Bioresource Technology 140 (2013) 131–137

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Bioresource Technology

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Comparison of sludge digestion under aerobic and anaerobic conditionswith a focus on the degradation of proteins at mesophilic temperature

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.04.081

Abbreviations: WAS, waste activated sludge; EEM, excitation–emission matri-ces; DOM, dissolved organic matter; TS, total solids; VS, volatile solids; HIX,humification index; HA, humic acid; FA, fulvic acid; HyI, hydrophilic.⇑ Corresponding author at: Institute of Waste Treatment and Reclamation, Tongji

University, Shanghai 200092, PR China Tel.: +86 21 65986104; fax: +86 2165981383.

E-mail address: solidwaste@tongji.edu.cn (P. He).

Liming Shao c, Tianfeng Wang a, Tianshui Li a, Fan Lü a, Pinjing He b,c,⇑a State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, PR Chinab Institute of Waste Treatment and Reclamation, Tongji University, Shanghai 200092, PR Chinac Centre for the Technology Research and Training on Household Waste in Small Towns & Rural Area, Ministry of Housing and Urban-Rural Development (MOHURD), PR China

h i g h l i g h t s

� Protein degradation mainly limited by hydrolysis under aerobic condition.� Protein degradation limited by hydrolysis and metabolism under anaerobic condition.� Humification degree of aerobic digested sludge was greater than anaerobic one.

a r t i c l e i n f o

Article history:Received 25 March 2013Received in revised form 19 April 2013Accepted 20 April 2013Available online 28 April 2013

Keywords:Waste activated sludgeAerobic digestionAnaerobic digestionProteinHumification

a b s t r a c t

Aerobic and anaerobic digestion are popular methods for the treatment of waste activated sludge. How-ever, the differences in degradation of sludge during aerobic and anaerobic digestion remain unclear. Inthis study, the sludge degradation during aerobic and anaerobic digestion was investigated at mesophilictemperature, focused on protein based on the degradation efficiency and degree of humification. Theduration of aerobic and anaerobic digestion was about 90 days. The final degradation efficiency of volatilesolid was 66.1 ± 1.6% and 66.4 ± 2.4% under aerobic and anaerobic conditions, respectively. The final deg-radation efficiency of protein was 67.5 ± 1.4% and 65.1 ± 2.6% under aerobic and anaerobic conditions,respectively. The degradation models of volatile solids were consistent with those of protein under bothaerobic and anaerobic conditions. The solubility of protein under aerobic digestion was greater than thatunder anaerobic digestion. Moreover, the humification index of dissolved organic matter of aerobic diges-tion was greater than that during anaerobic digestion.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Waste activated sludge (WAS) is an inevitable by-product of thebiological, physical and chemical processes in wastewater treat-ment plants (Appels et al., 2008). The treatment and disposal ofWAS is a problem of growing importance, representing up to 50%of the current operating costs of wastewater treatment plants (Ap-pels et al., 2011; Higgins and Novak, 1997). WAS is comprised of amicrobial consortium and organic and inorganic matter held to-gether in a matrix formed by exocellular biopolymers and cations(Murthy and Novak, 1999). Owing to the high water content andpresence of putrescible organic matter, WAS must undergo treat-

ment to guarantee its stability and reduce its corresponding vol-umes before final disposal.

Aerobic and anaerobic digestion are popular methods of WAStreatment (Hall, 1995). However, studies of these techniques con-ducted to date have focused on their ability to improve the dewa-tering characteristics and reduction efficiency of WAS (Devlin et al.,2011; Yang et al., 2011, 2010; Rasheda et al., 2010; Li et al., 2008).No matter aerobic or anaerobic digestion, the degradation of or-ganic matter is the basic process influencing the digestion effi-ciency. Novak et al. (2003) found that the concentration ofdissolved protein and polysaccharides was greater under anaerobicdigestion than anaerobic digestion. Moreover, Tomei et al. (2011a)found that dissolved proteins and polysaccharides showed obviousaccumulation under anaerobic digestion, but were notably reducedunder aerobic digestion. Tomei et al. (2011b) observed that theconcentration of protein and polysaccharides increased in theanaerobic phase, but decreased in the subsequent aerobic phase.By using the excitation–emission matrices (EEM) spectra method,Ramesh et al. (2006) showed that protein and humic substances

