94±0.04 SCB_D 1.64±0.11 15.34 1.00±0.05 1.77±0.04 ND ND 1.14±0.00 ND,not detected,under detection limits of the method. Codes:Lignocellulosic material:WS,wheat straw and SCB sugarcane bagasse. Pr therm ving: ite acid autoclaving:C.dilute alkali autoclaving and D.alkaline peroxide
3. Results and discussion 3.1. Composition of pretreatment liquids The presence and concentration of sugars, volatile solids and degradation compounds released into the liquid during pretreatments can significantly affect the anaerobic digestion of pretreated biomass. Table 2 shows these values for percentages of sugars (glucose plus xylose), volatile solids and degradation compounds formed and released into the liquid during the four pretreatments for both substrates. The pretreatment liquids of both substrates presented low sugars release, with values below 4.04 g/100 g raw material in all cases except for the liquids from the diluted acid pretreatment (B), which reached a higher content due to solubilization of hemicelluloses produced by this type of pretreatment. The experimental pretreatment conditions in this work are identical to those applied by Toquero and Bolado (2014) for ethanol production from wheat straw, so the sugar release results are also the same for this material. For most of the pretreatments tested, the sugar release was higher for WS than for SCB. In contrast, the concentration of volatile solids in the pretreatment liquids was greater, in all cases for SCB that for WS. The highest volatile solids concentration was found in the liquid phases from the basic pretreatment (C), despite the low sugar release in this pretreatment. This high volatile solids release may be related to the remarkable effect of the dilute alkaline pretreatment disrupting the lignin structure. Liu et al. (2015) observed a lignin reduction of up to 54.7% in wheat straw pretreatment with 50% KOH solution. Costa et al. (2014) extracted approximately 80% of the lignin content of sugarcane bagasse using alkaline pretreatment (130 C, 20 min, NaOH 1 M). The main degradation compounds found in the pretreatment liquids were organic acids and phenolics from lignin. Acetic acid and phenolic compounds appear in the liquid phase of all the pretreated samples. HMF and furfural were only detected in hydrolysates from acid pretreatment samples (B), where the formic acid concentration was very low or undetectable. Comparing both substrates, except for in the thermal pretreatment, which produced very low concentrations of inhibitors, the production of degradation compounds was higher for SCB than for WS. The highest concentrations of degradation compounds were detected in samples from the basic pretreatment (C), with 5.80 g/100 g of raw material for WS, and with 8.36 g/100 g of raw material for SCB, respectively. This pretreatment provided the highest releases of acetic acid and phenolic compounds, related to the previously noted high lignin removal effect. Sugars and volatile solids release, and degradation compounds concentrations obtained in this study are within the range of most of the results found in the literature (Bustos et al., 2003; Chandel et al., 2011). However, Sambusiti et al. (2013) reported the release of furfural (0.2–0.4 g/100 g VS raw material) in the thermal pretreatment of WS at 100 C and 160 C. Costa et al. (2014) obtained considerably higher sugar release (1.94 g/100 g raw material) and some HMF production from the thermal pretreatment of SCB, working with a higher temperature (150 C) but lower reaction time (30 min). Their experiments of SCB acid pretreatment, working at 120 C and 40 min with 0.63 M HCl, provided appreciably lower degradation compounds (0.001 g of 5-HMF and 0.8 g of furfural by 100 g of raw material). Rabelo et al. (2011) reported high sugar concentrations (1.504–2.904 g/L) with lower solid concentrations of sugarcane bagasse (4–8%) in a pretreatment with Ca(OH)2 at 90 C over a long period (90 h). They also obtained higher sugar concentrations (7.565–21.759 g/L) in an alkaline hydrogen peroxide pretreatment at 25 C with solids concentrations of between 4% and 15% over 1 h. These high sugar release results may be due to a higher concentration of H2O2 (7.36% (v/v). 3.2. Methane production 3.2.1. Test 1: BMP of untreated materials and of pretreated whole slurry The influence of pretreatments applied to both substrates was studied in terms of methane production, considering three parameters: methane yield, defined as the volume of methane gas produced per gram of volatile solid fed; biodegradability, defined as the percentage of the theoretical methane yield determined for raw substrates; and normalized production of methane (NP), defined as the ratio between the production of methane per gram of VS from treated and untreated substrates. Fig. 1 presents the cumulative methane production curves from test 1, working with whole slurry; the results of WS (a) and SCB (b) are shown together for comparison. Results from untreated substrates are also presented in this figure. By comparing the experimental values obtained in the BMP tests of raw WS with the theoretical methane yield (449 mL CH4/ g VS), it was determined that 48% of the VS were converted into methane. This result is very close to the 51% obtained with WS by Ferreira et al. (2013). For untreated SCB, the experimental biodegradability was slightly higher, reaching 52% of the theoretical value (420 mL CH4/g VS). Concerning the assays with whole slurry of pretreated materials, the highest methane yields were obtained for substrates pretreated by thermal autoclaving (A), both substrates followed comparable behaviors during the digestion process. The anaerobic biodegradability of WS_A1 increased up to 62% and the variable NP reached a value of 1.29. Thermal pretreatment also caused an increase in methane production from sample SCB_A1, which Table 2 Sugars (glucose plus xylose), volatile solids and degradation compounds (g/100 g of raw material) released to the liquid during pretreatments of wheat straw and sugarcane bagasse. Samplea Degradation compounds Sugars Volatile solids Formic acid Acetic acid HMF Furfural Total phenolics WS_A 1.48 ± 0.01 9.88 0.08 ± 0.01 0.13 ± 0.03 ND ND 0.42 ± 0.01 WS_B 17.74 ± 3.00 27.8 0.06 ± 0.00 0.72 ± 0.00 0.04 ± 0.00 1.10 ± 0.07 0.80 ± 0.00 WS_C 1.99 ± 0.12 33.42 0.25 ± 0.03 2.60 ± 0.14 ND ND 2.95 ± 0.00 WS_D 4.04 ± 0.07 12.74 0.79 ± 0.01 1.23 ± 0.06 ND ND 0.48 ± 0.01 SCB_A ND 10.43 0.01 ± 0.00 0.08 ± 0.00 ND ND 0.50 ± 0.01 SCB_B 21.42 ± 2.09 30.52 ND 4.11 ± 0.13 0.02 ± 0.00 1.22 ± 0.11 1.32 ± 0.00 SCB_C 0.98 ± 0.00 37.41 0.12 ± 0.01 4.30 ± 0.24 ND ND 3.94 ± 0.04 SCB_D 1.64 ± 0.11 15.34 1.00 ± 0.05 1.77 ± 0.04 ND ND 1.14 ± 0.00 ND, not detected, under detection limits of the method. a Codes: Lignocellulosic material: WS, wheat straw and SCB, sugarcane bagasse. Pretreatment: A, thermal autoclaving; B, dilute acid autoclaving; C, dilute alkali autoclaving and D, alkaline peroxide. S. Bolado-Rodríguez et al. / Bioresource Technology 201 (2016) 182–190 185
