microbial succession during a composting process as evaluated by denaturing gradient gel...

Microbial succession during a composting process asevaluated by denaturing gradient gel electrophoresis analysis

K. Ishii, M. Fukui and S. TakiiDepartment of Biological Science, Graduate School of Science, Tokyo Metropolitan University, Hachiohji, Tokyo, Japan

180/02/00: received 14 February 2000, revised 16 June 2000 and accepted 21 June 2000

K . I S H I I , M . F U K U I A N D S . T A K I I . 2000. Microbial succession during a laboratory-scale composting

process of garbage was analysed by denaturing gradient gel electrophoresis (DGGE)

combined with measurement of physicochemical parameters such as temperature, pH,

organic acids, total dissolved organic carbon and water-soluble humic substance. From the

temperature changes, a rapid increase from 25 to 58 �C and then a gradual decrease, four

phases were recognized in the process as follows; mesophilic (S), thermophilic (T), cooling

(C) and maturing (M). The polymerase chain reaction-ampli®ed 16S rDNA fragments with

universal (907R) and eubacterial (341F with GC clamp) primers were subjected to DGGE

analysis. Consequently, the DGGE band pattern changed during the composting process.

The direct sequences from DGGE bands were related to those of known genera in the

DNA database. The microbial succession determined by DGGE was summarized as

follows: in the S phase some fermenting bacteria, such as lactobacillus, were present with

the existing organic acids; in the T phase thermophilic bacillus appeared and, after the C

phase, bacterial populations were more complex than in previous phases and the

phylogenetic positions of those populations were relatively distant from strains so far in the

DNA database. Thus, the DGGE method is useful to reveal microbial succession during a

composting process.

INTRODUCTION

Recently, there has been growing interest in composting

because it is such an important process for the treatment of

organic waste, such as municipal solid waste, grass cuttings

and manure, with the resultant compost being used as soil

fertilizer. Composting turns easily degradable organic mat-

ter into stable matter containing a humic-like substance by

passing through a thermophilic stage (Gray et al. 1971;

Finstein and Morris 1975). This waste treatment is useful

in addressing environmental problems such as global

warming, due to the accumulation of man-made green-

house gases in the atmosphere, because the process emits

less CO2 than burning. Monitoring of the microbial succes-

sion is important in the effective management of the com-

posting process as microbes play key roles in the process

and the appearance of some microbes re¯ects the quality of

maturing compost (Macauley et al. 1993). Many early stu-

dies dealt with the isolation and description of various

microbes in compost by the classical culture method

(Webley 1947a; Webley 1947b; Forsyth and Webley 1948;

Shilesky and Maniotis 1969; Gray et al. 1971; Kane and

Mullins 1973; Finstein and Morris 1975; Strom 1985a;

Strom 1985b). Since this method cannot detect non-cultur-

able species, there is a possibility that organisms which are

dif®cult to isolate are predominant. Hardly- or non-cultur-

able organisms may exist, especially in a later phase of the

composting process, because easily utilizable substrates

have been depleted and only dif®cult substrates remain.

Indeed, information about bacteria in the later phases of

the composting process is scant (Miller 1996). In addition,

there has been a lack of investigation into anaerobic ther-

mophilic microbes that are dif®cult to culture, although

composting materials might contain anaerobic environ-

ments (Miller 1996). Recently, phospholipid fatty acid

(PLFA) analysis has been used to characterize microbes in

compost (Hellmann et al. 1997; Herrmann and Shann

1997; Carpenter-Boggs et al. 1998; Klamer and BaÊaÊth

1998). The results from these studies suggested the rapid

changes of different populations that occur during the com-

posting process. However, such results tend to be limited

to culturable microbes or large microbial taxa. Thus, new

methods, which can follow population changes in all poten-

Correspondence to: K. Ishii, Department of Biological Science, Graduate

School of Science, Tokyo Metropolitan University, Hachiohji, Tokyo 192±

0397, Japan (e-mail: [email protected]).

Journal of Applied Microbiology 2000, 89, 768ÿ777

= 2000 The Society for Applied Microbiology

tial organisms at the genus or species level, are required to

investigate microbial succession in the composting process.

