Bioresource Technology 101(2010)8713-8717 Contents lists available at ScienceDirect OPfE●UE EGOL○OY Bioresource Technology ELSEVIER journal homepage:www.elsevier.com/locate/biortech Comparative study of mechanical,hydrothermal,chemical and enzymatic treatments of digested biofibers to improve biogas production Emiliano Brunia.b,Anders Peter Jensen Irini Angelidaki. Department of Environmental Engineering Technical University of Denmark,Building 113.2800 Kgs.Lyngby,Denmark bProcess Development,Xergi A/S.Hermesvej 1.9530 Stevring.Denmark ARTICLE INFO ABSTRACT Article history Organic waste such as manure is an important resource for biogas production.The biodegradability of Received 30 April 2010 manures is however limited because of the recalcitrant nature of the biofibers it contains.To increase Received in revised form 15 June 2010 the biogas potential of the biofibers in digested manure,we investigated physical treatment(milling) Accepted 24 June 2010 chemical treatment(CaO).biological treatment (enzymatic and partial aerobic microbial conversion). Available online 16 July 2010 steam treatment with catalyst(H3PO4 or NaOH)and combination of biological and steam treatments (biofibers steam-treated with catalyst were treated with laccase enzyme).We obtained the highest meth- Keywords: ane yield increase through the chemical treatment that resulted in 66%higher methane production com- Treatment Lignocellulose pared to untreated biofibers.The combination of steam treatment with NaOH and subsequent enzymatic Biogas treatment increased the methane yield by 34%.To choose the optimal treatment,the energy require- Hydrolysis ments relative to the energy gain as extra biogas production have to be taken into account,as well as Enzymes the costs of chemicals or enzymes. 2010 Elsevier Ltd.All rights reserved. 1.Introduction plants,increasing the methane yield of lignocellulosic substrates by up to 25%(Hartmann et al.,2000).Steam treatment has been Anaerobic digestion of organic waste and residues combines optimized mainly for ethanol production.Glucose yields of 98% both sustainable treatment and renewable energy production. after enzymatic hydrolysis have been registered for wheat straw Some substrates such as lignocellulosic materials are resistant to steam-treated with H2SO4(Talebnia et al.,2010).Likewise,steam anaerobic digestion and can be converted into biogas only to a treatment with H3PO4 hydrolyzed hemicellulose and increased low extent.The low susceptibility of these materials to conversion the accessibility of cellulose for enzymatic hydrolysis (Geddes into biogas is a result of their composition and structure.Lignocel- et al.,2010).Steam treatment with NaOH has been reported to in- lulose is the complex and rigid matrix of plant cells,it is resistant crease the biogas production of sorted municipal solid waste by to enzymatic attack because of the tight association between 50%(Wang et al.,2009).Chemical treatments investigated include lignin,cellulose and hemicellulose.Cellulose and hemicellulose treatments with acids,bases and oxidants(Taherzadeh and Karimi. (carbohydrates composed of hexoses and mainly pentoses,respec- 2008).Chemical treatments with NaOH are among those that have tively)can be degraded in biogas processes.Lignin can however been investigated most (Tanaka et al,1997;Zheng et al.,2009). not be degraded under anaerobic conditions (Fernandes et al., Alkaline hydrolysis with NaOH has been successfully applied to 2009).In full-scale biogas plants digesting manure,the low digest- treat lignocellulosic materials such as straw or hardwood (Sun ibility of the biofibers contained in the manure causes a loss of and Cheng.2002).Some aerobic microorganisms(white-,brown-, methane production and limits the overall efficiency of the process soft-rot fungi)can selectively degrade lignin or hemicellulose. (Jin et al.,2009).Therefore,treatments facilitating the accessibility Srilatha et al.(1995)obtained a 33%methane yield increase for of holocellulose (cellulose and hemicellulose)are needed to in- orange processing waste by treatment with selected fungi strains. crease the biogas potential of biofibers in manure.Many treat- Similarly,Taherzadeh and Karimi(2008)reported improved enzy- ments for increasing the biodegradability of lignocellulosic matic hydrolysis (94%sugar recovery)when treating office paper material have been reported (Demirbas,2008).Mechanical treat- with selected aerobic bacteria.Commercially available enzymes ment (milling)increases the surface available for enzymatic attack have been used to treat the substrate for biofuels production and proved to be suitable for applications at full-scale biogas While enzymatic hydrolysis of holocellulose has been widely investigated mainly in connection with bioethanol production Corresponding author.Tel.:+45 4525 1429;fax:+45 4593 2850. (Talebnia et al.,2010),enzymatic hydrolysis or oxidation of lignin E-mail address:ria@er.dtu.dk (I.Angelidaki). for biogas production has not been sufficiently researched. 0960-8524/S-see front matter2010 Elsevier Ltd.All rights reserved. doi:10.1016j.biortech.2010.06.108