186 S.Bolado-Rodriguez et aL/Bioresource Technology 201 (2016)182-190 ·Untreated WS·WsAl+ws_Bl·Ws_C1×WsDI Uetreated WS-WS Al_model-----WS Cl model ----WS D1 model furfural concentrations were detected only in samples of this pre 300 (a) treatment.These degradation compounds released to pretreatment liquids could have caused the inactivity or death of the inoculum (Chen et al.,2008:Sezun et al.,2011).In contrast,Costa et al. 250 (2014)obtained lower inhibitory compound concentration (0.33 g/L of furfural and 0.31 g/L of 5-HMF)in the liquid phase of 200 acid pretreated SCB (136C,6.4 min,HCI 0.63 M).achieving a BMP of 122.2 L CH4/kg of substrate.Badshah et al.(2012)obtained a methane yield from acid treated SCB(2%H2SO4.121C,15 min) 150- of 173 L/kg VS,which showed an increase in methane of 18%com- pared to untreated SCB,probably due to the low furfural and no 100- HMF release in these experiments. Cumulative methane production curves from the anaerobic digestion of dilute alkali(C)pretreated materials followed a similar 50- trend for both substrates,with an acclimation period,according to Fig.1.At the end of the test,methane production from sample 0 WS_C1 did not increase in comparison with untreated WS,with 12 13 18 21 24 27 30 33 a NP ratio of 1.00,indicating no effect of this pretreatment in wheat Time (d) straw biodegradability.Chandra et al.(2012b),with an alkaline ·Untresed SCB·SCBA1·SCB8I·SCB CI SCB_D1 -Untreated SCBSCB_Al_model-SCB CI model pretreatment(4%NaOH,37C,120 h),observed a lower methane 300= production of 165.9 L/kg VS.Nevertheless,they reported an (b) increase of 111%in relation to its very low methane production 250 from untreated wheat straw(78.4 L/kg VS).Reilly et al.(2015) obtained a final biomethane potential yield increase from 260 to 313 mL/g VS,but working with very different conditions:0.08 M 200 Ca(OH)2.0.59%(w/v)calculated,pretreatment for 48 h at 20C of 3 mm milled WS particles. However,SCB_C1 showed a slight increase in methane produc- 150- tion compared to raw SCB,with a biodegradability of 55%and NP of 1.05.Similar results were reported by Rabelo et al.(2011)with Ca 100- (OH)2 pretreated SCB(4-8%solid concentrations.90C,90 h) where the methane yield was 180-148 mL/g VS and the biodegrad- 50- ability was 51-42%. The alkali pretreatment did not favour the anaerobic digestion of any of the tested substrates,reducing methane productions dur- 0- ◆ ing the process and resulting in a biodegradability similar or even 0 1215 18 21 24 27 lower than the untreated materials at the end of the experiment. Time(d) The high degradation compound concentrations generated by the high lignin solubilization in alkali pretreatment probably Fig.1.Experimental results and fitting curves of cumulative methane production affected the bacterial growth and the methanogenic activity of from untreated and whole slurry pretreated materials (test 1).(A)Thermal autoclaving:(B)dilute acid autoclaving:(C)dilute alkali autoclaving:(D)alkaline inoculum. peroxide pretreatments.(a)Wheat straw and (b)sugarcane bagasse. Finally,using the complete pretreated material from alkaline peroxide pretreatment(D).WS and SCB were seen to behave com- pletely differently when subjected to anaerobic digestion,as seen in Fig.1.After 17 days of approximately zero methanogenic activ- reached an anaerobic biodegradability of 58%and NP of 1.11.Costa ity,the anaerobic biodegradability of WS_D1 increased up to 51 et al.(2014).comparing the effect of hydrothermal,acid and during the last days of the experiment,and the variable NP reached alkaline pretreatments on sugarcane bagasse,also achieved the a value of 1.07,whereas biodegradability of sample SCB_D1 only best values of BMP for hydrothermal pretreatment.They obtained reached the value of 4%with a really low NP ratio of 0.08.The inhi- a methane production value slightly lower than that of our ther- bition in samples of both substrates,with no methane production mal pretreatment (197.5 mL CH4/g substrate)working at 200C. during the first days,probably proceeds from the presence of resid- 10 min. ual chemicals from pretreatment.The concentration of acetic acid The concentration of degradation compounds in liquid from and phenolic compounds is lower than the values obtained for thermal pretreatment was very low,with a production of possible basic pretreatment,where the inhibition was less remarkable. inhibitory compounds of 0.63 g/100g raw material for WS and of The alkaline peroxide pretreatment releases the highest formic 0.69 g/100 g raw material for SCB.Thus,the use of whole slurry acid concentrations and this value is higher for sugarcane bagasse, from pretreatment A was beneficial for the anaerobic digestion of so another possibility could be an inhibitory effect of formic acid two tested substrates,due to the increase in biomass biodegrad- on methane production.Some better results were reported ability and the production of low concentrations of inhibitory by Rabelo et al.(2011)with 36%of biodegradability in alkaline compounds,leading to an enhancement of methane production, peroxide pretreated SCB (4%w/w.7.36%(v/v)H202,pH 11.5, especially for WS. 25C,1h). In the case of substrates pretreated by dilute acid autoclaving In short,with the aim of using a whole slurry of pretreated (B),a clear negative influence in the digestion process was found materials in order to harness pretreatment liquids and to avoid a to the extent that the total inhibition of biogas production was separation step,the anaerobic digestion of samples from thermal observed.As shown in Fig.1,the anaerobic digestion of samples method A provided the highest methane production results.The WS_B1 and SCB_B1 resulted in zero biogas production.HMF and use of chemical pretreatments B,C and D resulted in similar or
reached an anaerobic biodegradability of 58% and NP of 1.11. Costa et al. (2014), comparing the effect of hydrothermal, acid and alkaline pretreatments on sugarcane bagasse, also achieved the best values of BMP for hydrothermal pretreatment. They obtained a methane production value slightly lower than that of our thermal pretreatment (197.5 mL CH4/g substrate) working at 200 C, 10 min. The concentration of degradation compounds in liquid from thermal pretreatment was very low, with a production of possible inhibitory compounds of 0.63 g/100 g raw material for WS and of 0.69 g/100 g raw material for SCB. Thus, the use of whole slurry from pretreatment A was beneficial for the anaerobic digestion of two tested substrates, due to the increase in biomass biodegradability and the production of low concentrations of inhibitory compounds, leading to an enhancement of methane production, especially for WS. In the case of substrates pretreated by dilute acid autoclaving (B), a clear negative influence in the digestion process was found, to the extent that the total inhibition of biogas production was observed. As shown in Fig. 1, the anaerobic digestion of samples WS_B1 and SCB_B1 resulted in zero biogas production. HMF and furfural concentrations were detected only in samples of this pretreatment. These degradation compounds released to pretreatment liquids