Enhanced methane production from anaerobic digestion of disintegratedand deproteinized excess sludge

Rong Cui & Deokjin Jahng*Department of Environmental Engineering and Biotechnology, Myongji University, 449-728, Yongin,Kyunggido, Republic of Korea*Author for correspondence (Fax: +82-31-336-6336; E-mail: djahng@mju.ac.kr)

Received 20 October 2005; Revisions requested 14 November 2005; Revisions received 13 January 2006; Accepted 13 January 2006

Key words: anaerobic digestion, deproteinization, excess sludge, sludge disintegration

Abstract

To improve biogas yield and methane content in anaerobic digestion of excess sludge from the wastewatertreatment plant, the sludge was disintegrated by using various methods (sonication, alkaline and thermaltreatments). Since disintegrated sludge contains a high concentration of soluble proteins, the resultingmetabolite, ammonia, may inhibit methane generation. Therefore, the effects of protein removal fromdisintegrated sludge on methane production were also studied. As a result, an obvious enhancement ofbiogas generation was observed by digesting disintegrated sludge (biogas yield increased from 15 to 36 ml/gCODaddedÆday for the raw excess sludge and the sonicated sludge, respectively). The quality of biogas wasalso improved by removing proteins from the disintegrated sludge. About 50% (w/w) of soluble proteinswere removed from the suspension of disintegrated sludge by salting out using 35 g MgCl2Æ6H2O/l and alsoby isoelectric point precipitation at pH 3.3. For deproteinized sludge, methane production increased by19%, and its yield increased from 145 ml/g CODremoved to 325 ml/g CODremoved. Therefore, the yield andquality of biogas produced from digestion of excess sludge can be enhanced by disintegrating the sludge andsubsequent protein removal.

Introduction

Excess sludge produced from activated sludgeprocesses increases with the expansion of popula-tion and industry. As an example, Korea’s an-nual production of excess sludge from municipalwastewater treatment facilities increased by 10%(w/w) every year from 1998 to 2003. As regula-tions on sludge disposal become increasinglystringent, processing and disposal of excesssludge are considered as one of the most seriousproblems encountered in wastewater treatmentfrom the viewpoint of environment, finance andtechnology (Weemaes et al. 2000).

Anaerobic digestion is one of the oldest andmost traditional processes for reducing the vol-ume of sewage sludge (Weemaes et al. 2000). It

attracts interest due to its capability of convert-ing volatile solids contained in the sludge tomethane (Xu et al. 2002), an alternative clean en-ergy source to limited fossil fuels. Generally, fourstages (hydrolysis, acidogenesis, acetogenesis, andmethanogenesis) are involved in anaerobic diges-tion, and the hydrolysis stage is considered asthe rate limiting step for excess sludge digestion(Tiehm et al. 2001, Navia et al. 2002, Kim et al.2003). This is because the hydrolysis of thesludge biomass is limited by the restricted acces-sibility of the extracellular enzymes produced bythe hydrolyzing bacteria to the intracellular poly-meric materials which are protected by cell mem-branes. To improve anaerobic digestion of excesssludge, mechanical breakdown (Choi et al. 1997,Nah et al. 2000), ozonation (Weemaes et al.

Biotechnology Letters (2006) 28: 531–538 � Springer 2006DOI 10.1007/s10529-006-0012-9

2000), ultra-sonication (Tiehm et al. 2001), alka-line dissolution (Navia et al. 2002) and a thermo-chemical treatment (Kim et al. 2003) have beenapplied to disrupt microbial cells. These pro-cesses improve volatile mass and biogas produc-tion, together with a shorter hydraulic retentiontime (HRT) and a solid retention time (SRT) inthe anaerobic digestion process.

Free ammonia is toxic to methanogenic bacte-ria and thus inhibits methane production (Gallert& Winter 1997). Poggi-Varaldo et al. (1997) ob-served that methane generation was ceased at aCOD (chemical oxygen demand)/N ratio lowerthan 50. Since C/N ratio of sewage sludge isaround 6–16 (Stroot et al. 2001), and the primarynitrogen sources are proteins that are hydrolyzedto yield ammonia in the digester, disintegrationof the sludge may decrease the soluble COD/Nratio and increase free ammonia, which thus mayinhibit methane production. In this study, there-fore, effects of protein removal from disinte-grated excess sludge on anaerobic digestion wereinvestigated.