Comparative study of mechanical, hydrothermal, chemical and enzymatic treatments of digested biofibers to improve biogas production Emiliano Bruni a,b , Anders Peter Jensen b , Irini Angelidaki a, * aDepartment of Environmental Engineering, Technical University of Denmark, Building 113, 2800 Kgs. Lyngby, Denmark b Process Development, Xergi A/S, Hermesvej 1, 9530 Støvring, Denmark article info Article history: Received 30 April 2010 Received in revised form 15 June 2010 Accepted 24 June 2010 Available online 16 July 2010 Keywords: Treatment Lignocellulose Biogas Hydrolysis Enzymes abstract Organic waste such as manure is an important resource for biogas production. The biodegradability of manures is however limited because of the recalcitrant nature of the biofibers it contains. To increase the biogas potential of the biofibers in digested manure, we investigated physical treatment (milling), chemical treatment (CaO), biological treatment (enzymatic and partial aerobic microbial conversion), steam treatment with catalyst (H3PO4 or NaOH) and combination of biological and steam treatments (biofibers steam-treated with catalyst were treated with laccase enzyme). We obtained the highest methane yield increase through the chemical treatment that resulted in 66% higher methane production compared to untreated biofibers. The combination of steam treatment with NaOH and subsequent enzymatic treatment increased the methane yield by 34%. To choose the optimal treatment, the energy requirements relative to the energy gain as extra biogas production have to be taken into account, as well as the costs of chemicals or enzymes. 2010 Elsevier Ltd. All rights reserved. 1. Introduction Anaerobic digestion of organic waste and residues combines both sustainable treatment and renewable energy production. Some substrates such as lignocellulosic materials are resistant to anaerobic digestion and can be converted into biogas only to a low extent. The low susceptibility of these materials to conversion into biogas is a result of their composition and structure. Lignocellulose is the complex and rigid matrix of plant cells, it is resistant to enzymatic attack because of the tight association between lignin, cellulose and hemicellulose. Cellulose and hemicellulose (carbohydrates composed of hexoses and mainly pentoses, respectively) can be degraded in biogas processes. Lignin can however not be degraded under anaerobic conditions (Fernandes et al., 2009). In full-scale biogas plants digesting manure, the low digestibility of the biofibers contained in the manure causes a loss of methane production and limits the overall efficiency of the process (Jin et al., 2009). Therefore, treatments facilitating the accessibility of holocellulose (cellulose and hemicellulose) are needed to increase the biogas potential of biofibers in manure. Many treatments for increasing the biodegradability of lignocellulosic material have been reported (Demirbas, 2008). Mechanical treatment (milling) increases the surface available for enzymatic attack and proved to be suitable for applications at full-scale biogas plants, increasing the methane yield of lignocellulosic substrates by up to 25% (Hartmann et al., 2000). Steam treatment has been optimized mainly for ethanol production. Glucose yields of 98% after enzymatic hydrolysis have been registered for wheat straw steam-treated with H2SO4 (Talebnia et al., 2010). Likewise, steam treatment with H3PO4 hydrolyzed hemicellulose and increased the accessibility of cellulose for enzymatic hydrolysis (Geddes et al., 2010). Steam treatment with NaOH has been reported to increase the biogas production of sorted municipal solid waste by 50% (Wang et al., 2009). Chemical treatments investigated include treatments with acids, bases and oxidants (Taherzadeh and Karimi, 2008). Chemical treatments with NaOH are among those that have been investigated most (Tanaka et al., 1997; Zheng et al., 2009). Alkaline hydrolysis with NaOH has been successfully applied to treat lignocellulosic materials such as straw or hardwood (Sun and Cheng, 2002). Some aerobic microorganisms (white-, brown-, soft-rot fungi) can selectively degrade lignin or hemicellulose. Srilatha et al. (1995) obtained a 33% methane yield increase for orange processing waste by treatment with selected fungi strains. Similarly, Taherzadeh and Karimi (2008) reported improved enzymatic hydrolysis (94% sugar recovery) when treating office paper with selected aerobic bacteria. Commercially available enzymes have been used to treat the substrate for biofuels production. While enzymatic hydrolysis of holocellulose has been widely investigated mainly in connection with bioethanol production (Talebnia et al., 2010), enzymatic hydrolysis or oxidation of lignin for biogas production has not been sufficiently researched. 0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.108 * Corresponding author. Tel.: +45 4525 1429; fax: +45 4593 2850. E-mail address: ria@er.dtu.dk (I. Angelidaki). Bioresource Technology 101 (2010) 8713–8717 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
8714 E.Bruni et aL/Bioresource Technology 101 (2010)8713-8717 Ligninases such as laccase,lignin peroxidase and manganese per- 2.2.2.2.Enzymatic treatment.The enzymes tested were laccase(E.C. oxidase are reported to delignify lignocellulose (Ohkuma,2003: 1.10.3.2)and a mixture of cellulases and hemicellulases with cellu- Hatakka,1994).For the present study,we have treated biofibers lase being the main activity (E.C.3.2.1.4).The enzymes were com- separated from digested manure with different methods with the mercial products DeniLite II S(laccase).Novozym 51003(laccase) aim to increase their methane potential.We tested chemical,bio- Novozym 342(cellulase)from Novozymes(Denmark).Laccase EN- logical,physical,hydrothermal treatments and combinations.The 204 from JenaBios(Germany).Treatments with DeniLite lI S and treatments considered have been selected due to their low input Novozym 342 were made at concentrations of 0.5,5.0.10.0. energy requirements.We evaluated each treatment based on the 20.0 U(g TS)and 