could have caused the inactivity or death of the inoculum (Chen et al., 2008; Sezˇun et al., 2011). In contrast, Costa et al. (2014) obtained lower inhibitory compound concentration (0.33 g/L of furfural and 0.31 g/L of 5-HMF) in the liquid phase of acid pretreated SCB (136 C, 6.4 min, HCl 0.63 M), achieving a BMP of 122.2 L CH4/kg of substrate. Badshah et al. (2012) obtained a methane yield from acid treated SCB (2% H2SO4, 121 C, 15 min) of 173 L/kg VS, which showed an increase in methane of 18% compared to untreated SCB, probably due to the low furfural and no HMF release in these experiments. Cumulative methane production curves from the anaerobic digestion of dilute alkali (C) pretreated materials followed a similar trend for both substrates, with an acclimation period, according to Fig. 1. At the end of the test, methane production from sample WS_C1 did not increase in comparison with untreated WS, with a NP ratio of 1.00, indicating no effect of this pretreatment in wheat straw biodegradability. Chandra et al. (2012b), with an alkaline pretreatment (4% NaOH, 37 C, 120 h), observed a lower methane production of 165.9 L/kg VS. Nevertheless, they reported an increase of 111% in relation to its very low methane production from untreated wheat straw (78.4 L/kg VS). Reilly et al. (2015) obtained a final biomethane potential yield increase from 260 to 313 mL/g VS, but working with very different conditions: 0.08 M Ca(OH)2, 0.59% (w/v) calculated, pretreatment for 48 h at 20 C of 3 mm milled WS particles. However, SCB_C1 showed a slight increase in methane production compared to raw SCB, with a biodegradability of 55% and NP of 1.05. Similar results were reported by Rabelo et al. (2011) with Ca (OH)2 pretreated SCB (4–8% solid concentrations, 90 C, 90 h) where the methane yield was 180–148 mL/g VS and the biodegradability was 51–42%. The alkali pretreatment did not favour the anaerobic digestion of any of the tested substrates, reducing methane productions during the process and resulting in a biodegradability similar or even lower than the untreated materials at the end of the experiment. The high degradation compound concentrations generated by the high lignin solubilization in alkali pretreatment probably affected the bacterial growth and the methanogenic activity of inoculum. Finally, using the complete pretreated material from alkaline peroxide pretreatment (D), WS and SCB were seen to behave completely differently when subjected to anaerobic digestion, as seen in Fig. 1. After 17 days of approximately zero methanogenic activity, the anaerobic biodegradability of WS_D1 increased up to 51% during the last days of the experiment, and the variable NP reached a value of 1.07, whereas biodegradability of sample SCB_D1 only reached the value of 4% with a really low NP ratio of 0.08. The inhibition in samples of both substrates, with no methane production during the first days, probably proceeds from the presence of residual chemicals from pretreatment. The concentration of acetic acid and phenolic compounds is lower than the values obtained for basic pretreatment, where the inhibition was less remarkable. The alkaline peroxide pretreatment releases the highest formic acid concentrations and this value is higher for sugarcane bagasse, so another possibility could be an inhibitory effect of formic acid on methane production. Some better results were reported by Rabelo et al. (2011) with 36% of biodegradability in alkaline peroxide pretreated SCB (4% w/w, 7.36% (v/v) H2O2, pH 11.5, 25 C, 1 h). In short, with the aim of using a whole slurry of pretreated materials in order to harness pretreatment liquids and to avoid a separation step, the anaerobic digestion of samples from thermal method A provided the highest methane production results. The use of chemical pretreatments B, C and D resulted in similar or Fig. 1. Experimental results and fitting curves of cumulative methane production from untreated and whole slurry pretreated materials (test 1). (A) Thermal autoclaving; (B) dilute acid autoclaving; (C) dilute alkali autoclaving; (D) alkaline peroxide pretreatments. (a) Wheat straw and (b) sugarcane bagasse. 186 S. Bolado-Rodríguez et al. / Bioresource Technology 201 (2016) 182–190
S.Bolado-Rodriguez et aL/Bioresource Technology 201(2016)182-190 187 lower biodegradability values compared to untreated substrates. liquid.WS_A2 and SCB_A2 were seen to behave similarly to the However,it is necessary to analyze the results of tests with a solid untreated biomass during digestion assays. fraction in order to compare the effect and efficiency of pretreat- In contrast to test 1,where there was complete inhibition,when ments on the biodegradability of both substrates. digesting the solid fraction from dilute acid autoclaving(B),some methanogenic activity,similar to that from the control occurred with both substrates.WS_B2 showed an anaerobic biodegradability 3.2.2.Test 2:BMP of solid fraction from pretreatments of 17%with NP of 0.36,whereas sample SCB_B2 reached a biodegrad- Cumulative methane production curves from test 2 are pre- ability of 18%with NP of 0.35.This low methanogenic activity can be sented in Fig.2,where the results from WS(a)and SCB(b)were attributed to the dilution of inhibitory compounds remaining in the displayed together for comparison.Experimental results for the solid fraction,which were in lower concentration than in test 1,when four tested pretreatments applied to each material are depicted using the pretreatment liquid,causing less inhibition. together,as well as results from untreated substrates. When solid fractions from dilute alkali pretreatment (C)were In this test,solid fractions from the thermal pretreatment (A) used,the highest methane yields of all trials were obtained from of both substrates provided lower results than those of test 1 and both of untreated substrates (Section 3.2.1).Thus,the anaerobic substrates.The anaerobic biodegradability of WS_C2 increased up to 64%achieving a NP of 1.32.This is the best result biodegradability of WS_A2 was only 40%,with a NP of 0.84.Some obtained from all assays,especially considering that in less than authors like Sambusiti et al.(2013)reported no difference in the methane production between the pretreated sample (100C. 10 days a production of approximately 240 mL CHa/g VS was reached,representing 84%of total production in this assay.Other 0.5 h)and untreated WS.Sample SCB_A2 provided similar results authors(Sambusiti et al.,2013)reported a higher methane produc- with an anaerobic biodegradability of 45%and NP of 0.9.Thus. tion of 341 L CH4/kg VS,NP of 1.67 and 85%biodegradability work- using solid fractions from pretreatment A resulted in slightly lower ing with the solid fraction of pretreated WS(10g NaOH/100 g TS. yields of methane production for both substrates,perhaps due to 100C,0.5 h).However,Taherdanak and Zilouei (2014)reported the small solubilization of biodegradable VS in the pretreatment methane production of 276 mL/g VS and NP of only 1.05 with pretreated WS(8%w/v NaOH,100C,60 min). Alkali pretreatment also caused an increase in methane produc- eated WSwsA2，wsB24WC2wsD2 tion from sample SCB_C2,which reached an anaerobic biodegrad- Ws C2 medel---WS_D2_modcl 300- ability of 68%and a NP of 1.30.but the degradation rate did not (a) increase for this substrate.In the first period of the experiment, methane production from pretreated SCB was very similar to that from raw material or from thermal pretreated solid.The increase in methane production was significant only after 15 days 200 of experimentation.Costa et al.