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2010 28: 800 originally published online 10 February 2010Waste Manag ResLin Yunqin, Wang Dehan and Wang Lishang

sludgeBiological pretreatment enhances biogas production in the anaerobic digestion of pulp and paper

  

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ISSN 0734–242XWaste Management & Research

2010: 28: 800–810DOI: 10.1177/0734242X09358734

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Biological pretreatment enhances biogas production in the anaerobic digestion of pulp and paper sludgeLin Yunqin, Wang Dehan, Wang LishangCollege of Natural Resources and Environment, South China Agricultural University, Guangdong Guangzhou, China

High efficient resource recovery from pulp and paper sludge (PPS) has been the focus of attention. The objective of this researchwas to develop a bio-pretreatment process prior to anaerobic digestion of PPS to improve the methane productivity. Active andinactive mushroom compost extracts (MCE) were used for pretreating PPS, followed by anaerobic digestion with monosodiumglutamate waste liquor (MGWL). Laboratory-scale experiments were carried out in completely mixed bioreactors, 1-L capacity with700 ml useful capacity. Optimal amount of active MCE for organics’ solubilization in the step of pretreatment was 250 A.U./gVSsludge.Under this condition, the PPS floc structure was well disrupted, resulting in void rate and fibre size diminishment after pre-treatment. In addition, SCOD and VS removal were found to be 56% and 43.6%, respectively, after anaerobic digestion, beingthe peak value of VFA concentration determined as 1198 mg acetic acid L–1. The anaerobic digestion efficiency of PPS with andwithout pretreatment was evaluated. The highest methane yield under optimal pretreatment conditions was 0.23 m3 CH4/kgVSadd,being 134.2% of the control. The results indicated that MCE bio-pretreatment could be a cost-effective and environmentallysound method for producing methane from PPS.

Keywords: pulp and paper sludge, anaerobic digestion, methane production, biological pretreatment, mushroom compost

AbbreviationsPPS pulp and paper sludge; TN total nitrogen; MCE mushroom compost extract; SCOD soluble chemical oxygen demand; MGWL monosodium glutamate waste liquor; VFA volatile fatty acid; MC mushroom compost; SV sludge settling ratio; A.U. activity unit; VSS volatile suspended solids; MnPs manganese peroxidases; TS total solids; CMCase carboxymethyl cellulose; VS volatile solids; ABTS 2,2’-azinobis-3-ethylbenthiazoline-6-sulphonate; OC organic carbon; DNS dinitrosalicylic acid; WAS waste-activated sludge; CMC carboxymethylcellulose;

MAS municipal activated sludge; BCTMP bleaching chemi-thermo-mechanical pulp; OFMSW organic fraction of municipal solid waste

IntroductionAnaerobic digestion of solid organic waste has gained increasedattention as a means of producing energy-rich biogas, destroy-ing pathogenic organisms and reducing problems associatedwith the disposal of organic waste. Anaerobic digestion is amultistage process including hydrolysis, acidogenesis, acetogen-esis and methanogenesis (Metcalf & Eddy 1991, Reynolds &Richards 1995). The hydrolysis step degrades both insolubleorganic material and high molecular weight compounds suchas lipids, polysaccharides, proteins and nucleic acids, into sol-uble organic substances (e.g. amino acids and fatty acids). Thecomponents formed during hydrolysis are further split duringacidogenesis, the second step. Volatile fatty acids (VFAs) areproduced by acidogenic (or fermentative) bacteria along withammonia (NH3), CO2, H2S and other by-products. The third

Corresponding author: Lin Yunqin, College of Natural Resources and Environment, South China Agricultural University, Guangdong Guangzhou510642, China.E-mail: yqlin@scau.edu.cnReceived 11 August 2009, accepted in revised form 30 November 2009Figure 1 appears in color online: http://wmr.sagepub.com

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stage in anaerobic digestion is acetogenesis, where the higherorganic acids and alcohols produced by acidogenesis are fur-ther digested by acetogens to produce mainly acetic acid aswell as CO2 and H2. This conversion is controlled to a largeextent by the partial pressure of H2 in the mixture. The finalstage (methanogenesis) produces methane by two groups ofmethanogenic bacteria: the first group splits acetate intomethane and carbon dioxide and the second group useshydrogen as electron donor and carbon dioxide as acceptor toproduce methane.

