Abstract
A methanogenic bioreactor that utilized wastepaper was developed and operated at 55°C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.
Waste utilization is a major concern in energy development and environmental improvement. In this regard, various attempts have been made to develop effective microbial digestion systems (6). We study the application of thermophilic methane fermentation in the reduction and utilization of municipal solid wastes. Although the operation of a thermophilic reactor consumes more electrical energy than that by a mesophilic reactor, it enables effective degradation of substrates and production of methane in a short fermentation period (1). We expect that the fermentation will contribute to the rapid and efficient processing of municipal solid wastes in urban districts. In Tokyo, half of the combustible waste consists of paper (22). Recently, utilization of the markedly increasing amount of shredded office paper has become a matter of great concern; it is nonrecyclable because shredded cellulose fiber is not suited for papermaking.
Conversion of cellulose to methane is mediated by four microbial populations: cellulolytic microbes, noncellulolytic saccharolytic microbes, syntrophic hydrogen-generating bacteria, and methane-producing members of the domain Archaea (2). Generally, the initial hydrolysis is the rate-limiting step in microbial conversion; the efficiency of cellulose degradation markedly affects the methane formation activity in the methanogenic microbial system (2, 11, 15). Therefore, characterization of the cellulose-digesting microbial population is important for the development of efficient fermentation systems.
This paper describes the isolation and characterization of a Clostridium sp. that effectively degrades cellulosic wastes. We operated a methanogenic bioreactor that primarily digested office paper and detected the presence of a group of effective cellulose-digesting clostridia by molecular ecological analysis. An attempt to cultivate and enrich the effective cellulose digesters enabled the successful isolation and identification of a member of this group; this member belongs to a new species that is closely related to Clostridium thermocellum and Clostridium straminisolvens.
MATERIALS AND METHODS
Microbial strains and medium composition.
Clostridium sp. strain EBR45 was obtained in this study. The strain has been deposited in the NITE Biological Resource Center (Kisarazu, Japan) and is available as Clostridium sp. strain NBRC101661. C. thermocellum JCM 9323 and C. straminisolvens IAM 15707 (10) were obtained from the Japan Collection of Microorganisms (RIKEN, Wako, Japan) and the IAM Culture Collection (Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan), respectively. Escherichia coli DH5α (Takara-shuzo, Kyoto, Japan) was used as a host for DNA manipulation. The M solution used for cultivation of Clostridium spp. was prepared by mixing 980 ml of basal solution with 10 ml each of vitamin solution and mineral solution. The basal solution had the following composition (in grams per liter; all of the chemicals were purchased from Kokusan, Tokyo, Japan, if not indicated otherwise): KH2PO4, 0.4; K2HPO4 · 3H2O, 0.4; NH4Cl, 1.0; MgCl2 · 6H2O, 0.1; yeast extract (Difco Laboratories, Detroit, Mich.), 0.2; NaHCO3, 6.0; cysteine-HCl · H2O, 0.5; Na2S · 9H2O (Yoneyama, Osaka, Japan), 0.25; and resazurin, 0.001. The vitamin solution had the following composition (in milligrams per liter): biotin, 2.0; folic acid, 2.0; pyridoxine hydrochloride, 10; thiamine HCl, 5.0; riboflavin, 5.0; nicotinic acid, 5.0; dl-calcium pantothenate, 5.0; vitamin B12, 0.1; p-aminobenzoic acid, 5.0; and lipoic acid, 5.0. The mineral solution had the following composition (in grams per liter): nitrilotriacetic acid, 4.5; FeCl2 · 4H2O, 0.4; CoCl2 · 6H2O, 0.12; AlK · (SO4)2, 0.01; NaCl, 1.0; CaCl2, 0.02; Na2MoO4 · 2H2O, 0.01; MnCl2 · 4H2O, 0.1; ZnCl2, 0.1; H3BO3, 0.01; CuSO4 · 5H2O, 0.01; and NiCl2, 0.02. M solution was supplied with various cellulosic substrates to prepare each culture medium. Mp medium contained 0.5 g (wt/vol) of cellulose powder (type D; Advantec, Tokyo, Japan) in 50 ml of M solution per flask. Mf medium contained a filter paper strip (1 cm by 8 cm; approximately 0.05 g; grade 1; Whatman, Brentford, United Kingdom) in 10 ml of M solution per tube. Mc medium contained 0.5% (wt/vol) cellobiose. Anaerobic cultivation was performed in the following anaerobic growth chambers: Concept 400 (Ruskin, West Yorkshire, United Kingdom) and Anoxomat Mark II (Mart Microbiology, Lichtenvoorde, The Netherlands). The systems were introduced with an N2-H2-CO2 (8:1:1) atmosphere and operated at 55°C. E. coli was cultured at 37°C in Luria-Bertani liquid medium (13) that had the following composition (in grams per liter): tryptone (Difco), 10; yeast extract (Difco), 5; and NaCl, 5. Agar was supplied at 1% when required.