(2014)found a high anaerobic biodegradability of the solid fraction of alkaline pretreated SCB (184C,47 min,0.80 M NaOH,3.2%(w/v)calculated).after the removal of lignin using NaOH solution,and water washing of the solid fraction.In this case the alkaline pretreated and subsequently 100 washed material generated 313.4 L CH4/kg substrate Thus,using only solid fractions from pretreatment C led to a significant enhancement of methane production,especially for WS,since the kinetic behavior of the inoculum was different for both substrates,probably due to the higher concentration of inhibitory compounds in pretreated sugarcane bagasse. 12 18 21 24 27 30 Lastly,the test on solid fractions from alkaline peroxide pre- Time(d) treatment (D)provided similar curves shapes for both substrates, SCB_D2 B_C2_model SCB_D2_mode typical for the degradation of two different substrates,and with a 300- lag period.In either case,starting with a methane production rate (b) similar to that from untreated raw material,there is a lag stage 250 from day 7 to day 20,reaching approximately 100-110 mL CH4/ g VS.and after this delay,the process of digestion restarts.This behavior could possibly be due to the presence of inhibitory com- 200- pounds and residual chemicals from pretreatment in samples of both substrates.The acclimation time for both substrates ends at 150- a very similar time to that found for the whole slurry of alkaline peroxide pretreated WS.After 30 days of the experiment,the 100- anaerobic biodegradability of WS_D2 was approximately 44%and the variable NP was 0.91,meaning a lower extent of reaction than untreated WS at this point of the experiment,whereas the biodegradability of sample SCB_D2 reached the value of 60%with E. a NP ratio of 1.16 at the same point in reaction time.This different effect on the tested substrates is opposite from that found in test 1. 2 15 18 21 24 77 30 3 when using the pretreatment liquid phase.The inhibitory com- Time(d) pounds'dilution effect in tests using the solid fraction allows for the microorganism's acclimation after a lag period,which was Fig.2.Experimental results and fitting curves of cumulative methane production from untreated materials and solid fraction after pretreatments(test 2).(A)Thermal not achieved in trials with SCB whole slurry.Once this acclimation autoclaving:(B)dilute acid autoclaving:(C)dilute alkali autoclaving:(D)alkaline period ends,the effect of alkaline peroxide pretreatment is higher peroxide pretreatment.(a)Wheat straw and (b)sugarcane bagasse. on SCB than on WS
lower biodegradability values compared to untreated substrates. However, it is necessary to analyze the results of tests with a solid fraction in order to compare the effect and efficiency of pretreatments on the biodegradability of both substrates. 3.2.2. Test 2: BMP of solid fraction from pretreatments Cumulative methane production curves from test 2 are presented in Fig. 2, where the results from WS (a) and SCB (b) were displayed together for comparison. Experimental results for the four tested pretreatments applied to each material are depicted together, as well as results from untreated substrates. In this test, solid fractions from the thermal pretreatment (A) of both substrates provided lower results than those of test 1 and of untreated substrates (Section 3.2.1). Thus, the anaerobic biodegradability of WS_A2 was only 40%, with a NP of 0.84. Some authors like Sambusiti et al. (2013) reported no difference in the methane production between the pretreated sample (100 C, 0.5 h) and untreated WS. Sample SCB_A2 provided similar results with an anaerobic biodegradability of 45% and NP of 0.9. Thus, using solid fractions from pretreatment A resulted in slightly lower yields of methane production for both substrates, perhaps due to the small solubilization of biodegradable VS in the pretreatment liquid. WS_A2 and SCB_A2 were seen to behave similarly to the untreated biomass during digestion assays. In contrast to test 1, where there was complete inhibition, when digesting the solid fraction from dilute acid autoclaving (B), some methanogenic activity, similar to that from the control occurred with both substrates. WS_B2 showed an anaerobic biodegradability of 17% with NP of 0.36, whereas sample SCB_B2 reached a biodegradability of 18% with NP of 0.35. This low methanogenic activity can be attributed to the dilution of inhibitory compounds remaining in the solid fraction, which were in lower concentration than in test 1, when using the pretreatment liquid, causing less inhibition. When solid fractions from dilute alkali pretreatment (C) were used, the highest methane yields of all trials were obtained from both substrates. The anaerobic biodegradability of WS_C2 increased up to 64% achieving a NP of 1.32. This is the best result obtained from all assays, especially considering that in less than 10 days a production of approximately 240 mL CH4/g VS was reached, representing 84% of total production in this assay. Other authors (Sambusiti et al., 2013) reported a higher methane production of 341 L CH4/kg VS, NP of 1.67 and 85% biodegradability working with the solid fraction of pretreated WS (10 g NaOH/100 g TS, 100 C, 0.5 h). However, Taherdanak and Zilouei (2014) reported methane production of 276 mL/g VS and NP of only 1.05 with pretreated WS (8% w/v NaOH, 100 C, 60 min). Alkali pretreatment also caused an increase in methane production from sample SCB_C2, which reached an anaerobic biodegradability of 68% and a NP of 1.30, but the degradation rate did not increase for this substrate. In the first period of the experiment, methane production from pretreated SCB was very similar to that from raw material or from thermal pretreated solid. The increase in methane production was significant only after 15 days of experimentation. Costa et al. (2014) found a high anaerobic biodegradability of the solid fraction of alkaline pretreated SCB (184 C, 47 min, 0.80 M NaOH, 3.2% (w/v) calculated), after the removal of lignin using NaOH solution, and water washing of the solid fraction. In this case the alkaline pretreated and subsequently washed material generated 313.4 L CH4/kg substrate. Thus, using only solid fractions from pretreatment C led to a significant enhancement of methane production, especially for WS, since the kinetic behavior of the inoculum was different for both substrates, probably due to the higher concentration of inhibitory compounds in pretreated sugarcane bagasse. Lastly, the test on solid fractions from alkaline peroxide pretreatment (D) provided similar curves shapes for