Denaturing gradient gel electrophoresis (DGGE) analy-

sis of polymerase chain reaction (PCR)-ampli®ed small

subunit (ssu) rRNA genes was applied to microbial ecology

and this method was able to detect microbes independent

of culture (Muyzer et al. 1993). The ssu rRNA was suitable

for inferring the phylogenetic position of a wide range of

organisms, including bacteria which are dif®cult to culture

and also anaerobes (Woese 1987; Amann et al. 1995).

Thus, this method may provide high-resolution informa-

tion about the succession containing non-culturable

microbes in the composting process.

The object of this study was to reveal the microbial suc-

cession during a composting process, especially later

phases, independent of the culture method. Therefore, we

aimed to investigate the succession of microbial populations

in the composting process in a laboratory system for 45 d

by DGGE analysis.

MATERIALS AND METHODS

Composting material

Garbage composed of mostly food scraps obtained from

restaurants was dried by steam. After being adjusted to

40% (w/w) water content by adding water, 20 kg of well-

mixed garbage was used for composting.

Composting facilities

Laboratory-scale composting was performed in a 30-l poly-

ethylene bin with a hole drilled in its side 5 cm above the

bottom. The composting material, on a stainless mesh ®xed

15 cm above the bottom, was aerated through the hole at a

rate of 10 l kgÿ1 hÿ1 with compressed air. Expanded plastic

material adiabatically covered the composting bin. The

temperature was measured 20 cm below the compost sur-

face. The water content was determined by the weight loss

of a 20-g composting subsample after drying at 105 �C for

20 h and water was added to the composting material in the

bin to adjust the water content to 40% (w/w). This water

content was decided by the ef®ciency of mineralization

(data not shown).

Water-dissolved component measurement

To each 0�5-g composting sample 1 ml ultra-pure water

was added, mixed well and centrifuged at 15 000 g at 4 �Cfor 10 min. The supernatant ¯uids were ®ltered

(MILLEX1-GP 0�22-mm ®lter unit; Millipore, Bedford,

MA, USA). The ®ltrates were used for the analysis of dis-

solved components during the composting process. The

total organic carbon (TOC) in the ®ltrates after diluting

1000� with ultra-pure water was measured by a TOC-

2000 (Shimadzu, Kyoto, Japan). Several organic acids and

amino acids in the ®ltrates were measured by capillary elec-

trophoresis (CIA; Waters, Milford, MA, USA). Brie¯y, the

®ltrates were mixed with 5 mol lÿ1 standard organic and

amino acids and diluted 10� with ultra-pure water. The

electrophoresis was performed at 200 V in supersaturated

sodium tetraborate buffer together with 1/10 volume of

CIA-Pak2 OFM Anion BT (Waters). The absorbance at

252 nm was measured for detection. The standard organic

and amino acids for measurement were oxalate, formate,

fumarate, maleic acid, succinate, a-ketoglutarate, malate,

citrate, tartarate, acetate, pyruvate, propionate, lactate, glu-

tamate, butyrate, levurinate, benzoate and asparaginate. In

order to measure the dissolved humic substance the absor-

bance at 440 nm of ®ltrates was measured. A standard

curve was made from humic acid (Wako, Osaka, Japan)

solution dissolved in 0�02 mol lÿ1 NaOH.

Direct bacterial count

Microbes in composting samples stained with 406-diami-

dino-2-phenylindole dihydrochloride (DAPI; Wako) were

observed and counted with an epi¯uorescence microscope

(BH2-RFCA; Olympus, Tokyo, Japan) as follows. Freeze-

dried composting samples (50 mg) were suspended in 1 ml

sodium phosphate buffer (50 mmol lÿ1; pH 7�6). The sus-

pensions were mixed well and centrifuged at 2000 g for 5

min to collect the cells. The supernatant ¯uids were dis-

carded and this washing procedure repeated three times.

The washed cells were then suspended in 920 ml 50 mmol

lÿ1 sodium phosphate buffer, pH 7�6, mixed with 30 ml

DAPI and left for 5 min in the dark. After mixing again, 1

ml of the solution was ®ltered through a 0�2-mm pore size

Nucleopore1 ®lter (Corning, New York, NY, USA). The

®lter was washed with sterilized water and observed under

the epi¯uorescence microscope using u.v. light. The num-

ber of bacteria was calculated by the average count of a

random ®ve ®elds on each ®lter.

