Microbial diversity analysis of long term operated biofilm configured anaerobic reactor producing hydrogen from wastewater under diverse conditions

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Microbial diversity analysis of long term operated biofilm
configured anaerobic reactor producing biohydrogen from
wastewater under diverse conditions
S. Venkata Mohan*, S. Veer Raghavulu, R. Kannaiah Goud, S. Srikanth, V. Lalit Babu,
P.N. Sarma
Bioengineering and Environmental Centre (BEEC), Indian Institute of Chemical Technology (IICT), Hyderabad 500 607, India
a r t i c l e i n f o
Article history:
Received 22 June 2010
Received in revised form
29 July 2010
Accepted 1 August 2010
Available online 20 September 2010
Keywords:
16S rDNA
PCR-DGGE
Phylogenetic analysis
Mixed culture
Dairy wastewater
Sequencing batch reactor (SBR)
a b s t r a c t
This communication provides an insight into the composition of the microbial community
survived in the biofilm configured anaerobic reactor operated for biohydrogen (H2)
production using wastewater as substrate under diverse conditions for past four years. PCR
amplified 16S rDNA product (at variable V3 region using universal primers 341F and 517R)
was separated by using denaturing gradient gel electrophoresis (DGGE) to identify the
diversity in microbial population survived. The phyologenetic profile of the bioreactor
showed significant diversity in the microbial community where major nucleotide
sequences were affiliated to Class Clostridia followed by Bacteroidetes, Deltaproteobacteria
and Flavobacteria. Clostridium were found to be dominant in the microbial community
observed. The controlled growth conditions, application of pre-treatment to biocatalyst,
operation with specific pH and variation in substrate composition are reasoned for the
robust acidogenic culture identified in the bioreactor. Most of the operational taxonomic
units (OTUs) observed in the bioreactor are capable to undergo acetate producing pathway,
feasible for effective H2production.
ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
1.Introduction
The rapid growth and industrialization of nations and bur-
geoning economies have resulted in a steep increase in fossil
fuel production and utilization. This has not only put a severe
strain on the already depleting fossil fuels but also resulted in
an alarming increase in pollution levels across the globe.
Thus, the rapid development of alternative, renewable,
carbon-neutral and eco-friendly fuels is of paramount
importance and biofuels perfectly fit this bill. In this realm,
biohydrogen (H2) production is considered as one of the
opportunistic and sustainable way to meet the future energy
demand. One of the possible biological approaches for
producing biohydrogen is to convert negative valued organic
waste under anaerobic fermentation into valuable sources
[1,2]. Much of the research on biohydrogen so far was
concentrated on the optimization studies, while, relatively
less stressappearsonthe community structure analysisofthe
biocatalyst (consortia) used [3,4]. Nature of the fermentation
products depends on the microbial diversity present in the
bioreactor [5]. Microbial community/diversity greatly influ-
ences the production of volatile fatty acids (VFA) which is one
of the crucial factor for H2production [6,7]. Generally, micro-
bial diversity gets influenced by the operating conditions used
viz., nature of substrate, substrate loading rate, hydraulic
retention time (HRT), operating pH, etc. Correlating the oper-
ation data with the microbial community profile will help to
understand the process happening in the bioreactor and
* Corresponding author. Tel.: þ91 40 27191664.
E-mail address: vmohan_s@yahoo.com (S. Venkata Mohan).
0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2010.08.008
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
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further facilitate to optimize the system conditions for effec-
tive output.