both substrates, typical for the degradation of two different substrates, and with a lag period. In either case, starting with a methane production rate similar to that from untreated raw material, there is a lag stage from day 7 to day 20, reaching approximately 100–110 mL CH4/ g VS, and after this delay, the process of digestion restarts. This behavior could possibly be due to the presence of inhibitory compounds and residual chemicals from pretreatment in samples of both substrates. The acclimation time for both substrates ends at a very similar time to that found for the whole slurry of alkaline peroxide pretreated WS. After 30 days of the experiment, the anaerobic biodegradability of WS_D2 was approximately 44% and the variable NP was 0.91, meaning a lower extent of reaction than untreated WS at this point of the experiment, whereas the biodegradability of sample SCB_D2 reached the value of 60% with a NP ratio of 1.16 at the same point in reaction time. This different effect on the tested substrates is opposite from that found in test 1, when using the pretreatment liquid phase. The inhibitory compounds’ dilution effect in tests using the solid fraction allows for the microorganism’s acclimation after a lag period, which was not achieved in trials with SCB whole slurry. Once this acclimation period ends, the effect of alkaline peroxide pretreatment is higher on SCB than on WS. Fig. 2. Experimental results and fitting curves of cumulative methane production from untreated materials and solid fraction after pretreatments (test 2). (A) Thermal autoclaving; (B) dilute acid autoclaving; (C) dilute alkali autoclaving; (D) alkaline peroxide pretreatment. (a) Wheat straw and (b) sugarcane bagasse. S. Bolado-Rodríguez et al. / Bioresource Technology 201 (2016) 182–190 187
188 S.Bolado-Rodriguez et aL/Bioresource Technology 201 (2016)182-190 As a result,it can be concluded that when anaerobically 3.3.Kinetics digesting the solid fraction from different pretreatments,only basic pretreatment increased the biodegradability of both tested sub- Two different models were tested to fit the methane production strates.Moreover,in the case of WS_C2,the time in which a satis- kinetic experimental results.The first order model considers the factory production of methane could be achieved was established hydrolysis reaction as the limiting step:and the modified Gom- as 10 days,greatly reducing the reaction time usually applied in pertz equation considers bacterial growth.and thus the inhibition such processes.Otherwise,the digestion of solids from thermal of the process,as the limiting step.Table 3 shows the kinetic and acid pretreatments resulted in a decrease of biodegradability parameters of the model that provided a best fit of methane pro- compared to untreated substrates,proving the remarkable inhibi- duction for each substrate and pretreatment,working with whole tory effect of degradation compounds and remaining chemicals slurry and with solid fraction.No kinetic results are provided for from these pretreatments.Finally,the anaerobic fermentation of methane production from the whole slurries of acid pretreated solid fractions from pretreatment D caused a different effect on WS,acid pretreated SCB,or alkaline peroxide pretreated SCB. both substrates,decreasing WS biodegradability but increasing because no methane production was found in any of these three SCB biodegradability,with a very similar lag period for both experiments.The fits using the calculated kinetic parameters of substrates. Table 3 are shown as curves in Figs.1 and 2. In order to calculate the final methane production balance,the Methane production from untreated and thermal pretreated losses of mass by solubilization during the pretreatment should be materials were fitted using the first order kinetic.Thermal pre- considered.As shown in Table 2,the four pretreatments solubilized treatment is a light treatment which gently opens the lignocellu- VS,and only part of the initial raw material was used for anaerobic losic structure,generating scarce amounts of degradation digestion.So,considering the VS in the initial raw material,none of compounds.As expected,the hydrolysis reaction is the limiting the pretreatments increased the biochemical methane potential of step in these anaerobic degradation experiments.The results are the studied raw materials by anaerobic digestion of the solid frac- very similar for both substrates.Almost identical methane poten- tion.The highest global methane yield was obtained for alkaline tials were obtained for untreated WS and SCB,in spite of the higher peroxide pretreatment of SCB with a NP of 0.98.Alkaline pretreat- cellulose content in SCB (Table 1).Kinetic hydrolysis coefficients ment provided global NP values of 0.88 for WS and 0.81 for SCB. are also very similar for both untreated materials,but fewer for Nevertheless,the chemical pretreatment of these materials can SCB.This difference for materials with a very close total lignin con- improve the economic viability of a global process,where materi- tent could be related to the higher acid insoluble lignin content of als solubilized during the pretreatment were valorized,and the bagasse(18.12%and 19.54%(w/w)in WS and SCB,respectively). residual solid fraction used for methane production,applying a Thermal pretreatment increased the methane production biorefinery concept.This alternative is especially promising for potential remarkably when the whole slurries were digested alkaline pretreatment where a high amount of lignin,which is of (around to 50%),but reduced hydrolysis coefficient.So,the increase interest in the chemical industry,is released into the liquid,and on methane production by the thermal pretreatment was observ- the residual solid retains high methane production potential able only from the 15th day of experimentation for both sub- (Costa et al.,2014). strates.The degradation of the thermally pretreated solid fraction Table 3 Kinetic model and parameters of fitting equations of cumulative methane production from raw and pretreated lignocellulosic biomass using pretreated whole slurry and solid fraction from pretreatment. Sample Kinetic modelb Bo(mL CH4g VS-1) k（d-l) (d) Rm(mL CH4g VS-1d-1) R28 WS RAW First order 224 0.100 0.9963 SCB RAW First order 222 0.083 09884 WS A1 First order 336 0.055 0.9976 WS_B1 WS_C1 Gompertz modified 278 3.8 8.8 09883 WS_D1 Gompertz modified 225 17.5 53.8 0.9924 SCR A1 First order 305 0.048 、 0.9976 SCB_B1 SCB_C1 Combined first order (1)+Gompertz (2) B(1:53 0260 13.2 93 0.9845 Ba(2):367 SCB_D1 WS_A2 First order 209 0.068 0.9976 WS_B2 First order 83 0.094 0.9969 WS_C2 First order 286 0.192 0.9975 WS D2 Combined first order (1)+Gompertz (2) Ba1y:110 0240 19 12.2 0.9562 B(2):128 SCB A2 First order 240 0.065 09786 SCB_B2 First order 80 0.091 0.9856 SCB_C2 First order 420 0.040 0.9847 SCB D2 Combined first order (1)+Gompertz(2) Ba(1)y117 0.191 19 27.1 0.9860 Ba(2:145 Codes:Lignocellulosic biomass:WS.wheat straw and SCB.sugarcane bagasse.Pretreatment:A thermal autoclaving:B.dilute acid autoclaving:C.dilute alkali autoclaving and D:alkaline peroxide.Fractions used:1.whole slurry and fraction. Equations of models:First order:Eq.(1).Gompertz modified:Eq.(2)and Combined First order (1)+Gompertz(2):Eq.(3) Bo:methane production potential (Eqs.(1-3)). coefficientin the first order kinetic model (Eq(1)) e lag time (Eq.(3)). Rm:maximum biogas production rate in the Gompertz model (Eqs.(2)and(3)). 8 R2:coefficient of determination