0.3,0.5,1.0.2.0 U (g TS),respectively.The effects on the measured methane potential. same concentrations were used for treatments with the combina- tion of the two enzymes.To elucidate the effect of the pH on enzy- 2.Methods matic treatment.the pH was adjusted to 4.0.5.5.6.0.7.0 by adding H3PO4.An additional experiment with DeniLite ll S alone at dosage 2.1.Materials 66 U(g TS)at pH 4.0 was made.The treatments with Novozym 51003 were made at 60 U(g TS),with and without the addition Biofibers separated from digested manure were used as the sub- of a mediator(syringaldazine 0.8 and 1.5 mmol I-1),at pH 5.5 and strate.The biofibers were collected from the effluent of Foulum 7.0.The treatments with Laccase EN-204 were made at 84 U biogas plant(Denmark)digesting cow and pig manure,maize si- (gTS)1,with and without the addition of a mediator (ABTS lage and industrial by-products.The biofibers were separated with 1.7 mmol I-1).The pH was adjusted to 5.0 with a citrate-phos- a 2-mm sieve and frozen at-18C.Batches of these biofibers taken phate buffer solution.All the enzymatic treatments were made at at different times were used for the treatments.For treatments 37C.Control experiments with enzymes without addition of biof- with enzymes,steam and a combination of these,the biofibers ibers and with biofibers but without enzymes were included.The were washed before being frozen. treatments lasted 20 h,under continuous mixing.Oxidative en- zymes such as laccase use oxygen as the electron acceptor,there- 2.2.Treatments fore the enzymatic treatments were made with continuous oxygen supply bubbling air with a membrane pump.The treated 2.2.1.Chemical treatment material was separated into a solid and a liquid fraction with a The biofibers were treated with calcium oxide(CaO)from Faxe 1-mm sieve. Kalk(Denmark).Cao was added to the biofibers to obtain concen- 2.2.3.Physical treatment trations of 6%,8%.10%w/w on wet weight (WW)basis.Controls As physical treatment,the size reduction of the fibers was without CaO addition were included.The treatments were made in duplicates,at 15C and lasted 25 days (Table 1).Every fifth tested.A kitchen blender Braun K600 was used to reduce the size day,the biofibers were mixed and samples were retrieved. of the fibers (2 mm,based on an approximate particle size distribution). 2.2.2.Biological treatment 2.2.4.Steam treatment 2.2.2.1.Partial aerobic treatment.The aerobic inocula used for the The effect of steam treatment was studied in combination with partial aerobic treatment were compost from garden waste from a catalyst.Phosphoric acid(H3PO4)and sodium hydroxide (NaOH) Arhus Affaldscenter (Denmark)and fungi collected from straw and maize silage stored outdoor in Foulum(Denmark)for a period were used as catalysts.Substrate concentration(12.9%TS),temper- of 6 months.Aerobic inoculum and biofibers were mixed in pro- ature (160C).time of treatment (15 min).Ww of substrate (200 g)and the concentration of the catalyst(4%w/w TS)were portions of 2%and 10%w/w on WW basis.Controls without inoc- the same for all steam treatments.The unit for steam treatment ulum were included.Atmospheric air was supplied from the was as described by Bruni et al.(in press). bottom of the biofibers-inoculum mixture with a Gude 215/8/24 compressor at a flow of 0(no aeration)and 280 ml(min kg TS)-1 2.2.5.Steam treatment followed by enzymatic treatment (TS is the total solids content of the mixture).The flow was mea- Enzymatic treatment was applied to the solid fraction of the sured with a flow meter Gallus 2100 G 1.6 TCE.A 2-mm sieve steam-treated material.The combined treatment was compared was used to avoid the blocking of the air tube and to ensure to a mere enzymatic treatment as control.The enzyme laccase homogenous air distribution.Experiments were carried out in Novozym 51003 was used for the combined treatment.The en- duplicates at 27C.The treatment lasted 20 days and sampling zyme dosage for the enzymatic treatment of the steam-treated and mixing took place every other day. material with H3PO4.NaOH and for the control experiment was 50,48 and 59 U(g TS)-,respectively.H3PO4 was used to adjust Table 1 the pH of the substrate and was dosed until pH 5.5 was reached, Treatments and corresponding conditions under continuous mixing.The enzymatic treatments lasted 20 h, at 37C,under continuous mixing and oxygen supply (bubbling Procedure Temperature Reaction (C) time air with a membrane pump). Chemical Cao 6%,8%.10%ww 15 0-25d WW 2.3.Methane potential assays Biological Partial aerobic Compost,fungi 27 0-20d Enzymatic Laccase,cellulase. 37 20h Methane potential was determined in batch assays (infusion hemicellulase bottles of 543 ml total volume,200 ml inoculum,at 52 C).as de- Phvsical Size reduction 2mm Steam H3P04 4%w/w TS 160 15 min scribed by Angelidaki et al.(2009).The inoculum for the batch as- NaOH says was the effluent from a thermophilic biogas plant(52C) Combined Steam+H3PO4. 160.37 15 min. digesting cow manure.For the samples with high TS content (un- laccase 20h treated biofibers or solid fraction from treated biofibers),10 g Steam +NaOH, laccase WW of substrate were used.Batches digesting the complete treated mixture of solid fraction and liquid fraction were prepared
