Bioresource Technology 175 (2015) 182–188

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Making lignin accessible for anaerobic digestion by wet-explosionpretreatment

http://dx.doi.org/10.1016/j.biortech.2014.10.0820960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Center for Bioproducts and Bioenergy, WashingtonState University, 2710 University Drive, Richland, WA 99354-1671, USA.Tel.: +1 (509) 372 7682; fax: +1 (509) 372 7690.

E-mail address: bka@wsu.edu (B.K. Ahring).

Birgitte K. Ahring a,⇑, Rajib Biswas a, Aftab Ahamed a, Philip J. Teller a, Hinrich Uellendahl b

a Bioproducts, Sciences & Engineering Laboratory (BSEL), Washington State University, USAb Section for Sustainable Biotechnology, Aalborg University Copenhagen, Denmark

h i g h l i g h t s

� The pretreatment of feedlot manure was performed using 4 bars oxygen.� Oxygen assisted wet-explosion pretreatment promotes lignin solubility.� 4.5 times higher methane yield observed as a result of the pretreatment.� 44.4% lignin in pretreated material was actually converted in the AD process.� Aliphatic compounds formed in the pretreatment were utilized by microbes.

a r t i c l e i n f o

Article history:Received 17 September 2014Received in revised form 16 October 2014Accepted 17 October 2014Available online 23 October 2014

Keywords:MethaneAnaerobic digestionSustainabilityWet explosion pretreatmentFeedlot manure

a b s t r a c t

Lignin is a major part of the recalcitrant fraction of lignocellulose and in nature its degradation occursthrough oxidative enzymes along with microbes mediated oxidative chemical actions. Oxygen assistedwet-explosion pretreatment promotes lignin solubility and leads to an increase biodegradation of ligninduring anaerobic digestion processes. The pretreatment of feedlot manure was performed in a 10 L reac-tor at 170 �C for 25 min using 4 bars oxygen and the material was fed to a continuous stirred tank reactoroperated at 55 �C for anaerobic digestion. Methane yield of untreated and pretreated material was70 ± 27 and 320 ± 36 L/kg-VS/Day, respectively, or 4.5 times higher yield as a result of the pretreatment.Aliphatic acids formed during the pretreatment were utilized by microbes. 44.4% lignin in pretreatedmaterial was actually converted in the anaerobic digestion process compared to 12.6% for untreatedmaterial indicating the oxygen assisted explosion promoted lignin degradation.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Management of the large amounts of manure generated fromlivestock production in United States is a major challenge for nat-ural resource sustainability. Feedlot manure for instance is one ofthe major anthropogenic sources, contributing to deterioration ofthe global atmosphere leading to ammonia (NH3) volatilizationas well as methane (CH4), nitrous oxide (N2O), nitric oxide (NO)and carbon dioxide (CO2) emission (Li et al., 2012; Vaddella et al.,2013). Feedlot manure will, however, if collected properly hold amajor potential for biogas production. This resource can be utilizedto make bioenergy which again can substitute use of fossil fuelsand reduce carbon dioxide (CO2) emission while managing the

impact of nutrients present in the material. Recent life cycleassessment has shown that manure-based biogas production hashigher reduction effect on greenhouse gas emissions than biomassbased liquid biofuels production (Wenzel and Thyø, 2007). Addi-tionally, nutrient-rich digestate improves the fertilizer quality ofmanure as a fertilizer. Rising prices of fertilizer have greatlyincreased commercial interest in using manure as part of fertiliza-tion of crops in today’s agriculture.

A series of reactions such as decomposition, hydrolysis, ammo-nia volatilization, nitrification, denitrification, fermentation occursafter manure is excreted by animals (Li et al., 2012). By aerobicdecomposition and microbial degradation, easily degradable vola-tile solids in the manure are lost as CO2, for example, during storageof manure at feedlot operations. Feedlot manure is collected, byscrapping from pens every 2–3 months, and the moisture contentwill only be between 25% and 40% as most of the moisture generallyhas evaporated. After collection, the manure is generally kept foranother 6 months in windrows where the manure is turned using

B.K. Ahring et al. / Bioresource Technology 175 (2015) 182–188 183

a windrow turning machine every 4–6 weeks to manage odors anddust. Dry manure is then transported to the agricultural fields forland application on agricultural fields. After several months of openair storage the easy degradable components such as carbohydratesand protein have been oxidized and the remaining organic matter ismainly lignin due to the lower degradability of lignin compared toother polymers in the manure. Consequently, the stored manurewill be highly recalcitrant to microbial degradation in an anaerobicdigester. The recalcitrant nature of the stored manure makesanaerobic digestion economically infeasible due to low biogas pro-duction. Thus, pretreatment of the feedstock is needed to unlock therecalcitrance of this feedstock before it undergoes anaerobic diges-tion, in order to improve the accessibility of the feedstock and,thereby, the biogas potential of this feedstock (Biswas et al.,2012; Carlsson et al., 2012; Hendriks and Zeeman, 2009).