As a result, it can be concluded that when anaerobically digesting the solid fraction from different pretreatments, only basic pretreatment increased the biodegradability of both tested substrates. Moreover, in the case of WS_C2, the time in which a satisfactory production of methane could be achieved was established as 10 days, greatly reducing the reaction time usually applied in such processes. Otherwise, the digestion of solids from thermal and acid pretreatments resulted in a decrease of biodegradability compared to untreated substrates, proving the remarkable inhibitory effect of degradation compounds and remaining chemicals from these pretreatments. Finally, the anaerobic fermentation of solid fractions from pretreatment D caused a different effect on both substrates, decreasing WS biodegradability but increasing SCB biodegradability, with a very similar lag period for both substrates. In order to calculate the final methane production balance, the losses of mass by solubilization during the pretreatment should be considered. As shown in Table 2, the four pretreatments solubilized VS, and only part of the initial raw material was used for anaerobic digestion. So, considering the VS in the initial raw material, none of the pretreatments increased the biochemical methane potential of the studied raw materials by anaerobic digestion of the solid fraction. The highest global methane yield was obtained for alkaline peroxide pretreatment of SCB with a NP of 0.98. Alkaline pretreatment provided global NP values of 0.88 for WS and 0.81 for SCB. Nevertheless, the chemical pretreatment of these materials can improve the economic viability of a global process, where materials solubilized during the pretreatment were valorized, and the residual solid fraction used for methane production, applying a biorefinery concept. This alternative is especially promising for alkaline pretreatment where a high amount of lignin, which is of interest in the chemical industry, is released into the liquid, and the residual solid retains high methane production potential (Costa et al., 2014). 3.3. Kinetics Two different models were tested to fit the methane production kinetic experimental results. The first order model considers the hydrolysis reaction as the limiting step; and the modified Gompertz equation considers bacterial growth, and thus the inhibition of the process, as the limiting step. Table 3 shows the kinetic parameters of the model that provided a best fit of methane production for each substrate and pretreatment, working with whole slurry and with solid fraction. No kinetic results are provided for methane production from the whole slurries of acid pretreated WS, acid pretreated SCB, or alkaline peroxide pretreated SCB, because no methane production was found in any of these three experiments. The fits using the calculated kinetic parameters of Table 3 are shown as curves in Figs. 1 and 2. Methane production from untreated and thermal pretreated materials were fitted using the first order kinetic. Thermal pretreatment is a light treatment which gently opens the lignocellulosic structure, generating scarce amounts of degradation compounds. As expected, the hydrolysis reaction is the limiting step in these anaerobic degradation experiments. The results are very similar for both substrates. Almost identical methane potentials were obtained for untreated WS and SCB, in spite of the higher cellulose content in SCB (Table 1). Kinetic hydrolysis coefficients are also very similar for both untreated materials, but fewer for SCB. This difference for materials with a very close total lignin content could be related to the higher acid insoluble lignin content of bagasse (18.12% and 19.54% (w/w) in WS and SCB, respectively). Thermal pretreatment increased the methane production potential remarkably when the whole slurries were digested (around to 50%), but reduced hydrolysis coefficient. So, the increase on methane production by the thermal pretreatment was observable only from the 15th day of experimentation for both substrates. The degradation of the thermally pretreated solid fraction Table 3 Kinetic model and parameters of fitting equations of cumulative methane production from raw and pretreated lignocellulosic biomass using pretreated whole slurry and solid fraction from pretreatment. Samplea Kinetic modelb B0 c (mL CH4g VS1 ) kH d (d1 ) ke (d) Rmf (mL CH4g VS1 d1 ) R2g WS RAW First order 224 0.100 – – 0.9963 SCB RAW First order 222 0.083 – – 0.9884 WS_A1 First order 336 0.055 – – 0.9976 WS_B1 – – – – – – WS_C1 Gompertz modified 278 – 3.8 8.8 0.9883 WS_D1 Gompertz modified 225 – 17.5 53.8 0.9924 SCB_A1 First order 305 0.048 – – 0.9976 SCB_B1 – – – – – – SCB_C1 Combined first order (1) + Gompertz (2) B0(1): 53 B0(2): 367 0.260 13.2 9.3 0.9845 SCB_D1 – – – – – – WS_A2 First order 209 0.068 – – 0.9976 WS_B2 First order 83 0.094 – – 0.9969 WS_C2 First order 286 0.192 – – 0.9975 WS_D2 Combined first order (1) + Gompertz (2) B0(1): 110 B0(2): 128 0.240 19 12.2 0.9562 SCB_A2 First order 240 0.065 – – 0.9786 SCB_B2 First order 80 0.091 – – 0.9856 SCB_C2 First order 420 0.040 – – 0.9847 SCB_D2 Combined first order (1) + Gompertz (2) B0(1): 117 B0(2): 145 0.191 19 27.1 0.9860 a Codes: Lignocellulosic biomass: WS, wheat straw and SCB, sugarcane bagasse. Pretreatment: A, thermal autoclaving; B, dilute acid autoclaving; C, dilute alkali autoclaving and D: alkaline peroxide. Fractions used: 1, whole slurry and 2, solid fraction. b Equations of models: First order: Eq. (1), Gompertz modified: Eq. (2) and Combined First order (1) + Gompertz (2): Eq. (3). c B0: methane production potential (Eqs. (1–3)). d kH: hydrolysis coefficient in the first order kinetic model (Eq. (1)). e k: lag time (Eq. (3)). f Rm: maximum biogas production rate in the Gompertz model (Eqs. (2) and (3)). g R2 : coefficient of determination. 188 S. Bolado-Rodríguez et al. / Bioresource Technology 201 (2016) 182–190
