Appl Microbiol Biotechnol (2001) 57:555–562 DOI 10.1007/s002530100806
O R I G I N A L PA P E R
Y. Ueno · S. Haruta · M. Ishii · Y. Igarashi
Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost
Received: 20 April 2001 / Received revision: 7 July 2001 / Accepted: 16 August 2001 / Published online: 22 September 2001 © Springer-Verlag 2001
Abstract Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/molhexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticum was isolated in the enrichment culture with or without added peptone, and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from Y. Ueno (✉) Kajima Technical Research Institute, Tobitakyu 2–19–1, Chofu-shi, Tokyo 182–0036, Japan e-mail:
[email protected] Tel.: +81-424-897066, Fax: +81-424-892896 S. Haruta · M. Ishii · Y. Igarashi Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113–8657, Japan
cellulose by microflora is performed by a consortium of several species of microorganisms.
Introduction Hydrogen is known as a clean energy resource and is one of the most important elements. The biological production of hydrogen by using wastewater and other biomass as raw materials has been attracting attention as an environmentally friendly process that does not consume fossil fuels. Hydrogen production by microorganisms can be divided into two main categories: one uses photosynthetic organisms cultured under anaerobic light conditions, and the other uses other anaerobic bacteria that produce hydrogen via fermentation metabolism. While conversion of biomass resources to hydrogen gas by fermentation has been extensively studied, most studies have been carried out with pure cultures of the isolated strains (Zeikus 1980; Heyndrickx et al. 1987; Taguchi et al. 1992; Rachman et al. 1998). Natural microflora, enriched from a natural population of bacteria, are used in various wastewater treatment processes since a sterilization process is not necessary and they can be adapted to different components in the wastewater. Although the most common processes for anaerobic wastewater treatment involve methane fermentation(Lettinga 1995), the present study was aimed at developing a hydrogen production process from the treatment of organic wastewater. Several reports have been published on hydrogen production during wastewater treatment (Minoda et al. 1983; Guwy et al. 1997; Sparling et al. 1997). In these studies, hydrogen production resulted from the inhibition of methane fermentation (Minoda et al. 1983; Sparling et al. 1997), but satisfactory stability and a sufficiently high molar yield were not obtained. We have found that anaerobic microflora in sludge compost converted cellulose to hydrogen with high efficiency (Ueno et al. 1995) and reported the stable production of hydrogen from industrial wastewater by the microflora in a chemostat culture
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(Ueno et al. 1996). However, the microbial population in the microflora was not revealed and the hydrogen-producers were not identified. In this study, we investigated the microbial population in hydrogen-producing microflora that was enriched from sludge compost by using an artificial medium as a wastewater model. Isolation of the microorganisms by the plating method and an analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rDNA (Muyzer and Smalla 1998) were both applied to investigate the microbial diversity involved in the production of hydrogen from cellulose.
Materials and methods Microflora and cultivation A sludge compost made by forced aeration was used as microflora. A CT medium with the following composition (g/l) was used: KH2PO4, 1.5; Na2HPO4.12H2O, 4.2; NH4Cl, 0.5; MgCl2.6H2O, 0.18; yeast extract (Difco, USA), 2.0; peptone (Wako Pure Chemicals, Japan), 5.0; cellulose powder (Funacel SF, Funakoshi, Japan), 10.0. Fifteen grams of the sludge compost was inoculated into 3 l of the CT medium in a 5-l jar-fermentor. The initial anaerobic conditions were established by replacing the gas phase with argon gas at the beginning. The cultivation was grown at 60°C with agitation from a magnetic stirrer at 200 rpm. The CT medium for the chemostat culture was supplied to the reactor using a peristaltic pump, which was controlled by a timer and a balance to maintain the hydraulic residence time (HRT) in the reactor at the required value. The pH value in the reactor was maintained at 6.4 by automatically titrating with 10 N NaOH. The medium to be fed into the reactor was stored at 4°C in a refrigerator before use. The broth in the reactor was periodically sampled and analyzed. The reactor was stirred at 200 rpm to maintain uniform conditions inside and to prevent the attachment of any microorganisms to the components inside the reactor.
