Photo-biohydrogen production potential of Rhodobacter capsulatus-PK from wheat straw
Biofilm microbial community of a thermophilic trickling biofilter used for continuous biohydrogen...
Transcript of Biofilm microbial community of a thermophilic trickling biofilter used for continuous biohydrogen...
Biofilm microbial community of a thermophilic trickling biofilterused for continuous biohydrogen production
Yeonghee Ahn a,*, Eun-Jung Park a, You-Kwan Oh b,c, Sunghoon Park b,c,Gordon Webster d,e, Andrew J. Weightman d
a Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Guseong-dong,
Yuseong-gu, Daejeon 305-701, Republic of Koreab Department of Chemical and Biochemical Engineering, Pusan National University, San 30 Jangjeon-dong, Kumjung-ku Pusan 609-735,
Republic of Koreac Institute for Environmental Technology and Industry, Pusan National University, San 30 Jangjeon-dong, Kumjung-ku Pusan 609-735,
Republic of Koread Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3TL, Wales, United Kingdom
e Cardiff School of Earth, Ocean and Planetary Sciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3YE, Wales, United Kingdom
Abstract
Molecular methods were employed to investigate the microbial community of a biofilm obtained from a thermophilic trickling
biofilter reactor (TBR) that was operated long-term to produce H2. Biomass concentration in the TBR gradually decreased as reac-
tor bed height increased. Despite this difference in biomass concentration, samples from the bottom and middle of the TBR bed
revealed similar microbial populations as determined by PCR-DGGE analysis of 16S rRNA genes. Nucleotide sequences of most
DGGE bands were affiliated with the classes Clostridia and Bacilli in the phylum Firmicutes, and the most dominant bands showed a
high sequence similarity to Thermoanaerobacterium thermosaccharolyticum.
� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Anaerobic; Hydrogen; DGGE; Microbial community; Thermophilic; Trickling biofilter
1. Introduction
Microbial H2 production can be either photosyn-
thetic or non-photosynthetic. Facultative or obligate
anaerobes perform non-photosynthetic (or fermenta-
tive) H2 production. Fermentative H2 production is light
independent and generally faster. To date, most studieson continuous processes for fermentative H2 production
have employed suspended culture systems such as a con-
* Corresponding author. Tel.: +82 42 869 3941; fax: +82 42 869 3910.
E-mail address: [email protected] (Y. Ahn).
ventional stirred tank reactor (CSTR) [1–8] since these
systems are relatively simple and easy to operate. How-
ever, washout of cells results in low biomass in these
systems.
Considering the low biomass in suspended systems,
employing biofilms would appear to be a better ap-
proach for fermentative H2 production since biofilmsaccommodate higher biomass and fermentation rate
[9–11]. Besides, a reactor employing a biofilm generally
shows more stable performance as biofilm cells are more
resistant to changes in environmental conditions (e.g.,
pH, temperature, organic load, etc.) [9–13].
Trickling biofilter reactors (TBR) employ biofilms
formed on supporting matrices packed inside the reac-
tors [14]. Biofilm in TBR can degrade organic com-
pounds in wastewaters that are trickled over the
biofilm. Thermophilic (45–65 �C) TBR can take advan-
tage of characteristics of biofilm and thermophilic bacte-ria to achieve high and stable production of H2.
Thermophilic bacteria show a higher degradation rate
of organic substances than mesophilic (30–40 �C) bacte-ria under anaerobic conditions [15]. Since H2 is less sol-
uble at high temperature, thermophilic TBR can reduce
partial pressure of H2 and alleviate inhibition of H2 pro-
duction [16,17]. Thermophilic TBR can also take advan-
tage of high temperatures of wastewaters dischargedfrom industries such as canneries, distilleries, and food
process plants.
Information on the microbial community in fermen-
tative H2-producing reactors is necessary to better
understand and improve the process. However, only a
few studies on the microbial community in such reactors
have been reported [5,18,19], although many studies
have reported physicochemical aspects of the process[4,6–11,20,21]. Despite many advantages of a TBR sys-
tem for H2 production, microbial community in this sys-
tem has not been reported. Therefore, the aim of this
study was to investigate the microbial community in a
thermophilic TBR that showed superior H2 production
rate and long-term stability of performance. This is the
first report on microorganisms in a thermophilic TBR
used for continuous H2 production.