Polymerase chain reaction (PCR)-denaturing gradient gel
electrophoresis (DGGE) is generally used to determine the
existing and dominant members of the microbial commu-
nity. The strength of DGGE as a screening method for
diversity lies in its ability to monitor the community struc-
ture in response to the changes in experimental parameters
by overcoming some of the limitations in cultural tech-
niques [8]. Full-cycle 16S rDNA analysis has allowed micro-
biologists to describe the diversity of individuals within the
population and to identify novel organisms [9]. The 16S
ribosomal DNA genes are useful for such studies as these
genes are present in all bacteria and comparison of
sequences of 16S ribosomal DNA genes has been well
established as a standard method for the identification of
bacteria [10]. The genes contain both conserved and variable
regions which can be used respectively for primer designing
in PCR amplification and to distinguish sequences from each
other. All the 16S rDNA genes are equally accessible for the
amplification, whether whole cell or extracted, physiological
state of the cell doesn’t effect the amplification and it is the
basic principle behind the success of this technique [10]. An
attempt was made in this communication to study the
microbial diversity persisted in anaerobic biofilm reactor
being operated for past four years to produce H2 from
different types of wastewaters under highly variable condi-
tions (Table 1) employing PCR-DGGE based 16S rDNA
molecular technique. The main focus of this study was to
interpret the relation among the persistent microbial
communities established during the long term operation of
the bioreactor with the function of bioreactor performance.
2. Materials and methods
2.1.Anaerobic consortia
Anaerobic mixed microflora from an operating laboratory
scale upflow anaerobic sludge blanket (UASB) reactor treating
chemical wastewater was used as parent inoculum for the
startup of the bioreactor. Prior to use, the seed inoculum was
sieved to remove any stone, sand and other coarse material.
Prior to inoculation, dewatered sludge was subjected to cyclic
pre-treatment sequence (four times) changing between heat-
shock (100?C, 2 h) and acid [pH 3 adjusted with orthophos-
phoric acid (88%), 24 h] treatment to restrain the growth of
methanogenic bacteria (MB) and at the same time to selec-
tively enrich the H2 producing acidogenic bacteria (AB)
[11e13]. The resulting enriched mixed culture was used as
parent inoculum for the startup of the bioreactors. During
operation occasionally, the reactors were subjected to chem-
ical pre-treatment by employing 2-bromoethane sulphonic
acid sodium salt (BESA, 0.2 g/l; 24 h).
2.2.Anaerobic bioreactor
Bench scale, biofilm configured, anaerobic reactor was
designed and fabricated in laboratory using ‘perspex’ material
to operate in upflow mode with a pre-defined volume of 1.8 l
and a gas holding capacity of 0.3 l (L/D ratio w9.7). Inert stone
chips (0.02e0.05 cm; void ratio w0.54) were used as packing
material for fixed bed to support the biofilm growth. Initially
the reactor was operated in suspended growth configuration
for a period of 320 days and subsequently shifted to biofilm
configuration. Peristalticpumps
programmedelectronictimerswereusedto regulate theFEED,
RECIRCULATION and DECANT operations. The bioreactor was
initially operated with designed synthetic wastewater [(mg/l)
sucrose, 3000; NH4Cl, 500; K2HPO4, 250; KH2PO4, 250; CaCl2, 5;
MgCl2, 300; FeCl3, 25; CoCl2, 25; MnCl2, 15; NiSO4, 16; CuCl2, 10;
ZnCl2, 10] as feed at an organic loading rate (OLR) of
1.8 kg COD/m3-day in fed-batch mode [periodic discontinuous
batch (PDBR)/sequencing batch (SBR)] with a total cycle period
of 24 h comprising 15 min FILL, 23 h REACT, 30 min SETTLE
and 15 min DECANT phases after adjusting the pH to 7 so as to
support the biofilm formation on the packing medium. At the
beginning of each cycle, immediately after withdrawal (earlier
sequence), a pre-defined volume (1.5 l) was fed to the reactor
during FILL phase and the reactor volume was circulated with
outlet-closed loop mechanism at recirculation volume to feed
volume ratio of 3 during the REACT phase to achieve
a homogeneous distribution of the substrate. Constant COD
removal and gas production (w5% variation) were considered
as the indicators for stabilized performance of the reactors.
Subsequently, reactors were shifted to real field wastewater
after adjusting the pH to 6.0 till stable performance was
attained in all the experimental variations studied (Table 1).