132 L. Shao et al. / Bioresource Technology 140 (2013) 131–137

declined in relative intensities after digestion, and that aerobicdigestion was more effective than anaerobic digestion. However,these studies lack a comparison of the degradation properties ofthe organic matter during different stages of WAS aerobic andanaerobic digestion, which is necessary to provide a fundamentalbasis for improving digestion efficiency by combined digestion.

WAS digestion primarily occurs via the degradation of macro-molecular organic matter. Protein, polysaccharides and humic sub-stances are the major components of macromolecular organicmatter in WAS (Wilen et al., 2003), among which protein is thedominant component, accounting for 50% of the total WAS organicmatter (Jimenez et al., 2013). The first-order rate coefficient forprotein is usually smaller than that of carbohydrate (Christ et al.,2000).

Owing to deficiencies in parallel comparison studies of the deg-radation of WAS organic matter, it is difficult to gain a deep com-prehension of the difference between aerobic and anaerobicdegradation of WAS organic matter. In this study, a parallel exper-iment method was used to measure the protein solubility and fluo-rescence EEM properties of dissolved organic matter (DOM). Theoverall objectives of this study were: (1) to investigate the aerobicand anaerobic digestion degradation of WAS protein at mesophilictemperature; (2) to present humification characteristics of sewagesludge during aerobic and anaerobic digestion at mesophilictemperature.

2. Methods

2.1. Materials

The same WAS was used for both digestion modes. WAS wasobtained from the aeration tank of a local domestic wastewatertreatment plant in Shanghai, China. The capacity of the plant was75,000 m3 d�1 and it employed an anaerobic–anoxic–oxic process.After being collected and passed through a 1.2 mm screen, WASwas centrifuged at 2000�g for 10 min. The sediments were thensuspended in the supernatant of sludge to adjust to the requiredconcentration. Mesophilic seeding sludge with a total solids (TS)of 14.6% and volatile solid (VS) was 88.8% of the TS was collectedfrom an anaerobic internal circulation reactor of a local paper mill,and crushed before seeding.

2.2. Aerobic and anaerobic digestion incubation

All batch operations were carried out in vessels with each effec-tive volume of 12 L (No. 2600-0012, Nalgene, USA). Table 1 showsthe characteristics of the mixture sludge in the vessels.

During aerobic digestion, continuous aeration was conductedusing an aeration pump, a gas-flow meter and two micro porous dif-fusers (placed at the bottom of the reactor). A gas flow meter wasconnected before the diffusers to control the ventilation rate atapproximately 1.2 m3 h�1 kg�1 (dry basis), which was selected toensure aerobic conditions. The moisture loss was then replenishedby adding distilled water to the reactor daily to maintain the originalvolume (subtracting the volume of sample). The reactors were thenincubated at 35 ± 1 �C for about 90 days and a peristaltic pump(120 rpm) was used to provide internal circulation for mixing.

Table 1Characteristics of the initial material in the vessels (mg/L, except pH).

pH VS TS

Aerobic 6.38 ± 0.00 17,260 ± 10 22.870 ±Anaerobic 6.97 ± 0.03 8890 ± 420 12.620 ±

For anaerobic digestion, after being inoculated with mesophilicseeding sludge at 10% (w/w) VS of the WAS, and diluted with waterat a ratio of 1:2 to ensure good mechanical mixing condition, thereactors were sealed and flushed with nitrogen gas for 1 min to in-duce anaerobic conditions. The reactors were then incubated at35 ± 1 �C for about 90 days, during which time they were mixedwith an airtight stirrer (240 rpm).

All tests were conducted in duplicate, and the data shown inthis paper were the averages based on two parallel experiments.

2.3. Analytical methods

Sludge samples were collected from the reactors using a peri-staltic pump at different intervals. In addition, the filtrate(0.45 lm, microfiber filter) of the supernatant (2000�g, 10 min)of sludge samples was collected as liquid samples.