Ligninases such as laccase, lignin peroxidase and manganese peroxidase are reported to delignify lignocellulose (Ohkuma, 2003; Hatakka, 1994). For the present study, we have treated biofibers separated from digested manure with different methods with the aim to increase their methane potential. We tested chemical, biological, physical, hydrothermal treatments and combinations. The treatments considered have been selected due to their low input energy requirements. We evaluated each treatment based on the effects on the measured methane potential. 2. Methods 2.1. Materials Biofibers separated from digested manure were used as the substrate. The biofibers were collected from the effluent of Foulum biogas plant (Denmark) digesting cow and pig manure, maize silage and industrial by-products. The biofibers were separated with a 2-mm sieve and frozen at 18 C. Batches of these biofibers taken at different times were used for the treatments. For treatments with enzymes, steam and a combination of these, the biofibers were washed before being frozen. 2.2. Treatments 2.2.1. Chemical treatment The biofibers were treated with calcium oxide (CaO) from Faxe Kalk (Denmark). CaO was added to the biofibers to obtain concentrations of 6%, 8%, 10% w/w on wet weight (WW) basis. Controls without CaO addition were included. The treatments were made in duplicates, at 15 C and lasted 25 days (Table 1). Every fifth day, the biofibers were mixed and samples were retrieved. 2.2.2. Biological treatment 2.2.2.1. Partial aerobic treatment. The aerobic inocula used for the partial aerobic treatment were compost from garden waste from Århus Affaldscenter (Denmark) and fungi collected from straw and maize silage stored outdoor in Foulum (Denmark) for a period of 6 months. Aerobic inoculum and biofibers were mixed in proportions of 2% and 10% w/w on WW basis. Controls without inoculum were included. Atmospheric air was supplied from the bottom of the biofibers–inoculum mixture with a Güde 215/8/24 compressor at a flow of 0 (no aeration) and 280 ml (min kg TS)1 (TS is the total solids content of the mixture). The flow was measured with a flow meter Gallus 2100 G 1.6 TCE. A 2-mm sieve was used to avoid the blocking of the air tube and to ensure homogenous air distribution. Experiments were carried out in duplicates at 27 C. The treatment lasted 20 days and sampling and mixing took place every other day. 2.2.2.2. Enzymatic treatment. The enzymes tested were laccase (E.C. 1.10.3.2) and a mixture of cellulases and hemicellulases with cellulase being the main activity (E.C. 3.2.1.4). The enzymes were commercial products DeniLite II S (laccase), Novozym 51003 (laccase), Novozym 342 (cellulase) from Novozymes (Denmark), Laccase EN- 204 from JenaBios (Germany). Treatments with DeniLite II S and Novozym 342 were made at concentrations of 0.5, 5.0, 10.0, 20.0 U (g TS)1 and 0.3, 0.5, 1.0, 2.0 U (g TS)1 , respectively. The same concentrations were used for treatments with the combination of the two enzymes. To elucidate the effect of the pH on enzymatic treatment, the pH was adjusted to 4.0, 5.5, 6.0, 7.0 by adding H3PO4. An additional experiment with DeniLite II S alone at dosage 66 U (g TS)1 at pH 4.0 was made. The treatments with Novozym 51003 were made at 60 U (g TS)1 , with and without the addition of a mediator (syringaldazine 0.8 and 1.5 mmol l1 ), at pH 5.5 and 7.0. The treatments with Laccase EN-204 were made at 84 U (g TS)1 , with and without the addition of a mediator (ABTS 1.7 mmol l1 ). The pH was adjusted to 5.0 with a citrate–phosphate buffer solution. All the enzymatic treatments were made at 37 C. Control experiments with enzymes without addition of biofibers and with biofibers but without enzymes were included. The treatments lasted 20 h, under continuous mixing. Oxidative enzymes such as laccase use oxygen as the electron acceptor, therefore the enzymatic treatments were made with continuous oxygen supply bubbling air with a membrane pump. The treated material was separated into a solid and a liquid fraction with a 1-mm sieve. 2.2.3. Physical treatment As physical treatment, the size reduction of the fibers was tested. A kitchen blender Braun K600 was used to reduce the size of the fibers (2 mm, based on an approximate particle size distribution). 2.2.4. Steam treatment The effect of steam treatment was studied in combination with a catalyst. Phosphoric acid (H3PO4) and sodium hydroxide (NaOH) were used as catalysts. Substrate concentration (12.9% TS), temperature (160 C), time of treatment (15 min), WW of substrate (200 g) and the concentration of the catalyst (4% w/w TS) were the same for all steam treatments. The unit for steam treatment was as described by Bruni et al. (in press). 2.2.5. Steam treatment followed by enzymatic treatment Enzymatic treatment was applied to the solid fraction of the steam-treated material. The combined treatment was compared to a mere enzymatic treatment as control. The enzyme laccase Novozym 51003 was used for the combined treatment. The enzyme dosage for the enzymatic treatment of the steam-treated material with H3PO4, NaOH and for the control experiment was 50, 48 and 59 U (g TS)1 , respectively. H3PO4 was used to adjust the pH of the substrate and was dosed until pH 5.5 was reached, under continuous mixing. The enzymatic treatments lasted 20 h, at 37 C, under continuous mixing and oxygen supply (bubbling air with a membrane pump). 2.3. Methane potential assays Methane potential was determined in batch assays (infusion bottles of 543 ml total volume, 200 ml inoculum, at 52 C), as described by Angelidaki et al. (2009). The inoculum for the batch assays was the effluent from a thermophilic biogas plant (52 C) digesting cow manure. For the samples with high TS content (untreated biofibers or solid fraction from treated biofibers), 10 g WW of substrate were used. Batches digesting the complete treated mixture of solid fraction and liquid fraction were prepared Table 1 Treatments and corresponding conditions. Procedure Temperature (C) Reaction time Chemical CaO 6%, 8%, 10% w/w WW 15 0–25 d Biological Partial aerobic Compost, fungi 27 0–20 d Enzymatic Laccase, cellulase, hemicellulase 37 20 h Physical Size reduction 2 mm – – Steam H3PO4 4% w/w TS 160 15 min NaOH Combined Steam + H3PO4, laccase 160, 37 15 min, 20 h Steam + NaOH, laccase 8714 E. Bruni et al. / Bioresource Technology 101 (2010) 8713–8717