Organic materials rich in lignin and crystalline cellulose arehighly recalcitrant to biodegradation under anaerobic conditions.This is why the city of Venice which is built on a wooden foundationburied in the deep sediment is still holding up. When feedingorganic materials such as manure or straw to anaerobic digesters,the inherent resistance of the lignocellulosic fraction in the rawmaterial will, however, limit the convertibility of the material. Asa result only between 40% and 50% of the feed stock will be con-verted to biogas and the rest leaves the reactor unused, which willreduce the profitability of the anaerobic digestion system and willleave half of organic material for further disposal. Various pretreat-ment processes such as thermal, chemical, thermochemical and bio-logical pretreatments are commonly used to destruct thelignocellulose matrix in biomass to facilitate hydrolysis of the carbo-hydrates into convertible sugar monomers (Alvira et al., 2010;Chiaramonti et al., 2012; Hendriks and Zeeman, 2009; Mosieret al., 2005). Recently, the wet explosion (WEx) pretreatmentmethod (Ahring and Munck, 2006; Biswas et al., 2014) showed tobe highly efficient for biomass raw materials accessible for directmicrobial degradation by anaerobic microbes (Biswas et al., 2012)or to enzymatic hydrolysis to sugars with a high product yield(Biswas et al., 2014, 2012; Rana et al., 2012) as well as yeast fermen-tation without detoxification (Biswas et al., 2013). During wetexplosion pretreatment oxygen is used as an oxidizing agent which,besides working on the carbohydrate to release xylose or xylooligo-saccharides, results in release of lignin degradation products such aslower molecular weight aliphatic acids and phenols. It was hypoth-esized that the oxidation of lignin as part of WEx pretreatment cre-ates lignin degradation products that can be utilized by anaerobicmicroorganisms in the anaerobic digester along with the otherdegradable substrates such as carbohydrates, proteins and fats.Lignin in it polymeric form is generally regarded as recalcitrant tobiodegradation under anaerobic conditions (Ko et al., 2009). Thisstudy shows a way to improve the convertibility of lignocellulosicmaterials during anaerobic digestion resulting in a far higher bio-gas/methane yield from the same raw material – in this case feedlotmanure – and much smaller organic fraction to be dealt with afterthe process. This study investigates the potential of converting lig-nin-rich feedlot manure for production of biogas and to investigatethe potential for using the wet explosion pretreatment for improv-ing the digestibility of the raw material. This study further investi-gates the fate of certain lignin degradation products producedduring the WEx pretreatment when exposed to anaerobic digestion.

2. Methods

2.1. Feedstock and inoculum

The feedlot manure was obtained from Easterday Ranches, Inc.,Kennewick, WA. Dry manure was collected from the site and used

for the experiments. The manure had already dried in the open airfor about 6 months upon collection from the pens by scrapping.Inoculum was used from a lab-scale digester running on liquidmanure under the same conditions.

2.2. Wet explosion pretreatment

Wet explosion pretreatment of the manure was carried out atWashington State University pilot-plant facility using a 10 L reactorwith an active volume of 4 L as previously described (Biswas et al.,2014). The system is equipped with a stirrer and a 100 L subsequentflash tank connected to the reactor. Temperature, pressure andsteering speed of the motor are regulated and recorded electroni-cally. Required amount of tap water was added with the dry manureto reach the solid concentration of 15% to perform the pretreatment.After charging the pretreatment reactor with the raw material, thereactor was hermetically closed. A total of 4 bar of O2 was addedto the reactor and treated at 170 �C for 25 min. The reaction was ter-minated by sudden pressure drop into the flash tank dropping thetemperature to about 100 �C and instant cooling of the flash tankwas initiated before collection of the material for further use.

2.3. Lab-scale experiments

2.3.1. Reactor set-upA custom made stainless steel 40 L continuous stirred tank reac-

tor (CSTR) with an active volume of 30 L was used. Feeding wasperformed with an automated system consisting of a peristalticpump (Watson-Marlow peristaltic pump, 600 series) connectedto the feed storage tank. Total biogas production was measuredby an electronic flow-meter (Agilent Technologies, CA) and regis-tered electronically to a data logger system. The anaerobic digesterwas operated under thermophilic conditions (55 ± 1 �C, pH7.7 ± 0.1) with a hydraulic retention time of 10 days. The pH wasmonitored manually by sampling using InLab� Micro combinationpH electrode with precise measurement values (±0.001 pH). Thestirring was performed intermittently, i.e., 5 min stirring at 14 Hzin every 2 h.