Determination of the TS content of sludge samples was con-ducted by drying the samples at 105 �C for 24 h, while the VS con-tent of the sludge samples was determined by heating the samplesat 600 �C for 2 h. Total organic carbon (TOC), inorganic carbon (IC)and total nitrogen (TN) of liquid samples were analyzed using a TC/TN analyzer (TOC-V CPN, TNM-1, SHIMADZU, Japan). Kjeldahlnitrogen (KN) and ammonia nitrogen (AN) were analyzed usingan auto Kjeldahl determination system (8400, FOSS, Sweden) forsludge samples and liquid samples. Protein was calculated by mul-tiplying the concentration of organic nitrogen (ON) (KN–AN) by6.25. Polysaccharides was measured by the Anthrone methodusing glucose as a standard (Gaudy, 1962).

2.4. Protocol for the extraction of sludge protein

Protein solubility determined by a thermochemical method wasused to characterize the degradability of protein of sludge duringaerobic and anaerobic digestion. The effect of thermochemical pre-treatment on WAS is to promote hydrolysis and to split complexorganic polymers into simpler constituent molecules (Stuckeyand McCarty, 1978; Whiteley et al., 2002). Thermophilic enzymatichydrolysis of sludge can further disrupt sludge extracellular poly-mer matrix, which resulted in enhanced solubilization of thesludge, including protein (Whiteley et al., 2002).

Briefly, sludge samples were centrifuged at 2000�g for 10 min,after which the sediments were diluted 15 times (m/m) using dis-tilled water. The diluted sediment was then mixed by vortexing for1 min (XW-80A, Shanghai, China), after which it was incubated at60 ± 1 �C in a thermostatic water bath for 5 h. Protein extractionefficiency was defined as the protein concentration of the liquidsample divided by the protein concentration of the diluted sedi-ment before incubation.

2.5. Fluorescence measurement

Fluorescence EEM was measured on a fluorescence spectropho-tometer (Cary Eclipse, Varian, USA) in scan mode. EEM spectrawere gathered from scanning emission spectra from 250 to500 nm at 2 nm increments by varying the excitation wavelengthfrom 200 to 450 nm at 10 nm increments.

In this study, the fluorescence regional integration (FRI) tech-nique was employed to analyze the five excitation–emission re-

AN KN Protein

10 197.7 ± 9.3 1523 ± 17 8283 ± 49420 121.1 ± 2.8 961.1 ± 27.3 5250 ± 153

L. Shao et al. / Bioresource Technology 140 (2013) 131–137 133

gions of EEM spectroscopy. The percentage of fluorescence re-sponse (Pi,n = Ui,n/UT,n) and normalized excitation–emission areavolumes (Ui,n) were calculated as described by Chen et al. (2003),where Ui,n is the normalized excitation–emission area volumesreferring to the value of region i. The humification index (HIX)was calculated using Eq. (1) from the emission intensity area atan excitation of 254 nm (linear interpolation) according to Ohno(2002):

HIX ¼X

I435!480=ðX

I300!345 þX

I435!480Þ ð1Þ

2.6. Isolation and characterization of DOM

The methods described by He et al. (2006) were adopted to frac-tionate organic materials into humic acid (HA), fulvic acid (FA) andhydrophilic (HyI) fractions of the DOM.

3. Results

3.1. Degradation efficiency of organic matter

Fig. 1A shows the evolution of aerobic degradation efficiency ofVS and protein. Throughout the aerobic digestion process, variationin the degradation efficiency of VS and protein was synchronous,and the degradation efficiency of protein was greater than VS. Onday 89, the degradation efficiency of protein and VS was stable at67.5 ± 1.4% and 66.1 ± 1.6%, respectively.

Fig. 1B shows the evolution of anaerobic degradation efficiencyof VS and protein. Throughout the anaerobic digestion process, var-iation of degradation efficiency in VS and protein was also synchro-nous; nevertheless, the degradation efficiency of VS was greaterthan that of protein. On day 94, the degradation efficiency of pro-tein and VS was stable at 65.1 ± 2.6% and 66.4 ± 2.4%, respectively.