Prior to feeding, influent pH of wastewater was adjusted to
6.0/7.0, using orthophosphoric acid (88%) or 1 M NaOH. After
feeding wastewater, the reactor was sparged with oxygen free
nitrogengasfor2 minto removepossibleoxygenaccumulated
in the headspace of the reactor to ensure complete anaerobic
condition. All the experiments were performed at mesophilic
(room) temperature (28 ? 2
experiments was estimated using a microprocessor based
pre-calibrated electrochemical H2sensor (ATMI GmBH Inc.,
Germany). Volatile fatty acids (VFA) and COD (closed dichro-
mate refluxing method) were determined according to the
standard methods [14].
integratedwith pre-
?C). H2 gas generated during
2.3.DNA extraction
The biofilm attached to the stone chips of the biohydrogen
reactor was scraped and washed with phosphate buffer
(50 mM) twice followed by centrifugation (1200 g for 5 min) to
remove suspended particles. Further, bacterial cells were
collected by centrifuging the supernant at 8100 g for 10 min
and the resulting pellet was suspended in 200 ml TE buffer.
Pellet (5 mg) was re-suspended in 0.5 ml of lysis buffer (pH 8,
Na2EDTA, 100 mM; TriseHCl, 100 mM; sodium phosphate
buffer 100 mM) mixed with NaCl (1.5 M) and hexadecyl-
trimethyl ammonium bromide (CTAB) (1%) followed by lyso-
zyme solution (50 mg/ml) and incubated at 37
30 min.50 ml of proteinase K solution (20 mg/l) was added and
the mixture was re-incubated at 37
further treated with 200 ml of 20% sodium do-decyl sulphate
(SDS). Subsequently, the suspension was agitated at 600 g at
65?C in water bath for 2 h with end-over-end inversion for
every 20 min and later centrifuged at 7800 g for 15 min.
?C for
?C for 30 min. It was
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The supernatant was extracted with an equal volume
of phenolechloroformeisoamyl alcohol (25:24:1, v/v) and
centrifuged at 9200 g for 15 min and the same step was
repeated once again. RNase solution (100 ml) was added to the
resulting aqueous phase and incubated at 37?C for 30 min.
The supernatant was mixed again with an equal volume of
chloroform/isoamylalcohol (24:1) solution and centrifuged at
10,900 g for 15 min. Later, the supernatant was mixed with 0.6
volume of isopropanol and kept at 4?C overnight. Further, the
DNA precipitate was centrifuged at 13,000 g for 20 min at 4?C
and then rinsed with 0.4 ml of 70% cold ethanol and subjected
for air-drying for 5 min, before being dissolved in 100 ml of
water [15,16].
2.4. PCR amplification
The variable V3 region of 16S rDNA was amplified by PCR with
primers to conserved regions of the 16S rRNA genes. The
nucleotide sequences of the primers were as follows: primer
341F, 50-AGG CCT AAC ACA TGC AAG TC-30; primer 517R, 50-
ATT ACC GCG GCT GCT GG-30; GC clamp was added to primer
63FGC, 50-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG
GCA CGG GGG GAG GCC TAA CAC ATG CAA GTC-30. The GC-
rich sequence attached to the 50-end of forward primer
prevents the PCR products from complete melting during
separation via DGGE [14]. Both primers have shown to anneal
with a majority of bacterial sequences in the ribosomal data-
base. All the PCR amplifications were conducted in 50 ml
volume containing 2 ml of total DNA having 100 ng/ml
concentration, 200 mM each of the four deoxynucleotide
triphosphates, 15 mM MgCl2, 0.1 mM of individual primers and
1 unit of Taq polymerase. An automated thermal cycler
(Eppendorf) was used for PCR amplification which was pro-
grammed for an initial denaturation at 96?C for 5 min, 35
cycles of denaturation (40 s at 94?C), annealing (50 s at 52.6?C)
and extension (1 min at 72?C) and a final extension (72?C for
8 min). Finally, the amplified PCR product was stored at 4?C.