Materials and methods

Sludge disintegration

Excess sludge [8000–14 000 mg/l as suspendedsolid (SS)] was taken from a local municipalwastewater treatment plant. For an ultrasonicpretreatment, 400–2000 ml of excess sludge (SS:11 850 mg/l) were sonicated with an ultrasonicprocessor at 0 �C for 20–140 min. For a thermaltreatment, 25 ml of excess sludge (SS: 13 900 mg/l)were autoclaved in an autoclave at 121 �C for10–120 min. Effects of temperature on the disin-tegration of excess sludge were examined by heat-ing excess sludge at 70, 90, 100, 110, and 121 �Cfor 40 min, respectively. For an alkaline treat-ment, pH of excess sludge (SS: 12 050 mg/l) wasadjusted to pH 8–13 with 1 M NaOH for 2 h.

After sludge disintegration, total COD(TCOD), soluble COD (SCOD), mixed liquorsuspended solids (MLSS), mixed liquor volatilesuspended solids (MLVSS) and soluble protein(S-protein) were analyzed to evaluate efficienciesof these disintegration methods. Degree of disin-tegration in terms of COD solubilization wasexpressed as follows:

COD Solubilization (%)

¼ SCODi � SCODa

TCODi � SCODi� 100ð%Þ

ð1Þ

SCODi and SCODa were the soluble COD’s be-fore and after the pretreatment, respectively.TCODi was the initial TCOD.

Protein removal

Three methods (salting out, isoelectric point pre-cipitation, and heating) were used to precipitatesoluble proteins from the suspension of disinte-grated sludge. Disintegrated sludge (ultrasonicatedat 1.15�105 kJ/kg VSS) was centrifuged at 393�gfor 30 min at 4 �C to collect the supernatant.

For salting out the soluble proteins, the super-natant was kept at 80 �C, and then a salting-outreagent was added under gentle agitation. Stocksolutions of MgCl2 Æ 6H2O (250 g/l), CaCl2(12 g/l), Mix A [50% (w/w) of CaSO4Æ2H2O, 30%of MgCl2Æ6H2O, and 20% of sodium citrate)(700 g/l)], and glucono-d-lactone (24 g/l) werediluted in the supernatant for the working concen-trations of 6–60 g/l, 0.2–0.4 g/l, 20–140 g/l, and0.8–2.2 g/l, respectively. For isoelectric point pre-cipitation, pH of the supernatant was adjusted topH 3.3, pH 6.7 or pH 9.9 with 2 M H2SO4 or 1 M

NaOH. Precipitated proteins were re-dissolved indistilled water at pH 4 or pH 10 for a mass bal-ance study of proteins. For the thermal treatment,the supernatant was heated at 98 �C for 2 h. For acombination of these three methods, superna-tant was pre-heated to 98 �C first, and thenMgCl2 Æ 6H2O (35 g/l) was added followed by apH adjustment (pH 3.3, pH 6.7 or pH 9.9).

Precipitated proteins were collected by centri-fugation at 393�g for 20 min. Deproteinizedsupernatant was mixed with the centrifuged pel-lets of disintegrated sludge and used as an anaer-obic digestion feed.

Batch anaerobic digestion

Batch anaerobic digestion was carried out in 3water-jacketed reactors. Each reactor had a totalvolume of 3000 ml and contained 1300 ml ofanaerobic sludge that were taken from an anaero-bic digester at the local municipal wastewatertreatment plant. About 200 ml of feed sludge (raw

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excess sludge, sonicated sludge, and sonicated anddeproteinized sludge) were added into each diges-ter. Temperature of digester was controlled at36 �C by circulating warm water through thewater jacket. Biogas was collected in a plastic masscylinder. The cylinder was filled with distilled wa-ter which was acidified to pH 1 with 1 M HCl toprevent absorption of CO2 and H2S. Compositionand volume of biogas, SCOD, MLSS, and volatilefatty acids were measured periodically.

Analytical methods

MLSS and MLVSS were measured followingthe Standard Methods. To determine the con-centration of SCOD, soluble total nitrogen(S-TN), and soluble proteins, a supernatantwas obtained by centrifuging the sample sus-pension at 393�g for 20 min. SCOD was mea-sured by the open reflux method, and theconcentration of proteins was measured by theLowry method using BSA as a standard pro-tein. S-TN was measured with the HACHmethod 10072 using the HACH DC/2500 spec-trophotometer.

