Isolation of hydrogen-producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor

Abstract

H2-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36 grown under aerobic conditions and
36 under anaerobic conditions) arbitrarily chosen from the agar plate cultures of a suspended sludge, 34 colonies (15 under
aerobic conditions and 19 under anaerobic conditions) produced H2 under anaerobic conditions. Based on various biochemical tests and microscopic observations, they were classified into 13
groups and tentatively identified as follows: From aerobic isolates,Aeromonas spp. (7 strains),Pseudomonas spp. (3 strains), andVibrio spp. (5 strains); from anaerobic isolates,Actinomyces spp. (11 strains),Clostridium spp. (7 strains), andPorphyromonas sp. When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H2 production yield in the batch cultivations after 12h (2.24–2.74 OD at 600 nm and 1.02–1.22 mol H2/mol glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic isolates, and ethanol,
acetate and propionate for the anaerobic isolates. This study demonstrated that several H2 producers in an anaerobic granular sludge exist in large proportions and their performance in terms of H2 production is quite similar.

Full text

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Biotechnology and Bioprocess Engineering 2003, 8: 54-57


Isolation of Hydrogen-producing Bacteria from Granular
Sludge of an Upflow Anaerobic Sludge Blanket Reactor

You-Kwan Oh2, Mi So Park1, Eun-Hee Seol1, Sang-Joon Lee3, and Sunghoon Park1,2*

1 Department of Chemical Engineering, 2 Institute for Environmental Technology and Industry, and
3 Department of Microbiology, Pusan National University, Pusan 609-735, Korea


Abstract H2-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36
grown under aerobic conditions and 36 under anaerobic conditions) arbitrarily chosen from the agar plate
cultures of a suspended sludge, 34 colonies (15 under aerobic conditions and 19 under anaerobic
conditions) produced H2 under anaerobic conditions. Based on various biochemical tests and microscopic
observations, they were classified into 13 groups and tentatively identified as follows: From aerobic
isolates, Aeromonas spp. (7 strains), Pseudomonas spp. (3 strains), and Vibrio spp. (5 strains); from
anaerobic isolates, Actinomyces spp. (11 strains), Clostridium spp. (7 strains), and Porphyromonas sp.
When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H2
production yield in the batch cultivations after 12 h (2.24 - 2.74 OD at 600 nm and 1.02-1.22 mol H2/mol
glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic
isolates, and ethanol, acetate and propionate for the anaerobic isolates. This study demonstrated that
several H2 producers in an anaerobic granular sludge exist in large proportions and their performance in
terms of H2 production is quite similar.

Keywords: hydrogen, granular sludge, UASB, fermentation



H2 is an efficient energy carrier with a high energy
content per unit mass. It is considered to be the cleanest
energy carrier because the combustion by-product is only
water and it does not produce a green house gas [1]. It can
also be used as a raw material for various industrial
applications, which is gaining increasing attention [2].
Microbial H2 production can be either photosynthetic or
non-photosynthetic. Photosynthetic H2 production is carried
out by algae [3] or photosynthetic bacteria [4]. Non-
photosynthetic or fermentative H2 production is performed
by facultative anaerobes [5,6] or obligate anaerobes [2,7].
The fermentative H2 process generally has a faster
production rate than the photosynthetic process and does not
rely on the availability of light. However, the H2 conversion
yield (mol H2/mol substrate) is lower than that obtained
using the photosynthetic process [1,8]. There have been a
large number of studies on fermentative H2 producers with
some emphasis on the H2 production rate and yield from
organic carbon [2,6,8,9]. Kumar and Das [8] reported that
Enterobacter cloacae IIT-BT 08 has a high specific H2
production rate of 29.6 mmol H2 (g cell)-1 h-1 and a high H2
conversion yield of 2.2 mol H2/mol glucose. Taguchi et al.
[2] reported that the H2 production yields of Clostri-


* Corresponding author
Tel: +82-51-510-2395 Fax: +82-51-512-8563
e-mail: shpark0@pusan.ac.kr