Nucleic acid extraction

To lyse the microbial cells in the composting samples, 0�5 g

glass beads (0�1 mm diameter), 0�4 ml 50 mmol lÿ1 sodium

phosphate buffer (pH 6�8), 0�03 ml 20% sodium dodecyl

sulphate, 0�05 ml 5 mol lÿ1 pyrophosphate, 0�6 ml TE (10

mmol lÿ1 Tris-HCl, pH 8�0, 10 mmol lÿ1 EDTA)-saturated

phenol and 0�1 g freeze-dried composting samples were

combined in a 2-ml plastic tube and shaken vigorously

(2000 rev minÿ1) on a beadbeater (Mikrodismembrator U;

B. Braun Biotech International, Melsungen, Germany) for

1 min. The tubes were centrifuged at 10 000 g for 5 min

769M IC R O B I A L S U C C E S S IO N D U R I NG C OM P O S T IN G

= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 768ÿ777

and the upper phase collected. The lower phases were

extracted twice by the same procedure. Nucleic acids in the

collected upper phase were then precipitated by adding 0�1volumes of 3 mol lÿ1 sodium acetate (pH 5�3) and two

volumes of 99�5% (v/v) ethanol and collected by centrifu-

gation at 10 000 g for 5 min. The nucleic acids were then

dissolved in 0�5ml TE buffer and further puri®ed by the

polyethylene glycol (PEG) precipitation method (Selenska

and KlingmuÈller 1991). The resultant solutions were quan-

ti®ed by absorbance at 260 nm.

Polymerase chain reaction

The PCR ampli®cations were performed in 50-ml volumes

containing approximately 100 ng template DNA, 1�Ex

Taq2 Buffer (Takara Shuzo, Shiga, Japan), 200 mmol lÿ1

dNTP, 25 pmol of each primer and 1�25 units Taq poly-

merase (TaKaRa Ex Taq2; Takara Shuzo). The PCR

cycling was performed using a PC800 (Astec, Fukuoka,

Japan). The temperature program was as follows: 25 cycles

at 94 �C for 0�5 min, 45 �C for 1 min and 72 �C for 1�5 min

with ®nal extension steps at 72 �C for 15 min. The PCR

products were quanti®ed by absorbance at 260 nm.

Polymerase chain reaction primers

A eubacterial 16S rRNA-targeted primer pair (341F with

GC-clamp, 907R) was used for PCR ampli®cation in this

study (Muyzer et al. 1997). The sequences were as follows:

GC-clamp, 50-CGCCCGCCGCGCCCCGCGCCCGTCC

CGCCGCCCCCGCCCG-30; 341F, 50-CCTACGGGAGG

CAGCAG-30 and 907R, 50-CCGTCAATTCCTTTRAG

TTT-30. The 341F without GC-clamp and 907R primers

were used for sequencing reactions.

Denaturing gradient gel electrophoresis

The PCR products were analysed by DGGE according to

Muyzer's protocol (Muyzer et al. 1997) followed by

sequencing. Denaturing gradient gel electrophoresis was

performed with a D-gene system (BioRad Laboratories,

Hercules, CA, USA). Similarly sized PCR products were

separated on a 1�5-mm thick vertical gel containing 8%

(w/v) polyacrylamide (37�5 : 1 acrylamide : bisacrylamide)

and a linear gradient of the denaturants urea and forma-

mide, increasing from 0% at the top of the gel to 80% at

the bottom. The 100% denaturant contained 7 mol lÿ1 urea

and 40% (v/v) formamide. The PCR products (10 ml) were

applied to individual lanes in the gel. Electrophoresis was

performed in a buffer (diluted 100�) of ready-made 50�Tris/Acetic acid/EDTA Buffer (BioRad Laboratories) and

200 V was applied to the submerged gel for 4 h at 60 �C.

After electrophoresis, the gels were stained in an aqueous

ethidium bromide solution (0�5 mg lÿ1) and photographed

on a u.v. (302 nm) transillumination table with a Polaroid

camera (CE-600; Nihon Polaroid, Tokyo, Japan). The

photographs were scanned and the image data downloaded

into a computer. The computerized images were then

inverted to negative images. Small pieces of selected

DGGE bands were excised from the DGGE gel with

Pasteur pipettes and DNA fragments in the gels were

washed and directly reampli®ed with the same primer. The

PCR products were con®rmed by DGGE as a single band

or not and, if isolated, then sequenced. Before sequencing,

the PCR products were puri®ed by a Qiaquick PCR puri®-

cation kit (QIAGEN, Hilden, Germany) according to the

manufacturer's instructions.