Operation of a cellulose-digesting bioreactor and analytical conditions.
A 4.0-liter anaerobic thermophilic (55°C) bioreactor with a 3.5-liter working volume was operated by continuous feeding of artificial municipal waste, which primarily consisted of office paper used for photocopying and a typical food ingredient in Japan (a dish called Makunouchi-bento). The wastes were first physically mashed, suspended in tap water, and supplied continuously (150 ml/day) with an automatic pumping system. The composition of the waste suspension was as follows (in grams per liter): total solids, 58; volatile total solids, 55; suspended solids, 50; volatile suspended solids, 48; chemical oxygen demand (dichromate method), 66; biochemical oxygen demand, 13; total sugar, 49; total cellulose, 32; pH 5.4. The composition analyses followed the standard protocol that has been conventionally used in water quality testing. The sludge was moderately agitated (ca. 100 rpm). The initial sludge was supplied from a methane fermentation reactor, which was operated at 55°C in a manner similar to that described above by feeding of artificial food waste. During the operation, the sludge and atmospheric space samples were collected every week and checked for pH and residual solid sugar content (for sludge) and methane content (for atmosphere). Residual solid sugar weight was measured by the phenol-sulfuric acid reaction (7). Methane content was measured by gas chromatography (model GC323; GL Science, Inc., Tokyo, Japan) according to the manufacturer's instructions.
Enrichment culture and analytical conditions.
All of the enrichment cultures were performed without shaking at 55°C in a 100-ml top-sealed bottle flask with a working volume of 50 ml. The methanogenic sludge collected from the bioreactor was added at 10% (vol/vol) to Mp medium (containing cellulose powder). After 7 days of cultivation, the resultant culture broth was added at 10% (vol/vol) to fresh Mp medium and cultured for another 7 days. Inoculation and cultivation at a 1% inoculation rate were then repeated four times successively in a similar manner. The resultant enriched culture was subjected to a comprehensive 16S rRNA gene sequencing study, as well as to a second enrichment step.
The second enrichment step was performed as follows. A 0.1-ml volume of the above-described culture was inoculated into 10 ml of Mf medium (containing a filter paper strip) prepared in a top-sealed tube and cultured at 55°C for 5 days without shaking. Next, a part of the substrate filter paper was retrieved from the culture broth, rinsed three times with 20 ml of sterile M solution, and inoculated into fresh Mf medium. This successive inoculation-and-cultivation process was repeated until the culture had a unique microbial content. The microbial content of the culture broth was examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis (see below). For colony isolation of Clostridium sp. strain EBR45, pure culture broth was inoculated onto solid Mc medium (containing 1.0% cellobiose and 1.5% agar) prepared in a petri dish and incubated at 55°C for 5 days with an Anoxomat anaerobic cultivation system.
Wastepaper digestion.
A 50-ml volume of M solution supplied with 0.3 g of photocopy paper (six pieces of photocopy paper each measuring 1 cm by 8 cm) was inoculated with a seed culture of Clostridium sp. strain EBR45, C. thermocellum, or C. straminisolvens at 1% and incubated for 3 days at 55°C. The seed culture was performed in 10 ml of Mc medium for 5 days at 55°C. Following incubation, the residual solid material was collected by centrifugation; washed sequentially with 30 ml each of (i) 0.9% NaCl, (ii) 5% NaOH, (iii) 0.5% acetic acid, and (iv) distilled water (steps ii and iii were repeated twice); and weighed after drying at 180°C for 2 h. For small-scale culture, 5 ml of M solution supplied with a single piece of the above photocopy paper was prepared in a test tube and similarly processed.
Physiological characterization of strain EBR45 and microscopic observation.
