KSCE Journal of Civil Engineering

Vol. 10, No. 5 / September 2006

pp. 319~323

Environmental Engineering

Vol. 10, No. 5 / September 2006 319

Microbial Activity and Population Structure of Anaerobic Sludge Alternately

Exposed to Mesophilic and Thermophilic Conditions

By Young Chae Song*, Jung Hui Woo**, Sang Jo Kwon***, and Sung Cheol Koh****

···································································································································································································································

Abstract

The microbial community structures of anaerobic sludge, alternately exposed to mesophilic and thermophilic condition, wereinvestigated and their microbial activities under mesophilic and thermophilic conditions. PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) profiles for the eubacterial and archaebacterial communities from thermophilic sludgealternately exposed to mesophilic condition (TSEM) and mesophilic sludge alternately exposed to thermophilic condition (MSET)had a few populations in common, these are probably thermophilic microorganisms. The population profiles for archaebacterialcommunities of TSEM and MSET were quite different from each other. For TSEM under thermophilic condition, the specificmethanogenic activity (SMA) was 159.6mL CH4/g VSS/d, which was higher than 106.3mL CH4/g VSS/d under mesophiliccondition. The SMA for MSET under both temperature conditions was as much as those of TSEM. The lag phase period for MSETin the batch culture was around 2 times longer than TSEM, but the lag periods for both sludges under thermophilic condition wereshorter than those under mesophilic condition. The acidogenic activity for both sludge types in MSET was slightly higher thanTSEM under the thermophilic and mesophilic conditions. The specific hydrolytic activity for the two sludge types was higher underthe mesophilic condition, and the hydrolysis potential of TSEM incubated under mesophilic condition was higher than other cases.

Keywords: anaerobic, sludge, thermophilic, mesophilic, methanogenesis, acidogenesis, PCR-DGGE

···································································································································································································································

1. Introduction

The performance of anaerobic digestion is significantly

dependent on the temperature conditions (Adrie and Bert, 1999;

Daniel and Lucie, 2001). The mesophilic temperature (31-38oC)

has been widely used for anaerobic stabilization of wastewater

sludge. However, mesophilic digestion usually requires a long

retention time of over 20 days, but is not so efficient in reduction

of volatile solids and deactivation of pathogenic organisms (Aoki

and Kawase, 1991; Han et al., 1997; Song et al., 2003). To

overcome these limitations, interest in thermophilic digestion

(49-57oC) with the higher metabolic rate has increased.

Although better performance in reduction of volatile solids, and

deactivation of pathogenic organisms, can be obtained from

thermophilic digestion, the effluent quality and ability to dewater

the residual sludge are poor, and requires additional energy to

heat the digester (Han et al., 1997). Especially, thermophilic

digestion is a little more sensitive to environmental changes (Han

et al., 1997). These characteristics of the mesophilic and

thermophilic digestions might be intrinsically attributable to the

differences of physiological characteristics of the two distinct

groups of anaerobic microorganisms under mesophilic and

thermophilic conditions. Therefore, the better understanding on

the anaerobic microorganisms could be lead to efficient

anaerobic digestion, taking advantages of both mesophilic and

thermophilic digestions by limiting their weaknesses (Han et al.,

1997; Song et al., 2003). Recently, the temperature tolerant or

temperature facultative anaerobic microorganisms, which are

highly active under both mesophilic and thermophilic temperature

ranges, were found (Vandenburgh et al., 2002). The temperature

tolerant anaerobic microorganisms could be a good breach to

develop a novel effective anaerobic digestion processes (Song et

al., 2003).

In this study, the microbial community structures of anaerobic

sludge, alternately exposed to mesophilic and thermophilic

condition, were investigated and their microbial activities

including hydrolysis, acidogenesis and methanogenesis under

mesophilic and thermophilic conditions.