from the jar-fermentor was immediately transferred to the anaerobic chamber, before being serially diluted with a PBS buffer containing following materials (g/l): NaCl, 8.0; Na2HPO4, 1.1; KCl, 0.2; KH2PO4, 0.2, and sodium cysteine-HCl, 0.25. An aliquot of the sample was streaked on to a BMC plate which contained the following materials (g/l): KH2PO4, 1.5; Na2HPO4.12H2O, 4.2; NH4Cl, 0.5; MgCl2.6H2O, 0.18; yeast extract (Difco, USA), 2.0; peptone (Difco, USA), 5.0; ball-milled cellulose (Funacel SF, Funakoshi, Japan), 5.0; cellobiose (Sigma, Germany), 2.0; and agar, 30.0. A BMC plate not containing peptone was used when the microorganisms were isolated from the enrichment culture without added peptone. After inoculating the plates, they were placed in the Gas-Pak anaerobic cultivation jar (BBL, UK) in the incubator at 60°C. After 96 h of cultivation, the microorganisms that had appeared on the plate were isolated and then purified by the anaerobic techniques of Hungate (1969) and Bryant (1972). To determine the fermentation products, the isolates were cultivated in a CTC medium at 60°C for 72 h. The CTC medium contained 2 g/l of peptone and 5 g/l of cellobiose in the CT medium. A CTC medium not containing peptone was used to determine the product formation by those strains that had been isolated from the enrichment culture without added peptone. The medium and buffer solution used for isolation of the microorganisms contained 1 ml/l of a 0.1% resazurin solution and were left in the anaerobic chamber for 3 days before being used, this being a sufficient time to reduce the color of resazurin. DNA extraction Cells in 1 ml of the broth were harvested in a tube by centrifugation (10,000g, 4°C, 5 min). Five hundred microliters of an extraction buffer containing 100 mM Tris-HCl (pH 9.0), 40 mM EDTA, 50 µl of 20% SDS, and 300 µl of benzyl chloride was added to the tube. The tube was vortexed and incubated at 50°C for 30 min, while shaking at 5 min intervals to keep the two phases mixed. Sodium acetate (3 M) was then added, and the tube was kept on ice for 15 min to remove the cell debris (Zhu et al. 1993). Crude DNA obtained by this method was purified by phenol extraction and ethanol precipitation. DNA prepared in this way was used as template DNA for the PCR experiment. Genetic fingerprinting of the isolates by RAPD
Analyses The evolved gas was collected in a bag (Omi Odor Air Service, Japan) and the volume was measured at room temperature by the water displacement method with graduated cylinders which had been filled with water of pH 3 or less in order to prevent dissolution of the gaseous components. The composition of the gas was determined by gas chromatography as described previously (Ueno et al. 1995). The sampled broth was filtered through a membrane of 0.45 µm pore size before the analysis. To determine the concentration of total suspended solids (SS) in the broth, the membrane was dried in an oven at 105°C for 3 h and then weighed after cooling in a desiccator. The cell mass in the broth containing cellulose was estimated from the nitrogen content in SS determined by the Kjeldahl method, and an empirical formula for a microbial cell (C5H7NO2). The filtrate was used for subsequent analyses. The amount of soluble total organic carbon (S-TOC) was determined with a TOC analyzer (TOC-5000, Shimadzu, Japan). The volatile fatty acids (C2-C5) and lactate were determined by liquid chromatography with an organic acid analyzing system (LC-10A, Shimadzu, Japan). Ethanol was determined enzymatically using a test kit (Boehringer-Mannheim, Germany). Isolation of the microorganisms All procedures were performed under anaerobic conditions inside an anaerobic chamber (Forma Scientific, USA). The broth sampled