2.3.2. Feeding strategiesBoth untreated and WEx treated feedlot manure were mixed

with 2% corn steep liquor (w/w) to supply additional nutrientsfor the microbes. Feed tank was filled with the designed feed mix-ture for 10 days feeding and preserved under controlled tempera-ture of 4 �C to avoid partial microbial degradation. Feeding waspreformed once a day with a constant organic loading rate of2.22 g-VS/L/Day. Calculated amount of feed was pumped into thedigester after vigorous mixing for 5 min to ensure a homogenizedfeed. During the start-up, the reactor was fed with untreated man-ure until day 22 while pretreated manure was used from day 23and onwards.

2.4. Analytical methods

2.4.1. Feedstock characterizationAnalyses of total solids (TS), volatile solids (VS), ash content of

biomass were carried out according to the standard methods(APHA/AWWA/WEF, 2005). Liquid samples were filtered througha Whatman No. 54 filter and the solid fractions were dried at37 �C for 48 h to reach a moisture content <10%. Solid sampleswere analyzed according to NREL to determine the carbohydrateand lignin (soluble and insoluble) content in both untreated andpretreated samples (Sluiter et al., 2011). The amount of total carbo-hydrates (sum of glucose, xylose and arabinose), acetate, solublelignin, insoluble lignin, and structural inorganics were analyzedafter the two-step hydrolysis. Soluble lignin was analyzed by

Table 1Compositional analysis of separated solid and liquid fractions of untreated andpretreated substrate before and after anaerobic digestion.

Untreated Pretreated

Feed Effluent* (day17)

Feed Effluent* (day75)

Solid fractionCarbohydrates (% of VS) 21.1 21.6 11.6 18.7Acetyl (% of VS) 0.0 0.3 1.3 0.0Soluble lignin (% of VS) 5.1 3.9 4.8 2.7Insoluble lignin (% ofVS)

73.8 74.2 82.2 78.6

Lignin:carbohydrate 3.74 3.61 7.49 4.34

Liquid fractionAcetic acid (g/L) 0.1 0.1 3.5 0.7Propionic acid (g/L) 0.2 0.2 1.7 1.1Isobutyric acid (g/L) 0.0 0.0 – 0.4Butyric acid (g/L) 0.1 0.1 1.4 0.5Isovaleric acid (g/L) 0.1 0.1 – 0.5Valeric acid (g/L) 0.1 0.1 – 0.5

* Insoluble solid fraction of the samples after filtration.

184 B.K. Ahring et al. / Bioresource Technology 175 (2015) 182–188

spectrophotometer (Jenway 6405 UV/Visible, NJ, USA) using awavelength of 320 nm within 6 h of the hydrolysis. Samples werequantified using HPLC equipped with refractive index and UV vis-ible detector, compounds were separated in an Aminex HPX-87Hcolumn (Bio-Rad, Hercules, USA) at 60 �C with 4 mM H2SO4 as aneluent with a flow rate of 0.6 mL/min.

2.4.2. Volatile fatty acids analysisSamples for volatile fatty acids (VFAs) analysis were taken at

least twice a week. Samples were always filtered (0:45 lm PTFEmembrane, Acrodisc� Syringe Filters, 13 mm, Pall� Life Sciences,USA) and diluted (25�) prior to VFAs analysis using Trace GC UltraGas Chromatographs (Thermo Scientific, West Palm Beach, FL)equipped with flame ionization detector (FID) and a Hewlett Pack-ard FFAP capillary column (30 m � 0.53 mm I.D., film thickness1.0 lm). Nitrogen was used as a carrier gas at 18 mL/min and theinjector port and detector temperature were 175 �C and 200 �C,respectively. The oven temperature was programmed from 115 �C(held for 3 min) to 125 �C at a rate of 5 �C/min and then increasing45 �C/min to 230 �C and held at final temperature for 2 min.