2. Materials and methods
2.1. Operation of TBR and sampling of biofilm
Biofilm samples were obtained from a bench-scale
(inner diameter 8 cm; length 75 cm; working volume,2 l) thermophilic TBR [22] packed with fibrous poly-
meric matrices (size, 2 cm · 1.5 cm · 1.5 cm; Model
DSRN, Dockil Felt Industry, Ltd., Seoul, Korea). Inoc-
ulum of the TBR was obtained from a bench-scale ther-
mophilic CSTR used for H2 production. Biomass
concentration and H2 production activity of the inocu-
lum were 0.72 g VSS l�1 and 49 mmol H2 l�1 d�1,
respectively. The CSTR was initially inoculated withheat-treated anaerobically digested sludge obtained
from a wastewater treatment plant. The feed for the
CSTR and the TBR was a glucose-based synthetic med-
ium [22]. Feed solution was purged with N2 gas and fil-
ter-sterilized before introduction into the reactor.
TBR was operated stably at 55–64 �C for 234 d. The
maximal volumetric H2 production rate and H2 yield
were 1050 ± 63 mmol H2 l�1 d�1 and 1.11 ± 0.12 molH2 mol�1 glucose, respectively [22]. Typical composition
of biogas from the reactor was as follows (v/v): H2,
53 ± 4%; CO2, 47 ± 4%. Methane in the biogas was
not detected throughout the normal operation of the
TBR, although methane was detected during the initial
start-up period. Major organic acid products were lac-
tate, n-butyrate, and acetate. Except the initial start-up
period, lactate concentration was the highest amongthe organic acids produced by the TBR during the
experiment [22].
The H2 production activity of the TBR was extremely
sensitive to oxygen and therefore opening the reactor to
take biomass samples was not possible during the oper-
ation of the TBR. Supporting matrix samples were ta-
ken from different heights of the TBR bed at the end
(day 234) of the reactor operation. Biofilm cells releasedfrom the supporting matrices were used for microbial
analyses. Volatile suspended solid (VSS) was measured
according to the Standard Methods [23] as described
previously [22].
2.2. DNA extraction
Two pieces of supporting matrices were placed into30 ml of phosphate-buffered saline (PBS; 0.13 M NaCl,
10 mM sodium phosphate buffer, pH 7.2) and vortexed
vigorously for 5 min to release cells from the matrices.
Cells were harvested and resuspended in 10 ml of PBS.
Total genomic DNA was extracted from the cells har-
vested from 1 ml of the biofilm resuspension. In addi-
tion, cells harvested from 8 ml of TBR inoculum were
also rinsed with PBS and used for DNA extraction. Asoil DNA isolation kit (Mo Bio Labs. Inc., Solana
Beach, CA) was used to extract DNA according to the
manufacturer�s instructions.
2.3. Polymerase chain reaction
The extracted DNA was used as template in polymer-
ase chain reaction (PCR) for DGGE analysis and meth-anogen detection. For DGGE analysis, 16 S rRNA gene
fragments were amplified with PCR primers 357f-GC
and 518r [24], producing �194-bp PCR products (corre-
sponding to positions 341–534, Escherichia coli number-
ing, [25]). PCR products of the correct size were
confirmed by agarose gel electrophoresis prior to
DGGE.
Two sets of mcrA gene primers were employed to de-tect methanogens; ME1 and ME2 [26], and the primers
developed by Luton et al. [27]. Gene mcrA encodes
a-subunit of methyl coenzyme M reductase, the key en-
zyme of methanogenesis. PCR conditions were as previ-
ously described [26,27]. Positive controls in the mcrA
PCR employed total DNA extracted from the reference
strains; Methanobacterium bryantii DSM 863T, Methan-
osarcina barkeri DSM 10131, Methanosaeta concilli
DSM 3671T, Methanospillium hungatei DSM 864T, and
Methanococcus jannashii DSM 2661T, belonging to the
Bed height (cm)
0 10 20 30 40
Bio
mas
s (g
VSS
l–1
)
0
5
10
15
20
25
30
Fig. 1. Abundance of microorganisms in the reactor bed. Bed heights
0 and 40 cm indicate bottom and top of the reactor bed, respectively.
