j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 1 2 e1 1 6

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Original Article

Hydrogen production by Pseudomonas stutzeri JX442762

isolated from thermal soil at Mettur power station, Salem
district, Tamil Nadu, India

S. Penislus Shiyan a, M. Krishnaveni b,*

a
Department of Biochemistry, Periyar University, Salem 636011, Tamil Nadu, India
b
Assistant Professor, Department of Biochemistry, Periyar University, Salem 636011, Tamil Nadu, India

article info abstract

Article history: Background: Carbohydrate based substrates presents a promising route of biological
Received 21 August 2012 hydrogen production compared with chemical routes. Pure substrates including starch,
Accepted 6 November 2012 sucrose as well as different organic waste materials can be used for hydrogen production.
Among a large number of microbial species, strict anaerobes and facultative anaerobic
Keywords: chemoheterotrophs, thermoanaerobacter species are efficient producers of hydrogen.
Biological hydrogen production Since hydrogen is a high-energy fuel than hydrocarbon fuel, it is essential to find an
Mango juice effluent alternative source.
SSKVM 2012 Objectives: To isolate hydrogen producing organism from thermal soil sample, to identify
Starch the organism as it is a cost effective way for biological hydrogen production. The hydrogen
Sucrose production was assessed in pure starch, sucrose and also in mango juice effluent obtained
from Krishnagiri dist., to find a better substrate.
Methodology: Morphological, biochemical characterization of the isolate was evaluated and
finally confirmed by 16S rRNA gene sequencing. Hydrogen production was measured by
simple water displacement method for the selected substrates at 70
C, initial pH 4.0 as
well as for the effluent.
Results: 16S rRNA gene sequencing confirms the organism as Pseudomonas stutzeri which
was deposited in gene bank under the accession number JX442762 and the identified strain
was named as SSKVM 2012. The G þ C content of the strain P. stutzeri was found to be
53 mol%. The obtained results showed that the maximum hydrogen production was
observed with pure starch (255.98  0.76 ml) but in sucrose it was found to be
212.82  0.57 ml. The mango juice effluent showed 190.03  0.81 ml hydrogen. Even though
it was less when compared to hydrogen production by starch and sucrose, mango juice
effluent could be a better substrate for the identified organism.
Conclusion: The identified strain confirms that it can use effluent as a good source for the
hydrogen production as its initial pH is 6.0 and also freely available in the environment.
Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.

* Corresponding author. Tel.: þ91 9894829823 (mobile).

