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Bioresource Technology 100 (2009) 676682

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Bioresource Technology
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Comparison of organic matter degradation and microbial community during thermophilic composting of two different types of anaerobic sludge
Kiyohiko Nakasaki a,*, Le Thi Hong Tran a, Yoshito Idemoto a, Michiharu Abe a, Analiza Palenzuela Rollon b
a b

Department of Materials Science and Chemical Engineering, Shizuoka University, 3-5-1, Johoku, Naka-ku, Hamamatsu 432-8561, Japan Department of Chemical Engineering, University of the Philippines, Diliman, 1101 Quezon City, Philippines

a r t i c l e

i n f o

a b s t r a c t
Changes in organic matter degradation and microbial communities during thermophilic composting were compared using two different types of anaerobic sludge, one from mesophilic methane fermentation, containing a high concentration of proteins (S-sludge), and the other from thermophilic methane fermentation, containing high concentrations of lipids and bers (K-sludge). The difference in the organic matter degradation rate corresponded to the difference in the organic matter constituents; the CO2 evolution rate was greater in the composting of S-sludge than of K-sludge; moreover, the NH3 evolution resulting from the protein degradation was especially higher in the composting of S-sludge. Then the differences in the microbial communities that contributed to each composting were determined by the PCR-DGGE method. Ureibacillus sp., which is known as a degrader with high organic matter degradation activity, was observed during the composting of S-sludge, whereas Thermobida fusca, which is a well known thermophilic actinomycete that produces enzymes for lignocellulose degradation, were observed during the composting of K-sludge. 2008 Elsevier Ltd. All rights reserved.

Article history: Received 2 May 2008 Received in revised form 23 July 2008 Accepted 24 July 2008 Available online 31 August 2008 Keywords: Anaerobic sludge Composting Microbial community structure Ureibacillus sp. Thermobida fusca

1. Introduction Anaerobic digestion has been used to treat various organic wastes including industrial wastewater, sewage sludge, agricultural wastes and the organic fraction of municipal solid waste (MSW). The residual sludge after anaerobic digestion, i.e., anaerobic sludge, can be treated further via composting in order to mineralize the remaining easily degradable portion of organics and to remove possible pathogens that may survive the anaerobic digestion process. After composting, the anaerobic sludge can be used on farmland as a high-quality hygienic fertilizer (Poggi-Varaldo et al., 1999). In order to manage the composting process efciently, the effects of various operational conditions have been investigated (e.g., Finstein and Morris, 1975; Golueke, 1977; de Bertoldi et al., 1983). Composting efciency is strongly affected by oxygen supply, since the composting efciency is directly linked to the composition and succession of the microbial community during the composting process. Sundberg and Jnsson (2008) demonstrated that increased aeration rate caused the higher microbial activity and increased pH at the biowaste composting and succeeded in shortening the time need to produce a suitable compost product. The most remarkable characteristic of the present study is that a drastic change of the microorganisms environment, from anaerobic to aerobic conditions, occurred at the start of composting. Therefore, microbial succession from anaerobic to aerobic microorganisms
* Corresponding author. Tel.: +81 53 478 1172; fax: +81 53 476 0095. E-mail address: tcknaka@ipc.shizuoka.ac.jp (K. Nakasaki). 0960-8524/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.07.046

was expected to occur as composting progresses. It is difcult, however, to ascertain this microbial succession by the traditional culture-dependent method of plating, since both aerobic and anaerobic incubations must be carried out simultaneously, which requires a great deal of time and labor. PCR-DGGE, a culture-independent molecular biological method, has been used to analyze the genetic diversity of the complex microbial communities in natural environments. In principal, both aerobic and anaerobic microorganisms can be determined at the same time by the PCR-DGGE method. By using the PCR-DGGE method, many researchers have tried to analyze the microbial diversity of composting, and various valuable ndings have been made (e.g., Ishii et al., 2000; Larsen and McCartney, 2000; Ishi and Takii, 2003; Takaku et al., 2006). However, to our knowledge there have been no studies of microbial succession during aerobic composting of anaerobic sludge. The purpose of the present study was to compare aerobic composting with two different types of anaerobic sludge in order to elucidate the changes in organic matter degradation and microbial community structure that occur with the progress of thermophilic composting as measured by the PCR-DGGE method. 2. Methods 2.1. Composting raw materials Two types of residual sludge derived from two different anaerobic digestion processes were used for composting. One type was derived from the methane fermentation process used in S-city

