www.fems-microbiology.org

FEMS Microbiology Letters 249 (2005) 31–38

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

Received 16 March 2005; received in revised form 23 May 2005; accepted 27 May 2005

First published online 14 June 2005

Edited by E. Baggs

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-

0378-1097/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2005.05.050

* Corresponding author. Tel.: +82 42 869 3941; fax: +82 42 869 3910.

E-mail address: yahn@kaist.ac.kr (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].

. Published by Elsevier B.V. All rights reserved.

32 Y. Ahn et al. / FEMS Microbiology Letters 249 (2005) 31–38

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.

Y. Ahn et al. / FEMS Microbiology Letters 249 (2005) 31–38 33

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

34 Y. Ahn et al. / FEMS Microbiology Letters 249 (2005) 31–38

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.

Y. Ahn et al. / FEMS Microbiology Letters 249 (2005) 31–38 35

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·).

36 Y. Ahn et al. / FEMS Microbiology Letters 249 (2005) 31–38

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

Y. Ahn et al. / FEMS Microbiology Letters 249 (2005) 31–38 37

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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