However, the application of anaerobic digestion to biosol-ids is often limited by very long retention times (20–30 days)and a low overall degradation efficiency of the organic drysolids (30–50%). Those limiting factors are generally associ-ated with the hydrolysis stage (Tiehm et al. 2001). Duringhydrolysis, cell walls are ruptured and extracellular polymericsubstances are degraded, resulting in the release of readilyavailable organic material for the acidogenic micro-organisms.This mechanism is particularly important in the digestion ofsludge, since the major constituent of its organic fraction arecells – a relatively unfavourable substrate for microbial degra-dation (Weemaes & Verstraete 1998, Graef & Andrews 1974).The cell envelope of micro-organisms is a semi-rigid structurewhich provides sufficient intrinsic strength to protect the cellfrom osmotic lysis. Microbial cell walls contain glycan strandscross-linked by peptide chains, causing resistance to biodegra-dation. Several authors (Elefsiniotis & Oldham 1994, Ferrer etal. 2006, Giraldo-Gomez 1990) have indeed identified hydrol-ysis as the rate-limiting step in anaerobic digestion of sludge.

Various sludge disintegration methods have hence beenstudied as a pre-treatment to avoid the limiting step. Thesepre-treatment methods that achieve a significant result in alysis or disintegration of sludge cell have the potential toenhance biogas production. Several methods have been studiedin the literature including thermal (Gavala et al. 2003, Bougrieret al. 2007, Roberts et al. 1999, Watts et al. 2005), chemical(Stephenson et al. 2003), ultrasonic (Dewil et al. 2006), andmechanical processes (Muller et al. 2003). In addition, bio-logical-microbial enzymes or bacteria (Miah et al. 2004, Guel-lil et al. 2001, Mayhew et al. 2002, Davidsson et al. 2007, Bol-zonella et al. 2007) and the combination of such processes hasbeen also used (Chulhwan et al. 2005). Among all pretreat-ment technologies, biological pretreatment has shown a greatimprovement in biogas production. Davidsson (2007) showedthat using prepared enzyme solutions in a pre-hydrolysis con-tact chamber with an HRT of 4 h increased methane produc-tion by 60% in subsequent anaerobic digesters during pilot-scale trials. Anthony (2005) found that 9 h of hydrolyticenzyme pretreatment of sisal pulp prior to anaerobic diges-tion produced a 26% higher methane yield compared to thecontrol treatment.

Pretreatments are thought to disintegrate the floc structureof sludge and extract both intracellular (within the microbialcell) and extracellular (within the polymeric network) mate-rials before sludge is sent to the digesters. In most of thestudies, pretreatments solubilize the waste activated sludge

(WAS), with a subsequently improvement of the anaerobicdigestion yield. While anaerobic digestion is commonly prac-ticed in the municipal sector, it has only little popularity inthe pulp and paper industry. To the best of our knowledge,there is no full-scale anaerobic digestion facility in the pulpand paper sector for the digestion of solid residues (Elliott &Mahnood 2007).

In the late 1980s and early 1990s, several investigationswere conducted to explore the use of anaerobic digestiontreating pulp and paper solid residues (Kowalczyk & Martyne-lis 1989, Poggi et al. 1997, Puhakka et al. 1988). The studieswere performed on both laboratory and pilot-scale systems.The results of these studies generally showed that anaerobicdigestion of pulp and paper biosolids could reduce the mass ofsolid waste by 30–70%, with the benefit of methane produc-tion. Otherwise, due to the large amount of slow biodegrada-ble organics (e.g. lignin) in pulp and paper sludge (PPS) andits high residence time (20–30 days), high operational andinvestment capital costs appeared to be the reason for the lackof anaerobic digesters in the pulp and paper mill industry. Anew technological advance that may reduce the requiredretention time is the development of pretreatment processes.Feasibilities of most of these pretreatment technologies havebeen demonstrated using municipal activated sludge (MAS).As PPS contains proteins (22–52%), lignin (20–58%), carbo-hydrates (0–23%), lipids (2–10%), and cellulose (2–8%) (Kyl-lonen et al. 1998), biological treatment of PPS is graduallybecoming the main management alternative instead of land-filling and incineration (Jokela et al. 1997). In addition,compared to MAS, PPS contains a higher volatile fractionwhich could make it more amenable to pretreatment tech-nologies.

In the pulp and paper industry, mechanical and chemicaltreatments are generally applied in the production process(Wu 2001). They act on the original material and destroypart of the molecular and cell structure. Lignin degraders innature are mainly white-rot fungi (Blanchette 1995), whichdegrade lignin by means of oxidative enzymes, like manganeseperoxidases (MnPs) and laccase (Hatakka 1994). Hence, bio-logical pretreatment is more suitable for PPS to enhancebiogas production before anaerobic digestion. However, fewstudies have reported on anaerobic digestion of PPS (Camp-bell et al. 1991, Puhakka et al. 1992) and there is practicallyno literature on its biological pretreatment.