2.4.3. Solvent extraction and analysisTo investigate degradation compounds in pretreated feed sam-

ple compared to untreated feed, organic solvent extraction wasperformed followed by GC–MS on both feed and subsequent efflu-ent samples of the reactor. Extractible compounds in bothuntreated and pretreated (feed and effluent) samples wereextracted with equal amount of ethyl acetate (5 + 5 mL). The mix-ture was vortex mixed for 2 min followed by centrifugation at10,000�g for 10 min. The filtered supernatant was used for GC–MS analysis. Low molecular weight (LMW) lignin compounds(MW < 850) formed during the pretreatment were characterizedin GC–MS (7890A GC-system with 5975C inert XL E1/C1 MSDmodel # G3174A, Agilent Technologies, Wilmington, DE, USA)equipped with DB-5MS capillary column(30 m � 0.250 lm � 0.25 lm). The CTC analytics CombiPAL roboticarm (G6500-CTC-LHS2.PAL system-CH001210757, CTC AnalyticsAG, Zwingen, Switzerland) was connected to GC–MS for auto sam-pling system as previously described elsewhere (Ahamed andAhring, 2011). The samples were analyzed in splitless mode usingHelium as a carrier gas and the average velocity was maintained at42.651 cm/s. The oven temperature was programmed with an ini-tial temperature of 30 �C for 1 min and ramp of 8 �C/min to 278 �Cholding for 1 min. The injector temperature was maintained at240 �C throughout the entire chromatographic separation. Themass spectrometer was operated in the full scan mode between25 and 1000 amu, the ion source temperature was at 230 �C andthe actual electron multiplier voltage was 1388.23 V. Initial identi-fication of unknown compounds was made through library com-parison using the NIST database. One blank ethyl acetate vial alsoanalyzed and the compounds found therein were subtracted fromthose appearing in the extracted samples. Tentative identificationof acids and lignin products was based on observed mass spectraldata as compared to those in the NIST database. The analysis wasrepeated at least twice with comparable results. The observedpeaks were in the dynamic range of the instrument and there isno dead time loss in the peak intensity. Thirteen commercial stan-dards (Sigma–Aldrich, St. Louis, MO, USA) of target compoundsidentified in the samples such as butanoic acid, pentanoic acid,hexanoic acid, n-hexadecanoic acid, benzenepropanoic acid, 2-methyl butanoic acid, tetradecanoic acid, heptanoic acid, dodeca-noic acid, 2-propyl phenol, 2-furanmethanol, 2,6-dimethylphenylisocyanate and n-decanoic acid were combined and prepared inethyl acetate with the concentrations of 10, 20, 30, 40 and50 lg/mL (ppm) of each compound. Qualitative analysis of abovestandards followed the exact method as samples and analyzed in

GC–MS. Further, values obtained from blank (ethyl acetate) werealso subtracted from standards and the final values were expressedin mg/L.

3. Results and discussion

3.1. Substrate characteristics

Feedlot manure was characterized in terms of moisture content,organic matter and ash content with the values of 53.4%, 17.4%(DM basis) and 82.6% (DM basis), respectively. High ash contentin the feedstock indicates the presence of inorganics such as soil,sand, and dust were mixed with the feedlot manure during thewindrow turning operations as well as atmospheric deposition.The untreated and pretreated feeds for the reactor and effluentsafter anaerobic digestion were characterized as depicted in Table 1.

Approximately three-quarter of the organic matter in feedlotmanure was composed of lignin in which only 6.5% was accountedfor as acid-soluble lignin while the rest was acid-insoluble. Acid-insoluble lignin can be described as high molecular weight(HMW) lignin, often molecular weight >850. Under anaerobic con-dition, biodegradation of HMW lignin is prevented unless lignin isfractionated into small molecules which can be transported intomicrobial cells. This is due to the inabilities of anaerobic microbesto produce extracellular enzyme required for lignin depolymeriza-tion under oxygen free environment (Hatakka, 1994; Ko et al.,2009).

In the solid fraction of pretreated feed, the percentage of acid-insoluble lignin was found to be 82.2% (VS basis) compared to73.8% (VS basis) in untreated feed. Similarly, acid-soluble ligninin solid fraction of pretreated and untreated feed was found 4.8%and 5.1%, respectively. However, significant difference wasobserved in lignin–carbohydrate ratio in pretreated and untreatedfeed to be 7.49 and 3.74, respectively (Table 1). This may be due topartial solubilization of carbohydrates (cellulose and hemicellu-lose) and lignin during WEx pretreatment, leaving comparativelyhigher fraction of lignin in the solid fraction. After the anaerobicdigestion of pretreated feed, the lignin–carbohydrate ratio in thesolid fraction of the effluent decreased to 4.34 from the initial valueof 7.49: however, in case of untreated feed, the values remainedalmost unchanged (Table 1). This variation in composition of thesolid fraction found in the effluent from the reactor being fed withuntreated and pretreated feedlot manure shows that conversion oflignin is taking place in the anaerobic digester. Volatile fatty acids