Sequencing and phylogenetic analysis

Sequencing reactions were carried out with an ABI

PRISM2 BigDye2 Terminator Cycle Sequencing Ready

Reaction Kit (Perkin Elmer, Foster City, CA, USA)

according to the manufacturer's instructions. The products

were then analysed by an automatic sequencer (model 377

A; Applied Biosystems, San Jose, CA, USA). Sequences

were compared with the compilation of 16S rDNA genes

available in the database (DDBJ, EMBL and GenBank) by

MPsrch (Smith and Waterman 1981) through the DNA

bank at the Ministry of Agriculture, Forestry and Fisheries

of Japan Internet site. Sequences were then aligned with a

CLUSTAL W Ver. 1.7 (Thompson et al. 1994) and dis-

tances determined by a neighbour-joining algorithm

(Saitou and Nei 1987) with the same software.

Phylogenetic trees were drawn with TREEVIEW (Page

1996).

Nucleotide sequence accession numbers

The sequences obtained in this study are available in

DDBJ under accession numbers AB029407±AB029419.

RESULTS

Physical and chemical changes in a composting

process

The temperature increased rapidly after mixing of the com-

post material, reached a maximum value (58 �C) within 9 d

and then gradually declined to ambient level over about 30

d (Fig. 1a). In the early stage, pH increased from 5�3 to 8�3and stabilized at the maximum after 20 d (Fig. 1a). The

changes in these parameters were similar to those in a typi-

cal composting process (Gray et al. 1971). This suggested

that the composting method in this study was as successful

as that using other methods.

770 K . IS H I I E T A L .


The composting process can be divided into four phases:

mesophilic, thermophilic, cooling down and maturing

(Gray et al. 1971; Godden et al. 1983). These four phases

were recognized in the temperature±time (and pH±time)

curves in the present process as follows: mesophilic (0±4 d;

S), thermophilic (4±13 d; T), cooling (13±32 d; C) and

maturing (32±45 d; M) (Fig. 1a).

The total dissolved organic carbon (TDOC) decreased

from 30 to around 15 mg-C g (wet weight)ÿ1 in the ®rst 4

d, rose once on day 9 and then decreased gradually (Fig.

1c). The initial concentrations of organic and amino acids

(mg-C g (wet weight)ÿ1) were as follows: formate, 0�1;malate, 0�6; citrate, 1�1; acetate, 1; lactate, 2�1; levurinate,

1�1; glutamate, 0�8 and asparaginate, 0�2. The above

organic and amino acids (except for acetate) declined in the

®rst 9 d and were exhausted after 9 d. In contrast, acetate

increased in the ®rst 9 d from 0�1 to 0�6 mg-C g (wet

weight)ÿ1 and was then exhausted after 13 d (Fig. 1b). The

sum of the organic acids measured decreased from 5�6 to

0�3 mg-C g (wet weight)ÿ1 for the ®rst 4 d and they were

exhausted after the C phase (Fig. 1c). The water-soluble

humic substance began to increase in the C phase and

maintained its maximum value of 11 mg-C g (wet

weight)ÿ1 until the end of the experiment (Fig. 1c). In con-

trast to TDOC, direct bacterial counts rapidly increased,

although declining to half the value of 4 d after 9 d, then

reaching their maximum and stabilizing at a constant order

(1011 cell g (wet weight)ÿ1) (Fig. 1c).

Microbial succession analysed by denaturing gradient

gel electrophoresis

Since compost contains much humic substance (Gray et al.1971), PCR ampli®cation of nucleic acids from composting

samples is dif®cult. After the dark-brownish colour of the

extracted nucleic acids solution was removed by PEG pre-

cipitation, the PCR ampli®cation was successful except for

the sample on day 0. The PCR ampli®cation of the sample

on d 0 failed because it might contain scarce nucleic acid

and various contaminants such as polysaccharides.

Denaturing gradient gel electrophoresis analysis of these

PCR products with a denaturing gradient ranging from 0

to 80% showed that all the bands were in the denaturing

range from 20 to 50%. Thus, we used a DGGE gel range

from 20 to 50%.