Fig. 2 shows the dual logarithmic relationship between timeand evolution of total residual efficiency [equal to (1 � degradationefficiency)] of organic matter (presented as VS) and protein duringthe aerobic and anaerobic digestion process. Under both aerobicand anaerobic conditions, the logarithmic relationship betweentime and evolution of total residual efficiency of VS and proteinwere almost linear. The degradation rule of VS was consistent withprotein, and followed the first-order kinetic reaction equation.When combined with the initial organic matter concentration (Ta-ble 1), the degradation of protein was show to have a major impacton sludge digestion.

3.2. Solid phase protein extraction efficiency and protein degradationefficiency

Fig. 3 shows the relationship between time and extraction effi-ciency of protein in the solid phase, which was contrasted with the

Fig. 1. Degradation efficien

degradation efficiency of protein. The results indicated that theextraction efficiency of solid phase protein characterized thehydrolysis ability of sludge protein. During the digestion process,the extraction efficiency of the solid phase protein first decreased,then increased, but then decreased again in the last stage, demon-strating the difference in the biodegradability of the diverse pro-teins in sludge.

During the aerobic process, the degradation rate of protein andthe extraction efficiency of solid phase protein were synchronous.After 40 days, the degradation rate of protein and the extractionefficiency of solid phase protein were maintained at low levels,indicating that the degradation of protein was mainly limited byhydrolysis. During the early stage of anaerobic digestion, the deg-radation rate of protein and the extraction efficiency of solid phaseprotein were synchronous. In the later stages of anaerobic diges-tion, the extraction efficiency of solid phase protein was main-tained at a high level, but the degradation rate was close to zero.These findings suggest that the degradation of protein was not onlylimited by hydrolysis, but also by other factors.

3.3. Evolution of carbon and nitrogen of liquid sample

The evolution of carbon and nitrogen in liquid samples is shownin Figure A1.

During the first 40 days, the dissolved organic carbon and nitro-gen were maintained at high levels under aerobic and anaerobicconditions, but the levels normalized by the initial feeding basiswere obviously higher during aerobic digestion, which indicatedthat the hydrolysis rate under aerobic conditions was higher thanthat under anaerobic conditions. After 40 days, the dissolved or-ganic carbon and nitrogen gradually declined during both aerobicand anaerobic digestion. Moreover, the inorganic carbon and nitro-gen gradually increased under anaerobic digestion.

Fig. 4 shows the distribution of different organic components ofliquid sample, as calculated on the basis of organic carbon. Thegeneral chemical formula of proteins and polysaccharides wasused to the calculated organic carbon content separately (Perrett,2007; Aminabhavi et al., 1990) .

During the aerobic process, the proportion of protein only in-creased during the first 4 days, after which it decreased gradually.During the aerobic process, the proportion of protein increased inthe first 18 days, then decreased gradually. These findings indi-cated that the solubility of protein under aerobic digestion is high-er than that under anaerobic digestion.

The evolution of polysaccharides was similar under aerobic andanaerobic digestion, and relatively stable throughout the entiredigestion period.

The proportion of other organic materials (except protein, poly-saccharide) differed significantly between aerobic and anaerobicdigestion. During the aerobic process, the proportion of other or-

cy of VS and protein.

Fig. 2. Evolution of VS and protein content.

Fig. 3. Extraction efficiency of solid phase protein and degradation efficiency of protein.

Fig. 4. Evolution of organic component of liquid samples.

Table 2Hydrophobic and hydrophilic fractionization of DOM after stabilization.

Organic carbon content (%) HyI HA FA

Aerobic (89 day) 16.6 ± 0.5 22.2 ± 0.4 61.2 ± 0.9Anaerobic (94 day) 28.4 ± 0.7 24.1 ± 0.3 47.5 ± 0.4

134 L. Shao et al. / Bioresource Technology 140 (2013) 131–137

ganic materials increased in the first 3 days, and then decreasedgradually. Based on the degradation characteristics of aerobicmetabolism, these materials should primarily be the metabolicintermediates. During digestion, DOM was gradually humified (Ta-

Fig. 6. Evolution of humification index.

