international journal of hydrogen energy 33 (2008) 6100–6108
Available at www.sciencedirect.com
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Improved hydrogen production by coupled systems
of hydrogenase negative photosynthetic bacteria
and fermentative bacteria in reverse micelles
Anita Singha,*, Krishna Misrab
a
Centre for Biotechnology, University of Allahabad, Allahabad 211002, India
Indo-Russian Center for Bioinformatics, Indian Institute of Information Technology, Allahabad 211011, India
b
article info
abstract
Article history:
Significant improvement in biological hydrogen production is achieved by the use of
Received 9 April 2008
coupled bacterial cells in reverse micellar systems. Two coupled systems (a) Rhodop-
Received in revised form
seudomonas palustris CGA009/Citrobacter Y19, and (b) Rhodobacter sphaeroides 2.4.1/Citrobacter
23 July 2008
Y19 bacteria have been immobilized separately in aqueous pool of the reverse micelles
Accepted 24 July 2008
fabricated by various surfactants (AOT, CBAC and SDS) and apolar organic solvents
Available online 11 September 2008
(benzene and isooctane). The gene for uptake hydrogenase enzyme has been manipulated
further for hydrogen generation. Mutants deficient in uptake hydrogenase (Hup) were
Keywords:
obtained from R. palustris CGA009 and R. sphaeroides 2.4.1, and entrapped with Citrobacter
Rhodopseudomonas palustris CGA009
Y19 in the reverse micellar systems. More than two fold increase in hydrogen production
Rhodobacter sphaeroides 2.4.1
was obtained by the use of Hup mutants instead of wild-type photosynthetic bacteria
Citrobacter Y19
together with Citrobacter Y19. Addition of sodium dithionite, a reducing agent to AOT/H2O/
Reverse micelle
isooctane reverse micellar system with the coupled systems of wild-type photosynthetic
Hup mutant
bacteria and fermentative bacterium Y19 effected similar increase in hydrogen production
rate as it is obtained by the use of mutants. CBAC/H2O/isooctane reverse micellar system is
used for the first time for hydrogen production and is as promising as AOT/H2O/isooctane
reverse micellar system. All reverse micellar systems of coupled bacterial cultures gave
encouraging hydrogen production (rate as well as yield) compared to uncoupled bacterial
culture.
ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.
1.
Introduction
Molecular hydrogen is an efficient and clean energy resource
which doesn’t produce pollutants such as CO2, CO and
methane; water is the only byproduct resulting from its
combustion and it can be stored compactly as metallic
hydride [1,2]. Current industrial methods of hydrogen generation such as electrolysis of water, coal gasification, steam
reforming of hydrocarbons, etc., are energy intensive and not
always environment friendly. Therefore, if hydrogen is to
replace fossil fuels in the future, it has to be produced
renewably with environmentally benign processes at large
scale [3–9]. Microbial hydrogen production using fermentative
bacteria, photosynthetic bacteria, cyanobacteria, or algae is an
environmentally friendly and energy saving process which
potentially can open a new avenue for the utilization of
* Corresponding author. Present address: Department of Biotechnology and Cell Biology, Rice University, 6100 Main Street, Houston, TX
77005, USA. Tel.: þ1 713 348 2580; fax: þ1 713 348 5154.
E-mail address: [email protected] (A. Singh).
0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2008.07.095
international journal of hydrogen energy 33 (2008) 6100–6108
renewable and inexhaustible energy resources, for example,
organic wastes [10–15].
Several groups of investigators have attempted to look for
the possibility to improve hydrogen production through
various immobilization techniques used in photobioreactors,
using polysaccharide gels like agar, agarose, alginate, ccarageenan, and chitosan combined with agar, etc. [16–18].
However, decreased H2 formation was observed during
immobilization in agar, agarose and c- carageenan matrices
as compared to free cells in liquid cultures. This was partly
attributed to the cell damage caused by excessive temperature
in the range of 4550 C and low light penetration in the gels
[19]. The profile of light penetration in photobioreactors has
also been studied [20,21].
In a reverse micellar system bacterial cells are fully
exposed to light owing to the transparency of the solutions
[22]. Reverse micelles are essentially spheroid aggregates
formed by dissolving a surfactant, e.g., sodium lauryl sulfate
(SDS), sodium bis-2-ethylhexyl-sulfosuccinate (AOT), cetylbenzyldimethyl-ammonium chloride (CBAC), cationic cetyltrimethyl ammonium chloride (CTAC) or bromide (CTAB) in
a polar organic solvent with a limited amount of water to
produce a macro-homogenous transparent solution, which
provides an artificial system that mimics many life systems,
mainly the biological membranous enzyme system. AOT/H2O/
isooctane and SDS/H2O/benzene reverse micellar system for
hydrogen producing bacteria have shown encouraging results
[23–26]. A reverse micellar system provides us a unique
immobilization technique to enhance the hydrogen production due to its homogeneity, transparency, compartmentalization and its ability to create anaerobic environment [22].
Therefore, a reverse micellar system is preferred over other
immobilization techniques to produce hydrogen from nitrogenase as well as hydrogenase enzyme.
In conventional methods, only photosynthetic bacteria are
used in a photobioreactor. However, various researchers used
coupled systems of photosynthetic and fermentative bacteria
to increase degradation of the substrates to improve hydrogen
production [27–30]. Furthermore, hydrogen production in the
presence of light is catalyzed by nitrogenase enzyme and the
oxidation of produced hydrogen is catalyzed by the presence
of uptake hydrogenase enzyme, thus, decreasing the overall
rate and yield of hydrogen [23,31]. Therefore, higher rate of
hydrogen production could be achieved by generating uptake
hydrogenase negative enzyme mutants through certain
specific mutations [32,33].
The scope of this paper is to show the effects of various
reverse micelles using coupled systems of bacteria on
hydrogen production. For this purpose, we have used coupled
systems of photosynthetic (Rhodopseudomonas palustris
CGA009; Rhodobacter sphaeroides 2.4.1) and fermentative (Citrobacter Y19) bacteria immobilized within AOT/H2O/isooctane,
CBAC/H2O/isooctane and SDS/H2O/benzene reverse micelles.
To the best of our knowledge, the coupled system of uptake
hydrogenase negative photosynthetic bacteria R. palustris
CGA009 with Citrobacter Y19 or uptake hydrogenase negative
photosynthetic bacteria R. sphaeroides 2.4.1 with Citrobacter Y19
has not been used for hydrogen production within a reverse
micellar system and one of the reverse micellar systems
CBAC/H2O/isooctane has not been reported for hydrogen
6101
production by coupled bacterial culture, so far. Mutants of
R. palustris CGA009 and R. sphaeroides 2.4.1 lacking uptake
hydrogenase activity were isolated and entrapped within
various reverse micellar systems together with Citrobacter Y19.
Furthermore, the effects of sodium dithionite as a reducing
agent and ethylene-diamine-tetraacetic acid (EDTA) as
a chelating agent have also been tested on the cultures of
wild-type R. palustris CGA009 and R. sphaeroides 2.4.1 entrapped together with Citrobacter Y19 within AOT/H2O/isooctane
reverse micelle.
2.
Methods and materials
2.1.
Chemicals
All chemicals used in the experiments were of AR Grade,
purchased from Merck India Ltd. Malate and vitamins were
purchased from Himedia Laboratories Pvt. Ltd., India and used
without further purification.
2.2.
Microorganisms and culture conditions
The photosynthetic purple non-sulphur (PNS) bacteria
R. palustris CGA009 and R. sphaeroides 2.4.1 were cultivated
photosynthetically at 30 C in Sistrom’s growth media [34]. In
order to increase hydrogen production, the growth medium
was modified by adding 30 mM of malate as a carbon source,
7 mM of glutamate as a nitrogen source, and by removing
succinic acid, aspartic acid, ammonium sulphate and
nitrilotriacetic acid. Furthermore, sodium molybdate
(Na2MO4$2H2O) was used in place of ammonium molybdate.
A fermentative bacterium Citrobacter Y19 was cultivated in
a mineral salt medium supplemented with 3.0 g yeast extract/
l and 30 mM glucose [35–37]. For maintenance of the bacterial
cells, the above media were solidified using 1.5% agar. The
agar plates were cultured at 30 C and stored at 4 C for several
weeks.
Batch cultivations for R. palustris CGA009, R. sphaeroides
2.4.1 and Citrobacter Y19 were performed at 30 C in a gyratory
incubator with a shaking speed of 250 rpm. An anaerobic
condition was developed in a serum bottle of 155 ml by filling
it with the media and flushing with argon gas (99.99%) till the
working volume of 50 ml was achieved. A tungsten lamp was
placed to maintain a luminance intensity of 6000 lx for
photosynthetic bacteria. The inoculum was cultivated in the
bottle and transferred anaerobically at the late-exponential
phase by a sterile disposable syringe. The culture was centrifuged at 10,000 rpm for 15 min. The pellets were resuspended
in the growth medium and used for entrapment studies.
2.3.