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under mesophilic conditions, where food wastes from the catering industry, i.e., cooking residues and leftovers from lunch boxes, were treated (S-sludge). The operation temperature and the solid retention time were 38 C and 14 days, respectively. The other type was collected from a thermophilic anaerobic digester in K-city, where a mixture of 70% organic fraction of MSW and 30% garbage from restaurants was used (K-sludge). The anaerobic system was operated with high solids content at 55 C. The solid retention time was adjusted to approximately 30 days. Some characteristics of the two different types of anaerobic sludge are shown in Table 1. Two experimental runs, runs I and II, were carried out using Ssludge and K-sludge, respectively as the raw material for composting. The anaerobic sludge was mixed with sawdust as a bulking agent and an inoculum with the trade name Alles G (Matsumoto Laboratory of Microorganisms Co. Ltd., Matsumoto, Japan) in a ratio of 5:14:1 to create a raw composting mixture. At the start of all of the experiments, the pH level was adjusted to around 8.0 by the addition of slaked lime, and the moisture content was adjusted to 60% by the addition of distilled water. 2.2. Composting operation The experimental system used in the present study was the same as that described in our previous paper (Nakasaki et al., 2004). A mini-reactor consisting of a cylinder (45 mm in diameter, 100 mm in depth) made of Pyrex glass was used. The air was rst introduced into a ask containing NaOH solution to eliminate CO2, then passed through a bubbler prior to reaching the reactor to saturate the air with moisture. The aeration rate was maintained at 5 mL/min throughout the experiment. It was ascertained that the aeration rate was sufcient for maintaining aerobic conditions in the preliminary experiment. The raw composting mixture was placed into the reactor at a wet weight of 12 g for runs I and II. Five reactors in total were used for the composting in each experimental runs I and II in order to measure the change in pH, moisture content, microbial cell density, and microbial diversity. The reactors themselves were placed in an incubator (Model LTI-1000; EYELA Co. Ltd., Tokyo, Japan) to regulate the composting temperature. The temperature was raised from room temperature to a set point of 60 C at a constant rate of 2 C/h, and the temperature of 60 C was then maintained until the eighth day, when the composting operation was stopped. The exhaust gas from the composting reactor was introduced into a 5-l plastic bag made of polyvinyl uoride (Tedlar BagTM; Omi Odoair Service Co. Ltd., Omihachiman, Japan) for 12 h, and the plastic bag was changed twice daily at 12-h intervals. The volume of exhaust gas captured in the plastic bag was measured, and the concentrations of CO2 and NH3 were analyzed with Kitagawa gas detector tubes (Komyo Rikagaku Kogyo K.K., To-