E-mail addresses: krishnavenim2011@gmail.com, logasarvesh@gmail.com (M. Krishnaveni).
0974-6943/$ e see front matter Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jopr.2012.11.024
 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 1 2 e1 1 6
1. Introduction
The current global demand for H2 was estimated to be
approximately 45 million tons/annum.1e3 Current estimates
indicate that the global energy demand would continue to
increase from 536 EJ in 2008, to 653, in 2020, and 812 EJ by
2035.4 On the other hand, the United Nations Statistics show
that the global CO2 emissions increased 44% between 1990
(20.69 billion metric tons) and 2008 (29.86 billion MT).5
Progressive depletion of non-renewable energy sources
worldwide, together with the fact that their use has resulted in
environmental deterioration and public health problems, has
led to development of new renewable energy harvesting
technologies.6,7 Hydrogen is considered an ideal alternative
fuel to the current energy scenario due to its high-energy
content and non-polluting nature.8e11 It is a clean and envi-
ronment friendly fuel that produces only water when com-
busted with oxygen. It is a high-energy fuel (122 kJ/g) than
hydrocarbon fuel.12 Approximately 95% of commercially
produced hydrogen comes from carbon containing raw
materials, primarily fossil in origin.13 Moreover, the petroleum
reserves of the world are depleting at an alarming rate.14 Due
to the depletion of fossil fuel and emission of greenhouse gas
(CO2) during conventional hydrogen production process, bio-
logical hydrogen production from biomass has been recog-
nized as an eco-friendly and less energy intensive process to
produce hydrogen compared to photosynthetic/chemical
processes.15 Thermophiles are organisms capable of living at
high temperature. These organisms do not only survive but
might even thrive in boiling water.16 The ability of thermo-
philic bacteria to grow at high temperature and to produce
stable extracellular enzymes was attributed to the probability
of increasing their enzyme excoriation and activity by means
of genetic manipulation. Therefore, these microorganisms
were the first candidates for massive enzyme production for
industrial applications.17 Thermophilic anaerobic fermenta-
tion processes hold tremendous potential for the forthcoming
generation as well as commercial production of hydrogen
fuel.18 Hence, in view of the above, we have isolated a Pseu-
domonas stutzeri from soil near thermal wells at Mettur power
station, Salem, Tamil Nadu, India. The identified strain was
studied for its ability to produce hydrogen using mango juice
effluent as a preliminary study, in order to reduce the cost of
hydrogen production by using synthetic source starch as well
as sucrose.
2. Materials and methods
2.1. Sample collection
Thermal soil samples were collected from soil near thermal
wells at Mettur power station, Tamil Nadu, India. One gram of
thermal soil was dissolved in 100 ml distilled water. Serial
dilution was carried out as per the standard procedure.19
Serial dilution technique was used to obtain pure cultures.
In order to be sure to obtain pure isolates, serial dilution steps
were repeated several times.
113
2.2. Cultivation of isolate
The isolate was cultivated in the solid nutrient agar medium con-
taining Peptone e1 g, Beef extract e 3.0 g, Sodium chloride e 5 g,
Yeast extracts e 2.0 g, Distilled water e 1000 ml, pH 7.4  0.2.
Sterile nutrient agar was taken in petri dishes. A sterilized loop
was dipped into the suspension of desired organism and was
streaked on the surface of solidified agar plate. The plates were
then incubated for 24e48 h to get the individual colonies.
Bacteria grows on the surface nutrient agar, and is clearly
visible as small colonies.
2.3. Isolation of strain
Thermal soil samples were inoculated in anaerobic liquid
basal medium consisting of (g/l): NH4Cl 0.5, Yeast extract 5,
K2HPO4 0.25, KCl 0.002, MgCl26H2O 0.125, NH4CO3 0.4, Peptone
1, NH4H2PO4 0.4, NaH2PO4 0.5. Trace element 1 ml, vitamin
solution 1 ml.20 Sucrose (10 g/l) was used as a carbon and
energy source. All the culture bottles were incubated at 70
C
for 3 days and sub cultured after 3 days of incubation. All the
sub cultures and diluted cultures were incubated at 70
C
under atmospheric pressure. Cells were observed under
a light microscope and pure isolate was routinely cultivated in
anaerobic liquid basal medium.
2.4. Morphological studies
Morphological characteristics were investigated. Gram stain-
ing was performed to confirm the gram reaction and spore
position. Motility was determined by hanging drop method.19
2.5. Conventional identification tests
All isolates were evaluated by conventional tests for catalase,
oxidase, indole, urease, methyl red, voges-proskauer, citrate
utilization, triple sugar iron, starch hydrolysis, hydrogen
sulphide and oxidative fermentative carbohydrate utilization.19
2.6. Identification of strain by using 16s rRNA gene
sequence
Genomic DNA was extracted from the isolate using Pure Fast
Bacterial Genomic DNA isolation kit. 1 mL of genomic DNA was
used as template and amplified by PCR using Master Mix Gene
kit (HELINI biomolecules Chennai, India) with the aid of 16S
rDNA primers (16S Forward Primer: 5-AGAGTRTGATCMTY
GCTWAC-3 16S Reverse Primer: 5-CGYTAMCTTWTTACGR
CT-3) with the programme consisted of denaturation at 94
C
for 1 min and subsequent 35 cycles of denaturation at 94
C for
30 sec, annealing at 60
C for 1 min, and extension at 72
C for
1 min followed by final extension at 72
C for 5 min. Amplified
product was sequenced using the Dye Deoxy Terminator Cycle
sequencing kit (HELINI biomolecules Chennai, India) as
directed in the manufacturer’s protocol. The nucleotide
sequencing of 16S rRNA gene of the isolate was compared with
other related sequences using FASTA programme. Further, the
nucleotide sequences of the isolate was aligned with closely
related sequence using CLUSTAL W mega version-5.
114 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 1 2 e1 1 6