2. Materials and Methods

2.1 Sources of Anaerobic Sludges

A two stage anaerobic digestion system for the stabilization of

sewage sludge, consisting of a mesophilic flow through digester

(28L of volume, 18days of HRT) and a thermophilic retention

digester (3L of volume, 2days of HRTs), was operated for over

100days at steady state. In the two stage digestion system, the

anaerobic sludge contained in each digester was alternatively

exposed to mesophilic and thermophilic conditions by

exchanging the sludge between both digesters, like described in

previous study (Song et al., 2003). The thermophilic sludge, which

was alternatively exposed to mesophilic condition (TSEM), from

the thermophilic digester and the mesophilic sludge, which was

alternatively exposed to thermophilic condition (MSET), from

*Professor, Division of Civil and Environmental Engineering, Korea Maritime University, Korea (Corresponding Author, E-mail: soyc@mail.hhu.ac.kr)

**Researcher, Research Institute of Marine Science and Technology, Korea (E-mail: sky-woo@hanmail.net)

***Researcher, Korea Atomic Energy Research Institute, Korea (E-mail: sangjo79@hanmail.net)

****Professor, Division of Civil and Environmental Engineering, Korea Maritime University, Korea (E-mail: skoh@mail.hhu.ac.kr)

Young Chae Song, Jung Hui Woo, Sang Jo Kwon, and Sung Cheol Koh

320 KSCE Journal of Civil Engineering

the mesophilic digester were used for this study. A mesophilic

anaerobic sludge (M-SS) was also obtained from a single stage

anaerobic digestion system (12.5L and 20days of HRTs),

operated for over 100days at steady state, but the sludge was

only used to compare the microbial community structure for the

TSEM and MSET. Table 1 summarized the characteristics of the

sludges from the anaerobic systems for the microbial community

analysis and their activity test.

2.2 Microbial Community Analysis

In order to compare the microbial community structure, nucleic

acid extractions for the anaerobic sludges were performed using

FastDNA SPIN Kit for Soil (BIO101 Laboratory, CA, U.S.A)

according to the manufacturer’s instructions with the following

modifications: 0.4 g of sludge sample was collected from each

reactor and transferred to 2 ml Lysing Matrix E Tube of the

FastDNA SPIN Kit for Soil. Each sample was mixed with 978 l

sodium phosphate buffer and 122 l MT buffer. Bead beating

was done at 2500 g for 80 seconds. The crude DNA extract

was purified by passing through a SPINTM Filter containing a

DNA-adsorbing silica matrix as described by the manufacturer.

Purified DNA was eluted from the matrix by 50 l sterile buffer

solution provided with the Kit. For community analysis of

Eubacteria and Archaea, a nested approach was used by utilizing

primers 27F, 1492R (Muyzer et al., 1993) and pRA46f, univ907

(Oureas et al., 1997) respectively. Amplification was performed

using a Perkin-Elmer DNA Thermo Cycler model (GeneAmp

PCR System 2400). A Eubacteria-specific amplification was

performed using primers F341-GC and R518 (Muyzer et al.,

1993), and the primers pARCH340f and pARCH519r were used

for the amplification of archaebactrial communities (Oureas et

al., 1997). DGGE was performed using the Bio-Rad DCode

system (Bio-Rad laboratories, Hercules, CA) according to the

manufacturer’s protocol.

2.3 Microbial Activity Test

The anaerobic microbial activity tests for the two kinds of

anaerobic sludges, TSEM and MSET, were performed in 125mL

serum bottles. For the tests, 25mL of the anaerobic sludge and

25mL of sewage sludge as the substrate were added into the

serum bottle, and 25mL of buffer solution containing sodium

carbonate of 1,500mg/L was also added. Then, the initial pH was

adjusted to 7.0-7.2 with 0.01N HCl and 0.01N NaOH, and

flushed with nitrogen gas to make oxygen free in the head space

of the bottle, and sealed with n-butyl rubber stopper and

aluminum crimps. For each anaerobic sludge, the prepared

bottles were incubated in the temperature controlled at 35oC and

55oC and darkened shaking incubators. The methane productions

from the test bottles were intermittently monitored by inserting a

graduated syringe through the rubber stopper. In order to monitor

the changes of the biochemical activities during the tests, more

bottles for each sludge were prepared under the same conditions,

and opened in duplicate at predetermined times. Control test

bottles were run in all experiments to determine background

biochemical activities.