E.Bruni et al/Bioresource Technology 101(2010)8713-8717 8715 adding 40g WW of the solid and liquid fractions with the same 300 proportions as in the treated material(on a WW basis).The meth- ane potential of inactivated enzymes(enzymes heated to 100C for 3 h)was measured.Blank batches containing only inoculum 240 and control batches with pure amorphous cellulose as substrate were included.The batch assays were done in triplicates 180 2.4.Analyses and calculations e 120 Total nitrogen TKN and ammonium nitrogen NH4-N(Kjeldahl-N method).TS,volatile solids (VS)were measured according to the standard methods(APHA,1998).The organic nitrogen content(ex- 60 pressed as proteins)was calculated from TKN and NH4-N as: organic N=(TKN-NH4-N)x 6.25 5 10 15 20 25 30 days The concentration of volatile fatty acids(VFA)in the biofibers was measured adding 20 ml of H3PO40.5 mol I'to 20 g of sample X no CaO added △6%Ca0 口8%Ca0o10%Ca0 and analyzing the VFA content of 1 ml liquid (Kaparaju et al.. 2009).A gas chromatograph equipped with a flame ionization Fig 1.Chemical treatment with CaO.specific methane yield of treated biofibers detector (FID)was used to monitor the methane production (Hansen et al.,2004).Gas measurements are reported in STP con- ditions(Standard Temperature and Pressure,273 K,101,325 Pa) Methane and energy yields are expressed as m3 CH4(t WW) 200 and kWh (t WW)-1,respectively,where WW indicates the wet weight of untreated biofibers.The thermal energy content of the 180 methane was calculated using the lower calorific value 50.1 MI (kg CH4)-.The mass balance was made on VS basis as described 160 by Cullis et al.(2004). 140 3.Results and discussion 120 3.1.Effects on the methane potential 100 Treatments with CaO and NaOH resulted in the highest meth- ane yield increases (Table 2).We obtained the highest methane yield(239 and 234 ml CH4 (g VS)1)by treating biofibers for 60 10 days with 6%and 8%CaO,respectively(Fig.1).The lower meth- ane yield that derived from the treatment with 10%CaO compared 40 to treatments with lower Cao dosages may be due to the formation of calcium-lignin complexes that caused lower lignin removal and consequently lower methane yield increase.Xu et al.(2010)re- ported a decrease of lignin solubilization because the treatment 20 60 80 100 with lime caused interaction between negatively charged lignin days molecules and positively charged calcium ions.Among the two cat- alysts used for steam treatment,NaOH resulted in the highest e-untreated biofibers -B-steam H,PO A-steam NaOH methane yield increase (49 ml CH(g VS))and had the highest conversion rate (Fig.2).Steam treatment with NaOH may have Fig.2.Steam treatment with catalyst,specific methane yield of whole treated converted part of the lignin into acetic acid (we detected acetic mixture(solid fraction hydrolysate). Table 2 acid,11.32%and 0.33%of TS in the steam-treated liquid fraction Effect on methane yield and in the steam-treated solid fraction,respectively),while steam Variation&of Variation of treatment with H3PO4 addition may have only reallocated lignin yield ml CHa yield mCHa (Kaparaju and Felby.2010).It is reported that oxidative treatments (g Vs)1 (t ww)-1b in alkaline conditions convert carbohydrates and lignin into car- Chemical Cao +59% +66% boxylic acids (Schmidt and Thomsen,1998).In this study,steam Biological Partial aerobic No effect No effect treatment with NaOH probably had a similar effect regarding ace- Enzvmatic No effect No effect tic acid formation.The presence of the acetic acid in the steam- Physical Size reduction +8% +10% Steam H3PO4 +6% +8% treated material explains the high conversion rate of this material NaOH +38% +26% into methane,as acetic acid can directly be utilized by aceticlastic Combined steam HaPO4+laccase +24% +18% methanogens.Steam treatment with H3PO4 addition increased the Steam NaoH+laccase +69 +34% methane yield of biofibers to a lower extent than the other treat- a VS of treated material. ments(8 ml CH(g VS)1).Our results showed a lower improve- b WW of untreated biofibers. ment of the biodegradability of biofibers compared to results
adding 40 g WW of the solid and liquid fractions with the same proportions as in the treated material (on a WW basis). The methane potential of inactivated enzymes (enzymes heated to 100 C for 3 h) was measured. Blank batches containing only inoculum and control batches with pure amorphous cellulose as substrate were included. The batch assays were done in triplicates. 2.4. Analyses and calculations Total nitrogen TKN and ammonium nitrogen NH4-N (Kjeldahl-N method), TS, volatile solids (VS) were measured according to the standard methods (APHA, 1998). The organic nitrogen content (expressed as proteins) was calculated from TKN and NH4-N as: organic N ¼ ðTKN—NH4-NÞ 6:25 The concentration of volatile fatty acids (VFA) in the biofibers was measured adding 20 ml of H3PO4 0.5 mol l1 to 20 g of sample and analyzing the VFA content of 1 ml liquid (Kaparaju et al., 2009). A gas chromatograph equipped with a flame ionization detector (FID) was used to monitor the methane production (Hansen et al., 2004). Gas measurements are reported in STP conditions (Standard Temperature and Pressure, 273 K, 101,325 Pa). Methane and energy yields are expressed as m3 CH4 (t WW)1 and kWh (t WW)1 , respectively, where WW indicates the wet weight of untreated biofibers. The thermal energy content of the methane was calculated using the lower calorific value 50.1 MJ (kg CH4) 1 . The mass balance was made on VS basis as described by Cullis et al. (2004). 