Mushroom production is the biggest solid-state fermenta-tion industry in the world (Moore & Chiu 2001). Corre-spondingly, 5 kg of mushroom compost is generated from theproduction of 1 kg of mushrooms (Semple et al. 2001). Highlevels of residual nutrients and enzymes are still left in mush-room compost (Eggen 1999, Semple et al. 2001), e.g. car-boxymethyl cellulose (CMCase), which represent the singleendoglucanase (1,4-β-D-glucan glucanohydrolases), mainlyused in cellulose degradation (Xiaohong, et al. 2004). Lac-cases may be also present in the compost, which are multi-copper oxidases that catalyze the one-electron oxidation ofseveral aromatic substrates with the simultaneous reduction

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of oxygen to two molecules of water (Karla et al. 2009). Thus,exploitation to recycle this waste material is carried out totreat lignocellulose-contaminated samples (Andrew et al.1995’ Tuomela et al. 2000).

The objective of this study was to evaluate the biogas pro-duction yield of a pretreated PPS (with mushroom compost)prior to the anaerobic digestion. This pretreated PPS was com-pared with untreated PPS (the check [CK]). Biogas productiv-ity, organic removal and reactor stability were examined.

Materials and MethodsMaterial collectionPPS samples were collected from the secondary clarifiers(normally settling tanks) of the Guangzhou Pulp & PaperPlant in China. There are two processes in this plant: one isbleaching chemi-thermo-mechanical pulp (BCTMP) madefrom Masson pine; the other is papermaking from wastepaper after de-inking. Waste water arises from three sections– pulping, papermaking and de-inking – and is usually dewa-tered to 60–70% moisture content at the end process ofwaste water treatment.

Seed sludge was obtained from the sewage tank (near Build-ing 4 at the South China Agricultural University, Guangzhou,China) and was acclimatized with PPS in the laboratory forabout 3 months. The substrate was activated sludge of 10%TS after acclimatization. In order to get the optimal C/Nratio, monosodium glutamate waste liquor (MGWL) wasapplied, which was collected from Ao-Sang MonosodiumGlutamate Factory (Guangzhou, China). For biological pre-treatment of PPS, the mushroom (Pleurotus ostreatus)compost, which can utilize a very broad spectrum of wastesubstrates (Chiu et al. 2000)), was obtained from Tai-HeMushroom Farm (Guangzhou, China) and its water extractwas used to pretreat PPS. Mushroom compost extract (MCE)was placed at the ratio of fresh mushroom compost to dis-tilled water of 1:4 in an orbital shaker (150 min–1, at ambienttemperature) for 5 h and then centrifuged (5000 rpm, atambient temperature) for 30 min. All kinds of samples werecollected prior to each experiment, stored in the refrigerator(0–4˚C) and analyzed for total solids (TS), volatile solids (VS),organic carbon (OC), total nitrogen (TN) and pH according tothe Standard Methods for the Examination of Water andWastewater (APHA 2005). The enzyme activity of MCE wasalso measured and expressed as CMCase and laccase activi-

ties (Yang & Ma 2006). Laccase activity was measured by amodified protocol with 2,2’-azinobis-3-ethylbenthiazoline-6-sulphonate (ABTS) substrate (Hofrichter et al. 1999, Galvezet al. 2000), and CMCase activity was measured by the dinit-rosalicylic acid (DNS) method with carboxymethylcellulose(CMC) as substrate (Miller 1959).

Experimental procedureThe first approach was the biological pretreatment of PPS.In this step, PPS was divided into six portions of identicalweight (61 g). The first portion, the control sludge, wasreturned to the refrigerator for storage. Portions 2–4 were allsolubilized by adding different doses of active MCE at theCMCase activity of 50 A.U./g VSsludge, 125 A.U./gVSsludge and250 A.U./gVSsludge, respectively. The fifth and sixth portionswere solubilized by adding inactive MCE (sterilized at 121˚Cfor 30 min) at the equivalent enzyme activity of 125 A.U./gVSsludge and 250 A.U./gVSsludge, which were compared totreatments with active MCE. The MCE dosages to each por-tion were 8 ml (active), 15 ml (active), 30 ml (active), 15 ml(inactive) and 30 ml (inactive), respectively, used at a freshmushroom compost to distilled water ratio of 1:4. All MCEswere diluted to 122 ml in order to soak PPS fully during pre-treatment. The solubilization was carried out in 1000-mlErlenmeyer flasks with a working volume of 700 ml at 37˚Cfor 4 h. Each reactor was kept under aerobic conditions andstirred to ensure sufficient dispersal of the added MCE.After pretreatment, the sludge for the control treatment wastaken out from the refrigerator and all portions were readyfor the anaerobic digestion stage.