©KSBB
dium beijerinckii AM21B isolated from termites ranged
from 1.3 to 2.0 mol H2/mol glucose. Oh et al. [5] reported
that Rhodopseudomonas palustris P4, although originally
isolated for CO-dependent H2 production, could perform
fermentative H2 production utilizing various organic
carbons.
In order to develop a cost-effective H2 production process,
the availability of efficient strains is very important. Thus
far, the isolation of the H2 producers has been mostly
dependent on the H2 production capability of each strain.
This requires laborious experiments for growing the many
individual cells under well-controlled conditions and
measuring their H2 production activity. This study focused
on isolating new H2-producing bacteria from the
biogranules of a UASB (upflow anaerobic sludge blanket)
reactor. The UASB reactor is currently being used to treat
high-strength organic wastewater and is known to contain
many acidogenic bacteria that are often H2 producers. The
anaerobic granules were homogenized, diluted, and plated
on agar, and cultivated under aerobic or anaerobic
conditions. Some colonies were isolated, identified by
various biochemical tests, and finally examined for their
individual H2 production capability in a liquid culture.
The UASB reactor (working volume, 4.5 L) was operated
at 35oC with a hydraulic retention time of 12 h in a
continuous mode. The feed contents (per liter) were: 2,200
mg glucose, 50 mg of a beef extract, 55 mg of a yeast
extract, 50 mg peptone, 50 mg urea, 2,800 mg NaHCO3, 66

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Biotechnol. Bioprocess Eng. 2003, Vol. 8, No. 1

mg Na2HPO4 ⋅ 2H2O, 44 mg K2HPO4, 36 mg KH2PO4, 30
mg K2SO4, 6 mg FeSO4 ⋅ 7H2O, 20 mg NH4Cl, 20 mg
MgSO4 ⋅ 7H2O, 1.34 mg CoCl2 ⋅ 6H2O, 76 mg CaCl2 ⋅ 2H2O,
0.05 mg H3BO3, 0.11 mg ZnSO4, 0.025 mg Na2MoO4 ⋅
2H2O, 0.038 mg MnSO4, 0.07 mg CuSO4 ⋅ 5H2O, 1 mg
EDTA, 0.07 mg NiNO3, and 2 mg AlCl3 ⋅ 2H2O [10]. The
granular sludge was disper-sed into an aqueous suspension
by vortexing it in PBS buffer (pH 7.2, 10 mM Na2HPO4,
and 0.13% NaCl) for 5 min. The suspension was diluted
serially, plated on an NB medium (pH 6.8, 0.3% beef
extract, and 0.5% peptone) and incubated at 30oC under
either aerobic or anaerobic conditions. The anaerobic
conditions were produced by placing the plates in an acrylic
desiccator container, and repeatedly vacuuming and purging
the container with argon (Ar) gas (99.999%). After
incubating for 24-36 h under either aerobic or anaerobic
conditions, 72 colonies (36 of each type) were selected
randomly and transferred to fresh NB plates at 12 colonies
per plate.
The 72 colonies were tested for H2 production, and 34
colonies (15 grown under aerobic conditions and 19 under
anaerobic conditions) were selected as the H2 producers.
They were subsequently characterized by gram-staining,
optical microscopy, the colony shape, the oxidase tests, and
the catalase tests (oxidase and catalase kits, bioMrieux,
France). The 34 colonies were also identified using an API
kit (bioMrieux, France). H2 production was examined in a
mineral salt medium [11] containing 100 mM phosphate, 3
g yeast extract/L and 5 g glucose/L. A 165 mL serum bottle