Error bars represent SDs of the mean; n = 5.
orders Methanobacteriales, Methanosarcinales, Methan-
omicrobiales, and Methanococcales, respectively. Nega-
tive controls were Pseudomonas putida DNA and no
added template DNA.
PCR mixtures contained 20 pmol of primers (synthe-
sized by MWG, Germany), 1 · reaction buffer, 0.5 mMeach dNTP, 3 mMMgCl2, 1.25 U TaqDNA polymerase
(Bioline Ltd., London, UK) and 1.0 ll of template DNA
made up to 50 ll with molecular grade water. The PCR
employed 1 ng of template DNA from each reference
strain. Five microliters of PCR products were analyzed
on 1% (w/v) agarose gel using a standard electrophoresis
protocol.
2.4. DGGE analysis
DGGE was performed using the PCR-amplified 16S
rRNA gene fragments to characterize the microbial com-
munity in the reactor. DGGE condition was optimized
by changing electrophoresis time to separate the ampli-
fied DNA fragments. PCR products were separated
using a DCode System (Bio-Rad, Hercules, CA) at200 V, 60 �C for 4 h. Samples (�200 ng) were loaded
on a 10% (w/v) polyacrylamide gel (acrylamide: N,N 0-
methylenebisacrylamide, 37.5:1, Bio-Rad) in 1· TAE
buffer (2 M Tris base, 1 M glacial acetic acid, 50 mM
EDTA, pH 8.0). The denaturing gradient in the gel was
generated by mixing two stock solutions of 10% poly-
acrylamide containing 40% and 60% denaturant: 100%
denaturant was 7 M urea and 40% (w/v) formamide.Denaturing gradient increased in the direction of electro-
phoresis. After electrophoresis, the gel was stained with
GreenStar� (Bioneer Co., Daejeon, Korea) and DNA
was visualized on a UV transillumination table. Digi-
tized DGGE images were obtained using Scion Image
software (Scion Co., Frederick, MD). DNA band inten-
sities of DGGE images were determined using SigmaGel
(Jandal Scientific, San Rafael, CA). Major DNA bandswere excised from DGGE gels and re-amplified by
PCR for nucleotide sequencing as described previously
[28]. Nucleotide sequences were analyzed using the CHI-
MERA CHECK program of the Ribosomal Database
Project II to screen for and eliminate chimeric sequences
[29]. Nucleotide sequences were also screened against
GenBank database using BLASTN (version 2.2.10) [30]
to identify the most similar sequences in the database.
2.5. Whole cell hybridization and DAPI (4 0,6-diamidino-
2-phenylindole) staining
Biofilm formed on the supporting matrices was used
for whole cell hybridization and DAPI staining. Biofilm
suspension was obtained as described above. An appro-
priate volume of biofilm suspension and TBR inoculumwas taken and cells were rinsed with PBS. Rinsed cells
were fixed and used for whole cell hybridization and
DAPI staining as described previously [31]. Fluores-
cently labeled probes (synthesized by ThermoHybaid
GmbH, Ulm, Germany) used in this study were
ARC915 for domain Archaea [32] and LGC354A-C
for part of Firmicutes [33]. Probes were 5 0-end labeled
with tetramethyl rhodamine. Hybridized or stained cellswere then visualized using a Zeiss Axiolab (Jena, Ger-
many) with a 50 W mercury lamp. Images were obtained
with a digital camera (Model AxioCam; Zeiss) mounted
on the microscope. Cell counting was done with at least
10 random microscopic fields with more than 100 cells
per field and MS Excel was used for statistical analysis.