The samples were verified on 1% agarose gel.
2.5.
screening
Denaturing gradient gel electrophoresis (DGGE)
DGGE has unique features, that it can differentiate mixture of
DNA,thoughtheydifferinsinglebasepairamongthem.Hence,
Table 1 e Long term operational details of anaerobic biofilm reactor during hydrogen production.
Days after startup Operating conditionsPurpose Reference
0a
Inoculated anaerobic culture acquired from
operating UASB reactor
Operated with design synthetic wastewater (DSW)
at neutral pH in batch mode with 24 h HRT
Operated with chemical wastewater (CCW,
single process) at neutral pH in batch mode
at 24 h HRT
Applied combined pre-treatment [heat-shock
(100?C, 2 h) and acid (pH 3.0, 24 h)]
Operated with DSW at variable OLRs at
acidophilic pH (6) in batch mode with 24 h HRT
Operated with dairy wastewater (DW)
at variable OLRs at acidophilic pH (6) in
batch mode with 24 h HRT
Reactor configuration changed to biofilm
configuration
Operated with DSW at neutral pH in batch
mode with 24 h HRT
Applied combined pre-treatment [heat-shock
(100?C, 2 h) and acid (pH 3.0, 24 h)]
Operated with DSW at acidophilic pH (6) in
batch mode with 24 h HRT
Operated with DSW at acidophilic pH (6) in
continuous mode with 24 h HRT
Operated with DSW at neutral pH in
continuous mode with 24 h HRT
Operated with at neutral pH in batch
mode with 24 h HRT
Shifted to DW at pH 6 in batch mode with
24 h HRT
Reactor operation hampered due to leakages
After repairing reactor was subjected to combined
pre-treatment (BESA, heat-shock and acidic)
Restarted with DW at pH 6 in batch mode
with 24 h HRT
Collected sample for microbial community
analysis
To facilitates adaptation of inoculated culture
e
95
To adopt microflora for complex wastewater
e
191
To selectively enrich acidogenic H2producing
bacteria
[11]
204[12]
316
To study the feasibility of H2production with
DW at variable organic loading conditions
[12]
432To study the influence of biofilm configuration
e
445 To facilitates adaptation of inoculated culture
and growth of biofilm on fixed bed
To selectively enrich acidogenic H2producing
bacteria
To study the process efficiency in biofilm
configuration
To study H2production during continuous mode
operation at pH 6
To study H2production during continuous mode
operation at pH 7
To study H2production during batch mode
operation at pH 7
e
514
e
519 [13]
575
[19]
624
[19]
689
[19]
712
e
969
1087
e
e
e
e
1120
ee
1211
ee
a Reactor operation started in March 2005.
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thistechniqueismoreappropriateforthecommunityanalysis
of mixed consortia. Further more, it is more sophisticated and
simple technique compared to the other molecular tools used
for community analysis [4]. DGGE was performed using the
DCode? Universal Mutation Detection System (BioRad).
Samples containing approximately equal amounts of PCR
amplicons (35 ml) were loaded onto 1 mm thick vertical gels
containing 6% (w/v) polyacrylamide with a linear gradient of
denaturants (urea and formamide). A denaturing gradient of
30e70% was applied to separate 16S rDNA fragments (where
100% denaturant is defined as 7 M urea and 40% (v/v) form-
amide). The gels were prepared in 1? TAE buffer (20 mM Tris,
10 mM acetic acid, 0.5 mM ethylenediamine tetraacetic acid
(EDTA), pH 8.0) which was also used as the electrophoresis
buffer. Electrophoresis was run at 60?C, initially for 1 h under
80 V and then for 12 h at a constant voltage of 100 V. After
electrophoresis, the gels were stained with ethidium bromide
(0.5mg/l)for15minfollowedbydestainingindistilledwaterfor
20 min. Images were captured using Molecular Imager (G:BOX
EF System; Syngene).
