380 D.Brown et aL/Bioresource Technology 124 (2012)379-386 Feedstocks for AD are influenced by accessibility and availabil- et al.,2012).Well mixed materials were loaded into 1-L glass reac- ity due to costs associated with collection and transportation(Fri- tors and incubated in a walk-in incubation room for up to 30 days gon and Guiot,2010:Li et al,2011).Lignocellulosic biomass can be at 37t 1 C.Duplicate reactors were run for each condition.Inocu- harvested at a high TS content,which results in lower transporta- lum without any feedstock addition was used as a control.Biogas tion costs per unit of solids compared to low TS feedstocks.Crop was collected in a 5-L Tedlar gas bag (CEL Scientific,Santa Fe residues,such as corn stover and wheat straw,are widespread Springs,CA,USA)attached to the outlet of the reactor.Biogas com- through much of the US.Switchgrass has the potential to be grown position and volume were measured every 2-3 days during the 30- in areas where common crops experience low production,such as day SS-AD. areas currently in the Conservation Reserve Program through the Natural Resources Conservation Service.Yard waste,which in- 2.3.Liquid anaerobic digestion (L-AD) cludes grass,leaves,and branches,is a major lignocellulosic source generated from households,municipalities,and landscaping com- Each feedstock was mixed with deionized(DI)water and efflu- panies.Although yard waste can be composted,the stored energy ent to obtain a mixture of 5%TS and an F/E ratio of 0.5 based on dry is lost in the form of respiration heat(Koch et al.,2010).The use of volatile solids (VS).The mixtures were loaded in 2-L glass jars and yard waste,leaves,maple,and pine may improve the overall eco- incubated in a walk-in incubation room on an Innova model 2300 nomics of SS-AD due to the low or zero cost associated with acquir- platform shaker (New Brunswick Scientific;Enfield,CT.USA)at ing these feedstocks (Jagadish et al..1998).Waste paper is paper 150 rpm for up to 30 days at 37+1C.Duplicate reactors were that either cannot be recycled and/or is used in some other way run for each condition.Inoculum without any feedstock addition such as a landfill cover.Paper is treated both chemically and ther- was used as a control.Biogas generated was collected in a 5-L Ted- mally during the paper-making process,effectively serving as a lar gas bag(CEL Scientific,Santa Fe Springs,CA,USA)attached to pretreatment and allows the breakdown of cellulose to occur.A the outlet of the reactor.Biogas composition and volume were number of these lignocellulosic biomass sources have been tested measured every 2-3 days during the 30-day L-AD. for methane production from L-AD systems (Turick et al.,1991: Gunaseelan,1997);however,the suitability of lignocellulosic bio- 2.4.Enzyme hydrolysis of lignocellulosic feedstocks mass as feedstocks and factors affecting methane production dur- ing SS-AD have not been studied. Cellulase (Spezyme CP)(Lot#4900857805,protein content Therefore,the objectives of the present study were to 82 mg/ml with activity of approximately 50 FPU/ml),and Multifect determine:(1)the methane yield and volumetric productivity of xylanase(Lot#4901063047,protein content 27 mg/ml with activ- switchgrass,corn stover,wheat straw,yard waste,leaves,maple, ity of approximately 25000 OSX/ml).were obtained from Genen- pine,and waste paper in L-AD and SS-AD systems using a single cor,now DuPont Industrial Biosciences (Palo Alto,CA,USA).All source of inoculum:and(2)the effect of the composition and enzy- enzymatic hydrolysis experiments were run in duplicate following matic digestibility of lignocellulosic biomass on methane yield the National Renewable Energy Laboratory(NREL)Laboratory Ana- from SS-AD. lytical Procedures(LAP)(Selig et al.,2008)at 50C with 0.05 M cit- rate buffer,pH 4.8,solid loading of 2%,and cellulase loading of 15 FPU/g solids supplemented with xylanase at a loading of 3750 OSX/ 2.Methods g solids on a rotary shaker (150 rpm for 72 h).The hydrolysate sample was filtered through a 0.2 um nylon membrane filter for 2.1.Feedstock and inoculum sugar analysis by HPLC as described in Section 2.5.The overall glu- cose and xylose yields of enzymatic hydrolysis (i.e.,enzymatic Corn stover,wheat straw,and switchgrass were collected in digestibility)were defined according to Cui et al.