3. Results and discussion
3.1. Abundance of biomass
Biomass amount (VSS) in the TBR gradually de-
creased as bed height increased from the bottom of the
bed (Fig. 1). VSS (g l�1) found at different heights (from
the bottom of the bed) was in the following ranges: 0 cm,24.1 ± 3.0; 14 cm, 19.6 ± 1.2; 26 cm, 17.7 ± 2.8; 40 cm,
1.6 ± 0.5. Biomass observed at the bottom was 26.6%
higher than that found at the height of 26 cm. Biomass
measured at the top of the bed was 93.4% less than that
found at the bottom of the bed. The decreasing trend
seems to be related with the flow rate of the biogas. As
the height increases, the gas flow becomes faster and a
higher shear results. The highest linear gas velocity whichwill be observed at the top of the reactor is estimated to
be 1.4 cm min�1 when the TBR is operated at a hydraulic
retention time (HRT) of 2 h and 20.6 g glucose l�1 [22].
A low biomass developed at the top section of the reactor
could be attributed to the high shear rate caused by
recirculation liquid flow and gas flow [14].
The biomass concentration (17.7–24.1 g VSS l�1) ob-
served in the TBR demonstrated that the system was
able to accommodate a higher biomass than previously
observed in the other systems used for fermentative H2
production [6–11]. However, the TBR showed no clog-
ging throughout the operational period. Since the TBR
was a Pyrex glass column, any clogging would have been
observed. Shear stress caused by fast biogas flow(1.4 cm min�1) and recirculation liquid flow (210 l d�1)
through the TBR seems to remove excess biomass [14]
as evidenced by biomass observed in the effluent from
the TBR. Effluents from the TBR showed biomass con-
centration in the range of 0.32–0.77 g VSS l�1 through-
out the experiment. No apparent clogging in the TBR
could have been due to slower growth of thermophilic
bacteria comprising the microbial community in theTBR. In contrast, a mesophilic TBR used for H2 pro-
duction exhibited clogging due to fast microbial growth
(data not shown).
3.2. DGGE profiles of microbial community
DGGE was performed to compare 16S rRNA gene
fragment profiles of biofilm cells obtained from two dif-ferent heights of the TBR bed. Despite the differences in
biomass concentrations with bed height, samples from 0
and 26 cm heights of the TBR bed revealed similar
DGGE profiles (Fig. 2), suggesting similar microbial
populations at these heights.
Fig. 2. DGGE profile of 16S rRNA gene fragments. The fragments
were PCR-amplified from the total DNA extracted from biofilm cells
in the TBR used for H2 production. Lanes: 1, biofilm sample taken
from the middle (26-cm high from the bottom) of the reactor; 2,
biofilm sample taken from the bottom of the reactor; 3, TBR
inoculum. Arrows indicate DNA bands that were excised and analyzed
for nucleotide sequences.
The DGGE profiles of TBR samples were also com-
pared with that of the inoculum and clear differences in
DGGE profiles were observed. TBR samples showed
two strongly stained distinctive bands along with some
additional weakly stained bands whereas the TBR inoc-
ulum showed at least eight distinctive bands with someother minor bands. The difference in DGGE banding
pattern suggests that long-term operation of the TBR
caused a change in the microbial community composi-
tion of the inoculated culture. The two strongly stained
bands observed in DGGE profiles of TBR samples sug-
gest the occurrence of strong enrichment culture in the
TBR.
Major bands of the TBR and inoculum samples werephylogenetically related to Firmicutes (Table 1). Major-
ity of the DGGE bands were affiliated with the classes
Clostridia and Bacilli in the phylum Firmicutes. In the
class Clostridia, DGGE bands closely related to the gen-
era Thermoanaerobacterium and Mitsuokella were
frequently observed. DNA bands affiliated with T. ther-
mosaccharolyticum appeared in biofilm cells of TBR
while DNA bands affiliated with M. jalaludinii appearedin the inoculum. H2 production activity of these two
bacterial species has previously been reported [34,35]
and 16S rRNA gene fragments or isolates affiliated
with these two bacteria have been found in other
anaerobic reactors used for thermophilic H2 production
[5–7].