2.6.DNA sequence and phylogenetic analysis
The middle portion of the selected DGGE band was excised
with a sterile pipette tip. The excised gel was incubated
overnight at 4?C in 50 ml of sterile distilled water. 10 ml of
eluted DNA was used as the template for PCR performed
under the conditions described as above, except that the
forwardprimer lackedthe GCclamp.A 5 ml sampleofeachPCR
product was subjected to agarose gel electrophoresis to
confirm product recovery and to estimate product concen-
tration. Five microlitres of each reaction mixture was again
subjected to DGGE analysis as described before to check the
purity and to confirm the melting behavior of the band
recovered. Some DNA samples still showed to contain mixed
products, as shown by multiple DGGE bands. In each of these
cases, the target band was excised from the recovered pattern
and reanalyzed with DGGE for getting single bands.
For sequencing analysis, amplified PCR products were sent
to MWG Biotech. All the 16S rDNA partial sequences were
aligned with those of the reference microorganisms in same
region of the closest relative strains available in the GenBank
database by using the BLASTN facility (http://www.ncbi.nlm.
nih.gov/BLAST/) and were also tested for possible chimera
formation with the CHECK CHIMERA program (http://www.
35.8.164.52/cgis/chimera.cgi?su¼SSU).
were further aligned with the closest matches found in the
GenBank Database with the CLUSTALW function of MEGA
version 4.0. Neighbor-joining phylogenetic trees were con-
structed with the Molecular Evolutionary Genetics Analysis
package (MEGA version 4.0) [17]. A bootstrap analysis with 500
replicates was carried out to check the robustness of the tree.
Bootstrap re-sampling analysis for the replicates was per-
formed to estimate the confidence of the tree topologies.
Thesesequences
2.7.Nucleotide sequence accession numbers
Sequences were submitted to the Nucleotide Sequence Data-
base to the Gene Bank public database under the accession
numbers from FN641894 to FN641902.
3. Results and discussion
3.1.
and substrate degradation
Performance of bioreactor-biohydrogen production
HBR3 reactor was specifically designed to study the feasibility
of biohydrogen production from the treatment of wastewater
employing anaerobic mixed consortia as biocatalyst (Table 1).
The bioreactor was initially operated in suspended growth
configuration for a period of 320 days and later shifted to
biofilm configuration. After inoculation, the bioreactor was
operated with design synthetic wastewater (DSW) for 99 days
at neutral pH to facilitate adaptation of the consortia to new
microenvironment. Subsequently, the feed was changed to
chemical wastewater (CCW) and operated at neutral pH to
enrich robust microflora which can sustain complex indus-
trial wastewater. During this phase of operation, negligible
amount of H2 production was observed despite of good
substrate degradation efficiency. After 191 days of startup, the
resident microflora present in the bioreactor was subjected to
combined pre-treatment employing heat-shock (100?C, 2 h)
and acid (pH 3.0, 24 h) methods so as to selectively enrich
acidophilic/acidogenic microflora. Since then, the bioreactor
was subjected to BESA treatment once in 90 days for 24 h.
Subsequently, the reactor was operated with dairy waste-
water (DW) at three OLRs of 2.4, 3.5, and 4.7 kg COD/m3-day at
acidophilic pH condition with 24 h retention time at room
temperature in fed-batch mode [12].
The operation data of bioreactor is depicted in Table 1. The
performance data showed the feasibility of molecular H2
production by utilizing wastewater as substrate. However, H2
generation rate differed significantly with the OLR of waste-
water used. Substrate (COD) removal efficiency varied
between 50 and 64.7%. Most part of the bioreactor operation
was under acidophilic condition except in two instances
where neutral pH was applied to study its specific role on the
H2production. The pH range of 5.5e6.0 is reported to be ideal
to avoid both methanogenesis and solventogenesis [2,18e20].