S.Bolado-Rodriguez et aL/Bioresource Technology 201 (2016)182-190 189 provided worse results than the untreated materials in terms of medium.The total methane potential for this experiment both methane potential and hydrolysis rate coefficient.Ferreira (Bo Boz)reached the theoretical methane yield for SCB.The same et al.(2013)used a first order equation to model the methane pro- theoretical methane potential value was achieved using the solid duction from untreated and steam exploded WS,demonstrating fraction of alkaline pretreated SCB,applying the first order model. that hydrolysis was the limiting step.For steam exploded WS,at The removal of liquid pretreatment,as for WS reduced inhibition 150C and 15 min,fitting parameters very similar to those from identified hydrolysis as the limiting step.Data fitted to a first order our thermal pretreatment,they obtained a methane potential of kinetic resulted in a very high methane potential but with a low 194.6 mL CH4/g VS and a hydrolysis coefficient of 0.085 d- hydrolysis kinetic coefficient. Very low values of methane production potential were obtained A strong inhibitory effect was found for all the alkaline peroxide from the solid fraction of both acid pretreated materials.The pretreated materials,probably generated by the degradation com- degradation compounds retained in the solid fraction probably pounds but also by some remaining peroxide.The Gompertz equa- inhibited the growth of most of the microorganisms.However, tion was used to fit the cumulative methane production from WS the small quantity of methane production obtained fitted ade- pretreated whole slurry with a methane potential very similar to quately to a first order kinetic,limited by the hydrolysis step. that of the untreated material.After a lag period of 17 days,the The microorganism species that were able to grow,biodegraded inhibition was completely defeated,reaching the highest degrada- the material with hydrolysis coefficients in a similar range to those tion rates.The 30 days period was not enough for microorganism for untreated materials. acclimation in the SCB whole slurry experiment. Alkaline pretreatment provided different results for both sub- The high inhibition of alkaline peroxide pretreatment also strates,probably attributable to the aforementioned differences affected the solid fraction experiments.Cumulative methane pro- in lignin structure and composition in tested materials.Basic pre- duction from both materials was fitted to a combination of the first treatment attacks mainly the lignin,and a higher concentration order kinetic and the Gompertz model,according to Eq.(3).Almost of phenolic compounds was found in SCB pretreatment liquid half of the total methane potential belongs to each kinetic model. (Table 2). Kinetic hydrolysis coefficients of first order reactions are the high- Methane production was limited by microorganism growth in est of all studied pretreatments,but the microorganisms require a the anaerobic degradation of WS pretreated whole slurry.The long lag period (19 days)for both substrates.Once the microorgan- Gompertz equation provided the best fit of these experimental isms are acclimated to the medium,very high biodegradation rates results,with a methane production potential higher than that of are also obtained from the Gompertz model fit. the untreated straw and a short lag period (around 4 days).How- The effect of inhibitory compounds released during pretreat- ever,the final methane production after 30 days of the experiment ment was evidenced for the four pretreatments studied for both was the same as that of the untreated WS.Therefore,alkaline pre- substrates but with different intensity.Experiments with low treatment increased the straw potential biodegradability by lignin inhibition fit to a first order kinetic model,but the hydrolysis rate solubilization,but this effect was counteracted by the reduction on coefficient decreases.Experiments with medium inhibition fit to hydrolysis rate by inhibition.Nevertheless,Reilly et al.(2015). the Gompertz model,since they are controlled by the microorgan- working with a whole slurry of Ca(OH)pretreated wheat straw ism growth.In these cases,after the lag period,when microorgan- (48 h,20C,7.4%(w/w)).obtained high anaerobic degradation isms are acclimated,a high degradation kinetic is usually achieved. rates.Their experiments achieved 202 mL-CH4/g VS after only Some combined models have been found,where a fraction of the 5 days of digestion. volatile solids is biodegraded by microorganisms able to grow in Anaerobic degradation of the alkaline pretreated solid fraction the inhibitory environment (first order kinetic)and the degrada- was fitted by a first order kinetic.Most of the inhibitory com- tion of another fraction of volatile solids requires microorganisms pounds were removed with the pretreatment liquid.The methane acclimation(Gompertz kinetic).Additional research work is neces- potential was close to that of the whole slurry pretreated material. sary in order to discover the methane production working in con- however lignin removal without inhibition remarkably increased tinuous bioreactors with acclimated microorganisms. the hydrolysis rate coefficient,obtaining the best methane produc- tion rate results.Liu et al.(2015)used the Gompertz model to fit cumulative methane production from the solid fraction of WS pre- 4.Conclusions treated with KOH.They obtained weak inhibition with low lag periods (1-2 days)for KOH loadings in the range from 2%to 50% Thermal pretreatment provided the lowest solubilization of w/w,for pretreatments at 20C during 48 h. sugars,VS and inhibitory compounds and the methane production The fit of cumulative methane production from alkaline pre- from whole slurry improved the raw material potential,with treated whole slurry SCB required a combination of both models. hydrolysis being the controlling step.Acid solubilized high concen- As observed for the solid fraction of acid pretreated SCB,only a trations of sugars,generating furfural and HMF with a strong inhi- small fraction of material was available for anaerobic degradation bition effect.Alkali solubilized lignin,generating acetic acid and from the starting time,according to a first order kinetic.Most of phenolic compounds;BMP experiments required an acclimation the microorganisms growth was inhibited,probably due to the period for whole slurry but obtained the highest methane produc- high acetic acid and phenolic compound concentration (Heiske tion from solid fractions.Alkaline-peroxide pretreatment caused et al..2013:Krishania et al..2013)and a lag period of 13 days higher inhibition in SCB than in WS,but high methane production was required.So.the Eq.(3)was used to fit the experimental rates were attained for solid fractions of both substrates after the results: acclimation period. B-Bo1·[1-exp(-kHt】+Bo2 Acknowledgements exp-exp me(-t)+1 (3) Bo2 This work was supported by the research unit UIC 071 of the regional government"Junta de Castilla y Leon-JCyL",Spain.Rodolfo In this case,Bo is the methane potential from microorganisms Travaini is also grateful to "Conselho Nacional de Desenvolvimento resistant to inhibitory compounds and Bo is the methane potential Cientifico e Tecnologico-CNPq",Brazil(238059/2012-0)for provid- from microorganisms able to acclimate in the pretreatment ing his Doctorate Scholarship