Mutant isolation
Hup mutants of R. palustris CGA009 and R. sphaeroides 2.4.1
were isolated by ethyl methyl sulphonate (EMS) mutagenesis
[38,39]. R. palustris CGA009 and R. sphaeroides 2.4.1 were grown
at 37 C till the late-exponential phase. At this stage, 0.1 ml
aliquots were withdrawn and mixed with 1 ml of 1% (v/v) EMS
in 0.1 M phosphate buffer (pH 7.4), and incubated at 37 C for
30 min. Then, 10 ml of the modified Sistrom’s medium were
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international journal of hydrogen energy 33 (2008) 6100–6108
inoculated with 0.1 ml of the mutagenized suspension and
grown in the light for 48 h. The cells were plated on the
modified Sistrom’s solid plates and incubated at 30 C for one
week.
2.4.
Screening for Hup mutants
The colonies were transferred onto a Whatman filter paper to
be used in the screening procedure. A stack of three filter
papers was put on top of the Whatman filter paper containing
the colonies. The filter papers were kept in a Petri dish and
soaked in 100 mM of potassium phosphate buffer (pH 9.4)
supplemented with 20 mM of oxidized methyl viologen. The
Petri dish was incubated under an atmosphere of 100%
hydrogen (traces of oxygen were eliminated with palladium
catalyst) in an air tight box. Hydrogenase positive colonies
started to turn blue after 0.5 h, while hydrogenase negative
colonies remained purple even after 5 h [40].
2.5.
Preparation of reverse micelles for
hydrogen production
The surfactants were added in powder form gradually to the
organic solvents taken in a flask and homogenized. SDS (1.2 g)
was added to benzene (250 ml); AOT (8.86 g) and CBAC (0.95 g)
were added to isooctane (250 ml) at 37 C. 1.0 ml of the
photosynthetic bacteria (R. palustris CGA009 or R. sphaeroides
2.4.1) and the fermentative bacteria (Citrobacter Y19) each,
grown during the late-log phase, along with 1.0 ml of the
fermentative bacterial culture medium having 30 mM glucose
were injected into the homogenates to fabricate reverse
micellar system. All bacterial cultures were grown in anaerobic environment and the culture media were carefully
flushed with Ar gas before use. The reverse micellar medium
containing entrapped bacterial cells was transferred to
hydrogen producing air tight setup. The setup was placed on
a magnetic stirrer in an upright position fitted with a clamped
stand and illuminated with a tungsten lamp of 600 lx intensity
at 37 C temperature. Hydrogen gas produced was collected in
the upper portion of the setup by displacement of the reverse
micellar organic medium.
In order to observe the effect of sodium dithionite on
hydrogen production, the AOT/isooctane reverse micellar
medium containing the coupled bacterial cells (R. palustris
CGA009/Citrobacter Y19; R. sphaeroides 2.4.1/Citrobacter Y19)
were transferred to the hydrogen producing setup along with
1.0 ml of 1.5 mM sodium dithionite. Furthermore, to study the
effect of EDTA on hydrogen production, the photosynthetic
bacteria were cultured in the modified Sistrom’s medium
having 0.5 mM EDTA. 1.0 ml of the photosynthetic bacteria
were entrapped within AOT/isooctane reverse micellar
medium along with 1.0 ml of Citrobacter Y19 and 1.0 ml of
0.5 mM EDTA.
2.6.
Assay of hydrogenase activities within
reverse micellar system
Hydrogenase activity in whole cells of photosynthetic bacteria
is commonly assayed by following the reduction of methylene
blue linked to H2 oxidation [41]. In this work, however, reverse
micelles were used instead of aqueous solution to trace the
hydrogenase activity linked to hydrogen production. Reverse
micelles were prepared by injecting slowly a Vortex-stirred
solution of 0.2 ml of the modified Sistrom’s media suspended
with the bacterial culture grown in the late-log phase and
0.2 ml of aqueous solution containing 0.15 mM MB and 20 mM
Tris–HCl buffer (pH 8.0) into 55 ml of AOT/isooctane solution.
Homogenization of the resulting solution was continued until
it became clear. The solution was flushed by argon gas for
1 min to create anaerobic condition. Whole solution was
placed into a capped vial and flushed by H2 gas for 1 min.
Hydrogenase activity was monitored spectrophotometrically
at 570 nm at 24 C.
2.7.
Measurement of protein content
Protein content of the bacterial cells was measured using
Lowry method. In this method, a bovine serum albumin (BSA)
is used as standard [42].
2.8.
Gas analysis
The concentration of the evolved molecular hydrogen was
measured by a gas chromatograph (Nikon) equipped with
a thermal conductivity detector and a stainless-steel column
(Carbosieve II). In the equipment argon gas is served as
carrier and pure hydrogen gas is used as standard. The
column temperature was maintained at 60 C and the
temperature of detector and injector was set to 70 C. The
volume of hydrogen was converted into the value at 37 C
and 0.1 MPa.
Stoichiometric maximum evolution of hydrogen was
calculated by assuming that hydrogen was evolved by
complete conversion of the carbon source in the catabolic
process. The yield of hydrogen evolution was calculated as
a percentage of the stoichiometric maximum. For example,
the stoichiometric maximum of hydrogen evolution from
30 mM glucose was 9.16 ml/ml-medium at 37 C and 0.1 MPa.
The complete conversions of the glucose and malate carbon
sources used in the present work are as follows:
ðGlucoseÞC6 H12 O6 þ 6H2 O / 6CO2 þ 12H2 [
ðMalateÞC4 H6 O5 þ 3H2 O / 4CO2 þ 6H2 [
3.
Results and discussion
3.1.
Hydrogen production by entrapped co-culture
of fermentative and photosynthetic bacteria
Tables 1–3 show the rates of hydrogen production, total
volume of hydrogen evolved and hydrogen yield by uncoupled
cells of R. palustris CGA009, R. sphaeroides 2.4.1 and Citrobacter
Y19 and coupled cells of R. palustris CGA009/Citrobacter Y19 and
R. sphaeroides 2.4.1/Citrobacter Y19 entrapped within different
reverse micellar systems of AOT/isooctane, CBAC/isooctane
and SDS/benzene at 37 C temperature, pH 7 and 600 lx light
6103
international journal of hydrogen energy 33 (2008) 6100–6108
Table 1 – H2 production in various reverse micellar systems and in aqueous medium using R. palustris CGA009 and R.
sphaeroides 2.4.1
H2 production rate
[mmol of H2
(mg protein)1 h1]
Fold enhancement
in rate w.r.t.
aqueous culture
Total amount of H2 evolved
[ml/ml-media]
Yield (%)
AOT/isooctane
3.00 0.80
3.64 0.50
w50
w52
1.15 0.01
1.47 0.02
25.20
32.00
CGA009
2.4.1
CBAC/isooctane
2.50 0.50
3.36 0.60
w42
w48
1.05 0.03
1.35 0.04
23.00
29.50
CGA009
2.4.1
SDS/benzene
1.80 0.40
2.52 0.30
w30
w36
0.80 0.01
1.01 0.01
17.50
22.00
CGA009
2.4.1
Aqueous culture
0.06 0.02
0.07 0.01
0.64 0.01
0.68 0.02
14.00
15.00
CGA009
2.4.1
Reverse micellar
systems
Carbon source: 30 mM
(standard deviation).
DL-malate.
Photosynthetic
bacteria
Temperature: 37 C. pH: 7 and light intensity: 600 lx. Each value is mean of at least three times replication
intensity. Both systems of coupled and uncoupled bacterial
cells produce more hydrogen in the AOT/isooctane reverse
micellar medium in comparison with the other reverse
micellar media. The hydrogen production rates of the coupled
systems (i.e. R. palustris CGA009/Citrobacter Y19 and R. sphaeroides 2.4.1/Citrobacter Y19) entrapped within all reverse
micellar systems (i.e. AOT/isooctane, CBAC/isooctane and
SDS/benzene) were 3–3.5 times higher when compared with
the hydrogen production rates of the uncoupled photosynthetic bacterial cells (i.e. R. palustris CGA009 and R. sphaeroides
2.4.1) and 1.5–2.5 times higher when compared with the
hydrogen production rates of fermentative bacterial cells
(Citrobacter Y19) entrapped in the same reverse micellar
systems. The hydrogen yield increased by a factor of 2 in the
coupled systems when compared with the uncoupled
systems. The evolved hydrogen gas was found to be 99.99%
pure.
Effectiveness of various reverse micellar systems over
aqueous system is measured for both photosynthetic and
fermentative bacteria and is presented in Tables 1 and 2.
Photosynthetic bacteria in the reverse micellar systems
showed 30–52 fold increase in the hydrogen production rate
and 5–17% increase in the hydrogen yield. Fermentative
bacterium in reverse micellar systems also showed similar
increase in the hydrogen production rate and hydrogen yield.
Furthermore, out of the two photosynthetic bacteria, R.
sphaeroides 2.4.1 was found to be more efficient in terms of
total hydrogen production.