Maintaining pH in the acidic redox range helps in the
suppression of MB, thus indirectly promoting H2producers
within the system. After operating in suspended mode with
DW, the bioreactor was modified to biofilm configuration by
placing stone chips as fixed bed supporting medium. The
consortia present suspension form was reused as inoculum
for startup of biofilm system. At this stage, the system was
operated with DSW at neutral pH to facilitate biofilm forma-
tion. After 514 days of startup, the biofilm microflora was
subjected to combined pre-treatment viz., heat-shock and
acid methods and the performance was assessed at OLR of
3.4 kg COD/m3-day under acidophilic condition. Configuration
of bioreactor showed marked influence on overall perfor-
mance [13]. Biofilm configured system registered relatively
efficient performance with respect to both H2production and
substrate degradation associated with higher VFA generation
over the corresponding suspended mode operation. Later, the
performance of biofilm reactor was evaluated by varying the
mode of operation (continuous and fed-batch) in concurrence
with the redox condition (acidophilic (pH 6.0) and neutral (pH
7.0)) (Table 1) [19]. Acidic pH (below 6) showed less substrate
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degradation efficiency than the corresponding neutral oper-
ation due to the reduced methanogenic activity. Maintenance
of acidic conditions in association with pre-treatment of bio-
catalyst has been observed to be effective for H2production.
Relatively higher levels of soluble metabolite production were
observed under acidic operation over the corresponding
neutral microenvironment, which corroborated well with the
H2production data. About 25% improvement in H2production
and substrate degradation efficiency was reported with batch
mode operation compared to the corresponding continuous
mode operation. Fed-batch mode of operation associated with
acidic pH showed highest H2production. On 969th days after
startup, the reactor showed leakage in joints at the lower/
basal part when hot water was introduced to pretreat the
biofilm. Choking of ports was also observed due to the settling
of sludge. Operation of bioreactor was stopped and after
rectification was restarted by applying pre-treatment on
1120th day. The bioreactor regained its performance rapidly
after the restart. During reactor operation, silicon tubes used
for recirculation resulted in occasional leaks which were
replaced immediately with new ones as and when required.
On 1211th days after startup, we have collected biofilm
samples for microbial community analysis.
3.2.Structure of microbial community in bioreactor
DGGE profiles generated from the universal bacterial primers
revealed the structural composition of communities in the
long term operated bioreactor system based on the V3 region
of16srRNA.Different banding
community structure as well as species diversity were
observed (image not given). Each of the distinguishable bands
in the separation pattern represents an individual bacterial
genus. The phyologenetic distribution was established with
a boot-strarp neighbour-joining method as shown in Fig. 1.
Dominant operational taxonomic units (OTUs) observed in the
bioreactor could be divided into four groups viz., Bacter-
oidetes, Clostridia, Flavobacteria and Deltaproteobacteria
(Table 2). Major bands were phylogenetically related to class
Clostridia (3 out of 8; HBR3-6, HBR3-7 and HBR3-8) followed by
patternsofmicrobial
Fig. 1 e Neighbor-joining trees constructed using Mega 4.0 showing phylogenetic relationships of 16S rDNA sequences from
bioreactor to closely related sequences from Gene Bank.
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Bacteroidetes (2 out of 8; HBR3-2 and HBR3-8), Deltaproteo-
bacteria (2 out of 8; HBR3-6 and HBR3-7) and Flavobacteria (1
out of 8; HBR3-1). The two strongly stained bands observed in
DGGE profiles suggest the occurrence of strong enrichment
culture in the of bioreactor samples (HBR3-3 and HBR3-4).
Majority of the DGGE bands were associated with the class
Clostridia [HBR3-6 (Clostridium carboxidivorans (AY170379.1,
98%), Clostridium drakei (AJ427628.1, 98% and Y18813.1 (partial),
98%)); HBR3-7 (Clostridium acetobutylicum (AY188847.1, 97%),
Clostridium sps (AY188846.1, 97%)); HBR3-8 [Eubacterium budayi
(NR024682.1, 97%), Eubacterium nitritogenes strain (NR024684.1
97%), Uncultured Firmicutes (CU919549.1, 97%)]. Clostridia
(gram-positive, low GþC) belongs to Fermicutes phyla are
generally wide spread in environmental samples and are
mostly with diverse metabolic activity. Many Clostridia species
are reported to be capable of producing H2, including Cl. ace-
tobutylicum [21], Clostridium butyricum [22], Clostridium kluyveri
[23] and Clostridium pasteurianum [1]. 16S rDNA fragments or
isolates affiliated with these bacteria have been found in
anaerobic reactors used for thermophilic H2production [24].