3. Results and discussion 3.1. Effects on the methane potential Treatments with CaO and NaOH resulted in the highest methane yield increases (Table 2). We obtained the highest methane yield (239 and 234 ml CH4 (g VS)1 ) by treating biofibers for 10 days with 6% and 8% CaO, respectively (Fig. 1). The lower methane yield that derived from the treatment with 10% CaO compared to treatments with lower CaO dosages may be due to the formation of calcium–lignin complexes that caused lower lignin removal and consequently lower methane yield increase. Xu et al. (2010) reported a decrease of lignin solubilization because the treatment with lime caused interaction between negatively charged lignin molecules and positively charged calcium ions. Among the two catalysts used for steam treatment, NaOH resulted in the highest methane yield increase (49 ml CH4 (g VS)1 ) and had the highest conversion rate (Fig. 2). Steam treatment with NaOH may have converted part of the lignin into acetic acid (we detected acetic acid, 11.32% and 0.33% of TS in the steam-treated liquid fraction and in the steam-treated solid fraction, respectively), while steam treatment with H3PO4 addition may have only reallocated lignin (Kaparaju and Felby, 2010). It is reported that oxidative treatments in alkaline conditions convert carbohydrates and lignin into carboxylic acids (Schmidt and Thomsen, 1998). In this study, steam treatment with NaOH probably had a similar effect regarding acetic acid formation. The presence of the acetic acid in the steamtreated material explains the high conversion rate of this material into methane, as acetic acid can directly be utilized by aceticlastic methanogens. Steam treatment with H3PO4 addition increased the methane yield of biofibers to a lower extent than the other treatments (8 ml CH4 (g VS)1 ). Our results showed a lower improvement of the biodegradability of biofibers compared to results Table 2 Effect on methane yield. Variation% of yield ml CH4 (g VS)1a Variation% of yield m3 CH4 (t WW)1b Chemical CaO +59% +66% Biological Partial aerobic No effect No effect Enzymatic No effect No effect Physical Size reduction +8% +10% Steam H3PO4 +6% +8% NaOH +38% +26% Combined steam + H3PO4 + laccase +24% +18% Steam + NaOH + laccase +69 +34% a VS of treated material. b WW of untreated biofibers. 0 60 120 180 240 300 0 5 10 15 20 25 30 days ml CH4 (g VS)-1 no CaO added 6% CaO 8% CaO 10% CaO Fig. 1. Chemical treatment with CaO, specific methane yield of treated biofibers. 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 days ml CH4 (g VS)-1 untreated biofibers steam + H3PO4 steam + NaOH Fig. 2. Steam treatment with catalyst, specific methane yield of whole treated mixture (solid fraction + hydrolysate). E. Bruni et al. / Bioresource Technology 101 (2010) 8713–8717 8715
8716 E.Bruni et aL/Bioresource Technology 101 (2010)8713-8717 10.00 3.3.Considerations for full-scale applications 8.00 Chemical treatment with Cao has a significant potential to de- crease the concentration of ammonia in the substrate.Because of 6.00 the risk of inhibition of the microorganisms involved in the biogas 4.00 process,removal of ammonia may be required at full-scale biogas 三 plants digesting substrates such as manure that have a high con- 2.00 tent of organic nitrogen or ammonia.Although the chemical treat- ment with CaO resulted in the highest methane yield gain and 0.00 increased the rate of NH-N volatilization,the advantage of this 10 15 20 25 0 treatment has to be evaluated carefully.The analysis will have to days take into account the costs of chemicals and the need for extra ×noCa0 added△6%Cao investments such as mixers (thorough mixing is required to ensure homogenous distribution of CaO on the biofibers)and storage (the Fig.3.Chemical treatment with Cao.NH4-N in the biofibers. storage volume is proportional to the reaction time of the treat- ment).Steam treatment with NaOH resulted in a lower methane yield increase compared to the chemical treatment with CaO.The obtained by other researches with H3PO4 on sugar cane bagasse low dosage of chemicals and the short reaction time make this and corn stover (Geddes et al.,2010:Um et al.,2003).This can treatment however very interesting for full-scale biogas processes. be explained with differences in the materials treated.In our study. The energy input for steam treatment may be available from waste the biofibers had previously been digested in a biogas reactor,thus heat at full-scale biogas plants equipped with gas engines the easily degradable organic material had already been removed (Pickworth et al.,2006).Considering economical and environmen- and we used a lower concentration of catalyst compared to some tal aspects,the catalyst NaOH is more expensive than Cao and for of the previous researches. reasons of soil pollution it is not desirable for the effluent of the Combined steam treatment using catalyst followed by treat- biogas process,should this be used as a fertilizer(Wyman et al. ment with laccase further increased the methane yield by 2005).Although the optimal dosage for NaOH would need to be 2.0+0.5 and 1.7+0.4 m3 CH4(t WW)-compared to steam treat- investigated,steam treatment with NaOH addition is preferred to ment alone with H3PO4 and NaOH,respectively,while enzymatic steam treatment