kyo, Japan). CO2 and NH3 analysis employing the Kitagawa gas detector tube has been used for the rapidity and simplicity of the measurement (Nakasaki et al., 2001), although a method using gas chromatography (GC) is more accurate than that using a gas detector tube to measure the gas concentration. We conrmed, however, that the difference in the analytical results was not large between the two methods in our preliminary experiment: 4.53 0.19% for the GC method and 4.5 0.3% for the detector tube method as the results of the evaluation test using approximately 4.5% CO2 gas standard. The CO2 and NH3 evolution rates during each 12-h period were determined by measuring the CO2 and NH3 concentrations and the exhaust gas volume. The cumulative CO2 and NH3 that had evolved up to a certain composting time were then calculated. The composting material inside the reactor was mixed by a sterilized spatula every 24 h. The compost sample was withdrawn from the mini-reactor and subjected to physicochemical and microbial analyzes. 2.3. Measurement of physicochemical parameters The pH value was measured with a pH meter (Model F-8; Horiba Co. Ltd., Tokyo, Japan) in a suspension of compost mixed with distilled water (1:9 w/w) by weight. The electrical conductivity of a 1:10 (w/w) compost:distilled water suspension was determined with a conductivity meter (Model DS-14; Horiba Co. Ltd., Tokyo, Japan). The moisture content was obtained by drying the sample at 105 C for three days in a drying oven (DS600; Yamato Scientic Co. Ltd., Japan). Total carbon and nitrogen of the solid compost sample and water extract of the sample (solid to distilled water ratio of 1:10 w/w) were determined using a Vario MAX CN analyzer (Elementar Analysesysteme GmbH, Hanau, Germany). 2.4. Microbial analyzes For the microbial analyzes, we measured the cell density of microorganisms by a dilution plating method on Trypticase-soy agar and determined the microbial succession by the PCR-DGGE method. The composition of the Trypticase-soy agar medium was as follows: trypticase peptone, 17 g; phytone peptone, 3 g; K2HPO4, 2.5 g; NaCl, 5 g; glucose, 2.5 g; agar, 20 g; distilled water, 1000 mL; pH 7.3. The incubation temperature was the same as the temperature of composting, 60 C, with an incubation period of three days. The cell density of the microorganisms was expressed in terms of colony-forming units per unit of dry weight of the composting material (CFU/g-DW). In the PCR-DGGE analysis, DNA was extracted from the compost samples and PCR was conducted using the DNA as a template. The PCR products were then subjected to DGGE analysis. Each step of the experimental procedure was as follows. 2.5. DNA extraction from compost sample

Table 1 Some characteristics of two different types of anaerobic sludge Run I (S-sludge) Moisture content (%) Initial pH (%) Ash content (%) Organic content (%) NFE (%) Proteins (%) Lipids (%) Fibers (%) C-content (%) N-content (%) C/N ratio () 84.1 6.88 27.8 72.2 18 43.3 9.2 1.7 41.6 6.21 6.7 Run II (K-sludge) 76 8.2 36.5 63.5 13.4 15.4 19.9 14.8 37.9 2.27 16.7

Organic constituents, NFE (nitrogen free extract), Proteins, Lipids, and Fibers were measured by the standard method for food analysis.

The DNA was extracted from the compost samples taken on days 0, 2, 4, 6 and 8 by the ISOIL method for the Beads Beating kit (Nippon Gene Co. Ltd., Toyama, Japan). A 0.1 g wet weight composting sample was put into the 2 mL plastic tube and suspended by adding 950 lL of the Lysis Solution BB together with 50 lL of the Lysis Solution 20S. The suspension was then shaken vigorously (5500 rev/min) on a beadbeater (Micro Smash; TOMY MEDICO., Ltd, Tokyo, Japan) for 45 s and centrifuged at 12,000 g for 1 min. The supernatant was collected and mixed with the Purication Solution. The mixture was deproteinized with chloroform and centrifuged at 12,000 g for 15 min. The top supernatant including DNA was precipitated by adding the Precipitation Solution and then centrifuged at 16,000 g for 15 min at 4 C, and washed with the Wash Solution. The pellet of DNA was puried by the addition of