2.4 Analysis and Calculation

The methane content in biogas was analyzed with a gas

chromatography (GowMac Series 580) using Porapak Q column

(6ft×1/8", Stainless steel) and a thermal conductivity detector.

The methane production was calculated with the procedure

described by Song (1995), and converted to STP state. The level

of COD (Chemical oxygen demand) and SS (Suspended solids)

for the samples from the opened bottles were measured

according to Standard Methods (1995) and the pH was measured

by pH meter (Orion 370). The compositions and levels of VFA

(Volatile fatty acids) were determined with a HPLC (High

performance liquid chromatography) (DX 500) equipped with a

ultraviolet detector using Aminex HPX-87H column.

The modified Gompertz equation, eqn(1), was used to describe

the biochemical reactions and kinetics in the batch anaerobic

culture (Lay et al., 1997).

(1)

where, P is the cumulative productions of methane in the

estimation of methanogenic activity test, and the hydrolyzed

monomer in the estimation of hydrolysis activity. In the later

case, the cumulative monomer was expressed as COD, which

was obtained from sum of the remaining soluble COD at time, t,

and the COD equivalent value of cumulative methane production.

Pu is the potential of the anaerobic reaction, is the lag-phase time,

and e is exp(1). Rm is the maximum reaction rate, and the specific

microbial activities for hydrolysis and methanogenesis were

calculated by Rm/VSS (mL CH4/g VSS/d, or mg COD/g VSS/d).

The model parameters were estimated using curve fitting

software, Curve Expert 1.3.

3. Results and Discussion

3.1 Population Communities

The communities of eubacteria and archaea from the anaerobic

sludge alternately exposed to thermophilic and mesophilic

conditions were simultaneously investigated.

The community profiles in the both sludges, which were MSET

(mesophilic sludge alternately exposed to thermophilic condition)

and TSEM (thermophilic sludge alternately exposed to mesophilic

condition), had a few populations in common (E6, E7, and E19),

P Pu exp expRm e

Pu

------------ t–– 1+=

Table 1. Characteristics of Anaerobic Sludges and Substrate Used

Content pHVSS

(mg/L)TCOD(mg/L)

SCOD(mg/L)

Alkalinity(mg/L as CaCO3)

M-SS (Mesophilic sludge-single stage) 7.12 13,410 13,118 1,773 2,866

TSEM (Thermophilic sludge alternately exposed to mesophilic condition) 7.30 16,700 20,932 1,084 2,780

MSET (Mesophilic sludge alternately exposed to thermophilic condition) 7.23 12,200 13,060 514 3,132

Substrate (Sewage sludge) 6.46 9,050 25,391 2,798 1,152

Microbial Activity and Population Structure of Anaerobic Sludge Alternately Exposed to Mesophilic and Thermophilic Conditions

Vol. 10, No. 5 / September 2006 321

these are probably heat tolerant microorganisms. However, the

populations (E17 and E48) were observed in MSET only.

Populations E6 and E7 were dominant in TSEM while E8 and

E19 were dominant in MSET. DNA sequence analysis data have

shown that E6 and E7 appeared to be closely related to the

species of acetate-oxidizing Desulfobacterum and Clostridium,

respectively. E17 and E19 turned out to be close to Syntrophus

sp. and a fatty acid oxidizing syntroph. The population E48 was

relatively closely related to Cytophaga marinoflava. Populations

E1 and E2 were only observed in the feed sewage sludge and

TSEM. They turned out to be closely to Cytophaga sp. and

Bacterioides sp., respectively. Archaebacterial communities

have also been analyzed using DGGE technique (Fig. 1(B)).

Dominant populations were mostly detected in the M-SS. All of

the rest samples from the seeding sludge and TSEM barely

showed distinctive bands. Essentially all the populations turned

out to be archaeons as taxa. The two bands (band # 1 and band 2)

in the M-SS matched the uncultured archaeon (95%) and

Haloferax sulfurifontis (98%), respectively. The archaeon was

reportedly involved in syntrophism in the sediment lake.