Genetic fingerprints of the isolated bacteria were obtained by a random amplified polymorphic DNA analysis (RAPD) (Williams et al. 1990). Test kits for the RAPD analysis was purchased from Amersham-Pharmacia (Germany), the conditions for PCR being according to the manufacturer's instructions. PCR conditions for the DGGE analysis The variable V3 region of 16S rDNA was enzymatically amplified by PCR with primers to conserved regions of the 16S rRNA genes (Muyzer et al. 1993). The nucleotide sequences of the primers were as follows: primer 357F, 5′-CCTACGGGAGGCAGCAG-3′; primer 517R, 5′-ATTACCGCGGCTGCTGG-3′; primer 357FGC, 5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′. Primer 357FGC contained the same sequence as primer 357F, but with a GC clamp. Combination of primers 357FGC and 517R generated a PCR-fragment of about 200 bp that was suitable for the subsequent DGGE analysis. PCR was performed with AmpliTaq Gold (Perkin-Elmer, Applied Biosytems, USA) using an automated thermal cycler (MP, TaKaRa Biomedicals., Japan) with initial denaturation at 95°C for 10 min being followed by a total of 30 cycles of 30 s of denaturation at 93°C, 30 s of annealing at 65°C (for the first 10 cycles), then at 60°C (for the next 10 cycles), and finally at 55°C (for the last 10 cycles). Each annealing step was followed by 1 min of extension at 72°C except for the final extension which was at 72°C for
557 5 min. The generated PCR product was then analyzed on 2% agarose gels with staining by ethidium bromide to determine the size, purity and concentration of DNA. DGGE conditions DGGE was performed with a DCode universal mutation detection system (Bio-Rad, USA). The PCR samples were applied directly to 6–12% (w/v) polyacrylamide gels in a 0.5× TAE buffer [20 mM tris-acetate (pH 7.4), 10 mM acetate, 0.5 mM Na2EDTA] with a denaturing gradient ranging from 20–60%. Denaturation of 100% corresponds to 7 M urea and 40% (v/v) formamide. The gradient gel was cast with a gradient delivery system (Model 475, Bio-Rad, USA). Electrophoresis was run at a constant voltage of 200 V and at 61°C. After 5 h of electrophoresis, the gel was stained with SYBR Green I (Molecular Probes, USA) at 10,000× dilution in 0.5× TAE for 30 min, rinsed with water and then photographed with Gel Print 2000i (Genetics Solutions, USA) equipped with a UV illuminator. DNA sequence After DGGE had been run, the bands of interest were excised from the gel, and DNA was extracted using a QIAEX II kit (Qiagen, Germany). The extracted DNA was then re-amplified by PCR and re-analyzed by DGGE to confirm the presence of a single band and its relative position compared with the initial run. The 16S rDNA sequence of the isolated microorganism was partially determined using a primer kit (Applied Biosystems, USA). PCR amplification for the sequence was performed according to the manufacturer's instructions. The sequencing reaction was run with a Big Dye terminator cycle sequencing kit (Perkin Elmer, USA). Sequencing was performed with an ABI Prism 377 DNA sequencer (Applied Biosystems, USA) after the PCR primers had been removed by a QIAquick PCR purification kit (Qiagen, Germany). A search of the GenBank database was conducted using the BLAST program (Karlin and Altschul 1990). Nucleotide sequence accession numbers The nucleotide sequences reported in this paper have been deposited in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases under accession nos. AB059451, AB059456 – AB059467, and AB059472 – AB059483.
Results Hydrogen production by anaerobic microflora in the chemostat culture A time course of the fermentation by anaerobic microflora is shown in Fig. 1. The product yields calculated from the amounts of metabolites and decomposed cellulose are listed in Table 1. Gas evolution began to occur after 18 h of cultivation. During 72 h of batch cultivation, a total of 4,050 ml/l-culture of gas had been produced. The composition of the gas was 57% hydrogen and 42% carbon dioxide, and no methane was detected. The pH value of the medium decreased from 6.6 to 5.2 with the progress of gas evolution and cellulose decomposition. Significant production of acetate, ethanol, propionate, and butyrate was observed with the batch cultivation, and the yield of hydrogen was 2.0 mol/mol hexose.