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70% ethanol with Ethachinmate and then allowed to dry in a vacuum for 20 min. To eliminate any contaminants, Microspin S-300 HR Columns (GE Healthcare UK Ltd., Buckinghamshire, England) were used in the nal purication step. 2.6. PCR of puried DNA PCR was performed with AmpliTaq GoldTM (Applied Biosystems, CA, USA) by an automated thermal cycler (TP240, TaKaRa Biomedicals, Shiga, Japan). A eubacterial 16S rRNA-target primer pair (518 F with GC-clamp, 907 R) was used: GC-clamp, 50 -CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC-30 (Muyzer et al., 2004), forward primer 518F (518-536), 50 - CAG CAG CCG CGG TAA TAC-30 (Edwards et al., 1989) and reverse primer 907R (907-926), 50 -CCG TCA ATT CCT TTR AGT TT-30 (Ishii et al., 2000). The temperature program was as follows: an initial 94 C denaturing step for 7 min, followed by 20 cycles of amplication (60 s of denaturation at 94 C, 60 s of annealing starting at 65 C and decreased by 1 C every two cycles to 56 C, and 120 s of extension at 72 C), and then 15 additional cycles of amplication (60 s at 94 C, 60 s at 55 C, and 120 s at 72 C) and a nal extension of 10 min at 72 C. The 518F without the GC-clamp and 907R primers was used for the sequencing reactions. 2.7. Denaturing gradient gel electrophoresis (DGGE) DGGE was performed using the Dcode DGGE Complete System (BioRad Laboratories, CA, USA). The PCR product was mixed with an equal volume of 2x gel loading dye (5 mL Bromophenol blue, 5 mL Xylene cyanol, 7 mL Lycelon and 2.5 mL distilled water) and applied onto 8% (w/v) polyacrylamide gels in 0.5x TAE (20 mM Tris-acetate; pH = 7.4, 10 mM acetate, 0.5 mM Na2EDTA) with a denaturing gradient ranging from 30% to 60%. One hundred percent of denaturant corresponded to 7 M urea and 40% (w/v) formamaide. The gradient gel was cast using a Gradient Delivery system, Model 475 (BioRad Laboratories). Electrophoresis was carried out at a constant voltage of 200 V and at 60 C. After electrophoresis for 3.5 h, the gel was stained with ethidium bromide (BioRad Laboratories) in 0.5x TAE for 30 min, then rinsed with water and photographed with a Epi-Light UV FA500 (AISIN, Saitama, Japan). 2.8. Sequencing of the DNA After DGGE, bands of interest were excised from the gel and DNA from each band was extracted using the QIAEX II GEL Extraction Kit (QIAGEN, Hilden, Germany). The extracted DNAs were then re-amplied and re-analyzed by DGGE to conrm that the single bands obtained were then sequenced by the standard procedures as follows. The sequencing reaction was carried out with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). Moreover, sequencing was performed using an ABI PRISM 3100 Genetic Analyzer System (Applied Biosystems, CA, USA). The sequence was compared to 16 S rRNA genes available in the database (DDBJ, EMBL and GenBank) and the genus including the relative strains of the sequences were determined using sequence match analysis in the ribosomal database project II (RDP-II) data and analysis services (Cole et al., 2007).
Fig. 1. The courses of the CO2 evolution rate and the cumulative emission of C, EC during composting in runs I and II. Error bars show 95% condence intervals for the averaged values (n = 3).

3. Results and discussion The courses of the CO2 evolution rate and the cumulative emission of C (EC) during runs I and II are compared in Fig. 1. The EC,

which corresponds to the degree of organic matter decomposition, was dened as the molar ratio of carbon lost as CO2 to the carbon contained in the anaerobic sludge. In run I, the CO2 began to evolve into exhausted gas after 12 h of composting and peaked at 36 h, then decreased as the composting progressed. The CO2 evolution rate of run I was larger than that of run II throughout the composting period except in the earliest stages. These results seemed to indicate that the easily degradable organic matter content in the S-sludge was greater than that of K-sludge. The nal values of EC attained for runs I and II were 51.6% and 42.2%, respectively. The slopes of the curves were gentle early in the composting, until the 8th day. It can therefore be deduced that the anaerobic sludges used in the present study would be rather stable, and that little of the easily biodegradable organic matter remained in the sludge, i.e., the organic matter in the raw materials for anaerobic digestion had been reduced and transferred to biogas including methane, carbon dioxide, and hydrogen during an anaerobic digestion process before the anaerobic sludges were produced. As was not shown here in detail, the pH dropped slightly to 7.88 during the rst two days in both runs I and II, probably due to an accumulation of organic acids associated with sludge degradation and/or to the consumption of ammonium ions by microorganisms for their growth. During the next 2 days, the pH value of run I increased once to 8.74 and nally reached 8.14 at the end of composting. In contrast, the pH value for run II never exceeded 8 until the composting experiment ceased. The nal pH value of run II was 7.25. The fact that the pH value was always larger in run I than in run II seems to be a result of the S-sludge containing a higher concentration of proteins (see Table 1). More NH3 would be produced with the decomposition of proteins in run I than in run II. The moisture content of the composting material is an important environmental variable, as it provides a medium for the transport of dissolved nutrients that are required by microorganisms, and the optimal moisture content for composting has been empirically determined to be in the range of 6040%. The moisture content for both experimental runs was gradually decreased from the initial value of 60% by drying with aeration; the moisture content