Eubacterium sps was reported to be anaerobic degraders of
amino acid either producing or utilizing H2or formate [1].
Fermicutes are reported to grow in acidogenic microenviron-
ment and form endospores in adverse conditions [25], which
substantiate its presence in the acidogenic reactors. Fig. 1
shows that OTUs HBR3-6 and HBR3-8 are most closely
related to each other, with a node bootstrap value of 99%
similarity, based on 186 nucleotides. These two OTUs are also
distantly related with OTU from HBR3-7. Similarity of OTUs
HBR3-6 and HBR3-8 is 78%, based on 186 nucleotides.
Next to class Clostridia, Bacteroidetes were dominant in
the bioreactors [HBR3-2 (Bacteroides massiliensis (FJ219917.1,
98%), Uncultured bacteriodes sps (FJ219913.1, 98% and
FJ219892.1, 98%)); HBR3-3 (Prevotella bryantii (Uncultured
bacterium clone; EU533427.1, 97%), Prevotella buccae (L16477.1,
99% and L16478.1, 97%))]. Bacteroidetes are gram-negative
anaerobes, able to grow on a variety of carbohydrates. Pre-
votella sps are generally pathogenic in nature which produces
H2under anaerobic conditions [1]. P. bryantii was reported to
have 40-fold higher degrading capacity than other strains [26].
Prevotella sps were characterized for their polysaccharide-
degrading activities with reducing sugars which constitutively
produce polysaccharide degrading enzymes. Prevotella sps
produce acetic, isobutyric, isovaleric and succinic acids as
metabolic end products from the fermentation of glucose,
sucrose and starch [27]. OTUs HBR3-1 showed 99% similarity
with OTUs HBR3-2 and HBR3-3, where as the later OTUs have
88% similarity among themselves.
Desulfovibrio putealis (EU732609.1, 93%), Desulfovibrio africa-
nus (EU659693.1 93%), Desulfovibrio (DQ839137.1, 93%), Desulfo-
vibrio paquesii (AY726757.3 96%)
(M34400.1, 98%)] sustained in the bioreactor which posses
sulfate-reducing property leading to acetate production. Some
Deltaproteobacteria (gram-negative) are of anaerobic genera
andmostofthemareknownsulfate-reducers.D.putealisandD.
gigas were reported as H2producers [28].These bacteria are
reportedtohavehydrogenasesintheirmembranewhichplays
vital role in H2production [29]. OTUs HBR3-4 and HBR3-5 have
shown 100% similarity among them. In the case of Flavobac-
teria, the specific OTUs were affiliated to Tenacibaculum
and Desulfovibrio gigas
Table 2 e Phylogenetic sequence affiliation and similarity to the closet relative of amplified 16 rDNA sequence excised from
DGGE.