Fig. 1 Time course of operation on cellulose fermentation by anaerobic microflora. The medium contained 5 g/l of peptone. Chemostat cultivation was started after 72 h of batch cultivation, the pH value being regulated at 6.4 by automatic titration with 10 N NaOH. Open circles Hydrogen, black circles carbon dioxide, open squares acetate, black squares propionate, open triangles butyrate, black triangles ethanol, open diamonds pH
After the batch cultivation, chemostat operation was started at 3 days of HRT with the pH value controlled at 6.4. The gas and metabolite formation was apparently stable after 6 days of operation. No change in the gas composition and metabolite formation compared with the batch cultivation was observed, although the proportions of the metabolite distribution were slightly different. Propionate formation increased, whereas butyrate formation decreased. Cellulose decomposition was im-
558 Table 1 Product yield from the fermentation of cellulose by anaerobic microflora Culture conditiona Batch Chemostatf Chemostatf
Nitrogen source NH4Cl and peptone NH4Cl and peptone NH4Cl
Yieldc (mol/mol-hexose)
Carbon recoverye
Cellulose decomposition (%)
H2
CO2
Acetate
Propionate Butyrate
Ethanol
Celld
2.0
1.5
0.8
0.2
0.4
0.5
0.3
1.34
49.9
2.0
1.5
0.9
0.3
0.3
0.6
0.2
1.23
90.4
1.0
0.4
0.5
N.P.b
0.3
0.5
0.1
0.68
72.5
a The chemostat culture was grown at pH 6.4 b Not produced c Cellulose was calculated as hexose [(C H O ) ] 6 10 5 n d Cells were calculated from an empirical fomula
e
for a microbial
Carbon recovery was calculated from the decomposed cellulose and produced metabolites in terms of the carbon content f Values were calculated from the data obtained at the steady state for the applied hydraulic residence time (=3 days)
cell (C5H7O2N)
proved to 90.4% by adjusting the pH value. The average hydrogen gas evolution rate with the chemostat culture was 29.6 mmol l-reactor–1 day–1, giving a hydrogen production yield of 2.0 mol/mol hexose. The carbon recovery calculated from the cellulose decomposition exceeded 1.0 with both the batch and chemostat cultures since the decomposition of peptone was not taken account for the calculation. Effect of the nitrogen source on hydrogen production The medium used contained either ammonium chloride (0.5 g/l) as an inorganic nitrogen source or peptone (5.0 g/l) as an organic nitrogen source. The fermentation yields from cultivation with the medium without peptone are shown in Table 1. The hydrogen production yield decreased remarkedly with decreasing formation of acetate and propionate (1.0 mol/mol-hexose), while the yields of butyrate and ethanol remained unchanged. PCR-DGGE analysis of the hydrogen-producing microflora The bacterial populations in the microflora were analyzed and compared by a PCR-DGGE analysis targeted at eubacterial 16S rDNA, the DGGE profiles being shown in Fig. 2. The major bands in the DGGE gels were excised and purified to determine the sequence. The results of the sequence affiliation determined by BLAST are shown in Table 2. It was found that several dominant populations were present under each culture condition, and that the microbial population differed according to the enrichment of the microflora. Most of the DGGE fragments obtained are related to those microorganisms that could proliferate under the applied thermophilic anaerobic conditions. Thermoanaerobacterium thermosaccharolyticum (bands 6, 8 and 12) and Clostridium cellulosi (bands 7, 9, 11 and 14) appeared in the enrichment culture with added
Fig. 2A–C Denaturing gradient gel electrophoresis (DGGE) profiles of the PCR-amplified 16S rDNA extracted from the enrichment culture of microflora. Lanes: A chemostat enrichment culture without added peptone, B batch enrichment culture with added peptone, C chemostat enrichment culture with added peptone. The numbered bands were excised, purified, and re-amplified to determine the sequence. The affiliation of each excised band is described in Table 2. The gradient concentration of the denaturant was 20–60%
peptone, while Clostridium thermocellum (bands 1 and 4) appeared in the enrichment culture without added peptone. There were two bands each affiliated with C. cellulosi (bands 11 and 14) and C. thermocellum (bands 1and 4) that were due to one or two mismatches in the sequences determined. They could thus be different at the strain level or be different PCR products from slightly different copies of the 16S rDNA gene within a single cell.