with HsPO4.due to the higher methane yield treatment alone did not improve the biodegradability significantly increase. (19.80.4 m3 CH(t WW)-).This suggests that the tight associa- The combined steam treatment with NaOH followed by laccase tion of lignocellulose did not allow effective enzymatic treatment. treatment resulted in extra methane production corresponding to Steam treatment can increase the porosity of the biofibers and can 68 kWh(t WW)-1,compared to untreated biofibers,and 16 kWh thus make the substrate more accessible for the enzymes.Interest- (t WW)-compared to biofibers that were treated with steam with ingly.treating the steam-treated material with laccase,without catalyst NaOH.Considerations should be made regarding the addi- adding a mediator increased the methane yield.Probably,laccase tional costs of the laccase treatment (separation of the solid frac- sufficiently oxidized lignin to an extent,which was high enough tion after steam treatment,pH adjustment and enzymatic to improve the biodegradability of the biofibers,although it is re- treatment)relative to the extra energy yield. ported that laccase in the absence of a mediator can only oxidize The methane yield increase due to mechanical treatment was small fractions of lignin(Widsten and Kandelbauer,2008).Similar lower than the one obtained by Angelidaki and Ahring(2000).This findings without addition of a mediator were obtained by Palonen underlines that although mechanical treatment is a straightfor- and Viikari(2004).It is possible that oxidizing substances.such as ward treatment that can be implemented at full-scale biogas solubilized or colloidal lignin,contained in the steam-treated plants,the costs of the energy input may be high in relation to material acted as mediators in the enzymatic reactions (Felby the extra energy yield (Taherzadeh and Karimi,2008). et al.,1997:Gronqvist et al.,2003). Our treatments with aerobic microorganisms did not improve the methane yield of the biofibers.We tested these microorgan- 3.2.Overall mass balance isms to aerobically treat the lignocellulose for a short time to initi- ate decomposition of the lignocellulosic structure and to increase Among the treatments tested,only steam treatment with NaOH its biodegradability,avoiding that aerobes achieve the oxidation caused mass losses (10%).This treatment formed the hydrolysate of the holocellulose.Other researchers applied treatments with (steam-treated)with the highest VS content (12%),while the microorganisms to substrates such as rice straw,office paper,agri- hydrolysate from steam treatment with H3PO4 contained approxi- cultural waste and kitchen waste.Positive results were reported mately 5%of the total steam-treated VS.Untreated biofibers con- from studies made with pure cultures of microorganisms (Dhouib tained 11.2*0.4g N/(kg WW)-1 as organic nitrogen.Steam et al.,2006;Kurakake et al.,2007;Schober and Trosch,2000:Srila- treatment with H3PO and NaOH released 19%and 53%of the or- tha et al.,1995),but the use of pure cultures of microorganisms ganic nitrogen into the hydrolysate,respectively.Untreated biofi- may not be possible for full-scale applications,unless the microor- bers did not contain VFA and we detected acetic acid only in ganisms can be cheaply cultivated at the biogas plant. biofibers treated with steam with NaOH.Based on of the overall ef- fect of the treatment on the mass and nitrogen distribution,on the acetic acid formation and on the methane yield increase,steam 4.Conclusions treatment with NaOH was more aggressive compared to steam treatment with H3PO4.The highly-reactive OH-anions that were We identified different methods to increase the methane yield released with steam treatment with NaOH reacted with different of biofibers from digested manure.Chemical treatment(CaO)and organic molecules,while the protons H'from steam treatment steam treatment with NaOH resulted in the highest methane yield with H3PO4 could hydrolyze mainly hemicellulose (Mousavioun increases (66%and 26%,respectively).Because steam treatment and Doherty,2010).Chemical treatment with Cao increased the with NaOH addition released 12%of the VS into the hydrolysate, rate of ammonia volatilization from the biofibers(Fig.3). the whole steam-treated mixture (solid fraction hydrolysate)
obtained by other researches with H3PO4 on sugar cane bagasse and corn stover (Geddes et al., 2010; Um et al., 2003). This can be explained with differences in the materials treated. In our study, the biofibers had previously been digested in a biogas reactor, thus the easily degradable organic material had already been removed and we used a lower concentration of catalyst compared to some of the previous researches. Combined steam treatment using catalyst followed by treatment with laccase further increased the methane yield by 2.0 ± 0.5 and 1.7 ± 0.4 m3 CH4 (t WW)1 compared to steam treatment alone with H3PO4 and NaOH, respectively, while enzymatic treatment alone did not improve the biodegradability significantly (19.8 ± 0.4 m3 CH4 (t WW)1 ). This suggests that the tight association of lignocellulose did not allow effective enzymatic treatment. Steam treatment can increase the porosity of the biofibers and can thus make the substrate more accessible for the enzymes. Interestingly, treating the steam-treated material with laccase, without adding a mediator increased the methane yield. Probably, laccase sufficiently oxidized lignin to an extent, which was high enough to improve the biodegradability of the biofibers, although it is reported that laccase in the absence of a mediator can only oxidize small fractions of lignin (Widsten and Kandelbauer, 2008). Similar findings without addition of a mediator were obtained by Palonen and Viikari (2004). It is possible that oxidizing substances, such as solubilized or colloidal lignin, contained in the steam-treated material acted as mediators in the enzymatic reactions (Felby et al., 1997; Grönqvist et al., 2003). 