Although nucleic acid samples were subjected to PCR±

DGGE more than three times, no difference in banding

pattern was observed among the PCR products from the

same sample. This showed reproducibility. The band pat-

tern in DGGE gel showed drastic changes over the com-

posting process and the higher the temperature became the

fewer the number of bands (Fig. 2). In addition, the num-

ber of bands increased as composting progressed after the

C phase. While the four phases were determined by tem-

perature and pH changes, DGGE band patterns were dif-

ferent within the same phases (Fig. 2).

Although DNA fragments from major bands in the

DGGE gel were successfully ampli®ed, those from minor

bands failed to be ampli®ed or isolated. This may be

because the major bands tailed and contaminated the minor

bands. Thus, it was dif®cult to isolate the minor bands

from DGGE gel, especially when many bands exist in a

lane. The sequences of successfully ampli®ed DNA frag-

ments were determined to deduce the phylogenetic af®lia-

tion of microbes in the composting process. These

sequence data showed the phylogenetic positions as seen in

Fig. 3. Consequently, analysis of the rDNA libraries, along

with the results from DGGE on the composting samples,

indicated the presence of putative bacteria closely related to

known genera. The similarities of these sequences to those

Fig. 1 Physical and chemical changes in the composting process.

(a) ., Temperature; &, pH. (b) ., Acetate; &, lactate. (c) .,

Total dissolved organic carbon; &, organic acids; ~, soluble

humic substance;� , direct bacterial counts



of most related strains in DDBJ and existing days of

DGGE bands are shown in Table 1.

In the S phase (0±4 d), the DNA sequences from DGGE

bands 4±4 : 1, 4±9 : 1 and 4±4 : 2 were closely related to the

fermenting bacteria Leuconostoc paramesenteroides,Pediococcus acidilactici and Staphylococcus piscifermentans,respectively (97�4, 98�2 and 99�1% similarity for each

recovered sequence over 500 nucleotides), concurrently

with high concentrations of organic acids, especially lactate

(Table 1, Figs 1b and 3). The sequence of DNA 4±13 : 1

was closely related to Bacillus coagulans and B. badius (99�5and 99�3%) that were closely related to each other (Table 1

and Fig. 3).

In the T phase (4±13 d), when the temperature showed

a maximum on day 9, the main DGGE band was only 4±

13 : 1 (B. coagulans or B. badius; Table 1 and Fig. 3). Other

major bands (4±9 : 2, 13±13 : 1 and 13±20 : 1) in this phase

were related to Bacillus sp., Virdibacillus proomii and

Gracilibacillus halotolerans, respectively (99�1, 94�7 and

94�4%), so the organisms represented by these bands might

belong to the Bacillaceae. The DNA sequence of band 13±

13 : 2 was related to Corynebacterium urealyticum (95�3%)

(Table 1 and Fig. 3).

In the C phase (13±32 d), DGGE bands in the T phase

died out and new bands appeared. The new bands were

named 24±45 : 1, 24±45 : 2, 24±45 : 3, 20±45 : 1 and 20±45 :

2, sequences of which were related to Sphingobacteriummultivorum, Alloiococcus otitis, Clostridium fervidus, Cl. ®li-

mentosum and Alcaligenes sp. NKNTAU, respectively (83�4,90�0, 88�2, 85�6 and 91�4%). While the similarity between

band 24±45 : 1 and S. multivorum was relatively low, it

might belong to the Cytophaga±Flavobacterium phylum.

Moreover, similarities between bands 24±45 : 3 and Cl. fer-vidus and bands 20 and 45 : 1 and Cl. ®limentosum were rela-

tively low; these bands might belong to the Clostridiaceae

and furthermore, the latter might belong to cluster XI or

XII de®ned by Collins et al. (1994). The DGGE pattern

and sequence data showed that the organisms in this phase

were different from those in the S phase.

The M phase (32±45 d) was very stable in all respects,

temperature, pH, direct counts, TDOC and soluble humic

substance (Fig. 1). Relative to earlier phases, the changes in

microbial communities re¯ected by the DGGE pattern

were stable and complex (Fig. 2). Only one DGGE band,

43±45 : 1, the DNA sequence of which was related to

Arthrobacter sp. (86�4%) appeared as late as 43 d.