The biogas composition (methane and CO2)was analyzed by GC equipped with a thermalconductivity detector (TCD) with the HP-Plot Qcolumn (30 m�0.32 mm�20 lm). Injector anddetector were at 60 and 250 �C, respectively. Thecolumn was increased from 30 �C (5 min) to100 �C at 20 �C/min. Carrier gas (helium) was at1.5 ml/min. About 50 ll gas sample was injectedinto the GC in the splitless mode. Retentiontimes for methane and CO2 were 2.7 and3.7 min, respectively.

Free fatty acids (acetic, propionic, butyric,valeric, caproic, and heptanoic acids) were as-sayed using a GC-flame ionization detector(FID) with the HP-INNOWAX column(30 m�0.25 mm�0.25 lm). Both injector anddetector were set at 220 �C. The oven wasincreased from 60 �C (4 min) to 170 �C at4 �C/min. Carrier gas (nitrogen) was at 0.9 ml/min. About 10 ll of a liquid sample was injected(splitless) into the GC. For a calibration, a stan-dard free fatty acid solution was made by dis-solving a pure fatty acid in distilled water at100 mg/l. For 1 ml of a standard free fatty acidsolution, 0.2 ml of 20% (v/v) H3PO4 solutionwere added, and the mixture was centrifuged for

10 min at 8010 � g and 4 �C. The supernatantwas injected into the GC. Retention times foracetic, propionic, butyric, valeric, caproic, andheptanoic acids were 17.6, 20.0, 22.6, 25.6, 28.4and 31.0 min, respectively.

Results and discussion

Sludge disintegration

Three methods (ultrasonic, thermal and alka-line treatments) were compared in terms of theefficiency of excess sludge disintegration. CODsolubilization and VSS reduction achieved byalkaline and ultrasonic treatments were shown inFigure 1. By alkaline treatment, significant dis-ruption of sludge (14% in terms of COD solubi-lization at pH 11.8) was observed as pHincreased (Figure 1a). However, NaOH doseincreased exponentially, whereas SCOD in-creased linearly for pH higher than pH 10(Figure 1a). Moreover, hydrolysis of excesssludge occurred immediately after the addition of

Energy input (kJ/kg VSS)

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Fig. 1. Disintegration of excess sludge. (a) Excess sludge waslysed using 1 M NaOH. (b) Excess sludge was disrupted by ultra-sonication.

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NaOH for pH lower than pH 12, but 20 min ofreaction time is needed to fully disintegratesludge for pH higher than pH 12 (data notshown). For ultrasonication, more sludge cellswere disrupted and more cellular materials(SCOD and S-TN) were released with the in-crease of energy input (Figure 1b). Increase ofSCOD was proportional to S-TN, and a rela-tively constant SCOD/S-TN value of 16 was ob-tained. Optimal energy input was 1.15�105 kJ/kgMLVSS, and 44.7% of COD solubilization wasachieved at this condition.

Thermal treatment of excess sludge was car-ried out in an autoclave, and effects of tempera-ture and heating time on sludge disintegrationwere studied. As shown in Figure 2, SCODincreased with the increases of heating time(Figure 2a) and temperature (Figure 2b), andabout 90 min were needed to achieve a maximumsludge disruption.

Protein removal

MgCl2 Æ 6H2O, CaCl2, glucono-d-lactone andMix A are commonly used precipitants to pro-duce Tofu from soy bean broth. The possibilityof using these reagents to precipitate soluble pro-teins released from the disintegrated excesssludge was studied. Observed maximum removalrates of soluble proteins were 49% (w/w), 35%,20% and 6% for MgCl2 Æ 6H2O (35 g/l), CaCl2(0.4 g/l), glucono-d-lactone (0.8 g/l) and Mix A(20 g/l), respectively. Thermal treatment, isoelec-tric precipitation and a combination of saltingout and these methods were also carried out. Asshown in Figure 3, thermal treatment did noteffectively remove proteins at pH 6.7 and pH 9.9,and isoelectric precipitation of proteins at pH 3.3achieved 52% (w/w) of protein removal.Although the combination of isoelectric precipi-tation and salting out further improved proteinremoval at pH 3.3, the efficacy was negligibleand cost ineffective. Therefore, isoelectric precipi-tation at pH 3.3 was chosen in this study toremove soluble proteins from the supernatant ofdisintegrated sludge.