All reverse micellar systems used in the study were
compatible with both fermentative and photosynthetic
bacterial cells. Solvents and surfactants in the reverse
micellar systems did not affect the viability of the bacterial
cells much and gave an appropriate microenvironment within
the water pool for hydrogen production. Coupling between the
fermentative and the photosynthetic bacteria within
the reverse micellar systems showed encouraging results as
the coupled systems produced higher amount of molecular
hydrogen in comparison with the uncoupled systems.
Fermentative bacterium produces hydrogen and organic acids
in the absence of light by degrading the carbohydrates used as
substrates. Photosynthetic bacteria utilize the resulting
organic acids as a carbon source to produce hydrogen.
Anaerobic bacteria gain both energy and electron by decomposing the carbohydrates. Further decomposition of organic
acids could not be possible by anaerobic bacteria in the positive free energy reaction. However, photosynthetic bacteria
available in the coupled system could use light energy to
overcome the positive free energy reaction and degrade
organic acids completely to produce hydrogen. Thus, the
demand of light energy by photosynthetic bacteria could be
reduced due to the combined use of anaerobic and photosynthetic bacteria for enhanced hydrogen production [43].
Since organic acids produced by Citrobacter Y19 are consumed
by photosynthetic bacteria at a slower rate, pH of the coupled
system would be in the acidic range, most likely in the range of
6–7 [37,53].
Table 2 – H2 production in various reverse micellar systems and in aqueous medium using Citrobacter Y19
Reverse micellar
systems
AOT/isooctane
CBAC/isooctane
SDS/benzene
Aqueous culture
Citrobacter Y19
H2 production rate
[mmol of H2 (mg protein)1 h1]
Fold enhancement
in rate w.r.t. aqueous culture
Total amount of H2
evolved [ml/ml-media]
Yield (%)
5.00 0.10
4.40 0.05
3.40 0.06
0.09 0.02
w56
w49
w38
2.02 0.01
1.80 0.03
1.40 0.01
1.07 0.01
22.00
19.70
15.28
11.67
Carbon source: 30 mM glucose. Temperature: 37 C. pH: 7 and light intensity: 600 lx. Each value is mean of at least three times replication
(standard deviation).
6104
CGA009/Y19
2.4.1/Y19
Carbon source: 30 mM glucose. Temperature: 37 C. pH: 7 and light intensity: 600 lx. Each value is mean of at least three times replication (standard deviation).
2.50 0.01
3.40 0.02
1.58
2.2
SDS/benzene
5.40 0.01
7.56 0.08
3.00
3.00
27.30
37.12
CGA009/Y19
2.4.1/Y19
3.94 0.03
5.32 0.02
1.8
2.45
8.00 0.20
10.81 0.10
CBAC/isooctane
3.2
3.22
43.01
58.10
CGA009/Y19
2.4.1/Y19
4.80 0.01
6.28 0.04
1.96
2.5
9.80 0.30
12.74 0.14
AOT/isooctane
3.3
3.50
52.50
68.50
Coupled systems
Yield (%)
Total amount of
H2 evolved [ml/ml-media]
Fold enhancement
w.r.t. Y19
Fold enhancement
w.r.t. 2.4.1
Fold enhancement
w.r.t. CGA009
H2 production rate
[mmol of H2 (mg protein)1 h1]
Reverse micellar
systems
Table 3 – H2 production in various reverse micellar systems using coupled systems of R. palustris CGA009/Citrobacter Y19 and R. sphaeroides 2.4.1/Citrobacter Y19
international journal of hydrogen energy 33 (2008) 6100–6108
Reverse micelles act as small microreactors in which the
activity of cellular enzymes is increased by bringing the
reactants together [44]. Thus, the enzymes responsible for
carbohydrate degradation and hydrogen production become
more active in the anaerobic water pool of reverse micellar
microreactor owing to compartmentalization of the cells
resulting in enhanced hydrogen production. Furthermore,
nitrogenase and hydrogenase enzymes are active only in
anaerobic environment and reverse micelles provide such
environment to these enzymes as the reverse micellar
aqueous pool itself has a very low concentration of dissolved
oxygen and the surrounding organic medium helps in
providing anaerobic condition to the aqueous pool. Free
bacterial cells in aqueous culture, however, are more exposed
to oxygen. The use of coupled system of photosynthetic/
fermentative bacteria also helps to maintain anaerobic
condition within the reverse micelles. This is achieved by
quenching the oxygen, produced during the metabolism of
nitrogenase, by glucose, a reducing agent, used for the growth
of fermentative bacteria. In this study it was observed that
AOT/H2O/isooctane reverse micellar system was the most
transparent and SDS/H2O/benzene system was the least
transparent [45]. Therefore, increased light availability for
nitrogenase enzyme of the bacterial cells in AOT/H2O/isooctane reverse micelles resulted in increased hydrogen
production rate (Tables 1–3). Hydrogen evolving systems,
AOT/isooctane and CBAC/isooctane gave very similar results
and were sustained up to 5.5 h Fig. 1 which could be further
improved by the addition of small amount of nutrient in the
media. In comparison, SDS/benzene reverse micellar system
produced less amount of hydrogen and sustained up to 5 h
only. It might be due to denaturation of the bacterial cells by
SDS.
3.2.
Hydrogen production by entrapped co-culture of
fermentative and Hup mutants of photosynthetic bacteria
In this section the effects on hydrogen production by mutated
versus wild-type cultures of photosynthetic bacteria coupled
with the fermentative bacterium within reverse micellar
systems are compared. Mutated photosynthetic bacteria
(Hup mutants) are devoid of uptake hydrogenase enzyme.
Hydrogen productions by mutated and wild-type cultures of R.
palustris CGA009 and R. sphaeroides 2.4.1 entrapped within
AOT/isooctane, CBAC/isooctane and SDS/benzene reverse
micellar systems with Citrobacter Y19 are shown in Table 4. All
the experiments were conducted at the temperature of 37 C
and pH 7 under the light intensity of 600 lx. A 2–2.4-fold
increase in hydrogen production rate and 4–14% increase in
hydrogen yield were observed in case of the coupled system of
mutated photosynthetic bacteria and the fermentative
bacterium when compared with the coupled systems of wildtype bacteria within various reverse micellar systems (Tables
3 and 4).
Presence of uptake hydrogenase (Hup) essentially breaks
down the molecular hydrogen into protons and electrons,
thereby, reducing the overall production of hydrogen. Therefore, mutants of photosynthetic bacteria devoid of uptake
hydrogenase were not able to oxidize the molecular hydrogen
produced by the nitrogenase enzyme [23]. Thus, enhanced
6105
2.10
2.00
10.80 0.02
15.25 0.24
SDS/benzene
Carbon source: 30 mM glucose. Temperature: 37 C. pH: 7 and light intensity: 600 lx. Each value is mean of at least three times replication (standard deviation).
CGA009/Y19
2.4.1/Y19
31.70
44.80
2.90 0.06
4.10 0.06
CGA009/Y19
2.4.1/Y19
51.60
69.80
2.20
2.2
17.60 0.40
23.80 0.12
CBAC/isooctane
2.4
2.20
4.73 0.04
6.40 0.08
CGA009/Y19
2.4.1/Y19
69.80
82.30
6.40 0.06
7.54 0.16
Mutated coupled systems
Yield (%)
Total amount
of H2 evolved [ml/ml-media]
Fold enhancement
w.r.t. CGA009/Y19
Fold enhancement
w.r.t. 2.4.1/Y19
23.76 0.24
28.10 0.40
Sodium dithionite, added as a reducing agent in the reverse
micellar systems, showed positive effect on hydrogen
production. Only AOT/isooctane reverse micelle was used for
this experiment as it was previously found to be the most
effective. Measurements of hydrogen production rate,
hydrogen yield and total amount of hydrogen evolved are
shown in Table 5. Fermentative bacterium Citrobacter Y19
coupled with wild-type photosynthetic bacteria R. palustris
CGA009 and R. sphaeroides 2.4.1 separately, were entrapped
within AOT/isooctane reverse micellar system at 37 C
temperature, pH 7 and 600 lx light intensity. Addition of
sodium dithionite in the system resulted in 2.3 and 2.2 fold
AOT/isooctane
3.3.
Effect of sodium dithionite as reducing agent on
hydrogen production by entrapped co-culture of
fermentative and photosynthetic bacteria
H2 production rate
[mmol of H2 (mg protein)1 h1]
hydrogen production was obtained as a result of decreased
oxidation of molecular hydrogen in the coupled system of
mutated photosynthetic bacteria.
Reverse micellar
systems
Fig. 1 – The time course profile of hydrogen production
within different reverse micellar systems by coupled
bacterial cells: (a) CGA009/Y19, and (b) 2.4.1/Y19.