Band numberOrganismBlast similarity
(%)
Accession number Phylogenetic affiliation
HBR3-1Tenacibaculum aiptasiae
Tenacibaculum sps
Tenacibaculum sps
Bacteroides massiliensis
Uncultured Bacteroides
Uncultured Bacteroides
Prevotella bryantii
(Uncultured bacterium clone)
Prevotella buccae
Prevotella buccae
Desulfovibrio putealis
(Bacterium enrichment culture)
Desulfovibrio africanus
Desulfovibrio sps
Desulfovibrio gigas
(D.gigas 16S ribosomal rRNA)
Desulfovibrio paquesii
Desulfovibrio gigas
Clostridium carboxidivorans
Clostridium drakei
Clostridium drakei (partial)
Clostridium acetobutylicum
Clostridium sps
Clostridium acetobutylicum
Eubacterium budayi
Eubacterium nitritogenes
Uncultured Firmicutes
97
97
97
98
98
98
97
FJ210798.1
EU651805.1
AB265190.1
FJ219917.1
FJ219913.1
FJ219892.1
EU533427.1
Flavobacteria
HBR3-2 Bacteroidetes
HBR3-3Bacteroidetes
99
97
93
L16477.1
L16478.1
EU732609.1HBR3-4 Deltaproteobacteria
93
93
98
DQ839137.1
EU659693.1
M34400.1HBR3-5Deltaproteobacteria
96
95
98
98
98
97
97
97
97
97
97
AY726757.3
DQ447183.2
AY170379.1
AJ427628.1
Y18813.1
AY188847.1
AY188846.1
AE001437.1
NR024682.1
NR024684.1
CU919549.1
HBR3-6 Clostridia
HBR3-7Clostridia
HBR3-8 Clostridia
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aiptasiae (FJ210798.1) and Tenacibaculum sps (EU651805.1 97%).
H2producing capability in Tenacibaculum sps has not been
reported so far as per the best of our knowledge. As expected,
aerobicand photosynthetic bacteriawereabsentin the reactor
because of the prevailing anaerobic-acidogenic microenviron-
ment. Despite the apparent similarities by major phylogenetic
groups, each fermentation type was unique in the bioreactor.
Characterization of community structure analysis in the
bioreactor demonstrated the successful establishment of
microbial community capable of harnessing H2. Bacteria
capable of producing H2are reported to distribute across 10 of
the 35 bacterial groups [1]. The controlled growth conditions,
selective enrichment of biocatalyst by applying periodic pre-
treatment, operation with specific pH and variation in
substrate composition along with substrate load might be the
probable reasons for lesser diversity observed in the biore-
actor. However, longer period of operation with diverse
operating conditions might result in survival of robust and
selectively enriched bacteria which are capable of producing
H2 under acidogenic conditions. Except few strains, our
results differ from previous reports on community charac-
terization in H2producing microflora [5,8,9,30]. The observed
difference in dominant populations might be due to the
difference in the parent inoculum used, operation conditions
adopted and the substrate type selected. Differences of
community structure reflect the differences in fermentation
type and the distribution and dominance of the fermentation
products depend on the dominant microorganisms present,
which can be controlled by handling the operating conditions.
Table 3 depicts some of the possible metabolites produced by
the microbial population observed in the bioreactor. Almost
all the OTUs present in the bioreactor can undergo acetate
producing pathway an important metabolite synthesized
during acidogenic H2producing process.
4.Conclusions
Microbial diversity in the long term operated biohydrogen
producing reactor under diverse operating conditions was
evaluated using PCR-DGGE. The phyologenetic distribution
showed significant diversity in microbial community, where,
the major nucleotide sequences of DGGE bands were affiliated
to Class Clostridia followed by Bacteroidetes, Deltaproteo-
bacteria and Flavobacteria. Clostridium are found to be
dominant in the bioreactor. Almost all the OTUs identified
from the bioreactor have capability to undergo acetate
producing pathway, which is an important metabolite
synthesized during acidogenic-hydrogen production process.
The diverse operating conditions (various substrates, pHs,
HRTs, and COD loading rates) employed during bioreactor
operation for past four years might helped to enrich robust
acidogenic-hydrogen producers. This study helps in devel-
oping specific mixed microbial culture which can efficiently
produce biohydrogen under diverse operating conditions with
simultaneously wastewater treatment.
Acknowledgements
The authors wish to thank the Director, IICT for his support
and encouragement in carrying out this work. SVR acknowl-
edges Indian Council for Medical Research (ICMR) and RKG, SS
and VLB thank the Council of Scientific and Industrial
Research (CSIR) for providing research fellowships.
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