The second part was a batch anaerobic digestion experi-ment with pretreated PPS, MGWL and seed sludge. Thechemical characterization of feedstock is presented in Table 1.The dosage of MCE and other waste are shown in Table 2. Allbioreactors were filled with the same weight of feedstock atthe amount of 700 g, including distilled water which wasadded at the end to keep the total amount up to 700 g. Thefeedstock for bioreactor A with no MCE was the control(CK). Anaerobic digestion of all six bioreactors were startedat the same time. The initial fermentation condition of anaer-obic digestion experiment was C/N = 20, TS = 3%, inocu-lum ratio = 10% of TSadded according to an earlier feasibilitystudy (Lin et al. 2009). The temperature of anaerobic diges-tion was always maintained at 37˚C. Methane yield, alkalin-

Table 1: General characterization of the different wastes used in the anaerobic test.

TS(%)

VS(% of TS)

pHOC

(%,b.d.w.a)TN

(%,b.d.w.)C/N ratio

CMCase activity (A.Ub/ml)

Laccases activity (A.U/ml)

Pulp and paper sludge 31.45 62.3 7.82 32.75 1.09 30.05 – –

Mushroom compost 23.71 – 5.86 – – – 57.19 10.07

Monosodium glutamate waste liquor

43.00 68.5 5.36 29.5 11.83 2.49 – –

Sludge inoculum 9.17 53.2 7.85 26.70 0.71 37.61 – –a b.d.w., based on dry weight.b Activity unit, 1 activity unit (A.U.) is the amount of enzyme that catalyses the reaction of 1 mmol of substrate per minute.

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ity, total and volatile solids (TS and VS), soluble COD(SCOD), pH and VFAs were measured during the period ofanaerobic digestion.

Anaerobic digestion experimental set-upTwelve, laboratory-scale, single-stage digesters were employed.Each bioreactor (used in pretreatment) had a gas-tight rub-ber stopper with an outlet equipped for methane collectionand was flushed with N2 for 5 min to replace the air (oxygen).The bioreactors were maintained at 37˚C in a water bath andshaken by hand several times per day to ensure sufficientmixing to prevent feedstock settling. The volume of methanegenerated in each bioreactor was measured by means of acylinder, which was connected to the bioreactor. To removethe produced CO2, NH3 and H2O, an absorption flask withCa(OH)2 powder and a collecting gas bottle with 3% NaOHsolution were placed between the reactor and the cylinder (Fig-ure 1). The methane produced displaced a measurable volumeof water from the collecting gas bottle, which was equivalent tothe methane volume (You et al. 2003). All experiments wereperformed in duplicate for 42 days.

Experimental analysesThe routine parameters were analyzed twice a week and allanalyses were done in triplicate. TS, VS, pH, SCOD and alka-

linity were determined according to standard methods (APHA2005), while SCOD was measured by the potassium dichro-mate method. VFAs were analyzed by a distillation-titrationmethod and the results expressed as acetic acid concentra-tion (He 1998). The sludge settling ratio (SV) was measuredby the sludge still-setting method (Chen et al. 2003). The con-tents of cellulose, hemicellulose and lignin were measured bythe modified method of Wang (1987).

Results and DiscussionPretreatment effect on chemical characteristics of PPSTable 3 shows the chemical characterization of PPS after bio-pretreatment. As organic matter in PPS was hydrolyzed duringpretreatment, the increase of SCOD from 1664 mg L–1 to 3811mg L–1 was noticed when MCE concentration was increased to250 A.U./gVSsludge (Table 2). Under this condition, a destruc-tion of sludge flocs was observed (Figure 2). On the otherhand, the concentration of volatile suspended solids (VSS)experienced a slight decrease between 13.0–16.1% after pre-treatment, which may be due to the progressive hydrolysis ofthe complex organic matter present in the feed. This resultwas consistent with the change in SCOD. Pumping filtrationwas needed to separate soluble substances away from thesludge before VSS was measured by drying samples in anoven until there was no further water in the sludge. Through-

Table 2: Composition of the feedstock used to pretreat PPS and fed to the bioreactors.

BioreactorA.U.