The methods used for physiological characterization of strain EBR45 followed the standard protocol that has been conventionally used in bacterial systematics (9). The optimum growth conditions were determined by cultivating strain EBR45 in Mc liquid medium. For acid production, strain EBR45 was cultured in M solution supplied with various substrates (see Table 1) at 1%. The organic acid content was measured with a high-performance liquid chromatography system (class VP; Shimadzu, Kyoto, Japan) equipped with an electric conductivity monitor (CDD-10A; Shimadzu). The samples were separated at 40°C with a Shimpack SPR-H column (Shimadzu) according to the manufacturer's protocol. The strain EBR45 cells were observed with an Axioskop 2 Plus optical microscope (Zeiss, Oberkochen, Germany) and a scanning electron microscope (model 3500N; Hitachi, Tokyo, Japan) according to the manufacturer's instructions. The specimen for scanning electron microscopy was prepared by the osmium fixation protocol and critical-point drying (9).
TABLE 1.
Physiological properties of Clostridium sp. strain EBR45 compared with those of C. thermocellum and C. straminisolvens
| Characteristic | Clostridium sp. strain EBR45 | C. thermocellum | C. straminisolvens |
|---|---|---|---|
| Gram reaction | Variable | − | NSa |
| Cell shape | Rod | Rod | Rod |
| Spore shape | Oval | Oval | Oval |
| Anaerobic growth | + | + | + |
| Optimum growth | |||
| pH | 7.5 | 7.3 | 7.5 |
| Temp (°C) | 55-60 | 60-64 | 50-55 |
| Flagella | + | + | + |
| Sulfate reduction | − | − | − |
| Acid production from: | |||
| Cellulose | + | + | + |
| Cellobiose | + | + | + |
| Glucose | − | + | − |
| Fructose | − | − | − |
| Galactose | − | − | NS |
| Glycerol | − | − | NS |
| Lactose | − | − | − |
| Maltose | − | − | NS |
| Mannitol | − | − | − |
| Mannose | − | − | − |
| Sorbitol | − | − | NS |
| Sucrose | − | − | − |
| Trehalose | − | − | NS |
NS, not shown in the previous report.
16S rRNA gene cloning, nucleotide sequencing, and phylogeny.
Total DNA was extracted from the cellular precipitate of the culture broth by using a bacterial genomic DNA purification kit (Edge BioSystems, Gaithersburg, MD). The resultant DNA was subjected to PCR with primers B8F (5′-AGAGTTTGATCCTGGCTCAG; nucleotides [nt] 8 to 27 by E. coli numbering) and B1500R (5′-AAGGAGGTGATCCAGCCGCA; nt 1508 to 1484 by E. coli numbering) for clostridia. PCR was performed on a T1 Thermocycler (Biometra, Göttingen, Germany) with Ex Taq polymerase (Takara-shuzo, Kyoto, Japan). The composition of the final 50-μl reaction mixture was as recommended by the manufacturer. The PCR protocol included an initial denaturation period of 5 min at 94°C; 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min; 72°C for 3 min; and incubation at 4°C until further processing. The PCR amplicons were purified with Gene Clean kit II (Funakoshi) and TA cloned onto pT7-Blue (Novagen). The ligated DNA was introduced into E. coli DH5α by the standard transformation method (13). Plasmids were extracted with the QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany). After checking the sequence heterogeneity by restriction fragment length polymorphism (RFLP) analysis with HaeIII restriction endonuclease, the nucleotide sequence of the 16S rRNA gene clone was determined with a BigDye Terminator v3.1 cycle sequencing kit on an ABI3100 automated DNA sequencer (Applied Biosystems, Foster City, CA).
Sequence data analyses.
The nucleotide sequences were compared with the sequences in the GenBank/EMBL/DDBJ nucleotide sequence databases by the BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST/) and the SEQUENCE_MATCH program and the Ribosomal Database Project (RDP) database (5). All of the nucleotide sequences were confirmed to be suitable for possible chimera construction with the CHECK_CHIMERA program at the RDP website (http://rdp8.cme.msu.edu/html/). The sequences were aligned by the ClustalW program (20). Neighbor-joining phylogeny (18) was constructed with the NJ plot program (16), and bootstrapping (8) was used to estimate the reliability of the phylogenetic reconstructions (1,000 replicates). The reference nucleotide sequences used in the tree construction were obtained from the GenBank/EMBL/DDBJ nucleotide sequence databases.