Nucleotide sequence of the most strongly stained
band (band 1–3) observed in the two TBR sampleswas most similar (98% similarity) to the 16S rRNA gene
of T. thermosaccharolyticum strain D120-70 (Accession
No. AF247003) [36] in the GenBank database. Another
dominant band 1–2 was also most closely related (98%
sequence similarity) with T. thermosaccharolyticum
strain D120-70. However, bands 1–2 and 1–3 differed
by 1 base in their nucleotide sequences which may repre-
sent different strains or different copies of the 16S rRNAgene in the same strain. Comparison of DGGE band
intensities in each lane showed reproducible data; con-
stantly, the intensities of bands 1–2 and 1–3 in the
TBR samples were 18.1 ± 0.3% and 23.9 ± 0.8%, respec-
tively (n = 3). Intensities of the bands 0–1 and 0–8 ob-
served in the TBR inoculum were 13.0 ± 0.1% and
11.9 ± 0.1%, respectively (n = 3).
Observing the two strong major bands affiliated withT. thermosaccharolyticum suggests that T. thermosac-
charolyticum related organisms are highly enriched un-
der the TBR conditions and that they play an
important role in H2 production. Stable and high H2-
production by the TBR was shown in the previous re-
port [22], despite various parameters (pH, temperature,
HRT, and COD load) applied to the reactor. Maintain-
ing high biomass of acidogenic bacteria such as T. ther-mosaccharolyticum in the reactor could be a basis for the
performance.
Table 1
Characteristics of 16S rDNA fragments obtained from DGGE gel
DGGE banda GenBank search result
Closest match
(Accession No.)
Isolated environment of
closest match
Sequence
similarity (%)
Taxonomic
description (class)
0–1 Uncultured bacterial clone w.4
(AY212556)
Manure 97 Bacteroides
0–6 Uncultured bacterial clone 3C3d-18
(AB034087)
Rumen 92 Clostridia
0–8 Mitsuokella jalaludinii strain M9
(AF479674)
Cattle rumen 96 Clostridia
0–11 M. jalaludinii strain M9
(AF479674)
Cattle rumen 98 Clostridia
0–12 M. jalaludinii strain M9
(AF479674)
Cattle rumen 94 Clostridia
0–13 Uncultured bacterial clone pPD6
(AF252319)
Compost 93 Bacilli
1–2 Thermoanaerobacterium
thermosaccharolyticum strain D120-70
(AF247003)
Extraction juice from sugar beet factory 98 Clostridia
1–3 T. thermosaccharolyticum strain D120-70
(AF247003)
Extraction juice from sugar beet factory 98 Clostridia
1–7 T. thermosaccharolyticum strain D120-70
(AF247003)
Extraction juice from sugar beet factory 97 Clostridia
4–1 Uncultured bacterial clone pPD6
(AF252319)
Compost 97 Bacilli
a Name of the DGGE bands in Fig. 2.
3.3. Whole cell hybridization
Whole cell hybridization and DAPI staining were em-
ployed to analyze microorganisms present at differentheight of the reactor bed and in the inoculum. DAPI
staining was employed to comparatively analyze mor-
phology of microbial community and enumerate total
cells while whole cell hybridization was performed to
analyze specific phylogenetic groups of microorganisms.
Epifluorescence microscopic observation of DAPI-
stained cells revealed that microorganisms in the TBR
inoculum had different morphology from those foundin TBR samples (Fig. 3). Long rod-shaped microorgan-
isms were frequently found in the samples containing
cells released from TBR biofilm. On the other hand,
the inoculum consisted of microorganisms with various
morphology, (long or short) rods and cocci. The result
of epifluorescence microscopy supported DGGE results
that samples obtained from TBR biofilm and TBR inoc-
ulum showed different microbial populations resultingfrom long-term operation of the TBR.
Although methane was not detected throughout the
normal operation of the reactor, we cannot eliminate
the possibility that the TBR might contain methanogens
as potential H2-consumers. Whole cell hybridization was
performed using probe ARC915 specific for the domain
Archaea where methanogens belong [32]. Cells hybrid-
ized with the ARC915 probe were not detected in the
samples obtained from the inoculum and TBR, suggest-
ing few or no methanogens in the samples. This result
was consistent with the gas analysis results that methanewas not detected throughout the normal operation of
the TBR.