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was maintained at a level higher than 40% until the 6th day and nally reached 37.7% for run I and 27.8% for run II. Therefore, we considered the moisture content of both composting runs to have been maintained in the optimal range mostly as a result of the composting. The initial EC value of run I was 6.13 ms/cm, and that value decreased to 2.19 ms/cm by the 8th day of composting. On the other hand, the initial EC value of run II was as low as 2.03, and it increased slightly to 2.52 by the 8th day of composting. We believe the EC value did not change so much in run II because the EC in run II was low enough from the start of composting. The initial C/N ratio of the solid compost samples was 30.6 for run I and 47.2 for run II, and the ratio reached similar values, 52.5 and 51.4, respectively, by the 8th day of composting. In this study, the raw composting material contained sawdust as a bulking agent, and it would not have been degraded in the high-rate composting with short reaction time used in this study. Therefore, we concluded that the relatively high value of the C/N ratio of the compost product was a result of the remaining sawdust. By contrast, the C/N ratio of the water extract of the compost samples was initially 6.6 for run I and 5.2 for run II, and the ratio declined further to 3.4 for run I and 3.7 for run II. As mentioned above, both the EC value and the two types of C/N ratio of the compost product for runs I and II showed similar values. In addition, when the compost product of run II was subjected to a germination assay according to the method advocated by Zucconi et al. (1981), the germination index (GI) obtained was 55.0%. A GI of 50% has been used as an indicator of phytotoxin-free composts (Zucconi et al., 1981; Bernal et al., 1998), and our measurements indicated that the compost product of this study was matured to a phytotoxin-free state in merely 8 days, which was faster than the composting of poultry manure at over 40 days (Kato et al., 2005), food waste at over 25 days (Chikae et al., 2006), and municipal solid waste at over 90 days (Said-Pullicino et al., 2007), although all these time periods were roughly estimated based on the gures supplied in the respective studies. We believe our results reect the fact that the raw composting materials of this study were the sludge after anaerobic fermentation and that most of the easily degradable organic components must have been degraded before the composting began. The courses of the NH3 evolution rate and the cumulative emission of N, EN during composting in runs I and II are shown in Fig. 2. The EN was dened as the molar ratio of nitrogen lost as NH3 to the nitrogen contained in the anaerobic sludge. The NH3 evolution rate peaked after 36 h of composting in run I and gradually decreased to a low level by the end of composting. Conversely, no clear peak of the NH3 evolution rate was observed in run II, and the value of the NH3 evolution rate was quite small, and far smaller than that of run I throughout the composting. The nal value of EN attained in run I was 23.5%, whereas the EN for run II was merely 6.8%. The EN for run II was relatively higher than that expected based on the NH3 evolution rate because the N content in the K-sludge was extremely low. It is well known that the lower C/N ratios will bring a higher decomposition rate and higher nitrogen loss, and the emission of ammonia is suppressed by adjusting the C/N ratio of the composting material. In the present case, it was also ascertained that the composting with the lower C/N ratio (run I) resulted in higher NH3 emission by comparing it with the composting that involved a higher C/N ratio (run II). Fig. 3 shows the relationship between the cumulative emission of C and N during the composting process of two different types of anaerobic sludge. The curves for the two runs did not coincide throughout the composting, and a greater amount of N was emitted in run I than in run II even if the same amount of C was emitted. These results may be dependent on the fact that the organic matter constituents of the two types of sludge differed signicantly.

Fig. 2. The courses of the NH3 evolution rate and the cumulative emission of N, EN during runs I and II. Error bars show 95% condence intervals for the averaged values (n = 3).