Microbial community structure analysis.
The microbial community structure of methanogenic sludge was examined by the PCR-based techniques with genomic DNA extracted from the sludge samples as templates. DNA extraction was performed with a QIAamp DNA Stool kit (QIAGEN) according to the manufacturer's protocol. For DGGE analysis, the partial 16S rRNA gene fragments were amplified by PCR with the following primers: 341F (5′-CCTACGGGAGGCAGCAG; nt 341 to 357 by E. coli numbering) with a 5′-terminal GC clamp and 534R (5′-ATTACCGCGGCTGCTGG; nt 534 to 518 by E. coli numbering) for eubacteria and CT110F (5′-AACGCGTGAGCAACCTGCC; nt 129 to 149 by E. coli numbering) and CT325R (5′-TCCCGTAGAGTCTGG; nt 349 to 334 by E. coli numbering) with a 5′-terminal GC clamp for the group III clostridia. The PCR protocol used was that described above.
DGGE was performed as originally described by Muyzer et al. (14) on the D-code Universal Mutation Detection System for DGGE (Bio-Rad). Samples containing approximately equal amounts of PCR amplicons were loaded onto 10% (wt/vol) polyacrylamide gels (37.5:1 acrylamide/bisacrylamide ratio) with a denaturing gradient ranging from 20% to 70% of the denaturant; 100% of the denaturant contained 7 M urea and 40% (vol/vol) formamide. Electrophoresis was carried out at 60°C and 200 V (180 min). Following electrophoresis, the gel was stained with SYBR Green (Molecular Probes) and visualized on an image analyzer (FluorImager; Amersham Pharmacia).
For specific detection of strain EBR45, primers 1F (5′-CATAACGAGGTGGCATCACTTTG; nt 282 to 304 by strain EBR45 numbering) and 1R (5′-CCTTGGGTACCGTCATTA; nt 572 to 555 by strain EBR45 numbering) were used for amplification of the specific 290-bp fragment. The amplicon was analyzed by agarose gel electrophoresis.
Nucleotide sequence accession numbers.
The 16S rRNA gene nucleotide sequences reported in this paper have been submitted to GenBank/EMBL/DDBJ under the accession numbers shown in Fig. 2.
FIG. 2.
Unrooted tree showing phylogenetic branches of the 16S rRNA gene clones obtained in this study. The 16S rRNA gene sequences derived from the first-step enrichment culture (in boldface) and the pure cells of two strains (inversed boxes) were aligned with those of known organisms and several uncultured clones obtained from the database. The Clostridiales group number that corresponds to each cluster is shown at the right. The tree, constructed by the neighbor-joining method, was based on a comparison of aligned positions of 1,200 nt (excluding deleted and ambiguously aligned sites). Each bootstrap value is expressed as a percentage of 1,000 replications. Values above 80% are given at branching points. The accession numbers of the sequences are in parentheses. Bar, 2% sequence divergence.
RESULTS
Bioreactor operation and microbial community structure analysis.
A thermophilic methanogenic bioreactor was operated by continuous feeding with artificial municipal waste which contained homogenized photocopy paper at approximately 3% (see Materials and Methods). Figure 1 shows the methane production and the residual solid sugar concentration during a 98-day operation, which included typical periods of high and low methane formation efficiency. The initial 14 days (days 0 to 14) was a nonfeeding period. After the restart of feeding, methane production started; the methane yield increased for 21 days (days 14 to 35) and then decreased. After 28 days of moderate production (days 42 to 70), methane formation activity was abolished (days 77 to 98). With a decrease in the amount of methane, there was an increase in the amount of residual solid sugar.
FIG. 1.
Methanogenic bioreactor operation and microbial community structure analyses. The graphs show the methane production (closed squares), solid sugar content (closed circles), and pH (closed triangles) of the fermentation sludge during the 98-day operation of the bioreactor. The two DGGE images and one agarose gel image demonstrate the transition of the microbial community structure in the fermentation sludge. Each lane corresponds to the operation date shown in the upper graph. DGGE analyses were performed with the universal primers for eubacteria and the specific primers for group III clostridia. DGGE separates the various 16S rRNA gene fragments according to nucleotide content (14). Specific detection of strain EBR45 was performed by PCR with primers specific for the bacteria. The specific 290-bp fragment was analyzed by agarose gel electrophoresis. L, liter(s).