The H2 production rate (1050 ± 63 mmol H2 l�1 d�1)
of the TBR is superior to the other fermentative H2-pro-
duction systems reported to date. The H2 yield
(1.11 ± 0.12 mol H2 mol�1 glucose) of the TBR is rea-
sonable but not superior [2,3]. In fermentative H2 pro-
duction, reduced end products such as alcohols andlactic acid represent hydrogen that has not been liber-
ated as hydrogen gas [16]. Thus, the production of re-
duced end products is associated with low H2 yield
[16,22,37]. Phylogenetically, lactic acid (producing) bac-
teria (LAB) can be divided into two groups [38,39]; most
LAB belong to Firmicutes, while some other LAB be-
long to Actinobacteria. In the phylum Firmicutes, the or-
der Lactobacillales contains the most important generaof LAB; Streptococcus, Lactobacillus, Lactococcus,
Enterococcus, Pediococcus, and Leuconostoc.
Among the major organic acids produced by the
TBR, lactic acid concentration was the highest [22].
TBR samples contained low number (less than 5% of to-
tal cells) of cells detected by the probes LGC354A-C, as
determined by whole cell hybridization (Fig. 3). The
Fig. 3. DAPI staining (A and D) and whole cell hybridization (B and E) of microorganisms with the probes LGC354A-C. A–C, inoculum of TBR;
D–F, cells released from biofilms formed on supporting matrix samples taken at the bed height of 26 cm; C and F, overlaid images of DAPI staining
and whole cell hybridization. All images were viewed by epifluorescence microscopy (1000·).
hybridized cells are likely to belong to members of the
Bacillales, considering the probes are specific for the
Bacillales and Lactobacillales orders of the Firmicutes
[33] and that DGGE analysis performed in this study re-
vealed 16S rRNA genes related to members of the
Bacillales but not Lactobacillales. Therefore, the results
of whole cell hybridization and DGGE suggest that
other microorganisms and not members of Lactobacill-ales play a role in producing lactate in the TBR.
3.4. PCR detection of methanogens
This study employed two different sets of mcrA-spe-
cific PCR primers to detect methanogens in the TBR
and inoculum samples. Repeatedly, no amplification
was obtained with the two sets of primers used (datanot shown), suggesting very few or no methanogens in
the TBR and inoculum samples, confirming the results
of the whole cell hybridization and gas analysis. How-
ever, all reference methanogens used in this study
showed PCR products of the correct sizes, except when
ME1/ME2 primers and M. concilli DSM 3671T total
DNA were employed, supporting a previous report that
these two sets of primers have different phylogenetic
coverage of methanogens [40].
However, the potential biases associated with DNA
extraction and PCR amplification [41,42] may limit thequantitative conclusions that can be drawn from the
PCR amplification of mcrA. Nonetheless, the results of
gas analyses and whole cell hybridization suggested that
H2 consumption by methanogens was negligible in the
TBR. However, possibility of H2 loss via CO2-reductive
acetogenesis [43] cannot be excluded considering the fact
that TBR showed 27.8% of conversion efficiency of glu-
cose to H2 (based on theoretical yield of 4 mol H2 mol�1
glucose when acetate is the sole byproduct [2,3]).
Now that we have our first insights into the microbial
community of a thermophilic TBR used for continuous
H2 production, it is clear from this study that bacteria
related to T. thermosaccharolyticum play a major role
in H2 production under the TBR conditions. Previous
studies [5–7,44] that employed different carbon sources
and reactor types also suggested the importance of these
bacteria in thermophilic H2 production. High concentra-tion of T. thermosaccharolyticum affiliated bacteria was
successfully maintained within the TBR to show supe-
rior H2 production rate. Considering that T. thermosac-
charolyticum can produce lactate by fermenting glucose
[34,45] and that lactate concentration was the highest
among the organic acids produced by the TBR, strate-
gies should be developed to avoid lactate formation to
increase H2 production yield since the pathway leadingto lactate is not related to H2 production [16].
Acknowledgment
This work was sponsored by Korea Research Foun-
dation Grant (KRF-2003-003-D00230).
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