Fig. 3. Relationship between the cumulative emissions of C, EC and N, EN for composting in runs I and II. Error bars show 95% condence intervals for the averaged values (n = 3).

The cell densities in run I at the beginning of the composting process were approximately 106 CFU/g-DW. This cell density drastically increased to a value greater than 108 CFU/g-DW by the 2nd day, and then leveled off at approximately 109 CFU/g-DW until the end of the process. While the cell density in run II at the start of the process was one order of magnitude higher than that in run I and it reached a similar value of 109 CFU/g-DW that lasted until the end of the process (detailed data not shown). It was considered that the different organic matter constituents of the anaerobic sludge would require different groups of microorganisms for their degradation. It is, however, difcult to distinguish the kinds of microorganisms in the composting in runs I and II by the appearance of their colonies. The PCR-DGGE analysis was expected to be effective to determine the differences in the microbial communities in these composting runs.

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K. Nakasaki et al. / Bioresource Technology 100 (2009) 676682 Table 2 Closely related sequences to denaturing gradient gel electrophoresis bands Band name Run I Day0 I-0-a I-0-b I-0-c I-0-d I-0-e Day 8 I-8-f I-8-g I-8-h Run II Day0 II-0-i II-0-j Day 6 II-6k II-6-l Closely related sequences (accession no.) Identity (%) Accession no. Genus containing related sequences

The courses of the PCR-DGGE pattern during composting in runs I and II are summarized in Fig. 4. The DGGE prole at the start of run I showed more variety than that of run II. This may correspond to the fact that the K-sludge was derived from thermophilic methane fermentation, and the thermophilic temperature will restrict the variety of microorganisms. In both types of composting, the DGGE patterns changed drastically from the 0th day to the 2nd day, indicating that microbial succession from anaerobic to aerobic microorganisms occurred immediately after the start of composting, although a characteristic band, II-0-j, which existed in the raw sludge and was still dominant until the end of the 60 C composting, may result from this sludge having been derived from thermophilic anaerobic digestion. The DGGE patterns did not change much after 4th day and became rather stable. Ishii et al. (2000) found more variety in the types of microorganisms that existed in the garbage composting. One of the reasons for the existence of a smaller variety of microorganisms in the present study is that the anaerobic sludge contains a smaller amount of easily degradable organic material than the garbage itself before anaerobic digestion. The changing pattern and number of bands differed for the two types of composting in runs I and II, indicating that the organic matter degradation of each required different kinds of microorganisms. Since the temperature was kept constant during the composting, the microbial succession that we measured here was thus affected by changes in the quality of organic materials, and the pH value resulted from organic matter degradation. Sequences corresponding to some prominent bands were determined and the results of the phylogenetic analyzes of these sequences are shown in Table 2. DNA purication and sequence analysis of band I-8-h did not succeed; the microbe corresponding to that band could not be identied. The sequence derived from band I-0-a was closely related to the genus Petrimonas, whose name was proposed by Grabowski et al.

Petrimonas sulfuriphila strain BN3 (AY570690) Uncultured bacterium (AB274510) Uncultured bacterium (EF174268) Sporosarcina aquimarina strain N3ZF-14 (EF619410) Tepidaerobacter syntrophicus strain: JL (AB106353) Ureibacilus koreensis HC148 (DQ348072) Uncultured bacterium (EF 174268) Unidentied

100 99 99 100 100

AB291607 AB291608 AB291609 AB291610 AB291611

Petrimonas Proteiniphilum unclassied Caldilineacea Sporosarcina Tepidanaerobacter

100 99 -

AB291612 AB291613 -

Ureibacillus unclassied Caldilineacea -

Bacteroides coprosuis strain PC139 (AF319778) Bacteroides coprosuis strain PC139 (AF319778) Symbiobacterium thermophilum IAM 14863 (AP006840) Thermobida fusca YX (CP000088)

100 99

AB291697 AB291698

Bacteroides Bacteroides

100

AB291699

Symbiobacterium

100

AB291700

Thermobida

The sample nomenclature follows the X-Y-Z pattern. X, Y, and Z show run No. of composting, the age in day after start of composting, and the band name, respectively.