/gVSsludge

Pulp and paper sludgeg

MCEa

mlMGWLb

gSludge inoculum

gDistilled waterc

ml

A 0 61 0 2 23 122 + 492

B 50 61 8 2 23 114 + 492

C 125 61 15 2 23 107 + 492

D 250 61 30 2 23 92 + 492

E 125 61 15 2 23 107 + 492

F 250 61 30 2 23 92 + 492a MCE, mushroom compost extract.b MGWL, monosodium glutamate waste liquor.c Distilled water was divided into two parts (connection with ‘ + ’); the first part was used to dilute MCE to 122 ml for PPS pretreatment and the latter part was added to each bioreactor to dilute total solids to 3% for PPS anaerobic digestion.

Fig. 1: The set-up of anaerobic digestion process.

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out the assays, the addition of MCE caused an increase inNH3-N of 36–42%, which may be produced by proteindecomposition. Such behaviour was also noticed by otherauthors (Nah et al. 1999). Alkalinity in the system is a result

of the presence of hydroxides, carbonates and bicarbonatesof elements such as calcium, magnesium or ammonia (Met-calf & Eddy 1991). Alkalinity helps to resist changes in pHcaused by the addition of acids. In the pretreatment experi-

Fig. 2: Microphotograph of PPS before (A) and after (B–F) pretreatment (A–F: Bioreactor A–Bioreactor F).

Table 3: Chemical characterization of PPS after pretreatment.

Bioreactor A B C D E F

Alkalinity (mg CaCO3·l–1) 504.2 ± 35.7 542.0 ± 17.8 579.8 ± 35.7 642.8 ± 17.8 516.8 ± 17.8 579.8 ± 35.7

SCOD (mg·l–1) 1664.0 ± 236.5 2254.8 ± 155.2 2328.6 ± 348.4 3810.7 ± 217.7 2015.8 ± 547.7 2874.9 ± 197.1

VSS (% of TSS) 39.9 ± 1.5 34.2 ± 1.3 34.7 ± 1.4 33.5 ± 1.1 34.0 ± 1.2 33.8 ± 1.6

NH3-N (g·l–1) 0.9 ± 0.3 1.7 ± 0.3 1.2 ± 0.3 1.2 ± 0.7 1.2 ± 0.4 1.2 ± 0.6

Svave (%) 12.5 ± 0.1 19.8 ± 0.1 18.5 ± 0.3 18.0 ± 0.3 18.5 ± 0.2 16.5 ± 0.3

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ment, alkalinity in bioreactors with pretreated PPS was foundto be higher than that of CK, in the range of 2.5–27.5%. Pre-treatment helped to solubilize carbonates and phosphates,provoking an alkalinity increment (Turovskiy & Mathai 2006).In addition, the increase of NH3-N also led to an increase ofalkalinity. Higher alkalinity affords higher buffer capacity toavoid the decrease of pH (Ren & Wang 2004).

The settling performance of sludge was expressed as SV.The SV of bioreactors with MCE were between 32–58%higher than CK, indicating that swelling happened to PPSafter pretreatment and surface area of complex organic mat-ter increased making PPS more susceptible to enzymatic attackby micro-organisms. In addition, the supernatant after pre-treatment was more turbid than that before pretreatment(not shown), indicating that colloids and dissolved solidsincreased in PPS after pretreatment, remaining in the super-natant, not being removed by settling.

Pretreatment effect on floc structure of PPSFigure 2 indicates that PPS floc structure was disrupted andthe crude fibre shortened after pretreatment. In addition, thevoid ratio of sludge and the size of the fine fibre decreasedwith the increase of active MCE dosage; the amount of finefibre was very low in PPS after pretreatment. This resultshows that most of the macromolecules present in PPS weredegraded into monomers (proteins and carbohydrates) byhydrolytic enzymes, facilitating transportation through thecell membrane and being taken up by micro-organisms. Sim-ilar reports in the literature were given by other authors(Azize 2005, Dey et al. 2006, Watson et al. 2004). In addition,the floc structures of bioreactors E and F attained morevoids than those of bioreactors C and D, because ofenzyme de-activation of MCE in the two latter reactors(see Figure 2). The sludge of these two bioreactors (E and F)attained less voids compared to CK, possibly due to the pHand carbon and nitrogen content change by inactive MCEapplication, which promoted the hydrolysis of macromole-cules in PPS.