Table 1. Identity and H2 production of the microorganisms isolated
55
(working volume, 50 mL) was used. After inoculation, the
bottle was flushed with Ar gas for 5 min and sealed with a
12 mm-thick butyl rubber septum and an aluminum cap.
The cultivation was performed at 30oC for 12 h in a
gyratory incubator with a shaking speed of 250 rpm. The
inoculum was cultivated in the same bottle and transferred
anaerobically during the late-exponential phase by a sterile
hypodermic disposable syringe.
The biomass concentration was measured at 600 nm with
a spectrophotometer (Lambda 20, Perkin-Elmer, USA). The
H2 gas was sampled from the headspace of the culture
bottles with a 250 µL pressure-lock gastight syringe
(1750SL, Hamilton Company, Reno, NV, USA) and
measured by a gas chromatograph (GC) equipped with a
thermal conductivity detector and a stainless steel column (6
x 1/8) packed with a Molecular Sieve 5A (80/100 mesh;
Alltech, Deerfield, IL, USA). Ar was used as the carrier gas
at a flow rate of 30 mL/min. The oven, injector and detector
temperature were at 80oC, 90oC and 120oC, respectively.
Organic acids and ethanol in the culture broth were also
analyzed by a GC equipped with a flame ionization detector
and a 0.25 mm (I.D.) x 30 m HP-INNOWax capillary
column (Agilent Technologies, Forster, CA, USA). N2 was
used as a carrier gas in this case at a flow rate of 1 mL/min.
The injection volume and split ratio were 1 µL and 30:1,
respectively. The injector and detector were kept at 220oC
and 260oC, respectively, while the column was held at
60oC for 2 min, heated to 200oC at 10oC/min
Cell growth and H2 production2)
Major metabolites (mg/L)
Isolation
condition
Colony
group1)
(number)
Microorganism
H2 production
(mL)
H2 yield (mol/
mol glucose)
Final biomass
(OD600)
Final
pH
Ethanol Acetate Propionate
A-1 (2) Aeromonas spp. 29.7 1.13 2.38 6.21 1,062 647 nd
A-2 (2) Aeromonas spp. 28.4 1.02 2.24 6.20 820 621 nd
A-3 (3) Aeromonas spp. 29.8 1.10 2.32 6.22 984 461 nd
A-4 (3) Pseudomonas spp. 29.6 1.09 2.37 6.21 919 529 nd
A-5 (2) Vibrio spp. 30.6 1.22 2.44 6.21 810 421 nd
Aerobic
A-6 (3) Vibrio spp. 29.5 1.12 2.43 6.21 1,180 896 nd
An-1 (2) Actinomyces spp. 33.0 1.21 2.54 6.26 450 348 323
An-2 (4) Actinomyces spp. 32.4 1.15 2.39 6.24 514 325 312
An-3 (3) Actinomyces spp. 31.2 1.16 2.38 6.22 410 387 349
An-4 (2) Actinomyces spp. 31.2 1.18 2.46 6.23 449 412 357
An-5 (6) Clostridium spp. 32.2 1.15 2.74 6.24 634 446 440
An-6 (1) Clostridium spp. 32.7 1.17 2.33 6.22 383 333 305
Anaerobic
An-7 (1) Porphyromonas sp. 29.7 1.08 2.60 6.21 391 275 272
nd, not detectable.
1) The 15 colonies (aerobic conditions) and 19 colonies (anaerobic conditions) were respectively classified into 6 and 7 groups according to the gram-
staining, microscopic observations, the colony shape, the oxidase tests, and the catalase tests.
2) Cell growth and H2 production of all strains was conducted for 12 h under anaerobic conditions.