Therefore, the population (band # 1) might have a syntrophic

relationship with the fatty acid-oxidizing eubacteria. The

remaining 7 bands (bands # 3, 4, 5, 6, 7, 8 and 9) in the MSET

matched the Methanoculleus sp. (99%), Methanoculleus

submarinus (92%), Methanoculleus sp. (90%), anaerobic

methanogenic archaeon E15-10 (85%), Methanosaeta sp. (87%),

Methanogenic archaeon F4/B-1 (100%), and Methanoculleus

submarinus (95%), respectively. The taxa that match bands # 3,

4, 5, 8, and 9 have been methanogens from a digester treating

wastewater or a lake sediment. Most of these archaeons appear

to be involved in methanogenesis in association with the

eubacterial anaerobes observed in the above (Fig. 1(A)).

Therefore, all these data indicate that the identified taxa are likely

to occur in the reactors in this study.

3.2 Methanogenic Activity

The effect of the temperature condition on the methanogenic

activity for the both anaerobic sludge was shown in Fig. 2 and

Table 2. For the MSET, the methanogenic potentials under

mesophilic condition were around 255.4mL CH4/g VS, which

was higher than that under thermophilic condition. For the

TSEM, the methanogenic potentials under mesophilic and

thermophilic conditions were 322.9 and 283.1 mL CH4/g VS.

The results indicate that the methanogenic potentials under

mesophilic condition were always higher than those under

thermophilic condition, but for both temperature conditions, the

Fig. 1. PCR-DGGE Profiles of 16S rDNA Fragments of TSEM

and MSET Eubacterial (A) and Archaebacterial (B) Com-

munities

Fig. 2. Cumulative Methane Production of the Sludges Under the Thermo- and Mesophilic Conditions

Table 2. Methanogenic Activity for Two Types of the Anaerobic Sludges Under Mesophilic and Thermophilic Conditions

Content Temp.Methane potential(mL CH4/g VS)

SMA(mL CH4/g VSS/d)

Lag phase time (hr)Correlation

(r)Stand.error

MSET35 255.4 110.3 20.64 0.993 11.44

55 237.1 155.5 9.41 0.992 11.32

TSEM35 322.9 106.3 9.32 0.989 5.995

55 283.2 159.6 4.99 0.992 4.479

Young Chae Song, Jung Hui Woo, Sang Jo Kwon, and Sung Cheol Koh

322 KSCE Journal of Civil Engineering

potentials of TSEM were higher than those of MSET. The

Specific methanogenic activity (SMA) for TSEM under

thermophilic conditions was 159.6mL CH4/g VSS/d, which was

higher than 106.3mL CH4/g VSS/d under mesophilic condition,

indicating higher metabolic rate of the sludge under thermophilic

condition.

Interestingly, the SMA for MSET under both temperature

conditions was as much as those of TSEM. This indicates that

the both sludge contain some temperature tolerant or facultative

anaerobic microorganisms, active to mesophilic, as well as

thermophilic condition. The lag phase period for MSET in the

batch culture was around 2 times longer than the MSET, but the

periods for the both sludges under thermophilic condition were

shorter than those under mesophilic condition. These results

suggest that metabolic rates of anaerobic sludge were dependent

on the temperature condition as well as microbial community

structures.

3.3 Acidogenic and Hydrolytic Activity

During the microbial activity tests, the trend of VFA

composition and SCOD in the serum bottles was shown in Fig.

3. Under both mesophilic and thermophilic conditions, the

composition of VFA for MSET was not different from the

TSEM, but the C3 (propionic acid as HAc) fraction of the VFA

for MSET was higher at thermophilic condition, and for TSEM,

at mesophilic condition. These suggest that acetogenic activity

for TSEM was higher at thermophilic condition, and for MSET,

at mesophilic condition. In the final stage of the activity test for

both sludges, the remaining VFAs under thermophilic condition

were slightly higher than those under mesophilic condition. This

indicates that the substrate affinity of methanogens under

mesophilic condition was higher than that under thermophilic

condition.

The hydrolytic activities of two kinds of sludge were estimated

by fitting the cumulative hydrolyzed monomer, which is the sum

of SCOD remained in the serum bottle and the equivalent COD

of cumulative methane production. The specific hydrolytic

activity of MSET was higher than that of TSEM, and under

thermophilic condition for both sludges, was higher than those

under mesophilic condition (Fig. 4). This was as a result of the

higher activity of hydrolytic enzyme under thermophilic condition.