The fermentation sludge was examined for the microbial community structure by PCR-DGGE analysis throughout the 98-day operation (Fig. 1). Two primer sets were used for the analysis; one was designed for the general detection of eubacteria, and the other was designed for the specific detection of group III clostridia. Evidence has indicated that group III contains effective cellulose-digesting strains (3, 10, 17, 21). The result revealed a marked transition of the bacterial population during the fermentation period. The wide detection of a eubacterial population with the universal primers revealed the presence of both types; one type is independent of fermentation efficiency, and the other occurs in specific periods. On the other hand, the organisms detected by Clostridium-specific primers mostly occurred in a phase-specific manner (Fig. 1). Among them, the bands that appeared in the high-performance period were expected to be derived from effective clostridia that have high cellulose-digesting activity.
Enrichment of cellulose-digesting bacteria.
To isolate the microorganisms that effectively digested cellulose in the bioreactor, a sludge sample was collected when effective methane production was observed (day 35) and used as a seed for an enrichment culture. For the enrichment, Mp medium containing cellulose powder as the sole carbon source was used (see Materials and Methods). The resultant enriched culture was assessed for microbial community structure by a comprehensive 16S rRNA gene sequencing study. As shown in Fig. 2, the 16S rRNA gene sequences that were determined could be categorized into various subgroups in the cluster of Clostridium and related genera. Some sequences, particularly those belonging to group IV, showed a marked divergence from the known species.
Isolation of Clostridium spp. from the enrichment culture.
To isolate effective cellulose-degrading Clostridium spp. from the above-described culture, we performed a second step of enrichment cultivation. The enrichment was performed with Mf medium, which was supplied with filter paper as the substrate, as well as the bait carrier of microbial cells (see Materials and Methods). The successive inoculation-and-cultivation procedure was expected to enrich the organisms that were tightly associated with the cellulose fibers. Each culture generation was checked for microbial content diversity by DGGE analysis with the bacterial universal primers. Successive cultivation was repeated until the culture had a pure microbial content.
We obtained monocultures of two different Clostridium spp.; one corresponds to EBR-02E-0045, and the other corresponds to EBR-02E-0046. Phylogenetic analysis (Fig. 2) has shown that the former belongs to cluster III, which includes C. thermocellum and C. straminisolvens, and the latter belongs to a novel group called the 16SX subgroup in the RDP database (http://rdp.cme.msu.edu/index.jsp). The sequence similarity percentages of EBR-02E-0045 against the 16S rRNA gene sequences of C. thermocellum and C. straminisolvens were 93.4% and 94.6%, respectively. EBR-02E-0045 was also close to Clostridium sp. strain JC3, which was recently identified in our different bioreactor fed with synthetic medium containing cellulose powder (19). EBR-02E-0046 did not cluster with the sequence of any known species, but it was closely related to the 16S rRNA gene sequences under accession numbers U27710 and AJ229251. The latter sequence corresponds to an isolate described by Chin et al. (4).
Further, we tried to purify the above-described organisms by colony isolation under anaerobic conditions (see Materials and Methods). The strain corresponding to EBR-02E-0045 was successfully isolated as a single colony, but the strain corresponding to EBR-02E-0046 did not form colonies. The former organism will hereafter be referred to as Clostridium sp. strain EBR45. Strain EBR45 showed effective growth after recovery from frozen storage for 1 month at −80°C. On the other hand, the putative Clostridium strain corresponding to EBR-02E-0046 was not viable after frozen storage. Therefore, we could not continue the characterization of that strain.
Characterization of Clostridium sp. strain EBR45.
The physiological properties of Clostridium sp. strain EBR45 are summarized in Table 1. In brief, the organism showed features similar to those that have been previously described for C. thermocellum (6) and C. straminisolvens (10). Similar to C. straminisolvens, but not C. thermocellum, strain EBR45 was unable to utilize glucose and was tolerant of oxygen. Strain EBR45 formed normal-sized colonies even in an atmosphere containing 4% O2. Fermentation balances of strain EBR45 and C. thermocellum JCM9323 cultured in Mc liquid medium containing 0.2% cellobiose for 3 days are shown in Table 2. Microscopic observation revealed that strain EBR45 cells exhibited a typical morphology of clostridia. It had rod-shaped vegetative cells (0.4 to 0.5 by 2.0 to 5.0 μm) and formed oval spores (Fig. 3A). The presence of flagella was also observed (Fig. 3B). Scanning electron microscopic observation revealed that strain EBR45 cells markedly associated with cellulose fibers (Fig. 3C and D). Notably, abundant cells were frequently found inside the fibers (Fig. 3D).