(2011). October 2009 from farms operated by the Ohio Agricultural Re- search and Development Center (OARDC)in Wooster,and Jackson, 2.5.Analytical methods OH.Fallen tree leaves were collected in October 2010 from the OARDC Wooster campus.Yard waste containing leaves and tree The extractive content of feedstock was measured according to branches was obtained in June 2011 from the OARDC Wooster the NREL Laboratory Analytical Procedure (Sluiter et al.,2008a). campus.Pine and maple wood with bark were obtained in June The extractive-free biomass and solid fractions before and after 2011 from the OARDC Wooster campus.The shredded waste paper digestion were further fractionated using a two-step hydrolysis used was classified as alternative daily cover for landfills and col- method based on the NREL Laboratory Analytical Procedure(Sluit- lected from the Solid Waste Management facility of Wayne County. er et al.,2008b).Monomeric sugars (glucose,xylose,galactose OH.These feedstocks were oven-dried at 40C for 48 h in a convec- arabinose,and mannose)and cellobiose in the acid hydrolysate tion oven (Precision Thelco Model 18,Waltham,MA)to obtain a were measured by an HPLC instrument(Shimadzu LC-20AB,MD. moisture content of less than 10%,ground with a hammer mill USA)equipped with a Biorad Aminex HPX-87P column and a (Mackisik,Parker Ford,PA)to pass through a 5 mm screen,and refractive index detector (RID).A deionized water flow rate of stored in air tight containers. 0.6 ml/min was used as the mobile phase.The temperature of Effluent from a mesophillic liquid anaerobic digester in Colum- the column and detector were maintained at 80 and 55 C,respec- bus,OH fed with municipal waste water sludge and food waste tively.The Standard Methods for the Examination of Water and was used as inoculum.The inoculum was kept in air-tight buckets Wastewater were used to analyze the TS and VS contents of feed- at 4C.Prior to use,the inoculum was acclimated at 37C for stocks,inoculum,and digestate taken at the beginning and end of 1 week. the AD process(Eaton et al.,2005).Total carbon and nitrogen con- tents in feedstock,effluent and digestion materials were deter- 2.2.Solid-state anaerobic digestion (SS-AD) mined using an elemental analyzer (Elementar Vario Max CNS, Elementar Americas,Mt.Laurel,NI.USA).VFAs and alkalinity were Each lignocellulosic biomass feedstock was mixed with the measured using a two-step titration method(McGhee,1968).Sam- inoculum using a hand-mixer(Black Decker,250-watt mixer, ples for pH,VFA,and alkalinity measurements were prepared by Towson,MD,USA)to obtain a feedstock to effluent (F/E)ratio of diluting a 5-g sample with 50 ml of DI water.The mixture was ana- 3(based on dry volatile solids)and TS content of 18-19%(Liew lyzed using an auto-titrator(Mettler Toledo,DL22 Food BeverageFeedstocks for AD are influenced by accessibility and availabil￾ity due to costs associated with collection and transportation (Fri￾gon and Guiot, 2010; Li et al., 2011). Lignocellulosic biomass can be harvested at a high TS content, which results in lower transporta￾tion costs per unit of solids compared to low TS feedstocks. Crop residues, such as corn stover and wheat straw, are widespread through much of the US. Switchgrass has the potential to be grown in areas where common crops experience low production, such as areas currently in the Conservation Reserve Program through the Natural Resources Conservation Service. Yard waste, which in￾cludes grass, leaves, and branches, is a major lignocellulosic source generated from households, municipalities, and landscaping com￾panies. Although yard waste can be composted, the stored energy is lost in the form of respiration heat (Koch et al., 2010). The use of yard waste, leaves, maple, and pine may improve the overall eco￾nomics of SS-AD due to the low or zero cost associated with acquir￾ing these feedstocks (Jagadish et al., 1998). Waste paper is paper that either cannot be recycled and/or is used in some other way such as a landfill cover. Paper is treated both chemically and ther￾mally during the paper-making process, effectively serving as a pretreatment and allows the breakdown of cellulose to occur. A number of these lignocellulosic biomass sources have been tested for methane production from L-AD systems (Turick et al., 1991; Gunaseelan, 1997); however, the suitability of lignocellulosic bio￾mass as feedstocks and factors affecting methane production dur￾ing SS-AD have not been studied. Therefore, the objectives of the present study were to determine: (1) the methane yield and volumetric productivity of switchgrass, corn stover, wheat straw, yard waste, leaves, maple, pine, and waste paper in L-AD and SS-AD systems using a single source of inoculum; and (2) the effect of the composition and enzy￾matic digestibility of lignocellulosic biomass on methane yield from SS-AD. 2. Methods 2.1. Feedstock and inoculum Corn stover, wheat straw, and switchgrass were collected in October 2009 from farms operated by the Ohio Agricultural Re￾search and Development Center (OARDC) in Wooster, and Jackson, OH. Fallen tree leaves were collected in October 2010 from the OARDC Wooster campus. Yard waste containing leaves and tree branches was obtained in June 2011 from the OARDC Wooster campus. Pine and maple wood with