559 Table 2 Affiliation of denaturing gradient gel electrophoresis (DGGE) fragments determined by their16S rDNA sequence
a Percentage
similarity to the closest relative according to the BLAST comparison
Sequence determined (bp)
Affiliation
Similaritya (%)
Accession no.
1 2 3 4 5 6
138 135 135 136 161 136
100.0 99.3 94.1 95.6 97.5 100.0
L09173 AB052397 L09175 L09173 AF329476 M59119
7 8
138 136
99.3 100.0
L09177 M59119
9 10 11 12
138 160 139 136
99.3 88.1 97.8 100.0
L09177 U34974 L09177 M59119
13 14 15
136 138 162
Clostridium thermocellum Symbiobacterium sp. Clostridium sp. Clostridium thermocellum Bacillus sporothermodurans Thermoanaerobacterium thermosaccharolyticum Clostridium cellulosi Thermoanaerobacterium thermosaccharolyticum Clostridium cellulosi Sulfobacillus disulfidooxidans Clostridium cellulosi Thermoanaerobacterium thermosaccharolyticum Ruminococcus albus Clostridium cellulosi Selenomonas noxia
Band
90.4 99.3 85.2
AF079847 L09177 AF287799
Table 3 Classification of the isolated strains by the partial sequences of 16S rDNA and genetic fingerprinting analyses Source for isolation and genetic fingerprinting patterna
Appearance Sequence Phylogenetically related organism frequency determined (no. of (bp) isolates/total no. of isolates)
Chemostat enrichment without added peptone D-1 7/23 137 Thermoanaerobacterium thermosaccharolyticum D-2 6/23 135 Thermobacteroides acetoethylicus D-3 10/23 133 Thermoanaerobacterium aotearoense
Similarity (%)b
Accession no.
Identical DGGE bandsc
98.5 100.0 99.2
M59119 X969336 X93359
6, 8, 12
Batch enrichment with added peptone R-1 8/29 502 R-2 4/29 501 R-3 6/29 500 R-4 4/29 532 R-5 2/29 499 R-6 2/29 500 R-7 1/29 503 R-8 1/29 510 R-9 1/29 501
Clostridium thermobutyricum Thermoanaerobacterium aotearoense Thermoanaerobacterium aotearoense Sulfobacillus disulfodooxidans Thermoanaerobacterium thermosaccharolyticum Clostridium cylindrosporum Thermoanaerobacterium thermosaccharolyticum Desulfotomaculum nigrificans Caloramator viterbiensis
99.8 98.2 97.9 92 97.8 98.3 99.4 99.2 91.1
Chemostat enrichement with added peptone 16/16 136
Thermoanaerobacterium thermosaccharolyticum
100.0
X72868 X93359 X93359 U34974 M59119 Y18179 M59119 AB026550 AF181848
M59119
10 6, 8, 12
6, 8, 12
a D-1 to -3 were determined by the fingerprint pattern from PCRDGGE. R-1 to -9 were determined by the fingerprint pattern of random amplified polymorphic DNA (RAPD). The sequences of D-1 and R-7 were identical, and were also identical to those of 16
isolates from the chemostat enrichment culture with added peptone b Percentage similarity to the closest relative by the BLAST analysis c Number of the DGGE band in Fig. 2
In the sample obtained from the chemostat enrichment culture with added peptone, band 13, which is closely related to Ruminococcus albus, was detected with approximately equal intensity to the signal obtained from C. cellulosi (band 14). R. albus is known as a cellulolytic hydrogen-producing microorganism, although it is a mesophile (Wolin 1974). A band affiliated with the Selenomonas species that produces propionate from carbohydrate (band 15), was detected (Scheifinger and Wolin 1973).