Table 4 – H2 production in various reverse micellar systems using coupled systems of mutated R. palustris CGA009/Citrobacter Y19 and mutated R. sphaeroides 2.4.1/
Citrobacter Y19
international journal of hydrogen energy 33 (2008) 6100–6108
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international journal of hydrogen energy 33 (2008) 6100–6108
Table 5 – Effect of sodium dithionite and EDTA on H2 production by R. palustris CGA009/Citrobacter Y19 and R. sphaeroides
2.4.1/Citrobacter Y19 coupled systems in AOT/isooctane reverse micellar system
H2 production rate
[mmol of H2 (mg protein)1 h1]
Total amount of H2 evolved
[ml/ml-media]
Yield (%)
Without sodium dithionite
or EDTA
9.80 0.30
12.74 0.14
4.80 0.01
6.28 0.04
52.50
68.50
CGA009/Y19
2.4.1/Y19
With sodium dithionite
22.54 0.15
26.90 0.20
6.10 0.12
7.23 0.16
66.50
79.00
CGA009/Y19
2.4.1/Y19
With EDTA
20.58 0.18
24.20 0.19
5.53 0.10
6.50 0.15
60.37
71.00
CGA009/Y19
2.4.1/Y19
Coupled systems
Carbon source: 30 mM glucose. Temperature: 37 C. pH: 7 and light intensity: 600 lx. Each value is mean of at least three times replication
(standard deviation).
increase of hydrogen production rate for the systems containing R. palustris CGA009 and R. sphaeroides 2.4.1, respectively; hydrogen yield increased by 14% and 10.50%,
respectively, for the two systems. Sodium dithionite essentially acts as an oxygen scavenger to produce hydrogen [46].
Sodium dithionite has been utilized by several researchers
and was found to be a good support for nitrogen fixation
[47,48]. Use of sodium dithionite does not affect the reverse
micellar structure.
3.4.
Effect of EDTA on hydrogen production by
entrapped co-culture of fermentative and
photosynthetic bacteria
Presence of hydrogenase enzyme in the photosynthetic
bacteria effectively reduces hydrogen production by utilizing
the molecular hydrogen produced by nitrogenase enzyme
[23,24,31]. Use of EDTA during culture of photosynthetic
bacteria inhibits the activity of hydrogenase enzyme [49]. The
effect of EDTA on hydrogen production by coupled systems of
wild-type photosynthetic bacteria and fermentative bacterium in a reverse micellar system is shown in Table 5. Two
photosynthetic bacteria R. palustris CGA009 and R. sphaeroides
2.4.1 and the fermentative bacterium Citrobacter Y19 are used
in the experiments. The coupled systems of bacteria are
entrapped within AOT/isooctane reverse micellar system at
37 C temperature, pH 7 and 600 lx light intensity. 0.5 mM
EDTA treatment of the coupled systems of bacteria resulted in
2.1 and 2.0 fold increase in hydrogen production rate as
compared to non-EDTA treated samples of R. palustris CGA009/
Citrobacter Y19 and R. sphaeroides 2.4.1/Citrobacter Y19 photosynthetic bacteria, respectively; hydrogen yield increased by
7.87% and 2.5%, respectively, for the two coupled systems.
Furthermore, hydrogenase activities for both photosynthetic
bacteria in EDTA and non-EDTA treated samples in the
reverse micellar systems are plotted in Fig. 2. The peak
observed in hydrogenase activity during 30–40 min in nonEDTA samples is possibly linked with the availability of Ni
ions in the media [50]. It is evident from the plot that the
hydrogenase activity is negligible in the presence of EDTA.
EDTA has been reported to suppress hydrogenase activity and
to increase nitrogenase activity by mobilization of iron within
the bacterial cells [51,52]. Also, the inhibitory effect of EDTA on
Hup enzyme formation is very likely due to a decrease in the
availability of Ni ions [49]. In addition use of EDTA does not
affect the reverse micellar structure and the viability of the
coupled bacterial cells.
4.
Fig. 2 – Effect of EDTA on hydrogenase enzyme activity of R.
palustris CGA009 and R. sphaeroides 2.4.1.
Conclusions
Multi-fold improvement in hydrogen production could be
achieved by the use of coupled systems of the photosynthetic
bacteria (wild-type or mutated) and the fermentative bacteria
entrapped in various reverse micellar systems. Compartmentalization of the bacterial cells and the enzymes in the
reverse micellar systems made the systems very efficient.
Moreover, CBAC/isooctane reverse micellar system which was
introduced in this study for hydrogen production gave
encouraging results by coupled bacterial cells. Furthermore,
increase in the hydrogen production obtained by using EDTA
is of the same order as that obtained by using of Hup
mutants. Addition of sodium dithionite in the coupled
systems as a reducing agent in the reverse micellar systems
has more than doubled the amount of hydrogen production.
The photosynthetic bacterium R. sphaeroides 2.4.1 has been
more effective than R. palustris CGA009 in all reverse micelles
whether used in coupled system or uncoupled system. Further
international journal of hydrogen energy 33 (2008) 6100–6108
studies are required to establish the reasons for 2.4.1 being
more effective, which would include identifying the gene
sequences of hydrogenase and nitrogenase enzymes as well
as the specific sites of oxidation and reduction reaction on
enzymes. From bioengineering view point, coupled bacterial
cells in reverse micellar systems exhibit very high hydrogen
production and may be suitable for development of bioreactors for industrial usage.
Acknowledgements
The authors thank Prof. C.S. Harwood of Department of
Microbiology, University of Iowa, Iowa City, USA for providing
the strains of Rhodopseudomonas palustris CGA009; Dr. Sunghoon Park of Department of Chemical Engineering, Pusan
National University, Pusan, South Korea for providing the
strains of Citrobacter Y19; Dr. Timothy J. Donohue of University
of Wisconsin, Madison for providing the strains of Rhodobacter
sphaeroides 2.4.1. This work was supported through the
research assistantship by University Grant Commission
(UGC), New Delhi in the form of a Senior Research Fellowship
to (A.S.).
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Isolation and Characterization of Benzene Degrading Bacteria from Gasoline
Contaminated Water
Anita Singh*, Pinaki Sar** and George N. Bennett*
*Department of Biochemistry and Cell Biology, Rice University 6100 Main St., Houston, TX 77005.
Phone:(713) 348-2580. Fax: (713) 348-5154. E-mail: [email protected], [email protected]
**Department of Biotechnology, Indian Institute of Technology, Kharagpur, 721302.
E-mail: [email protected]
ABSTRACT
In the present study, we have isolated AG3 and AG4
bacteria of the Bacillus group which are able to grow in
aerobic as well as anaerobic conditions. These bacteria
were isolated from a location near Rice University, Houston
by using BTEX (benzene, toluene, ethylbenzene, and
xylene) as the principal carbon source. They are Grampositive, and rod-shaped. The isolates utilized benzene as a
carbon source in both aerobic and anaerobic conditions.
Various carbohydrates and aromatic compounds were also
tested as substrates for the growth of these bacterial
isolates. Their 16S rDNA sequences indicate that they are
members of the Bacillaceae; AG3 belongs to Bacillus
cereus and AG4 belongs to Bacillus megaterium bacteria.
These bacteria isolates can potentially be utilized in
bioremediation of benzene in both aerobic and anaerobic
environment.
stable aromatic compounds, especially in ground and
surface waters. Microbial degradation of benzene in aerobic
environments has been successful, however, benzene is
poorly biodegraded in anaerobic conditions [6].
Pseudomonas species are common in aerobic
bioremediation of benzene [7]. Isolation, identification, and
genetic manipulation of a vast number of local bacterial
groups for the bioremediation of aromatic hydrocarbons
have been the focus of many researchers worldwide [4, 8].
However, there is still a need to isolate more microbial
species with novel enzymatic activities that are important
for environmental as well as biotechnological applications.
In this study, we describe the isolation and
characterization of two isolates. These two bacterial isolates
include Bacillus sp., which can utilize BTEX as the sole
source of carbon and energy. They were isolated from a
location near Rice University, Houston. They are also
capable of growing on other aromatic hydrocarbons as their
sole sources of organic carbon.
Keywords: BTEX, Benzene, Bacillus, Bacteria
2
1
INTRODUCTION
Hydrocarbon pollution of water bodies is widely
recognized as a serious environmental problem, since it not
only gives serious damage on fisheries but also causes
adverse effects on the natural environment and ecosystem
[1, 2]. Surface oil spills, leaking pipelines and underground
fuel storage tanks, improper waste disposal practices,
inadvertent spills, and leaching from landfills can lead to
subsurface contaminant plumes containing significant
amounts of the hazardous aromatic hydrocarbons. BTEX
(benzene, toluene, ethylbenzene, and xylene) compounds
are the major aromatic hydrocarbon components in many
petroleum products that are considered as a significant
threat to the health and environment. Some accidental oil
spills have activated developments of methods for
microbiological degradation of oil. Fortunately, many
microbial groups inhabiting contaminated sites have
developed interesting metabolic mechanisms for
detoxification and degradation of a wide range of aromatic
hydrocarbons [3-5].