TABLE 2.
Products of cellobiose fermentation by Clostridium sp. strain EBR45 and C. thermocellum
| Substrate or product | Amt degraded or produceda by:
|
|
|---|---|---|
| Strain EBR45 | C. thermocellum | |
| Cellobiose | 100 | 100 |
| H2 | 124 | 263 |
| CO2 | 125 | 155 |
| Ethanol | 149 | 149 |
| Formic acid | 37.0 | 44.3 |
| Acetic acid | 15.4 | 24.7 |
| Propionic acid | 0.50 | 0.30 |
| Lactic acid | 87.5 | 53.1 |
| Succinic acid | 0.50 | 5.10 |
| Carbon recovery | 0.63 | 0.59 |
| O/R balanceb | 0.68 | 0.63 |
| Balance of available H | 0.69 | 0.69 |
Results are mean values of duplicate tubes after 3 days of incubation and are expressed as millimoles per 100 mmol of cellobiose. Cellobiose was supplied at 0.2 g/10 ml in M medium.
O/R balance, ratio between oxidized and reduced carbon products.
FIG. 3.
Micrographs of Clostridium sp. strain EBR45. Phase-contrast micrographs of strain EBR45 cells showing the occurrence of a subterminal oval spore (A) and flagella (B). Scanning electron micrographs of strain EBR45 cells associated with the cellulose fibers supplied in the culture are also shown (C and D). Bars, 5 μm.
Specific detection of strain EBR45 in fermentation sludge.
To examine the occurrence of strain EBR45 in the original cellulose-digesting fermentation sludge, a PCR that specifically amplified the 16S rRNA gene of the organism was developed and used to study the sludge. As shown in Fig. 1 (bottom), the specific PCR product was obtained only from the sludge collected during the high fermentation efficiency period. This result confirms the specific occurrence of strain EBR45 during the period of high fermentation efficiency. Direct sequencing of the specific PCR product showed that it had a sequence identical to that of strain EBR45; this confirmed the specificity of the PCR primers.
Wastepaper digestion by strain EBR45.
The above results suggest that strain EBR45 has high digestion activity against photocopy paper, which was supplied in the waste suspension as a main cellulosic constituent and fed into the bioreactor. Further, to assess the activity, the organism was cultured in M medium supplied with waste photocopy paper (see Materials and Methods). As shown in Fig. 4A, strain EBR45 degraded approximately 79% of the substrate paper after 3 days of incubation. The degradation ratios of similarly processed C. thermocellum and C. straminisolvens were 74% and 68%, respectively. Strain EBR45 accumulated an orange-yellow pigment in the early growth phase and caused rapid disruption of paper strips into pieces (Fig. 4B). On the other hand, C. thermocellum and C. straminisolvens produced a pale yellow pigment and required a longer incubation time to achieve marked degradation of the paper. Scanning electron microscopic observation demonstrated that the above three organisms attached to cellulose fibers in similar manners (Fig. 4C). A notable feature that was commonly observed in the three organisms was the frequent occurrence of pits on the cellulose fibers; occasionally, many cells were found around and inside the pits. Pits were not observed when the organisms were cultivated on filter paper.
FIG. 4.
Photocopy paper digestion by strain EBR45. (A) Residual weight of substrate paper after digestion by strain EBR45 (45), C. thermocellum (CT), and C. straminisolvens (CS). Each 0.3 g of photocopy paper supplied in the medium was measured for residual dry weight after the 3 days of digestion by the clostridial strains. The data are means of two separate experiments. (B) The appearance of small-scale cultures of the three organisms in the medium supplied with photocopy paper (for conditions, see Materials and Methods). Photographs were taken after 1.5 days. (C) Scanning electron micrographs of clostridial cells associated with cellulose fibers. Photographs were taken after 1.5 days. Bars, 10 μm.