B.K. Ahring et al. / Bioresource Technology 175 (2015) 182–188 185

are the intermediate products formed in anaerobic digestion pro-cess and are ultimately converted into CH4 and CO2 (Ahringet al., 1995; Strong and Gapes, 2012). Acetic acid, propionic acidand butyric acid were found in the liquid fraction of the pretreatedfeed at a concentration of 3.5, 1.7 and 1.4 g/L, respectively. Theconcentration of the acids was decreased in the effluent indicatingthat most of the acids formed due to pretreatment and the anaer-obic digestion process was utilized in the digester.

3.2. Continuous stirred tank reactor experiments

Methane yields and pH from the continuous stirred tank reactor(CSTR) operation are presented in Fig. 1. The experiment was car-ried out for 86 days with a hydraulic retention time of 10 days.Methane content found in biogas over the whole operation periodwas fairly consistent around 70%, regardless of the feed (pretreatedor untreated feed). During the start-up, the digester was fed withuntreated feedlot manure mixed with 2% corn steep liquor (w/w). Methane yield during the period (day 1–22) varied from 26to 104 L/kg-VS/Day with a mean value of 70 ± 27 L/kg-VS/Day.The pH values for these days were relatively stable in the rangeof 7.4–7.9. Average total VFAs concentration in the reactor duringthe period was below 826 mg/L. After day 23 the reactor wasswitched to pretreated feedlot manure mixed with 2% corn steepliquor (w/w) and the reactor responded quickly to the addition ofpretreated feed. Methane yield increased considerably by severalfold indicating readily biodegradable compounds in the pretreatedfeed were converted into methane. This supported our hypothesisthat significant hydrolysis occurred during the wet explosion pre-treatment of the recalcitrant substrate which is otherwise difficultto hydrolyze under anaerobic conditions. On day 45, the reactorreached steady state with a consistent methane yield of320 ± 34 L/kg-VS/Day and continued at this level for the rest ofthe experiment (Fig. 1). The methane yield obtained during theperiod (day 45–86) using WEx-treated feedlot manure are favor-ably comparable to previous studies where raw and/or separatedmanure were used in different process conditions with the aimof improving yields (Castrillón et al., 2011; Kaparaju et al., 2009;Møller et al., 2007).

While pH values in the reactor were found 7.6 ± 1 until the day53, a sudden decrease in pH was observed after day 54 as displayedin Fig. 1. Between the day 54 and day 65, the pH remained low inthe reactor with an average value of 6.9 ± 1, before the pH reversed

Met

hane

yiel

d(L

/kg-

VS/D

ay)

0

100

200

300

400

0 10 20 30 40Days

Methane yield (L/kg-V

Feeding with pretreated manure (Day 23)

Fig. 1. The effect of wet explosion pretreatment on process performance showing metha55 �C. Initially the reactor was fed with untreated feedlot manure (day 1–22), after whic86).

to the previous state. This could be explained by the accumulationof degradation products from the pretreated feed including organicacids, phenolics, and aromatic aldehydes. In anaerobic digestionprocess carbon polymers are hydrolyzed and fermented into inter-mediates such as volatile fatty acids (VFAs). Besides phenolics andaromatic aldehydes, acids can also be formed during WEx pretreat-ment of lignocellulosic biomass as hemicellulosic side chains, deg-radation products as well as depolymerization of lignin (Biswaset al., 2014; Klinke et al., 2002). Although a slight decrease in meth-ane yields were observed during the period (day 54–65), pH andmethane production of the reactor was recovered by the processitself. This indicates that the microbes were capable of assimilatingthe degradation products overtime. VFAs concentration in the reac-tor was monitored during the period until the reactor showedfairly stable performance. VFAs concentrations in the reactorbetween the day 56 and 80 are presented in Fig. 2. There was asmall variation observed in acetic acid and propionic acid concen-tration during the period. Highest concentration of acetic acid was810 mg/L on day 61 during the period with low pH. However, iso-butyric, butyric, isovaleric and valeric acid concentrationsremained stable with the values of 383 ± 12, 456 ± 54, and499 ± 18 and 481 ± 12 mg/L, respectively. On day 78, acetic acidand propionic acid concentrations increased in reactor with thevalues of 797 and 785 mg/L, before the concentrations decreasedin the following days (Fig. 2). This indicates rapid utilization ofaccumulated acids in the digester. During that period, a peak meth-ane yield of 418 L/kg-VS/Day was observed on day 78.