Among BTEX, benzene is of major concern, because it
is soluble, mobile, toxic, carcinogenic and one of the most
2.1
MATERIAL AND METHODS
Isolation and cultivation of bacteria
The sample was collected from a location near Rice
University, Houston. Sample was mixed with sterile water
(1:1 wt.: vol.) on a vortex mixer for 1 min.; 0.1 ml of the
resulting supernatant was spread on the surface of solid
minimal medium [9]. Aerobic incubations with volatile
substrates (BTEX) took place inside glass desiccators with
the hydrocarbon supplied as vapor by saturating a piece of
filter paper (2 x 2 cm) with 100 µl of pure compound.
Plates were incubated at 30°C for 48 and 170 hours,
respectively. Of the colonies appearing on each plate, eight
were selected, picked and streaked for purification under
the same conditions as in the initial incubation. A rapidly
growing, visually distinct colony and a separate,
morphologically unique isolate were selected for further
analysis and purified by repeated plating.
For further studies, mineral medium [10] was used with
ATCC trace metal solution and Sigma BME 100X vitamin
solution.
2.2
Phenotypic testing
Isolates were Gram stained by using Sigma Gram
staining kit. Furthermore, oxidase test (Fluka) and
microscopy for shape were conducted. The growth of
isolates was tested at a temperature of 30°C. The
absorbance at 600 nm for the growth was measured with
spectrophotometer. The following compounds were tested
for growth in aerobic and anaerobic conditions as a carbon
source by HIMEDIA carbohydrate kit: L-arabinose,
fructose, glucose, dextrose sucrose, and glycerol. The
following aromatic compounds were also tested as the sole
carbon sources for growth in aerobic and anaerobic
conditions: 100µl of benzene, ethylbenzene, toluene and
xylene each on the solid plate, and 250 ppm of phenol and
Na-benzoate in the liquid medium. Isolates growth were
scored as positive or negative by comparing the turbidity of
the liquid medium and growth on solid plates after one
month of incubation at 300C temperature.
2.3 Identification of bacteria by 16S rDNA
sequencing
16S rDNA sequence was used to identify the isolates.
Genomic DNA was prepared with a DNA extraction kit
(ultra clean microbial DNA isolation kit, MO BIO
Laboratories). The 16S rDNA were amplified by PCR with
primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and
1492R (5’-CGGTTACCTTGTTACGACGACTT-3’) [11].
The temperature parameters used were as follows: hot start
at 940C for 5 min, 30 cycles of denaturation at 94 0C for 30
s, annealing at 580C for 30 s, and extension at 720C for 30 s
followed by one cycle at 720C for 7 min to complete
extension. The PCR assays were examined in 1.0% agarose
gels. The PCR fragments obtained were purified by using a
Qiagen QIAquick gel extraction kit according to the
manufacturer’s instructions. The amplified DNA was
cloned by using a TA cloning kit (Invitrogen USA). The
cloned genes were then sequenced with an automated
sequencer (ABI Prism Genetic Analyzer, Applied
Biosystems). The sequences were compared with the data
in GenBank using the basic local alignment search tool
(BLAST) from the National Center for Biotechnology
Information (NCBI).
3
3.1
in color; rod-shaped, aerobic/anaerobic, gram-positive and
oxidase-positive [Table 1 and Fig. 1].
The following aromatic hydrocarbons and carbohydrate
were utilized as sole sources of carbon by AG3 and AG4 in
mineral medium: benzene, ethylbenzene, toluene, xylene,
Na-benzoate, phenol, L-arabinose, fructose, glucose,
dextrose sucrose, and glycerol. Growth was not observed
for AG3 in sucrose and L-arabinose. AG4 utilized all tested
carbohydrates as a carbon source. For aromatic compounds
both isolates were able to grow in both aerobic and
anaerobic conditions on benzene, xylene, Na-benzoate and
phenol. AG3 was unable to grow in aerobic/anaerobic
conditions in toluene and ethylbenzene. AG4 was able to
grow in aerobic condition in toluene but unable to grow in
anaerobic condition. For ethylebenzene, AG4 did not show
any growth in both aerobic and anaerobic conditions.
3.2
Amplification and 16S rDNA sequencing
PCR amplification of 16S rDNA from strains AG3 and
AG4 resulted in 1.5 kb PCR fragments on the agarose gel
[Fig. 2]. The results were consistent with the results of the
BLASTN search and suggest high affiliation with genera of
the family Bacillaceae. Based on a BLASTN search of
GenBank, the closest matches to strain AG3 was Bacillus
cereus (96% sequence similarity) and AG4 Bacillus
megaterium (96% sequence similarity).
Many Bacillus strains for degradation of polycyclic
aromatic hydrocarbon compounds and other chemical classes
of petroleum origin have been previously reported [12, 13].
It seems likely that members of the Bacillaceae family
dominate in oil-contaminated soils or water.
4
CONCLUSIONS
Two strains, AG3 and AG4 were isolated and identified
as Bacillus cereus and Bacillus megaterium respectively.
These isolates are able to utilize benzene as a carbon source
in aerobic and anaerobic conditions. These isolates were
also capable of utilizing other aromatic compounds in both
environments. Further studies are needed to clarify their
exact taxonomic position and to explore their
biodegradation potential to other polycyclic aromatic
hydrocarbon.
RESULTS AND DISCUSSION
Phenotypic characteristics
Two bacteria (AG3 and AG4) were isolated from the
above mentioned location which were able to grow well
using BTEX as the sole source of carbon. Physiological
characteristics of the two isolates were examined. In
general, both isolates show identical results in the
phenotypic tests. Both bacterial isolates were straw yellow
5
ACKNOWLEDGEMENTS
Support of materials from the Army Research Office
W911NF0410179 is acknowledged. Dr. Sar was supported
by a BOYSCAST Fellowship from the Department of
Science and Technology of India.
6
TABLE AND FIGURES
(A)
Table 1: Biochemical and physiological characteristics of
AG3 and AG4 bacteria:
Characteristics
AG3
Color of bacterial Straw yellow
colonies
Morphology
Rod
Gram staining
+
Aerobic growth
+
Anaerobic growth +
Oxidase reaction
+
Carbohydrate utilization
Glucose
+
Sucrose
Dextrose
+
L-Arabinose
Glycerol
+
Aromatic compounds utilization
Benzene
+ve in aerobic /
anaerobic
Xylene
+ve in aerobic /
anaerobic
Toulene
- ve in aerobic /
anaerobic
Ethylebenzene
-ve in aerobic /
anaerobic
Na-benzoate
+ve in aerobic /
anaerobic
Phenol
+ve in aerobic /
anaerobic
AG4
Straw yellow
Rod
+
+
+
+
+
+
+
+
+
+ve in aerobic /
anaerobic
+ve in aerobic /
anaerobic
+ve in aerobic,
-ve in anaerobic
-ve in aerobic /
anaerobic
+ve in aerobic /
anaerobic
+ve in aerobic /
anaerobic
(B)
Figure 1: Picture of bacterial isolates under light
microscopy (A) AG3 bacterial isolates and (B) AG4
bacterial isolates.
Figure 2: Amplification of 16S rDNA gene using specific
primers 27F and 1492R from bacterial isolates AG3 (lane
1) and AG4 (lane 2), lane 3 is the control and M is 1.0 kb
DNA ladder marker.
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Enhanced Biodegradation of 2,3,4,5,6-pentachlorocyclohexan-1-ol -- a
β-HCH Metabolite -- in the Presence of α- and γ-HCH Isomers by
Pseudomonas aeruginosa ITRC-5
Anita Singh*, Ashwani Kumar** and Krishna Misra***
*Department of Biochemistry and Cell Biology, Rice University, Houston. [email protected]
**Environmental Biotechnology Section, Industrial Toxicology Research Centre, Lucknow.
[email protected]
***Indo-Russian Centre for Bioinformatics, Indian Institute of Information Technology, Allahabad.
[email protected]
ABSTRACT
Chlorinated insecticide hexachlorocyclohexane (HCH),
which is still used in many countries in agriculture and
forestry, leads to environmental problems. HCH consists of
a mixture of four isomers: α-, β-, γ-, and δ. A bacterium
Pseudomonas aeruginosa ITRC-5 degrades not only α-, γ-,
and δ- HCH isomers but also β-HCH which is the most
recalcitrant due to its chemical stability. The degradation of
β-isomer is accompanied with the formation of a metabolite
2,3,4,5,6-pentachlorocyclohexan-1-ol (PCCOL).
In the present study, effectiveness of ITRC-5 strain in
further degradation of PCCOL was evaluated. γ-HCH
grown ITRC-5 cells show higher degradation of PCCOL
than the t-HCH grown cells do. Furthermore, degradation
of PCCOL by γ-HCH grown cells is enhanced in the
presence of α- or γ-HCH isomers. Cloned genes of ITRC-5
bacterium showed 50% degradation of PCCOL in 16 hrs.
The ITRC-5, therefore, demonstrates potential for the
bioremediation of β-HCH and PCCOL
Keywords: Pseudomonas aeruginosa ITRC-5, βHexachlorocyclohexane, 2,3,4,5,6-pentachlorocyclohexan1-ol, Biodegradation.