provided worse results than the untreated materials in terms of both methane potential and hydrolysis rate coefficient. Ferreira et al. (2013) used a first order equation to model the methane production from untreated and steam exploded WS, demonstrating that hydrolysis was the limiting step. For steam exploded WS, at 150 C and 15 min, fitting parameters very similar to those from our thermal pretreatment, they obtained a methane potential of 194.6 mL CH4/g VS and a hydrolysis coefficient of 0.085 d1 . Very low values of methane production potential were obtained from the solid fraction of both acid pretreated materials. The degradation compounds retained in the solid fraction probably inhibited the growth of most of the microorganisms. However, the small quantity of methane production obtained fitted adequately to a first order kinetic, limited by the hydrolysis step. The microorganism species that were able to grow, biodegraded the material with hydrolysis coefficients in a similar range to those for untreated materials. Alkaline pretreatment provided different results for both substrates, probably attributable to the aforementioned differences in lignin structure and composition in tested materials. Basic pretreatment attacks mainly the lignin, and a higher concentration of phenolic compounds was found in SCB pretreatment liquid (Table 2). Methane production was limited by microorganism growth in the anaerobic degradation of WS pretreated whole slurry. The Gompertz equation provided the best fit of these experimental results, with a methane production potential higher than that of the untreated straw and a short lag period (around 4 days). However, the final methane production after 30 days of the experiment was the same as that of the untreated WS. Therefore, alkaline pretreatment increased the straw potential biodegradability by lignin solubilization, but this effect was counteracted by the reduction on hydrolysis rate by inhibition. Nevertheless, Reilly et al. (2015), working with a whole slurry of Ca(OH)2 pretreated wheat straw (48 h, 20 C, 7.4% (w/w)), obtained high anaerobic degradation rates. Their experiments achieved 202 mL-CH4/g VS after only 5 days of digestion. Anaerobic degradation of the alkaline pretreated solid fraction was fitted by a first order kinetic. Most of the inhibitory compounds were removed with the pretreatment liquid. The methane potential was close to that of the whole slurry pretreated material, however lignin removal without inhibition remarkably increased the hydrolysis rate coefficient, obtaining the best methane production rate results. Liu et al. (2015) used the Gompertz model to fit cumulative methane production from the solid fraction of WS pretreated with KOH. They obtained weak inhibition with low lag periods (1–2 days) for KOH loadings in the range from 2% to 50% w/w, for pretreatments at 20 C during 48 h. The fit of cumulative methane production from alkaline pretreated whole slurry SCB required a combination of both models. As observed for the solid fraction of acid pretreated SCB, only a small fraction of material was available for anaerobic degradation from the starting time, according to a first order kinetic. Most of the microorganisms growth was inhibited, probably due to the high acetic acid and phenolic compound concentration (Heiske et al., 2013; Krishania et al., 2013) and a lag period of 13 days was required. So, the Eq. (3) was used to fit the experimental results: B ¼ B01 ½1 expðkH tÞ þ B02 exp exp Rm e B02 ðk tÞ þ 1 ð3Þ In this case, B01 is the methane potential from microorganisms resistant to inhibitory compounds and B02 is the methane potential from microorganisms able to acclimate in the pretreatment medium. The total methane potential for this experiment (B01 + B02) reached the theoretical methane yield for SCB. The same theoretical methane potential value was achieved using the solid fraction of alkaline pretreated SCB, applying the first order model. The removal of liquid pretreatment, as for WS reduced inhibition, identified hydrolysis as the limiting step. Data fitted to a first order kinetic resulted in a very high methane potential but with a low hydrolysis kinetic coefficient. A strong inhibitory effect was found for all the alkaline peroxide pretreated materials, probably generated by the degradation compounds but also by some remaining peroxide. The Gompertz equation was used to fit the cumulative methane production from WS pretreated whole slurry with a methane potential very similar to that of the untreated material. After a lag period of 17 days, the inhibition was completely defeated, reaching the highest degradation rates. The 30 days period was not enough for microorganism acclimation in the SCB whole slurry experiment. The high inhibition of alkaline peroxide pretreatment also affected the solid fraction experiments. Cumulative methane production from both materials was fitted to a combination of the first order kinetic and the Gompertz model, according to Eq. (3). Almost half of the total methane potential belongs to each kinetic model. Kinetic hydrolysis coefficients of first order reactions are the highest of all studied pretreatments, but the microorganisms require a long lag period (19 days) for both substrates. Once the microorganisms are acclimated to the medium, very high biodegradation rates are also obtained from the Gompertz model fit. The effect of inhibitory compounds released during pretreatment was evidenced for the four pretreatments studied for both substrates but with different intensity. Experiments with low inhibition fit to a first order kinetic model, but the hydrolysis rate coefficient decreases. Experiments with medium inhibition fit to the Gompertz model, since they are controlled by the microorganism growth. In these cases, after the lag period, when microorganisms are acclimated, a high degradation kinetic is usually achieved. Some combined models have been found, where a fraction of the volatile solids is biodegraded by microorganisms able to grow in the inhibitory environment (first order kinetic) and the degradation of another fraction of volatile solids requires microorganisms acclimation (Gompertz kinetic). Additional research work is necessary in order to discover the methane production working in continuous bioreactors with acclimated microorganisms. 4. Conclusions Thermal pretreatment provided the lowest solubilization of sugars, VS and inhibitory compounds and the methane production from whole slurry improved the raw material potential, with hydrolysis being the controlling step. Acid solubilized high concentrations of sugars, generating furfural and HMF with a strong inhibition effect. Alkali solubilized lignin, generating acetic acid and phenolic compounds; BMP experiments required an acclimation period for whole slurry but obtained the highest methane production from solid fractions. Alkaline-peroxide pretreatment caused higher inhibition in SCB than in WS, but high methane production rates were attained for solid fractions of both substrates after the acclimation period. Acknowledgements This work was supported by the research unit UIC 071 of the regional government ‘‘Junta de Castilla y León – JCyL”, Spain. Rodolfo Travaini is also grateful to ‘‘Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq”, Brazil (238059/2012-0) for providing his Doctorate Scholarship. S. Bolado-Rodríguez et al. / Bioresource Technology 201 (2016) 182–190 189
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