Isolation of microorganisms from the enrichment culture of microflora Sixty-eight colonies were isolated from the broth obtained from the following three enrichment cultures of microflora: the chemostat culture without added peptone, and the batch and chemostat cultures with added peptone. To identify the identical strains in the isolated microorganisms, determination of the partial sequences of 16S rDNA or a fingerprinting analyses by DGGE and
560 Table 4 Fermentative characterization of representative strains Represen- Genetic tative fingerstraina printing W2–5 W2–7 W1–7 B40–2 C38–4 C-1 C38–3 C-4 C40–2 C41–3 C40–3 U40A-5
D-1 D-2 D-3 R-1 R-2 R-3 R-4 R-5 R-6 R-7 R-8 R-9
H2 formatione (mol/mol-glucose)
Cellulose Cellobiose decomdecompositionb positionc (mmol/l)
Lactate
Formate
Acetate
Propionate Butyrate Ethanol
– – – + – – – – – – – –
3.41 3.06 5.54 ND 8.60 9.81 8.63 10.38 ND 5.85 ND ND
NDd ND ND ND 2.20 1.72 2.20 1.55 1.16 2.52 ND 9.20
7.29 2.16 5.76 1.42 14.67 12.39 14.67 13.29 5.22 16.21 0.15 3.76
0.39 0.84 0.40 ND ND ND ND ND ND ND ND ND
10.3 6.1 10.5 11.2 26.7 25.1 26.8 19.2 4.8 22.5 1.5 9.6
Metabolite formation (mmol/l)
a
1.22 ND 0.16 10.40 9.49 7.76 9.17 6.63 0.04 5.11 0.04 ND
8.64 7.64 11.95 N.D 8.97 10.77 9.72 N.D 3.21 10.17 3.26 5.76
1.14 0.65 0.97 2.11 1.81 1.61 1.78 2.07 2.18 1.90 0.26 0.79
Fermentative characterization of the representative strain in each genetic fingerprint pattern is shown. Medium for determination is described in the Materials and methods section b CTC medium that contains cellulose powder substituted for cellobiose was used. Cellulolysis was checked in the test tube after 120 h of cultivation at 60°C. + decomposed, - not decomposed c Cellobiose concentration was determined by the phenol-H SO 2 4 method using glucose as standard
d Not detected e Hydrogen production
RAPD was applied. The strains identified and their frequency of appearance are shown in Table 3. Three distinct fingerprints were obtained from 23 colonies isolated from enrichment of the chemostat culture without added peptone, and 9 distinct fingerprints were obtained from 29 colonies isolated from enrichment of the batch culture with added peptone. Sixteen colonies that were isolated from enrichment of the chemostat culture with added peptone were grouped into one category by determining the partial sequence of 16S rDNA. The sequence of these 16 isolates corresponds to the sequence of fingerprinting patterns D-1 and R-7. The sequences obtained were compared with available database sequences using a BLAST search. T. thermosaccharolyticum was isolated from all the enrichment cultures. R-5 and R-7 were independent at the strain level since there were some minor mismatches in the determined sequences. The isolates with high frequency belong to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria (Cato and Stackebrandt 1989).