Fig. 4. The courses of the DGGE pattern, the inverted image of the gel stained by ethidium bromide, during composting in runs I and II. The number over each lane shows the age in days after the start of composting. The bands represented by letters were isolated and sequenced.

(2005). Most of the sequences afliated with the genus Petrimonas are registered as uncultured bacterium except for Petrimonas sulfuriphila BN3 (Grabowski et al., 2005). P. sulfuriphila BN3 is described as a mesophilic, anaerobic, fermentative bacterium. Therefore, it makes sense that the bacterium corresponding to band I-0-a, which is a relative of P. sulfuriphila BN3, dominated in the sludge digested under mesophilic, anaerobic conditions (S-sludge). The nucleotide blast of the sequences corresponding to band I0-b and I-0-c resulted in the nding that most of the related sequences were of uncultured bacteria (see Table 2). Sequence match analysis at the RDP-II web site revealed that the genus corresponding to the band I-0-b was Proteiniphilum and that the genus corresponding to band I-0-c was unclassied Caldilineacea. As in the case of Petrimonas, most of the sequences relating to the genus Proteiniphilum are designated as uncultured bacterium, except for culturable strains of Proteiniphilum acetatigenes TB107 and TB6-6, which have been observed in a methane fermentation reactor and described as proteolytic anaerobic bacteria (Chen and Dong, 2005). The family Caldilineacea is the lineage of phylum Chloroexi, which was formerly known as green non-sulfur bacteria and has been recognized as a typical bacterial cluster containing a number of diverse environmental clones with only a few cultured representatives (Kragelund et al., 2007). The most relative strain of the sequence corresponding to band I-0-c was anaerobic lamentous bacterium YMTK-2 (accession no. AB109438, identity 90%), which was isolated from an upow anaerobic sludge blanket (UASB) reactor as the rst cultured mesophilic species of Cloroexi subphylum I (Yamada et al., 2005). Sequence analysis of band I-8-g conrmed that it was identical to band I-0-c. Although the strain YMTK-2 was reported to be a strictly anaerobic microorganism, the bacteria corresponding to band I-0-c (or I-8-g) still remained dominant during the aerobic composting process. The reason why bacteria