1
INTRODUCTION
A technical mixture of hexachlorocyclohexane (t-HCH)
consisting of four major isomers α- (60-70%), β- (5-12%),
δ-HCH (6-10%) and 10-12% of γ-HCH (also known as
Lindane) remained a popular insecticide formulation and
was used extensively worldwide prior to the 1990s [1].
Although HCH has been banned or restricted in many
developed countries because of its toxic and persistent
nature, it is still in production and in use in many parts of
the world [2]. Agricultural soils and groundwater around
the world has been contaminated with HCH compounds to
various degrees [3]. Efforts have been made for the
remediation of soils and groundwater contaminated with the
toxic and persistent HCH isomers through biodegradation
processes [4-6].
In particular, β-HCH isomer has higher degree of
persistence compared to other HCH isomers and, therefore,
β-isomer residues have been reported predominantly from
soil, water, and food commodities [7-9]. It is the most
problematic of the HCH isomers as it is quite resistant to
biodegradation and is also suspected to cause endocrinal
disruption as well as breast cancer [10,11]. It is also
reported to have estrogenic effects in mammalian cells and
fish [2]. From various places around the world, β-HCH has
been reported to be found in human breast milk [3,12]. It
poses a serious threat as it has potential to be transferred
from one generation to the next via bioaccumulation in
various food chains.
Only a few bacterial strains have been found responsible
for the degradation of β-HCH isomer [13,14]. Nagata et. al.
(2005) reported that haloalkane dehalogenase linB in the
UT26 bacterial strain converted β-HCH to PCCOL, a
metabolite which could not be further degraded [15]. In a
recent study linB2 gene in Sphingomonas sp. BHC-A
bacterial strains converted not only β-HCH to PCCOL but
also PCCOL to tetrachlorocyclohexanediol [16].
The isolated bacterium Pseudomonas aeruginosa ITRC5 was reported to degrade all four major isomers (i.e. α, β,
γ, and δ) of HCH [17]. However, the degradation of
β−HCH isomer resulted in a dead-end product of PCCOL.
In the present study, we found that the ITRC-5 bacterial
strain grown in media containing γ-HCH and t-HCH was
able to further degrade PCCOL in aerobic condition. The
degradation of PCCOL was enhanced in the presence of
additional α- or γ-HCH. Furthermore, the cloned genes of
ITRC-5 expressed in E. coli DH5-α could successfully
degrade PCCOL.
2
2.1
MATERIAL AND METHODS
Chemicals
Technical HCH was obtained from India Pesticides
Limited, Lucknow, India. β-HCH and γ-HCH were
purchased from Riedel-de Haën, Germany. Silica gel TLC
plate, and all other chemicals of AR grade used in the
experiments were purchased from Merck India Ltd.
2.2
Microorganisms and culture conditions
The bacterium ITRC-5, isolated earlier in the ITRC lab
[17], was used in aerobic condition. It was grown in flasks
that were precoated with 200 ppm t-HCH and contained 50
ml of W+ medium [18]. The bacterium was incubated in the
flasks at 28°C with shaking at 180 rpm for 1 week. In order
to maintain fresh culture of the bacterium cells throughout
the experiments, 2 ml of the grown culture was transferred
to the fresh flasks and grown as above. E. coli DH5-α was
used in gene cloning experiments which was cultured in
Luria broth medium containing an appropriate
concentration of ampicillin antibiotic and incubated
overnight at 37°C.
2.3 Biodegradation of PCCOL by γ-HCH
and t-HCH grown cells
Three sets of 25 ml flasks were coated with 1 ppm of
PCCOL dissolved in acetone. Eight flasks in each set were
used. The flasks were kept open in a laminar hood at room
temperature. After evaporation of the acetone, 10 ml of
sterile W+ medium was added into each flask. While the
first set of flasks was kept un-inoculated, the second and the
third set of flasks were inoculated with 1 ml of the ITRC-5
cells previously grown in W+ medium with γ-HCH and tHCH, respectively, as the carbon source. The flasks were
inoculated in duplicate and were incubated for 6 days on a
rotary shaker (150 rpm) at 28°C. Two flasks from each set
were removed from the reaction setup at the interval of 2
days and the contents were extracted and analyzed by GC.
2.4 Biodegradation of PCCOL by “γ-HCH
grown cells” in the presence of α- and γ-HCH
In this experiment four sets of 25 ml flasks were coated
with 1 ppm of PCCOL dissolved in acetone. The first set
was used as a reference and therefore, kept un-inoculated.
The second set was inoculated with 0.2 ml of the “γ-HCH
grown cells” of ITRC-5 in the absence of α- or γ-HCH. The
flasks of sets 3 and 4 were coated additionally with 50 ppm
of α-HCH and γ-HCH, respectively. The flasks were kept
open in a laminar hood at room temperature. As acetone
evaporated from the flasks, 10 ml of sterile W+ medium was
added into each flask. The flasks were inoculated in
duplicate and were incubated for 6 days on a rotary shaker
(150 rpm) at 28°C. Two flasks from each set were removed
from the reaction setup at the interval of 2 days and the
contents were extracted and analyzed by GC.
2.5 Cloning of gene responsible for
degradation of PCCOL
ITRC-5 and DH5-α bacterial cells were used for
cloning procedure. ITRC-5 bacteria were incubated at 28°C
in 1 liter W+ medium containing 500 ppm of γ-HCH,
whereas DH5-α bacteria were incubated at 37°C in 50 ml
of Luria broth medium. pUC18 plasmid vector and
ampicillin (50 mg/ml) antibiotic were used in the gene
screening process. The alkaline lysis method was used to
isolate plasmid DNA of DH5-α and the DNA was further
dephosphorelated with calf enzyme [19]. Marmur’s method
was used to isolate genomic DNA from ITRC-5 strain [20].
Genomic library was constructed for ITRC-5 bacterial DNA
which was partially digested with Sau3AI restriction
enzyme. Isolated DNA fragments of 5 kb in size were
ligated with the dephosphorelated plasmid vector pUC 18
digested with Bam H1 restriction enzyme. The ligation
product was transfected into E. coli DH5-α and screened
for ampicillin resistance on agar plates. The library in DH5α was stored at 4°C.
2.6
In vivo assay for PCCOL degrading gene
ITRC-5 gene library was assayed for PCCOL degrading
gene. A small quantity of a colony was suspended in 1ml of
assay solution (1 ppm PCCOL, 100 ppm of each α, β, γ and
δ-HCH in 1/3rd LB medium). The suspension was incubated
at 37°C over night. Then 1 ml of hexane and acetone (9:1)
was added, and the mixture was vortexed for 3 min. After 5
min. the hexane and acetone layer was recovered. 1 µl of
this extract was analyzed by GC for PCCOL degradation.
2.7
GC Analysis
A gas chromatograph (GC, Netel Chromatograph; Michro
9100) fitted with 63Ni-ECD was used for this study. The
column, injector and detector temperatures were kept at
190°C, 250°C and 250°C, respectively. Nitrogen was used
as the carrier gas and maintained at a pressure of 15 psi.
The GC was calibrated with standard HCH isomers and
PCCOL.
3
RESULTS AND DISCUSSION
3.1 Biodegradation of PCCOL by γ-HCH
and t-HCH Grown Cells
The purified PCCOL was degraded by γ-HCH grown as
well as t-HCH grown ITRC-5 cells. The concentration of
PCCOL in the media decreased with increased time period
of incubation. TLC analysis of PCCOL degradation by γHCH is shown in Fig.1. After 6 days of incubation, approx
61% and 58% degradation of PCCOL was achieved by γHCH grown cells and t-HCH grown cells, respectively
[Fig.2].
In un-inoculated condition, a small degradation of
PCCOL was observed after 2nd day of incubation, as shown
in Fig. 2, which could be due to hydrolysis,
photodecomposition, volatilization, or other abiotic
transformations of PCCOL. Furthermore, under inoculated
condition, higher degradation of PCCOL was observed with
γ-HCH grown cells in comparison with t-HCH grown cells.
It is suspected that the genes responsible for PCCOL
degradation might be more active in the presence of γHCH.
3.2 Biodegradation of PCCOL in the
Presence of α- and γ-HCH by γ-HCH Grown
Cells
After 6 days of incubation of γ-HCH grown ITRC-5
bacterial cells, 75.5% degradation of PCCOL was observed
in the presence of additional 50 ppm γ- HCH whereas 71%
degradation was observed in the presence of α-HCH [Fig.
3]. The results show an increase in the degradation of
PCCOL by ~14.5% and ~10% in the presence of γ-HCH
and α-HCH, respectively. Presence of γ−HCH shows
higher degradation of PCCOL than the presence of α-HCH
isomer.
Increased degradation of PCCOL in the presence of αor γ-HCH could be due to additional carbon source
provided by α- or γ-HCH for the proliferation of ITRC-5
bacterial strain. Furthermore, ITRC-5 utilizes relatively
easily γ−HCH isomer than α-HCH isomer as a carbon
source owing to the difference in spatial arrangements of
chlorine atoms. This possibly explains the observed higher
degradation of PCCOL in the presence of γ-HCH than αHCH. No further degradation of PCCOL was observed after
6 days of incubation, possibly because of the depletion of
the growth supporting α- or γ- isomers by this time.