produced, with less butyrate. This lower formation of butyrate is a characteristic feature of Thermobacteroides acetoethylicus (Ben-Bassat and Zeikus 1981) and Thermoanaerobacterium aotearoense (Rainey et al 1996). On the other hand, the product formation by the strains isolated from the batch enrichment culture with added peptone differed with each strain. While some strains produced predominantly lactate and formate, most of the strains produced significant amounts of butyrate in addition to acetate and ethanol. The formation of butyrate was lower, or very low, in the organisms related to Clostridium cylindrosporum (C40–2), Desulfotomaculum nigrificans (C40–3), and Caloramator viterbiensis (U40A5). None of the strains produced a significant amount of propionate, as was observed for the microflora cultivated with added peptone. Significant formation of lactate was observed with some strains. This might have been due to a change in the metabolism of the cells caused by a difference in the culture conditions, since it has not been observed in fermentation by microflora. Only strain B40–2, which was identified as Clostridium thermobutyricum, demonstrated cellulose decomposition when the strains were cultivated in a CTC medium containing cellulose powder as the carbon source. It has been reported that C. thermobutyricum can decompose cellulose, and produce mainly butyrate with slight formation of acetate (Wiegel et al. 1989). The hydrogen production yield was estimated from the amount of metabolites formed. The yield of hydrogen produced by the strain obtained from the chemostat enrichment culture without added peptone was lower than the yield by the strain obtained from the enrichment culture with added peptone, as was observed for the yield from cultivation by microflora.
Physiological characterization of the isolates The fermentation products of a representative strain for each fingerprinting pattern were determined by using cellobiose as a substrate, and are shown in Table 4. All strains demonstrated gas evolution in durum tube in the test tube. The end-product formation varied at the strain level, but most strains produced mainly acetate. The product formation by the strains isolated from the chemostat enrichment culture without added peptone was similar: predominantly acetate and ethanol were
yield was estimated from the amount of acetate and butyrate formed using following equations: C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2; C6H12O6 → 2CH3CH2CH2COOH + 2CO2 + 2H2
561
T. thermosaccharolyticum and T. aotearoense were isolated from the enrichment cultures with and/or without added peptone. When these strains, which were isolated from the enrichment culture without added peptone, were cultivated in the CTC medium containing peptone, the product formation was no different from that in the medium not containing peptone. Butyrate formation did not increase. In contrast, the product formation by the strains that were isolated from the enrichment culture with added peptone, was similar to that in the medium not containing peptone (data not shown). This indicates that they might have had a physiological difference at the strain level, although they are identical at the species level. This specific property could have been accumulated through the enrichment process of chemostat cultivation.
Discussion In this study we have demonstrated hydrogen production by anaerobic microflora with a relatively high production yield (2.0 mol/mol hexose) by both batch and chemostat cultivations (Table 1). This yield is comparable with that reported thus far using pure culture strains (Wood 1961; Wolin 1974). Under all applied culture conditions, microorganisms closely related to T. thermosaccharolyticum were isolated, and detected by PCR-DGGE analysis with strong intensity. This result implies that this organism participates in fermentation reactions. T. thermosaccharolyticum is a saccharolytic organism which produces hydrogen (Skerman et al. 1980). However, the strain related to T. thermosaccharolyticum isolated in the present experiment did not show cellulolytic activity (Table 4). Therefore the organism seems to have a symbiotic relationship with cellulolytic organisms. C. thermocellum, C. cellulosi and R. albus-like species were detected by PCRDGGE, although the isolation of these organisms was not successful using the method applied. It can be inferred from these results that these microorganisms are strongly interrelated with each other and function as hydrogen producers in the microflora. Non-cellulolytic bacteria can grow in a cellulosedegrading co-culture, which indicates that at least some soluble sugars are released by cellulolysis or are otherwise available as a growth substrate for other microorganisms. Cellulolytic microbes produce enzymes that depolymerize cellulose, thereby producing cellobiose, cellodextrins, and some glucose. These sugars are fermented by cellulolytic and other saccharolytic microorganisms. (Leschine 1995). By keeping the cellobiose concentration low, and thus preventing any inhibition of the cellulase system by this product of cellulose hydrolysis, non-cellulolytic cellobiose-fermenters such as T. thermosaccharolyticum may play a very important role in this process. The observation that microorganisms related to T. thermosaccharolyticum appeared with high frequency in the chemostat culture strongly supports this speculation (Table 3).