3.3. Biodegradability of substrate

Carbohydrates (mainly cellulose and hemicellulose) and lignincomposition of the solid fractions before and after the digestionwere measured for both raw and pretreated substrate to determinethe biodegradability of the recalcitrant fibers (Fig. 3). Volatile soliddestruction for the raw untreated substrate was found to be 11.9%compared to 40.3% for WEx pretreated substrates. This indicatesthat the wet explosion pretreatment improved the conversion ofthe raw material. It is noteworthy that major part of the lignin(44.4%) was converted when the reactor was fed with pretreatedsubstrate compared to (12.6%) for untreated feed (Fig. 3). Depoly-merization of lignin occurs under the pretreatment conditions(170 �C, 25 min and 4% O2) due to the breakage of the predominantbeta-aryl ether bonds between phenylpropanoid units liberating

7.0

7.5

8.0

8.5

9.0

pH

50 60 70 80 90

S/Day) pH

ne yield and pH during continuous anaerobic digestion of feedlot manure in CSTR ath the feedlot manure used was pretreated by wet explosion pretreatment (day 23–

Fig. 2. VFA concentrations during the period (day 56–80) while the reactor was fed with pretreated feedlot manure and reached steady state.

Fig. 3. Mass of the volatile solids with carbohydrate and lignin composition ofuntreated feed and effluent (day 17) and WEx pretreated feed and effluent (day 75).

186 B.K. Ahring et al. / Bioresource Technology 175 (2015) 182–188

phenolic and non-phenolic lignin fragments that can be accessibleand utilized as substrate by the microbes of interests (Yokoyamaand Matsumoto, 2010). Although lignin is known to be highlyrecalcitrant to microbes such as bacterial and fungi, several degra-dation pathways have been explored after lignin modification(Abdel-Hamid et al., 2013; Bugg et al., 2011). Jayasinghe et al.observed higher methane production rates in enzyme amendedsamples of lignin rich substrate due to the lignin depolymerizationby lignin peroxidase (LiP), manganese peroxidase (MnP) enzymes(Jayasinghe et al., 2011). Strong oxidants such as LiP with highredox potential are able to oxidize non-phenolic structures of lig-nin while oxidation of phenolic structures of lignin is catalyzedby MnP (Mn-dependent enzyme) (Bugg et al., 2011). A studyshowed microbes in anaerobic digester are able to utilize ligninoxidative products as a carbon source (Jayasinghe et al., 2011);however, higher resistance was found to high molecular or poly-meric lignin. Our study showed significant improved depolymer-ization of lignin as a result of pretreatment. It was hypothesizedthat during wet explosion pretreatment, biodegradable fraction-ates are formed by the oxidation of lignin which promotes micro-bial utilization of lignin, which can act as carbon source for theanaerobic culture.

Wet explosion pretreatment of feedlot manure creates lignindecomposition products of which some can be either beneficial

or detrimental for growth of anaerobic consortia. A list of com-pounds were detected in GC–MS analysis and identified as listedin Fig. 4. Among the cell wall decomposition products, aliphaticcompounds and phenolics were detected in the pretreated feedand effluent samples (Fig. 4A and B). While tested extracted sam-ples for untreated feed and effluent, none of these types of com-pounds was detected (Fig. 4C and D) indicating no originations ofsuch compounds in raw samples. It was observed that most ofthe aliphatic compounds formed in WEx treated feed samples(Fig. 4A) were not detected in the samples of digester effluent(Fig. 4B). This indicates that conversion of aliphatic compoundsoccurred in the digester which formed during the wet explosionpretreatment. Hence, it was observed that the aromatic phenoliccompounds retained in effluent samples, which had not been con-verted or utilized in this process. Although phenolics are reportedto inhibit the microbial degradation of biomass (Hernandez andEdyvean, 2008; Klinke et al., 2002), previous studies suggest thatphenolics can be converted into methane with some limitations(Barakat et al., 2012; Hernandez and Edyvean, 2008). Anaerobicconsortia tend to acclimatize aromatic substrate with an initiallag phase, although little is known about the mechanism andfavorable condition required to facilitate the lignin conversionprocess.