Fig. 5. Evolution of normalized excitation–emission area volumes.

L. Shao et al. / Bioresource Technology 140 (2013) 131–137 135

ble 2 and Fig. 6). In the anaerobic process, the proportion of otherorganic materials decreased in the initial stage, to about 2% on day18, after which they increased and remained stable. Based on thedegradation characteristics during anaerobic metabolism, solubleorganic matter should consist of simple anaerobic fermentationproducts formed in the initial stage, such as organic acid. Owingto imbalance between hydrolysis and methanization metabolism,soluble organic matter was accumulated and eventually convertedto methane. After 40 days of digestion, the majority of DOM con-sisted of refractory soluble microbial products. When comparedwith aerobic digestion, the degree of humification of DOM waslow (Fig. 6).

3.4. Fluorescence properties of the DOM

According to Chen et al. (2003), the EEM spectra can be dividedinto five regions (Region I: tyrosine-like organic compounds; Re-gion II: tryptophan-like organic compounds; Region III: fulvicacid-like materials; Region IV: soluble microbial byproduct-likematerials; Region V: humic acid-like materials). Regions I and IIare related to protein, region IV is related to soluble microbialbyproduct-like material, and regions III and V are related to humicsubstances.

Fig. 5 shows the evolution of Ui,n. During the initial period of theaerobic and anaerobic process, Ui,n related to protein and solublemicrobial byproduct-like material first increased, then declinedand remained stable. The rising level and decreasing time of Ui,n

was larger during anaerobic digestion than aerobic digestion.These findings were consisted with the aforementioned evolution

of organic matter in liquid sample, which once again demonstratedthat the degradation ability of hydrolysate of aerobic digestion wasgreater than that of anaerobic digestion. In the latter period of aer-obic digestion, Ui related to humic material obviously increased,but this value remained stable during the latter stage of anaerobicdigestion. These findings are in accordance with the results shownin Table 2, which demonstrate that the degree of humification dur-ing aerobic digestion was greater than that of anaerobic digestion.

As shown in Figure A2, the evolution of FRI parameters duringthe process of aerobic and anaerobic digestion could be dividedinto two stages. During the first 18 days, the FRI parameters of eachregion were stable. After 18 days, the FRI parameters of regions re-lated to protein declined and remained stable. Moreover, the FRIparameters of regions related to humic substances first increased,then remained stable.

Throughout the digestion process, the FRI parameters of regionsrelated to the proteins of aerobic digestion were smaller than thoseduring anaerobic digestion. Moreover, the FRI parameters of re-gions related to humic substances of aerobic digestion were largerthan those observed during anaerobic digestion.

Similarly, the degree of humification during aerobic digestiondiffered from that during anaerobic digestion (Fig. 6). The HIX ofDOM of aerobic digestion sludge and anaerobic digestion sludgeshowed a similar variation trend. In the first 18 days, HIX waslow, after which it increased and then stabilized.

Throughout the digestion process, the HIX of DOM of aerobicdigestion was greater than that of anaerobic digestion.

4. Discussion

4.1. Differences in degradation of organic matter and protein in sludgebetween aerobic and anaerobic digestion

The variations in the degradation efficiency of VS and proteinwere synchronous during the aerobic and anaerobic process. Dur-ing aerobic digestion, the degradation efficiency of protein wasgreater than that of VS, which was opposite to that observed dur-ing anaerobic digestion. Moreover, the concentration of dissolvedprotein was greater during aerobic digestion that in anaerobicdigestion in the initial stages. During the digestion process, theEEM representing the dissolved protein of aerobic digestion wasalways lower than that of anaerobic digestion, indicating that thehydrolysis rate and the degradation rate of hydrolysate of aerobicdigestion was greater than during anaerobic digestion.

These findings inferred that the digestion degradation of sludgeprotein significantly influenced the biological digestion degrada-tion efficiency of sludge organic matter. The degradation efficiencyof organic matter of aerobic digestion was greater than that ofanaerobic digestion, which was mainly influenced by the differentcharacteristics of hydrolysis and the degradation of protein.