3.3 Screening of Clones with PCCOL
Degradation Activity
The gene library of ITRC-5 was constructed in DH5-α.
The clones responsible for PCCOL degradation activity
were screened by GC analysis. Out of 200 clones that were
tested for insert DNA using plasmid isolation method, 100
clones showed positive results. Fig.4 shows 4.5kb, 4.5kb,
5kb, 5kb and 4.5kb insert DNA with plasmid DNA band in
lane 1, 2, 5, 6 and 7, respectively. Only three clones out of
100 positive clones showed PCCOL degradation activity.
These three clones showed approx. 50% degradation of 1
ppm PCCOL in 16 hrs. The degradation activity was not
detected in other clones having insert DNA possibly due to
lack of PCCOL degrading gene. The gene responsible for
the degradation of PCCOL has not been characterized in the
present paper.
pUC18 plasmid vector in this research has not been
used for functional assay. Although pUC18 is not equipped
with promoter, regulator sequences, large genomic DNA
fragment of ITRC-5 up to 5 kb size used in this study may
contain gene as well as all things needed for expression of
protein. Therefore, the gene involved in the degradation
pathway of PCCOL could be expressed by three clones.
The gene of ITRC-5 responsible for the degradation of
PCCOL might be very similar to LinB2 gene. ITRC-5 has
full copy of LinB gene designated as LinB1 and LinB2
gene [21]. Jun et al. (2007) reported that LinB2 gene in
Sphingomonas sp. BHC-A was involved in PCCOL
degradation pathway [16].
4
CONCLUSIONS
In conclusion, the present study suggests that an
effective remediation of persistent β-HCH isomer and its
metabolite PCCOL can be achieved by the addition of
ITRC-5 bacterium. Furthermore, degradation activity of
ITRC-5 bacterium is induced by the additional α-HCH and
γ-HCH isomers in the medium. Further experiments are
necessary to characterize the PCCOL degrading gene which
seems to have very strong degrading activity. These
findings are very important as it is one step further in the
elucidation of probable β-HCH biodegradation pathway.
5
ACKNOWLEDGEMENTS
This work was supported through the research
assistantship by the University Grant Commission (UGC),
New Delhi in form of a Senior Research Fellowship to
Anita Singh. We would also like to thank Industrial
Toxicology Research Centre (ITRC) for providing
logistical support for this research.
6
FIGURES
Figure 1: TLC shows biodegradation of PCCOL metabolite
(M1) after 0 time, 2, 4 and 6 days of incubation. Heptane
and 5% acetone were used as solvents for run. Rf value: αHCH 0.63, γ-HCH 0.47, β-HCH 0.32, δ-HCH 0.15, M1
0.05.
REFERENCES
Figure 2: Degradation of PCCOL metabolite (M1) under
un-inoculated (UI) and inoculated (I) conditions.
Figure 3: Biodegradation of PCCOL under un-inoculated
(UI) and inoculated (I) conditions. Biodegradation of
PCCOL under inoculated conditions in the presence of
additional γ-HCH (I+γ), α-HCH (I+α) by γ-HCH grown
cells, is also shown.
Figure 4: Lane 1, 2, 5, 6 and 7 shows pUC18 plasmid
vector with insert DNA from ITRC-5.
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Improvement of Hydrogen Production by Immobilized Rhodopseudomonas palustris
CGA009 Using Reverse Micelles as Microreactor
Anita Singh* and Krishna Misra**
* Department of Biochemistry and Cell Biology, Rice University, Houston, TX. [email protected]
**Indo-Russian Center for Bioinformatics, Indian Institute of Information Technology, Allahabad.
ABSTRACT
In the present study, the bacterium Rhodopseudomonas
palustris CGA009 has been immobilized separately in an
aqueous pool of the reverse micelles fabricated by various
surfactants (AOT, CBAC and SDS) and apolar organic
solvents (benzene and isooctane). All reverse micellar
systems of bacterial culture gave encouraging hydrogen
production (rate as well as yield) compared to the aqueous
system. An average of 50 fold increase in hydrogen
production rate was observed in case of AOT/isooctane
reverse micellar system as compared to the aqueous culture.
CBAC/isooctane reverse micellar system is used for the
first time for hydrogen production and is as promising as
AOT/isooctane reverse micellar system. All these reverse
micellar media were screened to reduce the inhibitory effect
of NH4+ on hydrogen production. Studies of the effects of
pH, temperature and light intensities on hydrogen
production in reverse micelles show optimal hydrogen
production at 37°C temperature, 600 lx light intensity and 7
pH.
Keywords: Rhodopseudomonas palustris CGA009,
Reverse micelles, AOT, Biological hydrogen production.
1
INTRODUCTION
Hydrogen production from biological sources is gaining
major emphasis recently since these processes are less
energy intensive and comparatively cost effective than any
other thermochemical or electrochemical processes. Among
different alternative routes of biological hydrogen
production microbial hydrogen production has already been
investigated with both photosynthetic [1,2] and
fermentative microorganisms [3,4].
Various groups of investigators have attempted to look
for the possibility to improve hydrogen production through
immobilization techniques using polysaccharide gels like
agar, alginate and chitosan combined with agar etc. [5,6].
The major disadvantage of polysaccharide gel
immobilization technique is inhomogeneous entrapment of
bacteria, so that few bacterial cells suffer from nutritional
exhaustion as well as low light penetration and this results
in decreased hydrogen production.
In a reverse micellar system, bacterial cells are fully
exposed to the light owing to the transparency of the
solutions [7]. Therefore, a reverse micellar system is
preferred over other immobilization techniques to produce
hydrogen. Reverse micelles are essentially spheroid
aggregates formed by dissolving a surfactant in a polar
organic solvent with a limited amount of water to produce a
macro-homogenous transparent solution, which provides an
artificial system that mimics many life systems. AOT/
isooctane and SDS/benzene reverse micellar system for
hydrogen producing bacteria have shown encouraging
results [8-10].
The scope of this paper is to show the effects of various
reverse micelles using bacteria on hydrogen production. For
this purpose, we have used photosynthetic bacteria
CGA009
immobilized
within
AOT/isooctane,
CBAC/isooctane and SDS/benzene reverse micelles. To the
best of our knowledge, one of the reverse micellar systems
CBAC/isooctane has not been reported for hydrogen
production by this bacterial culture. All these reverse
micellar systems were also screened to reduce the
inhibitory effect of NH4+ on hydrogen production.
Furthermore, effects of pH, temperature and light intensities
on hydrogen production in reverse micelle have also been
studied.
2
2.1
MATERIAL AND METHODS
Chemicals
All chemicals used in the experiments were of AR
Grade, purchased from Merck India Ltd., L-malic acid and
vitamins were purchased from Himedia Laboratories Pvt.
Ltd., India and used without further purification.
2.2
Microorganism and Culture Conditions
The photosynthetic bacteria R. palustris CGA009 was
cultivated photosynthetically at 300C in Sistrom’s growth
media [11]. In order to increase hydrogen production, the
growth medium was modified by adding 30 mM of malate
as a carbon source, 7 mM of glutamate as a nitrogen source,
and by removing succinic acid, aspartic acid, ammonium
sulphate and nitrilotriacetic acid. Furthermore, sodium
molybdate (Na2MO4.2H2O) was used in place of
ammonium molybdate. Batch cultivations for CGA009 was
performed at 300C in a gyratory incubator with a shaking
speed of 250 rpm. An anaerobic condition was developed in
a serum bottle of 155 ml by filling it with the media and
flushing with argon gas (99.99%) till the working volume
of 50 ml was achieved. A tungsten lamp was placed to
maintain a luminance intensity of 6000 lx. The inoculum
was cultivated in the bottle and transferred anaerobically at
the late-exponential phase by a sterile disposable syringe.
The culture was centrifuged at 10,000 rpm for 15 min. The
pellets were resuspended in the growth medium and used
for entrapment studies.
2.3 Preparation of reverse micelles for
hydrogen production
The surfactants were added in powder form gradually to
the organic solvents taken in a flask and homogenized.
sodium lauryl sulfate (SDS) (1.2 g) was added to benzene
(250 ml); sodium bis-2-ethylhexyl-sulfosuccinate (AOT)
(8.86 g) and cetylbenzyldimethyl-ammonium chloride
(CBAC) (0.95 g) were added to isooctane (250 ml) at 370C.
2.0 ml of the bacteria CGA009, grown during the late-log
phase, was injected into the homogenates to fabricate
reverse micellar system. The reverse micellar medium
containing entrapped bacterial cells was transferred to
hydrogen producing air tight setup. The setup was placed
on a magnetic stirrer in an upright position fitted with a
clamped stand and illuminated with a tungsten lamp of 600
lx intensity at 370C temperature. Hydrogen gas produced
was collected in the upper portion of the setup by
displacement of the reverse micellar organic medium.