Precipitated proteins are desired to be reusedsince they contain useful enzymes and nutrients.For example, Jung et al. (2002) recovered a pro-tease from proteins obtained by salting out ofdisrupted excess sludge with ammonium sulfateand used it for hydrolysis of milk proteins. Pro-teins obtained by the isoelectric precipitation atpH 2.8 were resolubilized in distilled water by

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Fig. 2. Disintegration of excess sludge by thermal treatment.(a) Excess sludge was autoclaved at 121 �C for 0–130 min. (b)Excess sludge was treated at 20–121 �C for 40 min.

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Fig. 3. Protein removal by salting out (35 g/l of MgCl2 Æ6H2O), thermal treatment (98 �C for 2 h), and isoelectric pre-cipitation (pH 3.3, pH 6.7, and pH 9.9).

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increasing the pH of the solution. As shown inFigure 4, precipitated proteins were redissolvedand almost all of proteins were solubilized byincreasing the solution pH to pH 10. If treatedproperly, these excess sludge-originated proteinsmight serve as an ingredient of animal feeds.

Anaerobic digestion of excess sludge

Effects of disintegration of excess sludge usingthermal and ultrasonic treatments on anaerobicdigestion are shown in Figure 5. Three days of lagphase were observed for the control, whereas bio-gas was immediately produced from ultrasonic-(1.15�105 kJ/kg MLVSS) and thermally- (121 �C,40 min) treated sludges. This was attributed to therelease of soluble cellular materials by disruptingmicrobial cells contained in the excess sludgeswhich enable anaerobes to consume them immedi-ately rather than hydrolyze the cell wall first.Compared with thermally-treated sludge, a little

higher biogas yield was observed during the first4 days for the ultrasonicated sludge (Figure 5a).This was due to a higher degree of sludge disrup-tion for sonicated sludge [48.1% (w/w) disruptionby sonication versus 17.4% disruption by thermaltreatment], which resulted in a higher concentra-tion of initial SCOD (Figure 5b). By feedingthe disrupted sludge, biogas yield for the first3 days of anaerobic digestion increased from3.65 ml/g CODaddedÆday (control) to 30.2 ml/gCODaddedÆday (sonicated sludge). Patterns ofSCOD uptake were in accordance with biogasgeneration, and little change in SCOD concentra-tion was observed for all reactors after about4 days (Figure 5b). This supports the hypothesis

14.080 g supernatantof sonicated sludge( 128.9 mg protein)

pH 2.8393 × g, 20 min.

Supernatant3.164 g protein/l(41.8 mg protein)

Pellet(87.1 mg protein)

20 ml DW atpH 4.0

393 × g, 20 min.

Supernatant0.222 g protein/l(4.3 mg protein)

Pellet(82.8 mg protein)

19 ml DW atpH 10.0

393 × g, 20 min

Supernatant4.768 g protein/l(89.7 mg protein)

Fig. 4. Mass balance of protein recovery. Isoelectric-precipi-tated proteins were re-dissolved in distilled water by increas-ing solution pH to pH 10 with 1 M NaOH.

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Fig. 5. Effect of sludge disintegration on biogas yield (a) andSCOD uptake (b) in anaerobic digestion. Excess sludges werethermally treated at 121 �C for 40 min and sonicated at1.15�105 kJ/kg MLVSS, respectively. Excess sludge withoutany treatment was used as a control.

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that hydrolysis of sludge is the rate limiting stepfor the anaerobic digestion of excess sludge. Sincethe cell wall of the sludge behaves as a physico-chemical barrier to exoenzymes and hydrolysis,the availability of intracellular organic materials tothe anaerobes is restricted, which limits the globalefficiency of the digestion process (Lin et al. 1997).By feeding disintegrated sludge, it was thoughtthat the impact of this rate limiting step was sig-nificantly reduced and a higher biogas yield wasobtained.