DISCUSSION

For DGGE analysis in this study, the 16S rRNA gene was

targeted because it has large databases, the contents of

which have been increasing, and it is suitable for inferring

phylogenetic relationships (Woese 1987; Amann et al.1995). There is not an accepted value of percentage iden-

tity at which two 16S rRNA genes are concluded to belong

to the same genus or species. It can be quite different for

Fig. 2 Inverted image of the denaturing gradient gel electrophoresis gel stained by ethidium bromide. The number under each lane shows

the sampling day. The band nomenclature follows the A±B :C pattern. A and B show the existing period from A days to B days. C is the

serial number for the bands with the same existing periods

772 K . IS H I I E T A L .


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Bac

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different genera (Fox et al. 1992). In this study, sequence

data obtained from the early phase of the composting pro-

cess had high similarities to most relative sequences in the

database although those from the later phase had relatively

low similarities indicating the scarcity of sequence data

related to microbes in the later phase so far (Table 1). This

suggested that microbes which appeared in the early phase

were often isolated and well investigated; on the other

hand, those in the later phase were dif®cult to culture and/

or were not well investigated. While bacteria existing after

the C phase in the composting process might be dif®cult to

isolate, the phylogenetic positions of those microbes are of

use to select media for the isolation of composting micro-

organisms.

In the DGGE pattern the number of bands decreased as

the temperature rose. This suggests that temperature is a

signi®cant factor in determining the relative advantage of

some population over another. This supported the conten-

tion that temperature is the dominant physicochemical

parameter controlling microbial activity during composting

(McKinley and Vestal 1984). The band patterns were dif-

ferent between the low temperature phases, mesophilic and

maturing, suggesting that the environments in these phases

were different. The difference between these environments

was observed as being due to TDOC, pH and the concen-

tration of organic acids and humic substance in this study

(Fig. 1).

In the S phase, the DNA sequences from the DGGE

bands fell within the cluster of Gram-positive fermenting

bacteria, concurrent with high concentrations of organic

acids (Table 1, Figs 1b and 3). This suggested that the

microbes appearing in this phase might have fermenting

ability enabling them to be isolated. This was supported by

Golueke's suggestion that fermenting bacteria dominated at

the beginning of the composting process (Golueke et al.1954). Usually fermenting bacteria rapidly use easily

degradable substrates and proliferate. This was observed in

the results of a decrease in TDOC, accumulation of organic

acids and an increase in bacterial cell numbers (Figs 1b and

1c).

In the T phase, fermenting bacteria disappeared and dif-

ferent microbes appeared as the pH and temperature

increased. The microbes proliferating in this phase were

related to bacillus. This was supported by previous studies

showing that 87% of isolated thermophilic bacteria in com-

posting were identi®ed as Bacillus spp. (Strom 1985b).

Interestingly, when the temperature showed a maximum

on day 9, there was only one major DGGE band, named

4±13 : 1, whose sequence was related to that of B. coagulansor B. badius (Table 1 and Fig. 3). Bacillus coagulans isolated

from compost by Strom had the ability to grow at 65 �C(Strom 1985b). Also, the acidic environment early in this

phase is suitable for B. coagulans (closely related to the

sequence 4±13 : 1) which requires a slightly low pH value

(6�0) for the initiation of growth (Sneath et al. 1986).

These factors support the dominance of B. coagulans in

this phase. The increase in pH in this phase might be

caused by ammonia production, as evidenced by the strong

ammoniacal odour. This suggested that proteolysis

occurred in this phase. This was supported by the report

of Riffaldi et al. (1986) that proteolytic and ammonia-pro-

ducing bacteria proliferated when the temperature was

high, but were soon extinct. Substantial strains of B. coagu-lans have a proteolytic ability (Sneath et al. 1986). In sum-

mary, the fermentation of carbohydrate interchanged with

proteolysis by thermophilic B. coagulans relatives in the T

phase of this composting system.

Fig. 3 Neighbour-joining tree of partial 16S rRNA sequence

(approx. 550 bp) recovered by denaturing gradient gel

electophoresis bands. Accession numbers appear before the genus

name. The neighbour-joining tree was constructed as described in

the text. Each sequence, except for those from this study, was

obtained from the DDBJ. The numbers on the branches refer to

bootstrap values for 1000 times; only those above 500 are shown

774 K . IS H I I E T A L .


In the C phase, the DGGE patterns and sequence data

showed that the organisms in this phase were different

from those in the S phase. The DGGE bands in the T

phase faded out and new bands appeared. These additional

bands were related to S. multivorum, A. otitis, Cl. fervidus,Cl. ®limentosum and Alcaligenes sp. NKNTAU. The meta-

bolic characters of these microbes could not be inferred

from the phylogenetic positions because the phylogenetic

distance between the sequence of the band and those of

these organisms was, respectively, low (Fig. 3). They

might, at least, degrade the remaining complex compounds

since easily degradable compounds inducing a temperature

increase were lost. The appearance of obligate anaerobes,

such as clostridium, in this phase suggested that an anaero-

bic microenvironment developed within the aggregates of

composting materials.