2.4 Determination of optimum temperature,
pH and light intensity for maximum hydrogen
production
To study the effect of temperature on reverse micellar
systems for the hydrogen production 5 sets of experiments
were conducted. 2.0 ml late-log phase culture was
entrapped within each set of AOT/isooctane reverse
micelles at 600 lux light intensity. Using hot plate cum
stirrer and water bath the temperature of the systems was
maintained at 25, 30, 35, 37 and 40°C and the hydrogen
production rate was measured every 30 min. For
determining the pH dependence of the H2 production,
bacterial culture was suspended within several different
buffer solutions. These were as follows (all in 100 mM):
acetate buffer (pH 4.0 and 5.0), phosphate buffer (pH 6.0 to
7.0), and trizma base buffer (pH 8.0 and 9.0). 2.0 ml
volume of suspended culture was entrapped within the
reverse micelles formed by AOT/isooctane and the set-ups
were exposed to 600 lux intensity and maintained at 37°C,
the hydrogen production rate was measured every 30 min.
To determine the effect of light intensity, late-log phase
culture was entrapped within AOT/Isooctane reverse
micelles at different light intensity from 100 to 2500 lux.
Temperature of the micellar solution was maintained at
37°C and hydrogen production in each case was recorded
every 30 min. Dry weight of the organisms was taken to
express the results.
2.5 Determination of the effect of NH4+ on
hydrogen production by CGA009
To determine the effect of NH4+ on hydrogen
production, culture was grown in modified sistrom’s
medium by replacement of glutamate with ammonium
sulphate. Five sets of experiments were conducted. In each
set culture was grown in above media with varying
concentration of ammonium sulphate i.e. 3mM, 6mM,
9mM, 12mM and 15mM. 2.0 ml volume of suspended
culture and 1.0 ml of above media was entrapped within the
various reverse micellar system of AOT/isooctane,
CBAC/isooctane and SDS/benzene. The set-ups were
exposed to 600 lux light intensity and maintained at 37°C
temperature. The hydrogen production rate was measured
every 30 min.
2.6
Measurement of protein content
Protein content of the bacterial cells was measured
using Lowry method. In this method, a bovine serum
albumin (BSA) is used as standard [12].
2.7 Gas analysis
The concentration of the evolved molecular hydrogen
was measured by a gas chromatograph (Nikon) equipped
with a thermal conductivity detector and a stainless-steel
column (Carbosieve II). In the equipment argon gas is
served as carrier and pure hydrogen gas is used as standard.
The column temperature was maintained at 600C and the
temperature of detector and injector was set to 700C. The
volume of hydrogen was converted into the value at 370C
and 0.1 MPa.
Stoichiometric maximum evolution of hydrogen was
calculated by assuming that hydrogen was evolved by
complete conversion of the carbon source in the catabolic
process. The yield of hydrogen evolution was calculated as
a percentage of the stoichiometric maximum. For example,
the stoichiometric maximum of hydrogen evolution from 30
mM malate was 4.58 ml/ml-medium at 370 C and 0.1 MPa.
3
RESULTS AND DISCUSSION
3.1 Hydrogen production by entrapped
CGA009 in reverse micelles
Table 1 show the rates of hydrogen production, total
volume of hydrogen evolved and hydrogen yield by the
bacterial cells of CGA009 entrapped within different
reverse
micellar
systems
of
AOT/isooctane,
CBAC/isooctane and SDS/benzene at 370C temperature, pH
7 and 600 lx light intensity. The bacterial cells produce
more hydrogen in the AOT/isooctane reverse micellar
medium in comparison with the other reverse micellar
media. The hydrogen production rates of the CGA009
entrapped within all reverse micellar systems (i.e.
AOT/isooctane, CBAC/isooctane and SDS/benzene) were
30–50 times higher when compared with the hydrogen
production rates of the aqueous culture The hydrogen yield
increased by a factor of 2 in the reverse micellar systems
when compared with the aqueous culture. The evolved
hydrogen gas was found to be 99.99% pure.
All reverse micellar systems used in the study were
compatible with the photosynthetic bacterial cells. Solvents
and surfactants in the reverse micellar systems did not
affect the viability of the bacterial cells much and gave an
appropriate microenvironment within the water pool for
hydrogen production. Reverse micelles act as small
microreactors in which the activity of cellular enzymes is
increased by bringing the reactants together [13]. Thus, the
enzymes responsible for carbohydrate degradation and
hydrogen production become more active in the anaerobic
water pool of reverse micellar microreactor owing to
compartmentalization of the cells resulting in enhanced
hydrogen production. Furthermore, nitrogenase and
hydrogenase enzymes are active only in anaerobic
environment and reverse micelles provide such
environment to these enzymes as the reverse micellar
aqueous pool itself has a very low concentration of
dissolved oxygen and the surrounding organic medium
helps in providing anaerobic condition to the aqueous pool.
Free bacterial cells in aqueous culture, however, are more
exposed to oxygen. In this study it was observed that AOT/
isooctane reverse micellar system was the most transparent
and SDS/benzene system was the least transparent [14].
Therefore, increased light availability for nitrogenase
enzyme of the bacterial cells in AOT/isooctane reverse
micelles resulted in increased hydrogen production rate
[Table 1]. Hydrogen evolving systems, AOT/isooctane and
CBAC/isooctane gave very similar results.
3.2 Effect of temperature, pH and light
intensity on hydrogen production in reverse
micellar system
The results in Fig. 1 show that, by increasing the
temperature from 25 to 37°C, hydrogen production rate
gradually increases. At temperatures higher than 37°C, the
rate decreased. At pH 7 optimal hydrogen production was
observed. At higher or lower pH the rate decreased
significantly [Fig. 2]. Also note that hydrogen was not
produced at pH 4 . The evolution of hydrogen was affected
significantly by the light intensity. The evolution rate of
hydrogen increased exponentially with the light intensity
from 100 lux to 600 lux. Hydrogen production rate remains
constant up to 1500 lux light intensity [Fig. 3]. Further
increase in light intensity caused a decrease in the rate of
hydrogen production.
3.3 Effect of NH4+on hydrogen production in
reverse micellar system
The results shown in Fig. 4 indicate that at the initial
concentration of ammonium sulphate i.e., 3 mM, larger
amount of hydrogen was evolved by all the reverse micelles
(AOT/isooctane, CBAC/H2O/isooctane and SDS/benzene)
than by the normal aqueous culture. Comparison within the
reverse micelles indicates that the maximum hydrogen
evolution was obtained by AOT/isooctane and lowest
hydrogen production by SDS/benzene. However, when
ammonium sulphate concentration was increased to 15
mM, hydrogen evolution rate was much depressed. In all
the 3 systems, maximum evolution appears at 3mM
concentration of NH4+ ion.
4
CONCLUSIONS
Under the experimental conditions during the present
work, reverse micelle system AOT/isooctane at 37°C, pH
of 7.0, light intensity of 600 lux and an optimal
concentration of 3 mM NH4+ shows highest hydrogen
evolution by bacterial cells. CBAC/isooctane reverse
micellar system also gave an encouraging amount of
hydrogen by bacterial cells.
On the basis of above results, we believe that the
entrapment of whole cells within these reverse micellar
systems can be applied to the construction of bioreactor for
hydrogen production
5
ACKNOWLEDGEMENTS
The authors thank Prof. C.S. Harwood of Department of
Microbiology, University of Iowa, Iowa City, USA for
providing the strains of CGA009. This work was supported
through the research assistantship by University Grant
Commission, New Delhi in the form of a Senior Research
Fellowship to A.S.
6
TABLE AND FIGURES
Figure 1: Effect of temperature on hydrogen production by
CGA009 in AOT/isooctane reverse micelle at pH 7 and 600
lux light intensity.
Table 1: H2 production in various reverse micellar
systems and in aqueous medium using CGA009.
Figure 2: Effect of pH on hydrogen production by CGA009
in AOT/isooctane reverse micelle at 37oC and 600 lux light
intensity.
Reverse
Micellar
Systems
H2
production
rate [mmol
of H2 (mg
protein)-1
h-1]
AOT/
isooctane
CBAC/
isooctane
SDS/
benzene
Aqueous
culture
3.00 ± 0.80
Fold
enhance
ment in
rate
w.r.t.
aqueous
culture
~50
Total
amount of
H2
evolved
[ml/mlmedia]
Yield
(%)
1.15 ±0.01
25.20
2.50 ± 0.50
~42
1.05 ±0.03
23.00
1.80 ± 0.40
~30
0.80 ±0.01
17.50
0.06 ± 0.02
1
0.64 ±0.01
14.00
Note: Carbon source: 30 mM DL-malate. Temperature:
370C, pH: 7 and light intensity: 600 lx. Each value is mean
of at least three times replication (±standard deviation).
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Figure 3: Effect of light intensity on hydrogen production
by CGA009 in AOT/isooctane reverse micelle at 37oC and
pH 7.
Figure 4: Effect of NH4+ concentration on hydrogen
production in different reverse micellar systems and in
aqueous culture by CGA009 at temperature 37oC, pH 7 and
600 lux light intensity.
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