bark were obtained in June 2011 from the OARDC Wooster campus. The shredded waste paper used was classified as alternative daily cover for landfills and col￾lected from the Solid Waste Management facility of Wayne County, OH. These feedstocks were oven-dried at 40 C for 48 h in a convec￾tion oven (Precision Thelco Model 18, Waltham, MA) to obtain a moisture content of less than 10%, ground with a hammer mill (Mackisik, Parker Ford, PA) to pass through a 5 mm screen, and stored in air tight containers. Effluent from a mesophillic liquid anaerobic digester in Colum￾bus, OH fed with municipal waste water sludge and food waste was used as inoculum. The inoculum was kept in air-tight buckets at 4 C. Prior to use, the inoculum was acclimated at 37 C for 1 week. 2.2. Solid-state anaerobic digestion (SS-AD) Each lignocellulosic biomass feedstock was mixed with the inoculum using a hand-mixer (Black & Decker, 250-watt mixer, Towson, MD, USA) to obtain a feedstock to effluent (F/E) ratio of 3 (based on dry volatile solids) and TS content of 18–19% (Liew et al., 2012). Well mixed materials were loaded into 1-L glass reac￾tors and incubated in a walk-in incubation room for up to 30 days at 37 ± 1 C. Duplicate reactors were run for each condition. Inocu￾lum without any feedstock addition was used as a control. Biogas was collected in a 5-L Tedlar gas bag (CEL Scientific, Santa Fe Springs, CA, USA) attached to the outlet of the reactor. Biogas com￾position and volume were measured every 2–3 days during the 30- day SS-AD. 2.3. Liquid anaerobic digestion (L-AD) Each feedstock was mixed with deionized (DI) water and efflu￾ent to obtain a mixture of 5% TS and an F/E ratio of 0.5 based on dry volatile solids (VS). The mixtures were loaded in 2-L glass jars and incubated in a walk-in incubation room on an Innova model 2300 platform shaker (New Brunswick Scientific; Enfield, CT, USA) at 150 rpm for up to 30 days at 37 ± 1 C. Duplicate reactors were run for each condition. Inoculum without any feedstock addition was used as a control. Biogas generated was collected in a 5-L Ted￾lar gas bag (CEL Scientific, Santa Fe Springs, CA, USA) attached to the outlet of the reactor. Biogas composition and volume were measured every 2–3 days during the 30-day L-AD. 2.4. Enzyme hydrolysis of lignocellulosic feedstocks Cellulase (Spezyme CP) (Lot#4900857805, protein content 82 mg/ml with activity of approximately 50 FPU/ml), and Multifect xylanase (Lot#4901063047, protein content 27 mg/ml with activ￾ity of approximately 25000 OSX/ml), were obtained from Genen￾cor, now DuPont Industrial Biosciences (Palo Alto, CA, USA). All enzymatic hydrolysis experiments were run in duplicate following the National Renewable Energy Laboratory (NREL) Laboratory Ana￾lytical Procedures (LAP) (Selig et al., 2008) at 50 C with 0.05 M cit￾rate buffer, pH 4.8, solid loading of 2%, and cellulase loading of 15 FPU/g solids supplemented with xylanase at a loading of 3750 OSX/ g solids on a rotary shaker (150 rpm for 72 h). The hydrolysate sample was filtered through a 0.2 lm nylon membrane filter for sugar analysis by HPLC as described in Section 2.5. The overall glu￾cose and xylose yields of enzymatic hydrolysis (i.e., enzymatic digestibility) were defined according to Cui et al. (2011). 2.5. Analytical methods The extractive content of feedstock was measured according to the NREL Laboratory Analytical Procedure (Sluiter et al., 2008a). The extractive-free biomass and solid fractions before and after digestion were further fractionated using a two-step hydrolysis method based on the NREL Laboratory Analytical Procedure (Sluit￾er et al., 2008b). Monomeric sugars (glucose, xylose, galactose, arabinose, and mannose) and cellobiose in the acid hydrolysate were measured by an HPLC instrument (Shimadzu LC-20AB, MD, USA) equipped with a Biorad Aminex HPX-87P column and a refractive index detector (RID). A deionized water flow rate of 0.6 ml/min was used as the mobile phase. The temperature of the column and detector were maintained at 80 and 55 C, respec￾tively. The Standard Methods for the Examination of Water and Wastewater were used to analyze the TS and VS contents of feed￾stocks, inoculum, and digestate taken at the beginning and end of the AD process (Eaton et al., 2005). Total carbon and nitrogen con￾tents in feedstock, effluent and digestion materials were deter￾mined using an elemental analyzer (Elementar Vario Max CNS, Elementar Americas, Mt. Laurel, NJ, USA). VFAs and alkalinity were measured using a two-step titration method (McGhee, 1968). Sam￾ples for pH, VFA, and alkalinity measurements were prepared by diluting a 5-g sample with 50 ml of DI water. The mixture was ana￾lyzed using an auto-titrator (Mettler Toledo, DL22 Food & Beverage 380 D. Brown et al. / Bioresource Technology 124 (2012) 379–386