However, the hydrolytic potential from TSEM under mesophilic

condition was the higher than the other cases.

Fig. 3. VFA Production Profiles (Left) from the Anaerobic Activity Tests of (a) the MSET and (b) TSEM, and Soluble COD (Right) Under

Mesophilic and Thermophilic Conditions

Fig. 4. Hydrolytic Activity of Two Anaerobic Sludges under Mesophilic and Thermophilic Conditions

Microbial Activity and Population Structure of Anaerobic Sludge Alternately Exposed to Mesophilic and Thermophilic Conditions

Vol. 10, No. 5 / September 2006 323

4. Conclusions

The biochemical activities of the mesophilic and thermophilic

sludge alternately exposed to thermophilic and mesophilic

conditions, respectively, were dependent on the temperature

condition, as well as their microbial community structures. The

anaerobic sludge was easily adapted to thermophilic condition

than mesophilic one. The methanogenic activity under thermophilic

condition was higher than under mesophilic condition, which

was not affected by sludge types. The methanogenic potential of

the thermophilic sludge was higher than that of the mesophilic

sludge, but the potential for both sludges under mesophilic

condition was higher than those under thermophilic condition.

The substrate affinity of the sludge was not considerably affected

by the microbial community structures, but the affinity was

slightly higher under mesophilic condition than thermophilic

condition. The hydrolytic activity under thermophilic condition

was higher than the mesophilic condition, and the hydrolytic

potential of the thermophilic sludge was higher under mesophilic

condition. These results would be useful for development of a

novel efficient anaerobic digestion process using thermophilic or

temperature facultative anaerobic microorganisms.

Acknowledgement

This work was supported by the academic research program

(Grant No. 2001-S-0159) of Korea Energy Management

Corporation.

References

Adrie, V. and Bert, H. (1999). “Effect of temperature on hydrolysis rates

of selected biowaste components.” Bio. Tech., Vol. 69, No. 3, pp.

249-254.

Aoki, N. and Kawase, M. (1991). “Development of high performance

thermophilic two-phase digestion process.” Wat. Sci. Tech., Vol. 23,

pp. 1147-1156.

Daniel, I. M. and Lucie, M. (2001). “The effect of temperature on

slaughterhouse wastewater treatment in anaerobic sequencing batch

reactors.” Bio. Tech., Vol. 76, No. 2, pp. 91-98.

Han, Y., Sung, S., and Dague, R. R. (1997). “Temperature-phased

anaerobic digestion of wastewater sludge.” Wat. Sci. Tech., Vol. 36,

No. 6-7, pp. 367-374.

Lay, J.J., Li, Y.Y., and Noike, T. (1997). “Influences of pH and moisture

content on the methane production in high-solids sludge digestion.”

Wat. Res., Vol. 13, No. 6, pp. 1518-1524.

Song, Y.C. (1995). High-rate Methane Fermentation of the Organic

Solid Waste. Ph. D thesis, Dept. of Civil Engineering, KAIST.

Song, Y.C., Kwon, S.J., and Woo, J.H. (2003). “Mesophilic and

thermophilic temperature co-phase anaerobic digestion compared

with single-stage mesophilic- and thermophilic digestion of sewage

sludge.” Wat. Res., Vol. 38, pp. 1653-1662.

Song, Y.C., Woo, J.H., Kwon, S.J., Park, I.S., and Yoo, J.S. (2003).

Influence of Temperature on the Biochemical Activity of the sludges

from the Thermophilic-Mesophilic Cophase Anaerobic Digestion

System, Korean Society of Env. Eng., Oct..

Van Lier, J. B. (1996). “Limitation of thermophilic anaerobic

wastewater treatment and the consequences for process design.”

Antonie van Leeuwenhoek, Vol. 69, pp. 1-14.

Vandenburgh, S.R. and Ellis, T.G. (2002). “Effect of varying solids

concentration and organic loading on the performance oftemperature

phased anaerobic digestion process.” Water Environ. Res., Vol. 74,

No. 2, pp. 142-148.

(Received February 17, 2006/Accepted August 10, 2006)