3.2. Overall mass balance Among the treatments tested, only steam treatment with NaOH caused mass losses (10%). This treatment formed the hydrolysate (steam-treated) with the highest VS content (12%), while the hydrolysate from steam treatment with H3PO4 contained approximately 5% of the total steam-treated VS. Untreated biofibers contained 11.2 ± 0.4 g N/(kg WW)1 as organic nitrogen. Steam treatment with H3PO4 and NaOH released 19% and 53% of the organic nitrogen into the hydrolysate, respectively. Untreated biofi- bers did not contain VFA and we detected acetic acid only in biofibers treated with steam with NaOH. Based on of the overall effect of the treatment on the mass and nitrogen distribution, on the acetic acid formation and on the methane yield increase, steam treatment with NaOH was more aggressive compared to steam treatment with H3PO4. The highly-reactive OH anions that were released with steam treatment with NaOH reacted with different organic molecules, while the protons H+ from steam treatment with H3PO4 could hydrolyze mainly hemicellulose (Mousavioun and Doherty, 2010). Chemical treatment with CaO increased the rate of ammonia volatilization from the biofibers (Fig. 3). 3.3. Considerations for full-scale applications Chemical treatment with CaO has a significant potential to decrease the concentration of ammonia in the substrate. Because of the risk of inhibition of the microorganisms involved in the biogas process, removal of ammonia may be required at full-scale biogas plants digesting substrates such as manure that have a high content of organic nitrogen or ammonia. Although the chemical treatment with CaO resulted in the highest methane yield gain and increased the rate of NH4-N volatilization, the advantage of this treatment has to be evaluated carefully. The analysis will have to take into account the costs of chemicals and the need for extra investments such as mixers (thorough mixing is required to ensure homogenous distribution of CaO on the biofibers) and storage (the storage volume is proportional to the reaction time of the treatment). Steam treatment with NaOH resulted in a lower methane yield increase compared to the chemical treatment with CaO. The low dosage of chemicals and the short reaction time make this treatment however very interesting for full-scale biogas processes. The energy input for steam treatment may be available from waste heat at full-scale biogas plants equipped with gas engines (Pickworth et al., 2006). Considering economical and environmental aspects, the catalyst NaOH is more expensive than CaO and for reasons of soil pollution it is not desirable for the effluent of the biogas process, should this be used as a fertilizer (Wyman et al., 2005). Although the optimal dosage for NaOH would need to be investigated, steam treatment with NaOH addition is preferred to steam treatment with H3PO4, due to the higher methane yield increase. The combined steam treatment with NaOH followed by laccase treatment resulted in extra methane production corresponding to 68 kWh (t WW)1 , compared to untreated biofibers, and 16 kWh (t WW)1 compared to biofibers that were treated with steam with catalyst NaOH. Considerations should be made regarding the additional costs of the laccase treatment (separation of the solid fraction after steam treatment, pH adjustment and enzymatic treatment) relative to the extra energy yield. The methane yield increase due to mechanical treatment was lower than the one obtained by Angelidaki and Ahring (2000). This underlines that although mechanical treatment is a straightforward treatment that can be implemented at full-scale biogas plants, the costs of the energy input may be high in relation to the extra energy yield (Taherzadeh and Karimi, 2008). Our treatments with aerobic microorganisms did not improve the methane yield of the biofibers. We tested these microorganisms to aerobically treat the lignocellulose for a short time to initiate decomposition of the lignocellulosic structure and to increase its biodegradability, avoiding that aerobes achieve the oxidation of the holocellulose. Other researchers applied treatments with microorganisms to substrates such as rice straw, office paper, agricultural waste and kitchen waste. Positive results were reported from studies made with pure cultures of microorganisms (Dhouib et al., 2006; Kurakake et al., 2007; Schober and Trösch, 2000; Srilatha et al., 1995), but the use of pure cultures of microorganisms may not be possible for full-scale applications, unless the microorganisms can be cheaply cultivated at the biogas plant. 4. Conclusions We identified different methods to increase the methane yield of biofibers from digested manure. Chemical treatment (CaO) and steam treatment with NaOH resulted in the highest methane yield increases (66% and 26%, respectively). Because steam treatment with NaOH addition released 12% of the VS into the hydrolysate, the whole steam-treated mixture (solid fraction + hydrolysate) 0.00 2.00 4.00 6.00 8.00 10.00 0 5 10 15 20 25 30 g NH4-N (kg VS)-1 days no CaO added 6% CaO Fig. 3. Chemical treatment with CaO, NH4-N in the biofibers. 8716 E. Bruni et al. / Bioresource Technology 101 (2010) 8713–8717
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