136 L. Shao et al. / Bioresource Technology 140 (2013) 131–137

The hydrolysis rate of protein is significantly affected by theavailable electron donor, and the hydrolysis rate under aerobicconditions is greater than that under anaerobic conditions (Henzeand Mladenovski, 1991). Amino acids are the products of proteinhydrolysis, and denomination is the initial step of the degradationof protein. The main metabolic pathway of deamination is as fol-lows (Sawers, 2009):

(1) Oxidative deamination–amino acid oxidases

NH3+

C CH

O-O

ROC C O-O

R+ 1/2 O2 + NH4+

(2) Oxidative deamination–NAD-linked dehydrogenases

NH3+

C CH

O-O

ROC C O-O

R+ NAD+ + NH4++ H2O + NADH + H+

(3) Elimination

NH3+

C CH

O-O

CH

HOR

H2O NH3+

C C O-O

CH

RNH2

+C C O-O

CH

RH

H2O OC C O-O

CH

RH

+ NH4+

Metabolic pathway (1) only occurs under aerobic conditions, while

metabolic pathways (2) and (3) occur under both aerobic andanaerobic conditions, where the limiting factors are the NAD+ con-centration and microbial activity.

There are more pathways of amino acid metabolism for aerobicdigestion than anaerobic digestion. Owing to lower amount of lim-iting factors under aerobic condition, it is conducive for the degra-dation of dissolved protein, which was manifested by the lowconcentration of dissolved protein (Fig. 4A). When compared withaerobic digestion, there are more limiting factors for amino metab-olism under anaerobic conditions. Low concentration of dissolvedprotein showed that the hydrolysis is the limiting factor. On thecontrast, Fig. 3B (after 30 day) indicated that hydrolysis is not syn-chronous with the degradation of protein (via amino metabolism)under anaerobic conditions.

4.2. Differences in humification between aerobic and anaerobicdigestion

The experiments showed that during the whole digestion pro-cess, the degree of humification of aerobic digestion was greaterthan that of anaerobic digestion at corresponding times.

A greater degree of humification is associated with more complexand condensed aromatic structures and/or more conjugation in ali-phatic chains, which are resistant to degradation (He et al., 2011).There are two main pathways for humification of organic matter,(1) degradation and accumulation of recalcitrant material (Chefetzet al., 2000, De Leeuw and Largeau, 1993), (2) oxidation and polyphe-nolic condensation (Martin and Haider, 1971; Veeken et al., 2000).During the aerobic and anaerobic digestion process, the mutationsof Ui,n and HIX were fitted with the turnover from rapid degradationto slow degradation. The increased degree of humification may becaused by the degradation and accumulation of recalcitrant mate-rial. When compared with anaerobic digestion, oxidation and poly-phenolic condensation easily occur under the aerobic conditions.Under anaerobic conditions, humic substances act as electron accep-

tors for microbial respiration and oxidize organic matter in the envi-ronment (Lovley et al., 1996, 1998; Benz et al., 1998).

Overall, the degree of humification of aerobic digested sludgewas greater than that of anaerobic digested sludge owing to theirbeing more biosynthetic pathways and fewer metabolic pathways.

5. Conclusions

1. Under aerobic or anaerobic conditions, the degradation of VSwas consistent with that of protein, and followed the first-orderkinetic reaction equation respectively.

2. After stabilization of aerobic digestion, degradation of proteinwas mainly limited by hydrolysis under aerobic conditions.

3. After stabilization by anaerobic digestion, degradation of pro-tein was limited by hydrolysis and lack of a metabolic pathwayunder anaerobic conditions.

4. After stabilization, the degree of humification of mesophilic aer-obic digested sludge was greater than that of anaerobic digestedsludge.

Acknowledgements

The work thanks the support from 973 Program(2012CB719801), NSFC project (20977066), and the Key SpecialProgram on the S & T for the Pollution Control and Treatment ofWater Bodies (No. 2011ZX07303-004-03).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.04.081.

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