3.2. Identification of strain by using 16S rRNA gene

sequence

The 16S rRNA gene sequence of isolate confirms that the

Fig. 1 e Phylogenetic tree showing position of P. Stutzeri
among closely related members.
2.7. Hydrogen production by P. stutzeri using synthetic
source e starch, sucrose and also using mango juice effluent
The hydrogen production by P. stutzeri was analysed for the
synthetic sources selected i.e. starch and sucrose. In order to
find the effect of starch and sucrose, these sugars were
taken 7.5 g in 1500 ml, 5.0 g in 1000 ml, 3.75 g in 750 ml, 2.5 g
in 500 ml. Similarly, the amount of hydrogen produced by
utilizing the mango juice effluent was also studied. For this
study 1500 ml, 1000 ml, 750 ml and 500 ml mango juice
effluent (waste water) were used. Mango juice effluent was
collected from the Maaza juice production unit located at
Krishnagiri, Krishnagiri District, Tamil Nadu. The starch and
sucrose soluble medium were inoculated with P. stutzeri and
its pH was maintained at 4.0, at temperature 70
C. Since,
the effluent’s initial pH is 6.0, when effluent was inoculated
with the identified organism P. stutzeri, the strain starts
producing hydrogen immediately. The influence of pH
change on hydrogen production was observed to find the
maximum hydrogen production. The hydrogen produced
was measured by simple water displacement method for
a period of 5 days.21
3. Results
3.1. General properties of strain SSKVM 2012
P. stutzeri SSKVM 2012 is found to be thermophilic, rod shaped,
gram negative, anaerobic with an optimum growth at 70
C.
The strain is alkaliphilic and able to grow at wide range of pH
from 5.5 to 9.0. There was no growth observed at pH 4.0epH
5.0 or below. Further pH in the range of 6.5e8.5 was found to
be a favourable for the strain to produce hydrogen. The strain
hydrolyses starch and found to produce hydrogen sulphide.
organism isolated was P. stutzeri. The sequence of P. stutzeri
(HM209781.1) had 99% identity to Pseudomonas xanthomarina
(HQ848111.1) and Pseudomonas knackmussii (JN646015.1) and
these two sequences grouped together in a phylogenetic tree
(Fig. 1). The sequence reported in this paper has been depos-
ited in the genbank under the accession number JX442762 and
the strain identified from the thermal soil sample was named
as SSKVM 2012.
3.3. Hydrogen production by P. stutzeri from starch and
sucrose
The hydrogen production from starch, sucrose measured by
water displacement method is shown in Table 1. Initial pH
of the soluble starch, sucrose medium was maintained at
pH 4.0 and at 70
C. No hydrogen production was observed
at initial pH 4.0 to pH 5.0. The maximum hydrogen
production observed for starch was 255.98  0.76 ml,
195.87  0.82 ml, 176.84  0.64 ml, 125.83  0.64 ml. Simi-
larly, the sucrose showed 212.82  0.57 ml, 194.85  0.69 ml,
191.85  0.76 ml, 177.92  0.78 in 7.5 g/1500 ml, 5.0 g/
1000 ml, 3.75 g/750 ml, 2.5 g/500 ml respectively. Among the
different concentrations used 7.5 g starch showed highest
hydrogen production.
The hydrogen production from effluent is shown in
Table 2. The initial pH of the mango juice effluent was found to
be pH 6.0. The effluent was inoculated with culture P. stutzeri
and the study was performed at 70
C. The maximum
hydrogen production observed was 190.03  0.81 ml,
186.13  0.57 ml, 144.96  0.72 ml, 104.93  0.64 ml in 1500 ml,
1000 ml, 750 ml, 500 ml mango juice effluent at pH 8.0. The
hydrogen production was found to be low when compared to
starch and sucrose but the effluent is recycled to an useful
product and signifies eco-friendly environment. Water
displacement methods can be more effective as pressure is
released, but gases can disproportionally dissolve based on
their different solubilities in the solution, making it difficult to
determine the produced gas composition.

Table 1 e Hydrogen production by P. stutzeri using starch and sucrose at 70
C.

pH H2 (ml) in 7.5 g/1500 ml H2 (ml) in 5.0 g/1000 ml H2 (ml) in 3.75 g/750 ml H2 (ml) in 2.5 g/500 ml

Starch Sucrose Starch Sucrose Starch Sucrose Starch Sucrose

4.0 0 0 0 0 0 0 0 0
5.0 0 0 0 0 0 0 0 0
5.5 85.10  0.21 46.74  0.45 69.83  0.14 42.43  0.24 65.60  0.36 40.48  0.26 54.75  0.23 39.46  0.24
6.0 160.16  0.44 75.80  0.51 130.57  0.35 68.56  0.29 120.70  0.49 60.65  0.45 99.53  0.26 54.62  0.38
6.5 200.06  0.49 88.72  0.55 150.39  0.13 80.64  0.39 130.75  0.52 79.72  0.45 105.83  0.49 76.77  0.54
7.0 219.95  0.52 136.80  0.57 185.80  0.64 129.65  0.39 145.78  0.56 132.78  0.60 112.92  0.52 122.76  0.56
7.5 249.93  0.64 198.83  0.55 190.78  0.68 184.67  0.49 155.84  0.61 177.85  0.67 115.09  0.62 154.86  0.69
8.0 255.98  0.76 212.82  0.57 195.87  0.82 194.85  0.69 176.84  0.64 191.85  0.76 125.83  0.64 177.92  0.78
8.5 100.03  0.42 193.80  0.56 95.76  0.64 189.60  0.49 89.73  0.49 178.77  0.67 76.46  0.26 169.90  0.76
9.0 96.16  0.32 81.72  0.54 89.13  0.44 72.64  0.33 68.68  0.42 80.71  0.58 54.80  0.25 64.65  0.38

Each experiment is representative of three independent variables.