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corresponding to the band I-0-c (or I-8-g) existed in the aerobic condition of composting has not been claried yet; however, the subphylum I of Chloroexi contains the most diverse environmental clones among the four subphyla (Sekiguchi et al., 2003). Furthermore, recent molecular biological studies have revealed a remarkable abundance of microbes belonging to Chloroexi subphylum I, also in the activated sludges (Bjrnsson et al., 2002; Juretschko et al., 2002; Kindaichi et al., 2004); therefore, the predominance of bacteria corresponding to band I-0-c (or I-8-g) throughout the composting process is a plausible phenomenon. Since it was suggested previously that populations belonging to Chloroexi subphylum I contributed to the degradation of carbohydrates and other cellular components, such as amino acids in the sludge (Yamada et al., 2005), the bacteria corresponding to band I-0-c (or I-8-g) may have contributed to the degradation of the protein fraction primarily contained in the S-sludge. DGGE bands I-0-d and I-0-e were closely related to Sporosarcina aquimarina (identity 100%) and Tepidaerobacter syntrophicus (identity 100%), respectively. S. aquimarina was reported to be a facultative anaerobic bacteria (Yoon et al., 2001), and the bacteria corresponding to band I-0-d became a minor population during the composting process. In a recent study, three strains of T. syntrophicus were isolated from sludges digested anaerobically under thermophilic conditions, and these bacteria were described to be strictly anaerobic bacterium (Sekiguchi et al., 2006). The fact that band I-0-e faded out during the aerobic compositing process was consistent with the nding that the bacterium corresponding to this band was a relative of a strictly anaerobic bacterium. The sequence of band I-8-f was closely related to Ureibacillus koreensis HC148 (identity 100%), as shown in Table 2. Ureibacillus spp. are often detected in the composting processes and are well known by their high ability for organic matter degradation, although U. koreensis has not yet been detected in the composting process. Vargas-Garcia et al. (2006) investigated the inuence of Ureibacillus inoculation on the production of humic-like substances during the composting of horticultural wastes, and found out that the strain included in Ureibacillus thermosphaericus was effective for inducing a higher humication level. Kim et al. (2006) succeeded in isolating two strains whose close relatives were U. thermosphaericus and Ureibacillus terrenus from cotton compost and proposed a new name for the two stains, Ureibacillus suwonensis sp. nov. The bacteria corresponding to the band I-8-f would have contributed to the vigorous organic matter degradation in the thermophilic composting of protein-rich anaerobic sludge, S-sludge. Bands II-0-i and II-0-j were determined to be close relatives of Bacteroides coprosuis, which is a typical Bacteroides species. Bacteroides spp. were identied in the microbial granules from a UASB reactor, and uorescence in situ hybridization (FISH) ascertained that Bacteroides spp. were concentrated at a depth of approximately 800 lm below the surface of the granule (Tay et al., 2002). Hernon et al. (2006) indicated that the dominating fermentative bacteria of mesophilic sludge were of the Bacteroidetes and Spirochaetes classes. Saddler and Khan (1979) found that a mesophilic anaerobe, a member of the Bacteroidaceae family isolated from a cellulose-enrichment culture, digested cellulosic materials while producing H2, CO2, cellobiose, glucose, and acetic acid as the sole volatile acid in an anaerobic digestion system. The genus Bacteroides is well-known for comprising some of the bacteria present in anaerobic digesters, and its major role in the fermentation system is to break down macromolecules such as protein, starch, cellulose, ber, and chitin (Ponpium et al., 2000). The sequence of the bands II-6-k and II-6-l that appeared at the later stages of composting in run II were closely related to those of Symbiobacterium thermophilum (identity 100%) and Thermobida fusca YX (identity 100%), respectively. S. thermophilum is a thermophile, which shows normal growth only in co-culture with its sup-

porting bacteria (Ueda et al., 2004). Symbiobacterium species have been frequently obtained from compost, soil, animal feces, and the contents of intestinal tracts, as well as from feeds (Ueda et al., 2001). Thermobida fusca, a thermophilic actinomycete, is known to produce enzymes for the degradation of lignocellulose. In addition, a certain strain of T. fusca isolated from composted horse manure was found to produce a number of lignocelluloses degrading hydrolases when grown on cellulose or hemicellulose as the carbon source (Beki et al., 2003). It is well known that cellulose in the composting materials begins to be degraded in the later stages of composting after easily degradable organic materials are almost completely consumed. Thermobida fusca observed in the present composting would contribute to the degradation of lignocellulosic materials in the later stages of composting of K-sludge which contained signicant amount of bers. 4. Conclusions The effects of the organic constituents of compost raw material on the microbial community during thermophilic composting were investigated by using two different types of anaerobic sludge. The difference in the organic matter degradation rate seemed to correspond well to the difference in the organic matter constituents. The NH3 evolution that resulted from the protein degradation was especially higher in the composting of anaerobic sludge with a higher concentration of proteins. The cell densities of thermophilic microorganisms measured by a culture-dependent method were similar when composting was performed with different raw materials; however, the kinds of microorganisms contributing to the organic matter degradation for each composting as measured by the culture-independent method differed signicantly. Two characteristic microorganisms were identied; one was Ureibacillus sp., which is known as a degrader with high organic materials degradation activity, and which was identied in the composting of anaerobic sludge containing a high percentage of proteins, and the other was Thermobida sp. which is known as a thermophilic actinomycete possessing high lignocellulose degradation activity, and which was identied in the composting of anaerobic sludge containing high concentrations of bers and lipids. It can thus be concluded that the microbial community structure corresponded well to the quality of organic matter constituents of anaerobic sludge used in the composting. Acknowledgement We are grateful to the ASEAN University NetworkSoutheast Asia Engineering Education Development Network (AUNSEEDNet). References
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