Further, simultaneous acclimatization to other selected aro-matic substrates might occur once microbial populations accli-mated to a particular aromatic substrate (Healy and Young,1979). The compounds which could be extracted by organic sol-vent extraction of pretreated feed and effluent are quantified anddisplayed in Fig. 5. The concentration of most compounds was sig-nificantly decreased in the effluent indicating microbial utilizationof the compounds. However, 2,6-dimethylphenyl isocyanate and 2-propyl phenol remain unutilized while other compounds such asbutanoic acid, pentanoic acid, hexanoic acid, n-hexadecanoic acid,benzenepropanoic acid, 2-methyl butanoic acid, tetradecanoicacid, heptanoic acid, dodecanoic acid, 2-furanmethanol, and n-decanoic acid were found in a significantly lower concentrationin the effluent samples of the reactor when fed with pretreatedfeed (Fig. 5). Among the aliphatic acids, butanoic acid was foundin highest concentration (3221 mg/L) in the pretreated feed sampleand the post digestion concentration was found to be 52 mg/L.From the relative abundance (Fig. 4A and B), it was observed thatthe phenolic compounds such as phenol, 4-methyl phenol, 4-ethylphenol and 2-propyl phenol were remained the in the digestereffluent samples. However, aliphatic acids formed during wet

Fig. 4. GC–MS analysis of the samples after organic solvent extraction; (A) WEx pretreated feed sample; (B) effluent from the digester fed with pretreated feed (day 75); (C)untreated feed sample and (D) effluent from the digester fed with untreated feed (day 17).

Fig. 5. Quantification of the GC–MS identified compounds in pretreated feed andeffluent samples (day 75).

B.K. Ahring et al. / Bioresource Technology 175 (2015) 182–188 187

explosion pretreatment of lignin rich biomass can be converted inanaerobic digestion process without any process disturbance.Besides, the anaerobic digestion process can be useful to obtainfairly purified aromatic compounds from biomass that might havefurther implication to develop lignin-based bioproducts.

4. Conclusions

This study unambiguously demonstrates that oxygen assistedwet-explosion pretreatment promotes lignin solubilization and

forms certain lignin degradation products that can be utilized bymicrobes in anaerobic digestion process, thus help improving theoverall degradability of feedlot manure. The pretreatment of feed-lot manure under elevated temperature of 170 �C for 25 min using4 bars O2 enhanced the lignin degradation up to 44.4% under ther-mophilic conditions in continuous anaerobic digestion processcompared to only 12.6% for untreated feedlot manure, resultingin 4.5 times higher methane yield compared to the untreatedsubstrate, suggesting enhanced conversion of lignin in anaerobicdigestion process.

Acknowledgements

The authors would like to thank the reviewers for their con-structive and very helpful comments. Further we thank Dr. KeithThomsen, for his help with substrate collection and communica-tion with the ranchers.

References

Abdel-Hamid, A.M., Solbiati, J.O., Cann, I.K.O., 2013. Insights into lignindegradation and its potential industrial applications. Adv. Appl. Microbiol. 82,1–28.

Ahamed, A., Ahring, B.K., 2011. Production of hydrocarbon compounds byendophytic fungi Gliocladium species grown on cellulose. Bioresour. Technol.102, 9718–9722.

Ahring, B.K., Munck, J., 2006. Method for Treating Biomass and Organic Waste withthe Purpose of Generating Desired Biologically Based Products. WO2006032282A1.

Ahring, B.K., Sandberg, M., Angelidaki, I., 1995. Volatile fatty acids as indicators ofprocess imbalance in anaerobic digesters. Appl. Microbiol. Biotechnol. 43, 559–565.

188 B.K. Ahring et al. / Bioresource Technology 175 (2015) 182–188

Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J., 2010. Pretreatmenttechnologies for an efficient bioethanol production process based onenzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861.

APHA/AWWA/WEF, 2005. Standard methods for the examination of water andwastewater. In: Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., Franson,M.A.H. (Eds.), American Public Health Association/American Water WorksAssociation/Water Environment Federation, Washington, DC.

Barakat, A., Monlau, F., Steyer, J.-P., Carrere, H., 2012. Effect of lignin-derived andfuran compounds found in lignocellulosic hydrolysates on biomethaneproduction. Bioresour. Technol. 104, 90–99.

Biswas, R., Ahring, B.K., Uellendahl, H., 2012. Improving biogas yields using aninnovative concept for conversion of the fiber fraction of manure. Water Sci.Technol. 66, 1751–1758.

Biswas, R., Uellendahl, H., Ahring, B.K., 2013. Conversion of C6 and C5 sugars inundetoxified wet exploded bagasse hydrolysates using Scheffersomyces (Pichia)stipitis CBS6054. AMB Express 3, 42.

Biswas, R., Uellendahl, H., Ahring, B.K., 2014. Wet explosion pretreatment ofsugarcane bagasse for enhanced enzymatic hydrolysis. Biomass Bioenergy 61,104–113.