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56

and maintained at 200oC for 1 min.
Table 1 shows the isolated strains along with their growth
and H2 production rates. The strains were classified into 13
groups based on the various biochemical tests and
microscopic observations. There were 6 groups in the
aerobic isolates and 7 groups in the anaerobic isolates.
Further identification by API analyses showed that the
aerobic isolates were Aeromonas spp. (7 strains),
Pseudomonas spp. (3 strains), and Vibrio spp. (5 strains),
and the anaerobic isolates were Actinomyces spp. (11
strains), Clostridium spp. (7 strains), and Porphyromonas sp.
It should be noted that, although 3 groups of Aeromonas, 2
groups of Vibrio, 4 groups of Actinomyces, or 2 groups of
Clostridium were identified to be the same genus according
to API tests, their biochemical characteristics were not
identical. Among the aerobic isolates, Aeromonas spp. and
Vibrio spp. belong to the γ-purple bacteria and
Pseudomonas spp. belong to the β-purple bacteria. The
photosynthetic or fermentative H2 production by some
purple bacteria has been well documented [12,13]. Among
the anaerobic isolates, fermentative H2 production by
Clostridium spp. has been studied most extensively; while
the H2 production by Actinomyces spp. and Porphyromonas
sp. to the best of the author’s knowledge has not been
detected. Further investigations including 16S rDNA
analyses are cur-rently underway.
Table 1 also summarizes the batch fermentation results
with the new isolates when glucose was used as the carbon
source. A high phosphate buffer of 100 mM and a relatively
low glucose concentration of 5 g/L were employed in this
experiment because the medium pH usually decreases
drastically due to the accumulation of organic acids as
fermentation proceeds and a lower pH suppresses H2
production significantly (data not shown). The final pH of
the culture broth was at approximately 6.2, indicating that
the fermentation conditions were appropriate. Regardless of
the screening conditions or the type of microbial species, all
isolates could produce H2 during anaerobic cultivation. The
major by-products were ethanol and acetate for the aerobic
isolates, and ethanol, acetate and propionate for the
anaerobic isolates. No detectable propionate was found in
the fermentation broth of the aerobic isolates, suggesting
that there are distinctive differences in the carbon
metabolism between the aerobic and anaerobic isolates.
The final cell density and H2 production yields of all the
species were approximately 2.24-2.74 A600 and 1.02-1.22
mol H2/mol glucose, respectively. The cell density is
primarily dependent on the initial glucose concentration and
is not a key factor in evaluating the performance of a H2
producer. In contrast, the H2 production yield for glucose is
the most critical in determining the economy of the
biological production of H2. Since one of the motivations of
this study was obtaining some new isolates with a high H2
production yield or at the least observing some significant
variation in the H2 yield among the isolates, observation of
the same H2 yield was somewhat disappointing.
Nevertheless, it is believed that these results are important
at least in the following two aspects. First, the H2 yield (mol
H2/mol glucose) of the present isolates was not low and was
generally similar to that of most other reported strains (the
Biotechnol. Bioprocess Eng. 2003, Vol. 8, No. 1
glucose concentration in the parenthesis indicates the initial
concentration used in the experiments). The production
yields are as follows: Rhodopseudomonas palustris P4, 1.33
(glucose, 5 g/L) [5]; Enterobacter aerogenes strain HO-39,
1.0 (glucose, 10 g/L) [6]; E. cloacae IIT-BT 08, 2.2
(glucose, 10 g/L) [8]; Citrobacter intermedius, 0.27-1.14
(glucose, 7.7 g/L) [14]; C. freundii, 1.29 (glucose, 7.7 g/L)
[9]; Clostridium beijerinckii AM21B, 1.3-2.0 (glucose, 10
g/L) [2]; and Cl. butyricum strain SC-E1, 1.4–2.2 (glucose,
10 g/L) [15]. However, there remains the possibility of
finding a superb strain from the 34 isolates by carefully
optimizing the culture conditions such as the glucose
concentration, temperature, and the H2 partial pressure for
each strain because the conditions giving the maximum H2
yield tend to be species-specific. Secondly, and more
importantly, most H2 producers from the UASB reactor have
a similar H2-to-glucose yield and further efforts at trying to
identify a high-yield strain from UASB granules are not
rewarding. This suggests that further screening work of H2
producers should be conducted with microbial consortia that
are distinctively different from the UASB granules.
In conclusion, extensive screening for H2-producing
bacteria from anaerobic granular sludge was carried out.
Among the 72 colonies chosen, 34 exhibited H2-pro-ducing
capability under anaerobic conditions with glucose as the
carbon source. This suggests that the UASB granule is a
rich source of fermentative H2-producing bacteria.



Acknowledgements This work was supported by Korean
Science and Engineering Foundation though the Institute for
Environmental Technology and Industry (IETI), Pusan National
University, Pusan, Korea (Project no: R12-1996-015-08001-0).



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[Received December 6, 2002; accepted February 12, 2003]