Since about 40–50% (w/w) of the dry weight ofa microbial cell is occupied by proteins (Atkinson& Mavituna 1991), and it was reported that a highconcentration of ammonia could inhibit methano-genesis (Gallert & Winter 1997, Poggi-Varadoet al. 1997), it was anticipated that methane gener-ation could be improved by removing proteinsfrom the solubilized sludge. To verify this, anaero-bic digestion with a sonicated and deprotei-nized sludge was carried out. Characteristics ofexcess sludge, sonicated sludge and protein-removed sludge are shown in Table 1. Excesssludge was disintegrated by ultrasonic treatmentfirst. After sonication, 44.7% (w/w) of sludge wassolubilized in terms of COD, which caused theconcentration of soluble proteins to increase from0.07 to 4.71 g/l. Around 35.6% (w/w) of solubleproteins were precipitated and removed by adjusting

pH of the supernatant to pH 3. The supernatantwas then mixed with the original centrifuged pel-lets and pH of the mixture was adjusted to pH 8with 1 M NaOH. This mixture was used as a feedfor anaerobic digestion. Sludge without sonicationand protein removal was used as a control.SCOD/S-protein ratio decreased from 2.48 to2.18 g SCOD/g protein after sonication dueto release of soluble intracellular proteins and re-increased to 2.38 g SCOD/g protein after theprotein removal. Soluble fatty acids (C2–C7)which were major substrates for methanogenesissignificantly increased due to the disruption ofsludge, and only 21% (w/w) of fatty acids, mostlyacetic and propionic acid, were removed togetherwith the protein. Biogas generations were com-pared in Figure 6, and biogas generation ratesduring the first 4 days were calculated and com-pared in Figure 7.

By feeding disrupted sludge, biogas genera-tion was significantly improved (Figure 6–7).For the first 2 days, generation of biogasfrom the sonicated and deproteinized sludge wasslightly higher than that of the sonicated sludge(Figure 6a). After 2 days, biogas productionfrom the sonicated sludge became faster thanthat from the protein-removed sludge. This wasdue to that substrates for methanogenesis(SCOD and fatty acids) contained in the depro-

( )

Table 1. Characteristics of sludges used for anaerobic digestion (Unit: mg/l).

Item Control Sonicated sludge1 Sonicated and deproteinized sludge2

MLSS 14 100 4950 ND3

MLVSS 10 900 3750 ND

SCOD 166 10 260 7190

TCOD 22 960 22 960 12 020

S-Protein 67.0 4707.6 3020.2

SCOD/S-protein 2.48 2.18 2.38

PH 8.0 8.0 8.0

Acetic acid 2.5 124.1 94.7

Propionic acid 6.5 149.5 106.5

Butyric acid 0 36.6 30.5

Valeric acid 0.6 47.3 38.5

Caproic acid 0.2 6.7 13.2

Heptanoic acid 0.3 4.5 5.2

Total Fatty acid 10.1 368.8 288.5

1Excess sludge was sonicated at 1.15�105 kJ/kg MLVSS.2Soluble proteins in the suspension of sonicated sludge were removed by isoelectric precipitation at pH 3. Protein-removed suspensionwas mixed with the original centrifuged pellets of the sonicated sludge, and pH was adjusted to pH 8 before feeding to the digester.3Not determined.

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teinized sludge were consumed quickly for thefirst 2 days (Figure 6d). By feeding the deprotei-nized sludge, 73.4 and 178.2% increase of biogasand methane generations, respectively, wereobserved (Figure 7). To clarify the effect ofprotein removal on methanogenesis, methaneproduction was calculated based on COD re-moval. As a result, methane generation increasedfrom 145.2 ml/g CODremoved to 325.5 ml/gCODremoved, indicating that methane yield wasenhanced by 124.2% through deproteinization.By improving methane generation, methane con-tent in the biogas produced from deproteinizedsludge also increased from 55.6% (v/v) (control)to 74.8% (Figure 6b), which suggested that

biogas quality was significantly improved bydigesting the deproteinized sludge.

Conclusion

Optimal conditions for disintegration were90 min heating at 121 �C, 1.15�105 kJ/kgMLVSS and pH 12 for thermal, ultrasonic andalkaline treatments, respectively. 44.7% (w/w)sludge COD materials were solubilized after dis-integration by ultrasonication, and 50% (w/w)soluble protein were removed from the soni-cated sludge by isoelectric precipitation at pH3.3 or salting out with MgCl2 Æ 6H2O at 35 g/l.An obvious enhancement of biogas generationwas observed by feeding disintegrated sludgeand the quality of biogas was also improved byremoving proteins from the disintegrated sludge.Therefore, it was concluded that anaerobicdigestion of excess sludge could be enhanced (interms of biogas yield and biogas quality) bydisintegrating sludge and subsequent proteinremoval.

Acknowledgement

This work was supported by a grant (Code#20050401-034-750-142-02-00) from BioGreen 21Program, Rural Development Administration,Republic of Korea.

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