Bugg, T.D.H., Ahmad, M., Hardiman, E.M., Rahmanpour, R., 2011. Pathways fordegradation of lignin in bacteria and fungi. Nat. Prod. Rep. 28, 1883–1896.

Carlsson, M., Lagerkvist, A., Morgan-Sagastume, F., 2012. The effects of substratepre-treatment on anaerobic digestion systems: a review. Waste Manag. 32,1634–1650.

Castrillón, L., Fernández-Nava, Y., Ormaechea, P., Marañón, E., 2011. Optimization ofbiogas production from cattle manure by pre-treatment with ultrasound andco-digestion with crude glycerin. Bioresour. Technol. 102, 7845–7849.

Chiaramonti, D., Prussi, M., Ferrero, S., Oriani, L., Ottonello, P., Torre, P., Cherchi, F.,2012. Review of pretreatment processes for lignocellulosic ethanol production,and development of an innovative method. Biomass Bioenergy 46, 25–35.

Hatakka, A., 1994. Lignin-modifying enzymes from selected white-rot fungi:production and role from in lignin degradation. FEMS Microbiol. Rev. 13,125–135.

Healy, J.B., Young, L.Y., 1979. Anaerobic biodegradation of eleven aromaticcompounds to methane. Appl. Environ. Microbiol. 38, 84–89.

Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility oflignocellulosic biomass. Bioresour. Technol. 100, 10–18.

Hernandez, J.E., Edyvean, R.G.J., 2008. Inhibition of biogas production andbiodegradability by substituted phenolic compounds in anaerobic sludge. J.Hazard. Mater. 160, 20–28.

Jayasinghe, P.A., Hettiaratchi, J.P., Mehrotra, A.K., Kumar, S., 2011. Effect of enzymeadditions on methane production and lignin degradation of landfilled sample ofmunicipal solid waste. Bioresour. Technol. 102, 4633–4637.

Kaparaju, P., Ellegaard, L., Angelidaki, I., 2009. Optimisation of biogas productionfrom manure through serial digestion: lab-scale and pilot-scale studies.Bioresour. Technol. 100, 701–709.

Klinke, H.B., Ahring, B.K., Schmidt, A.S., Thomsen, A.B., 2002. Characterization ofdegradation products from alkaline wet oxidation of wheat straw. Bioresour.Technol. 82, 15–26.

Ko, J.-J., Shimizu, Y., Ikeda, K., Kim, S.-K., Park, C.-H., Matsui, S., 2009. Biodegradationof high molecular weight lignin under sulfate reducing conditions: lignindegradability and degradation by-products. Bioresour. Technol. 100, 1622–1627.

Li, C., Salas, W., Zhang, R., Krauter, C., Rotz, A., Mitloehner, F., 2012. Manure-DNDC: abiogeochemical process model for quantifying greenhouse gas and ammoniaemissions from livestock manure systems. Nutr. Cycling Agroecosyst. 93, 163–200.

Møller, H.B., Nielsen, A.M., Nakakubo, R., Olsen, H.J., 2007. Process performance ofbiogas digesters incorporating pre-separated manure. Livestock Sci. 112, 217–223.

Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M.,2005. Features of promising technologies for pretreatment of lignocellulosicbiomass. Bioresour. Technol. 96, 673–686.

Rana, D., Rana, V., Ahring, B.K., 2012. Producing high sugar concentrations fromloblolly pine using wet explosion pretreatment. Bioresour. Technol. 121, 61–67.

Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2011.Determination of structural carbohydrates and lignin in biomass. In: LaboratoryAnalytical Procedure (LAP), National Renewable Energy Laboratory, Golden,Colorado, p. 18.

Strong, P.J., Gapes, D.J., 2012. Thermal and thermo-chemical pre-treatment of fourwaste residues and the effect on acetic acid production and methane synthesis.Waste Manag. 32, 1669–1677.

Vaddella, V.K., Ndegwa, P.M., Ullman, J.L., Jiang, A., 2013. Mass transfer coefficientsof ammonia for liquid dairy manure. Atmos. Environ. 66, 107–113.

Wenzel, H., Thyø, K.A., 2007. Life Cycle Assessment of Biogas from Maize Silage andfrom Manure. Institute for Product Development, Lyngby, Denmark.

Yokoyama, T., Matsumoto, Y., 2010. Revisiting the mechanism of b-O-4 bondcleavage during acidolysis of lignin. Part 2: detailed reaction mechanism of anon-phenolic C6-C2 type model compound. J. Wood Chem. Technol. 30, 269–282.