After the C phase, the DGGE band patterns were more

stable and more complex than in earlier phases (Figs 2 and

3). Only one DGGE band related to Arthrobacter sp. was

added later in the M phase. Most of the species in this

genus were known soil bacteria. These results suggested

that the environment of the later phases in the composting

process was similar to an oligotrophic environment such as

that of soil. Since information on bacterial taxa after a tem-

perature drop was scarce in previous studies because of the

dif®culty of isolation, this provides important documenta-

tion on bacterial communities after the C phase in the com-

posting process and could become a guide for isolation.

The advantage of PLFA analysis was its ability to quan-

tify indicators of organisms as a biomass without cultiva-

tion. The results obtained by this method mainly showed

that fungi and actinomycetes proliferated when the tem-

perature was low and that thermophilic bacteria dominated

when it was high (Hellmann et al. 1997; Herrmann and

Shann 1997; Klamer and BaÊaÊth 1998). The microbial suc-

cession in this study partially disagreed with the reports

using PLFA analysis with respect to the absence of fungi

and the scarcity of actinomycetes detected after the C

phase. Ampli®cation of eukaryotic rRNA genes was

attempted using eukaryotic speci®c primer sets and the

PCR products were only con®rmed from a 4-d sample

(data not shown). There are three possible reasons for the

absence of fungi and the scarcity of actinomycetes. Firstly,

actinomycetes and fungi were generally suitable for drier

conditions than bacteria (Scott 1957). The surface of the

composting materials in this study was wet because the

composting materials were supplied with water every day.

Secondly, fungi are favoured by C : N ratios higher than

those for bacteria and actinomycetes (Grif®n 1985).

Generally, garbage has lower C : N ratios than ®eld waste

and wastepaper. Thirdly, since the growth rates of fungi

and actinomycetes were slower than those of bacteria, they

were not suitable for a laboratory-scale composting system

that progressed more rapidly than ®eld-scale composting

(Godden et al. 1983). In the T phase, acetate was abundant

and the temperature was high. These environments are sui-

table for thermophilic methanogens and former researchers

had detected ether lipids using PLFA analysis (Hellmann

et al. 1997). We tried to amplify the archaeal rRNA gene

using archaea-speci®c primer sets, but the PCR product

was not recognized (data not shown).

The DGGE combining sequence analysis requires the

following re®nements for a more accurate estimation of

microbial succession. Firstly, an excessively complex

microbial population is not suitable for analysis because

sequence analyses of the vast number of DGGE bands

were too laborious and bands on the DGGE gel were often

dif®cult to isolate. Compost is, however, a suitable subject

for DGGE analysis of bacterial populations because of its

relatively simple community structure as shown in Fig. 2.

Secondly, weak bands on DGGE gel were dif®cult to iso-

late and reamplify, because they tended to be contaminated

by strong bands. The only solution to this problem is to

carefully excise the target band as precisely as possible.

Furthermore, DGGE pro®ling cannot provide accurate

quantitative information because of the PCR ampli®cation

bias of genes extracted from mixed microbial populations

(Farrelly et al. 1995; Suzuki and Giovannoni 1996; Polz

and Cavanaugh 1998). Some researchers, however, were

able to ®nd dominant microbes by using the series of

diluted template DNA for PCR ampli®cation (Ferris et al.1997; évreaÊs et al. 1997).

It is unknown whether there are differences in the

microbial succession for different composting processes.

However, composting has speci®c conditions, such as a

temperature increase, humic substance production and the

solid matrix of the substrate. Consequently these conditions

must limit organisms to the speci®c microbes that generally

appeared in the composting process. The results presented

here will help to elucidate general composting processes.

For example, these results are of use to select media for the

isolation of composting micro-organisms. Further study is

needed to clarify microbial nutritional capacities in the

composting process by DGGE combined with the cultiva-

tion method.

ACKNOWLEDGEMENTS

The authors would like to thank Dr Kazuyoshi Suzuki and

Ms Maki Itagaki for their advice and help.

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