 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 1 2 e1 1 6 115

Table 2 e Hydrogen production by P. stutzeri using mango juice effluent at 70
C.

pH H2 (ml) in 1500 ml H2 (ml) in 1000 ml H2 (ml) in 750 ml H2 (ml) in 500 ml

6.0 0 0 0 0
6.5 98.16  0.27 93.13  0.21 84.05  0.33 86.06  0.16
7.0 119.90  0.36 115.16  0.38 100.23  0.40 91.03  0.42
7.5 149.85  0.47 120.23  0.40 110.20  0.43 95.90  0.43
8.0 190.03  0.81 186.13  0.57 144.96  0.72 104.93  0.64
8.5 183.98  0.76 136.03  0.49 115.96  0.56 98.83  0.44
9.0 66.90  0.17 58.08  0.15 70.00  0.36 44.05  0.21

Each experiment is representative of three independent variables.

H2 production are its availability, cost, carbohydrate content

4. Discussion
Biological H2 production is the most challenging area of
biotechnology with respect to environmental problems. A
challenging problem in establishing biohydrogen as a source
of energy is the renewable and environment friendly genera-
tion of large quantities of H2 gas. However, two major aspects
need indispensable optimization, viz. a suitable renewable
biomass/wastewater and ideal microbial consortia that can
convert this biomass efficiently to hydrogen gas. It is a natural,
though transient, by-product of several microbial driven
biochemical reactions, mainly in anaerobic fermentation
processes. Dark-fermentation is a ubiquitous phenomenon
under anoxic or anaerobic conditions. The oxidation of the
substrate by bacteria generates electrons which need to be
disposed off in order to maintain the electrical neutrality
under the anaerobic or anoxic conditions other compounds,
such as protons, act as the electron acceptor and are reduced
to molecular hydrogen.22,23
The bacteria which grows at 65e80
C are called as extreme
thermophiles. The G þ C content of the P. stutzeri was 53 mol%
which was higher than the reports of Isaac KO Cann24 for the
species Thermoanaerobacterium polysaccharolyticum and Ther-
moanaerobacterium zeae. This strain grew well on starch and
sucrose at 70
C. No hydrogen evolution was observed at pH
4.0e5.0 in both starch and sucrose. The hydrogen production
was low at 5.5e6.5 in starch and sucrose. The low or no
hydrogen production at low pH could be partially attributing
to strong decrease in hydrogenase activity25 and this enzyme
is also highly sensitive to oxygen.23 The strain preferred high
temperature for optimum hydrogen production. The
hydrogen production in the medium was pH dependent and
occurred within the wide range of pH 6.0e9.0. In dark-
fermentation processes, this problem is compounded by the
fact that the gas produced is a mixture of primarily H2 and
CO2, but may also contain other gases such as CH4, H2S, or
ammonia (NH4). Moreover, the H2 content of the gas mixture
may be low (>50%). Maximal H2 production rates were
observed during exponential growth phase, which was in the
order of 8e12 h, within a total growth cycle of approximately 2
days.23 Thus no hydrogen evolution was found after 42 h of
fermentation. The hydrogen production obtained is however
variable and depends greatly on the bacterial consortium and
culture medium.26
Raw materials add to the cost of biohydrogen production
processes. The main criteria for the selection of a substrate for
and biodegradability.27 Cost reduction is achieved by either
using the low cost substrate or finding a means to effectively
utilize the 67e85 % of the unused spent media.28 Commer-
cially produced food products, such as corn and sugar are not
economical for H2 production.29 However, solid organic
wastes from agricultural crops, industrial processes and
domestic waste water represent a valuable resource for the
energy production. Starch based wastewater has great
potentiality for the H2 production.30 Disposal of these wastes
is an economic load on the society. Therefore, its utilization in
the generation of an energy carrier will greatly add to econo-
mize the process. From this we can conclude, that the
production of biohydrogen had showed great promise in
converting waste like mango juice effluent.
Conflicts of interest
All authors have none to declare.
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