Anaerobic digestionMethane yield

Bioreactors fed with pretreated PPS presented higher meth-ane yield, and the methane yield increased as the dosage ofMCE increased (Figure 3). The fractional increase of meth-ane yield in bioreactors B, C and D was 12%, 13% and 34%over the control treatment (bioreactor A), respectively. Fur-thermore, the bioreactor with active MCE produced moremethane than that with inactive MCE. The reason was thatpretreated sludge with active MCE hydrolyzed more organicmatter into the solution, so that anaerobic micro-organismswere able to use it immediately. However, the fractional incre-ment of methane in bioreactors E and F was only about 8%compared to CK (bioreactor A), mainly due to the de-acti-vated MCE. Nevertheless, their methane yield was still higherthan that of CK, as inactive MCE was also used as substrate.Similar results were reported by Rintala (1994). Moreover,the increment of methane production rate in bioreactor Ewas higher than that of bioreactor F. The reason may be that ahigher addition of MCE caused a disequilibrium in C:N:P ratiofor methanogenic bacteria. Additionally, the methane produc-tion rate in each bioreactor with pretreated PPS reached themaximum on day 11, in the range of 90–102 ml day–1, com-pared to the peak value on day 19 with 80 ml day–1 for thecontrol treatment. This result clearly indicates that bio-pre-treatment by MCE could increase methane yield and accel-erate the methane yield peak during anaerobic digestion.

The peak value of methane yield was 0.23 m3/kgVSadd atthe standard temperature and pressure in bioreactor D,which was higher than that from PPS and organic fraction ofmunicipal solid waste (OFMSW) studied by other authors(Jokela et al. 1997, López Torres et al. 2008), but it was lowerthan that produced from pretreated waste activated sludge(WAS) (Table 4). On the one hand, the organic carbon (OC)of PPS is mainly present in cellulose, hemicellulose andlignin (Table 5), which are macromolecules and have a com-plex structure. OC in PPS is more difficult to degrade thanthat in WAS. On the other hand, taking economics into con-

Fig. 3: Cumulative methane production (STP) for each bioreactor with PPS pretreated by different amounts of MCE. Filled diamonds, CK; filledtriangles, 50 A.U./gVSsludge (active); crosses, 125 A.U./gVSsludge (active); asterisks, 250 A.U./gVSsludge (active); filled circles, 125 A.U./gVSsludge

(inactive); + , 250 A.U./gVSsludge (inactive). STP, standard conditions of temperature and pressure.

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sideration, the pretreatment alternative studied in this workshows positive results, being affordable from the economicpoint of view, considering the low cost of MCE, which isindeed another kind of solid waste produced in mushroomfarmland.

Organics removal

The bioreactors presented various rates of substrate removalin terms of SCOD, VS, and cellulose, hemicellulose andlignin content. The bioreactors fed with pretreated sludgeshowed a faster substrate removal rate. As for SCOD removal,the results showed the following removal efficiencies: biore-actor A, 55%; bioreactor B, 67%; bioreactor C, 56%; biore-actor D, 60%; bioreactor E, 69%; and bioreactor F, 71%,respectively. Pretreatment offered the advantage of obtain-ing a higher removal yield of organics, especially for bioreac-tors E and F.

As shown in Figure 4, SCOD of each bioreactor first roseand then decreased, and they all reached their peak valueson the 15th day. During the first 15 days of anaerobic diges-tion, the insoluble organic fraction of PPS was largely hydro-lyzed and solubilized by acidogenic bacteria, causing thegreat increase of SCOD. A similar result has been alreadyreported by others (Wang et al. 2005, Yu et al. 2002).

As for VS removal efficiency, it moved in the followingrange: bioreactor A, 30%; bioreactor B, 33%; bioreactor C,37%; bioreactor D, 44%; bioreactor E, 36%; and bioreactorF, 40%, respectively (Table 5). Pretreatment offered littleimprovement in VS removal efficiency. The greatest VSremoval efficiency happened in bioreactor D, because moreactive enzymes (active MCE) were present to break theorganic structures.

SCOD removal in this study was generally higher thanthose of other studies, but VS removal was a little lower

Table 4: Comparison of the methane yield under different conditions.

Tiehm et al. (2001)

Chulhwan et al. (2005)

Chulhwan et al. (2005)

Lopez Torres et al. (2008)

This study

Waste WAS WAS WAS OFMSW PPS

Pretreatment Ultrasonica Biologicalb Thermochemicalc Chemicald Biologicale

CH4 yield (m3 CH4/kg VSadd) 0.30 0.29 0.52 0.15 0.23a Waste activated sludge (WAS), ultrasonic disintegration.b Waste activated sludge (WAS), aerobic bacteria and acidogenic process with selected bacteria.c Waste activated sludge (WAS), thermal treatment with NaOH.d Organic fraction of municipal solid waste (OFMSW), chemical hydrolysis with Ca(OH)2.e Pulp and paper sludge (PPS), biological treatment with mushroom compost.

Table 5: Percentage of VS, cellulose, hemicellulose and lignin in PPS before and after anaerobic digestion (% of TS).