DISCUSSION
The thermophilic methanogenic bioreactor used in this study converted wastepaper into methane. During the high-performance period, the conversion proceeded effectively and there was almost no residual sugar in the sludge. However, the high-efficiency period did not have a long duration and was subsequently followed by the breakdown phase. Similar breakdown events had occasionally been observed during the long-term continuous operation of the bioreactor. It is possible that the breakdown occurs because of some physiological alteration that affects the microbial activities in the fermentation sludge, although the details remain unknown. In our fermentation system, acidification of the fermentation sludge appears to precede the reduction of the activity of effective cellulose digesters.
A comprehensive 16S rRNA gene sequencing study of the enriched culture revealed the existence of a wide variety of clostridia and related bacteria that include strains that have a phylogenetic position that is distinct from those of known species. This result raises the possibility that these organisms can be cultured with the synthetic medium and some of them may remain unknown because of the lack of colony-forming ability or nonviability after storage. We assume that the organism corresponding to EBR-02E-0046 is an example of such an organism; the strain belongs to the 16SX subgroup, which consists of several uncultured clones (http://rdp8.cme.msu.edu/html/) (4, 12). The group of clostridia may serve as an indicator for future studies to develop new culture and storage techniques which will enable efficient isolation and characterization of unknown strains.
With regard to the isolation of Clostridium sp. strain EBR45, successive inoculation and cultivation with filter paper as a bait carrier was quite effective; this could probably be attributed to the characteristic property of the organism of being tightly associated with cellulose fibers. In fact, electron microscopic observation demonstrated that the cells of this organism are frequently distributed inside the cellulose fiber structure. Burrell et al. (3) recently reported that the strains of Clostridium group III (strain EBR45 belongs to this group) are often found to be associated with cellulose fibers, while those belonging to Clostridium group XIVa are distributed in the planktonic phase. A successive inoculation method would be effective in enriching the strains belonging to the former group of clostridia.
The 16S rRNA gene-based phylogeny showed that strain EBR45 belongs to the same subgroup as C. thermocellum and C. straminisolvens but has a phylogenetic position distinct from that of these two species. Although the physiological properties of strain EBR45 were almost identical to those described for the two known clostridia, the low sequence similarity percentages suggest that the organism belongs to a new species. The 16S rRNA gene clone library obtained from the enriched culture contained the clones that were closely related to strain EBR45 (EBR-02E-0444, EBR-02E-0445, and EBR-02E-0448 in Fig. 2) but not those clustering with C. thermocellum and C. straminisolvens. Strain EBR45 and its relatives may adapt better to the growth conditions supplied with the wastepaper than the other two species. Previous reports have stated that C. thermocellum was isolated from soil and feces (6) and C. straminisolvens was isolated from a straw digester (10).
The adaptability of strain EBR45 to fermentation conditions was also demonstrated by its specific detection in the original methanogenic sludge. This showed that strain EBR45 is a member of the probable effective clostridial group that specifically occurred during the high-performance fermentation period. We also performed the same monitoring study for another period of reactor operation and obtained a similar result (unpublished data). Since the decrease in the population of strain EBR45 appears to be linked to the initiation of breakdown, the precise characterization of the factors that inhibit the growth of strain EBR45 may help in understanding the reason behind the breakdown phenomena that occasionally occur in the microbial conversion system.
Strain EBR45 exhibited a marked digestion activity against waste office paper even in the pure cultivation system (Fig. 4). Our preliminary experiment showed that the cellulase activity of this organism was almost at the same level as that of C. thermocellum (unpublished data). Therefore, some other specific properties of this organism may be related to its high wastepaper-degrading activity. A marked feature of strain EBR45 that was observed during wastepaper-digesting cultivation was the accumulation of a yellow-orange pigment. Yellow pigment formation is a characteristic property of clostridia; in C. thermocellum, the pigment has been shown to have a carotenoid-like structure and has been implicated in cellulolysis (6). The pigment produced by strain EBR45 may be a similar compound and may have some relationship with the cellulolytic function of this organism. We anticipate that future improvement of the fermentation system will develop an efficient utilization system for municipal cellulosic wastes.
Acknowledgments
We thank Ichiro Hongo for technical assistance.
This study was supported by the 21st Century COE program of MEXT, Japan, and the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
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