Not only T. thermosaccharolyticum, but also C. cellulosi (Yanling et al. 1991) and C. thermocellum (Lamed et al. 1988) have the capability to produce hydrogen via fermentation metabolism. The hydrogen production observed with the chemostat cultivation of microflora would have been performed by both these latter microorganisms. From the batch culture, not only T. thermosaccharolyticum, but also several microorganisms that can produce hydrogen were isolated (Table 3). In particular C. thermobutyricum and T. aotearoense were isolated with high frequency, although these organisms were not found by the PCR-DGGE analysis. This could have been due to a peculiar specificity of the isolation medium having selectivity for bacteria with specific genotypes for a catabolic enzyme. The level of butyrate formed was higher from the batch cultivation than from the chemostat cultivation (Table 1) indicating that these microorganisms also contributed to hydrogen formation from cellulose in the batch culture. The variety of isolated species by chemostat cultivation was fewer, with only T. thermosaccharolyticum being solely isolated from the chemostat enrichment culture (Table 3). Microorganisms inhabiting the sludge compost that was used as the inoculum still remained in the batch culture broth. With the stable-pH chemostat cultivation, the microorganisms that adapt to the applied culture conditions can continue to proliferate in the reactor. Those microorganisms with an insufficient growth rate for the applied HRT cannot exist in the reactor, and would be washed out from the reactor with the present experimental system. Hydrogen-producing microorganisms should differ between batch and chemostat cultivation, even if the same hydrogen production yields were obtained; the variation of hydrogen producers was less with chemostat cultivation. On the other hand, in spite of the fact that Thermobacteroides acetoethylicus and Thermoanaerobacterium aotearoense were isolated from the chemostat enrichment culture without added peptone (Table 3), they were not detected by the PCR-DGGE analysis (Table 2 and Fig. 2). This indicates that these organisms certainly existed in the microflora for chemostat cultivation, although the concentration of the populations might have been lower than that of Thermoanaerobacterium thermosaccharolyticum. The DGGE profiles display the numerically dominant populations and therefore only provide information on the richness of species when the populations are equally abundant. Estimating the microbial abundance based solely on the concentration of a PCR product might therefore be impossible. However, a change in the intensity of a particular DGGE band from an artificial environment such as a chemostat enrichment culture may be used to infer that there has been a change in the size of a population. Preparation of the anaerobic microflora is crucial to recovering hydrogen gas with high efficiency. Hydrogen gas can be produced if surplus electrons in the reaction form and then reduce protons by hydrogenase. The for-
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mation of acetate and butyrate accompanies reducing power, whereas the formation of lactate, propionate and ethanol consumes reducing equivalents. The distribution of fermentation products depends on microorganisms that are dominant in the microflora. The proliferation of acetate and butyrate producers in microflora can be expected in order to recover hydrogen gas with high efficiency. Hydrogen-producing acetate/butyrate fermentation has recovered 2.6 mol/mol hexose of hydrogen gas from industrial wastewater (Ueno et al. 1996). Therefore, acetate/butyrate formation is advantageous for the formation of hydrogen. To our knowledge, this is the first study specifically directed at establishing the enrichment of hydrogen-producing microflora. The present study is unique in that both a culture-dependent approach and molecular approach were applied for a microbial community analysis. Quantification of the predominant organisms in the microflora is needed to gain a deeper insight into the population dynamics. A physiological study of hydrogen-producing microorganisms would also be crucial to the development of a process for hydrogen production by anaerobic microflora. Acknowledgements The authors thank Miss Ikuko Sakamoto for her analytical support in the experiments. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO).
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