BioreactorBefore anaerobic

digestion

After anaerobic digestion

A B C D E F

VS 62.9 ± 1.1 44.0 ± 0.8 42.2 ± 0.8 39.6 ± 0.8 35.2 ± 0.6 40.3 ± 0.9 37.8 ± 0.6

Cellulose 23.4 ± 0.9 18.0 ± 0.7 14.0 ± 0.6 13.4 ± 0.6 9.2 ± 0.5 13.5 ± 0.8 11.3 ± 0.6

Hemicellulose 8.6 ± 0.9 17.0 ± 1.0 16.9 ± 1.0 17.1 ± 0.9 14.2 ± 0.9 14.0 ± 0.9 14.3 ± 1.1

Lignin 16.5 ± 1.8 15.8 ± 1.6 14.8 ± 1.5 15.6 ± 1.6 14.4 ± 1.4 15.5 ± 1.6 14.9 ± 1.5

Fig. 4: SCOD changes in effluent for each bioreactor with PPS pretreated by different amount of MCE. Filled diamonds, CK; filled triangles, 50A.U./gVSsludge (active); crosses, 125 A.U./gVSsludge (active); asterisks, 250 A.U./gVSsludge (active); filled circles, 125 A.U./gVSsludge (inactive); + , 250A.U./gVSsludge (inactive).

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compared to other authors (Table 6). These results may beattributed to different materials and different pretreatmentprocesses. Regarding this study, PPS was pretreated by bio-solid waste MC (mushroom compost) and anaerobic diges-tion technology could re-use three kinds of solid wastes(PPS, MC and MGWL) at the same time with a very lowcost.

Additionally, the cellulose content of PPS in each bioreac-tor decreased after anaerobic digestion (Table 5). The degra-dation efficiencies of cellulose were: 22.9% (bioreactor A),40.0% (bioreactor B), 42.7% (bioreactor C), 60.4% (bioreac-tor D), 42.4% (bioreactor E), and 51.7% (bioreactor F),respectively, increasing with the increased active MCEamount to pre-treat PPS. The results show that adding MCEto pretreat PPS would be favourable to degrade celluloseduring anaerobic digestion, as cellulose was contained inactive MCE (Table 1) and nutrients were contained in inac-tive MCE for micro-organism growth (Rintala et al. 1994).The degradation effect was higher with active MCE com-pared to inactive MCE due to the de-activation of hydrolasesand micro-organisms after sterilization. However, there wasonly a small difference in lignin content in PPS after anaero-bic digestion among each bioreactor, and the lignin contentof the original PPS only varied by a small amount afteranaerobic digestion. This fact indicates that it was difficult todecompose lignin even though MCE was applied. Otherwise,the hemicellulose content of PPS in each bioreactor increasedafter anaerobic digestion, mainly due to the synthesis ofsmaller molecules (e.g. D-glucose, glucomannose) produced

in the hydrolysis step; the same result was obtained previ-ously by others (Lin et al. 2009).

VFA concentration

As shown in Figures 4 and 5, the VFA concentration in eachbioreactor followed the same trend as SCOD, increasing tothe peak value due to the degradation of organics by acidog-enic bacteria and then decreasing to the minimum due tomethane production by methanogenic bacteria (Dearman &Bentham 2006). For this experiment, VFA concentration var-ied in the range of 380–1200 mg L–1. The peak value of VFAconcentration in each bioreactor was almost obtained on the8th day, which indicates that the start-up of anaerobic diges-tion was much faster. The VFA concentration in each bioreac-tor showed the following trend: D > C > B > E > F > A,indicating that PPS pretreated with MCE would be easier todegrade by acetogenic bacteria, as a result of the pretreat-ment effect for producing VFA in anaerobic digestion withactive MCE being more efficient than that with inactiveMCE. The VFA concentration orders were similar to themethane production sequence (Figure 3), indicating that therewas no VFA accumulation in bioreactor C during anaerobicdigestion, and the peak value of VFA concentration in biore-actor C was not inhibitory for methane generation.

pH values and alkalinity

Fermentative micro-organisms can function in a wider range ofpH between 4.0 and 8.5 (Hwang et al. 2004). pH values in thisexperiment remained in the range 7.4–8.6 during anaerobic

Table 6: Removal of organics under different pretreatment methods.

Reference Pre-treatment Comments during anaerobic digestion

Kim (2003) Thermal Increase of VS reduction by 30%

Baier (1997) Mechanical Increase of 19% in VS degradation

Tanaka (2002) Alkaline/NaOH 60% increase of overall SS reduction

Weemaes (2000) Ozonation Increase of COD degradation up to 64%

Neis (2000) Ultrasonic Improved VS destruction ranged from 40% to 55%

Fig. 5: VFA changes in effluent for each bioreactor with PPS pretreated by different amount of MCE. Filled diamonds, CK; filled triangles, 50A.U./gVSsludge (active); crosses, 125 A.U./gVSsludge (active); asterisks, 250 A.U./gVSsludge (active); filled circles, 125 A.U./gVSsludge (inactive); + , 250A.U./gVSsludge (inactive).

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digestion (Figure 6), as PPS and seed sludge were determinedto be alkaline substrates (Table 1). The pH curve was similarfor all six bioreactors (Figure 6), with a drop on days 0–8 andrising on days 9–32. During the initial anaerobic digestionphase, the pH dropped with a simultaneous increase in VFAconcentration until reaching the lowest pH values on day 8,corresponding to a pH slightly above 7.4. At the same time, theVFA concentration reached a peak value. With VFA consump-tion, pH values increased steadily to 8.6 between days 9–32.

pH is not an effective parameter to measure the stabilityof an anaerobic process when there is a high buffering capac-ity. Small changes in pH may occur due to large changes inprocess performance (Björnsson et al. 2000). Under this condi-tion, alkalinity is a better option to measure process perform-ance directly. The alkalinity of a steady-state anaerobic systemranges between 1000–5000 mg CaCO3 L–1 (Ren & Wang 2004).In this experiment, during the initial digestion stage in days0–4, the alkalinity of each bioreactor increased sharply from236 mg CaCO3 L–1 to 1178 mg CaCO3 L–1, an average value(Figure 7), reflecting that the initial process was quite unsta-ble. Between days 5–11, the alkalinity increased steadily to themaximum of 1580 mg CaCO3 L–1 on average (Figure 7); then,the alkalinity decreased slowly between days 12–32, main-taining a value higher than 1000 mg CaCO3 L–1 during thisstage, assuring a steady-state of the process. An increase inalkalinity is normally due to the activity of methanogenicbacteria, which can produce alkalinity in the form of carbon

dioxide, ammonia and bicarbonate (Turovskiy & Mathai2006). In addition, the change curves of pH and alkalinitywere the same for all treatments and there was only a little dif-ference of values among the six treatments (Figures 6 and 7),which implies that the values of pH and alkalinity for eachtreatment were closely related to the feedstock supplies ofPPS, MGWL and sludge inoculum, instead of the MCE dos-age applied to the bioreactor (see Table 2).

ConclusionsSix bioreactors were employed to evaluate the methane pro-ductivity when pre-treating PPS with MCE (active and inac-tive) prior to anaerobic digestion at retention times of 42 daysand 37˚C. Bioreactor A was fed with original PPS (as a con-trol); bioreactors B, C and D were fed with PPS pretreatedwith different amounts of active MCE, respectively; bioreac-tors E and F were fed with PPS pretreated with differentamount of inactive MCE, respectively. PPS after bio-pre-treatment presented higher SCOD, VSS, SVave, NH3-N andalkalinity values. The performance of each bioreactor, e.g.organics (VS) removal, VFA concentration and methane pro-duction yield had the trend D > C > B > E > F > A (CK).VS and SCOD removal efficiencies improved in the range33–44% for VS and 56–71% for SCOD after pretreatment.

This study shows that the best bio-pretreatment was the useof 250 A.U./gVSsludge active MCE prior to anaerobic digestion,increasing methane productivity by 34% with a peak value of

Fig. 6: pH value changes in effluent for each bioreactor with PPS pretreated by different amount of MCE. Filled diamonds, CK; filled triangles, 50A.U./gVSsludge (active); crosses, 125 A.U./gVSsludge (active); asterisks, 250 A.U./gVSsludge (active); filled circles, 125 A.U./gVSsludge (inactive); + , 250A.U./gVSsludge (inactive).

Fig. 7: Alkalinity changes in effluent for each bioreactor with PPS pretreated by different amount of MCE. Filled diamonds, CK; filled triangles, 50A.U./gVSsludge (active); crosses, 125 A.U./gVSsludge (active); asterisks, 250 A.U./gVSsludge (active); filled circles, 125 A.U./gVSsludge (inactive); + , 250A.U./gVSsludge (inactive).

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0.23 m3/kgVSadd at a lower cost compared with other pretreat-ments. Conversely, inactive MCE pretreatment was the worseoption for increasing methane productivity, enhancing it byonly 1–8%.

AcknowledgementsThe authors would like to thank the Natural Science Fund ofChina and the Natural Science Fund of Guangdong Provincefor financially supporting this research.

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