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i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 2 1 0e2 1 2

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Original Article

Bio-hydrogen production by Pseudomonas stutzeriKF532951

Sigamany Soniagandhi, Marimuthu Krishnaveni*

Department of Biochemistry, Periyar University, Salem 636011, India

a r t i c l e i n f o

Article history:

Received 26 July 2013

Accepted 21 August 2013

Available online 17 September 2013

Keywords:

Alternate fuel

Biomass

Gas chromatogram

Pseudomonas

Soil

* Corresponding author. Tel.: þ91 9894829823E-mail address: logasarvesh@gmail.com (

0976-1209/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijcas.2013.08.004

a b s t r a c t

Aim: Hydrogen production by biological means is more advantageous as it utilizes

renewable sources and produces very low or no carbondioxide. Since, lignocellulosic

feedstocks are cheaper source, the present study was aimed to isolate Pseudomonas stutzeri

from soil sample at country bricks chamber furnace.

Methods: Final confirmation of the strain was done by 16S rRNA sequencing. The GC con-

tent was calculated. The isolated strain was inoculated in hydrogen production medium

containing sugarcane bagasse replacing glucose. After the incubation period of 16 days, the

gas collected was studied for hydrogen production through gas chromatography technique.

Results: According to sequencing and phylogenetic tree results, our isolate was found to be

P. stutzeri KF532951. The GC content of our strain was above 53%. Our strain could produce

up to 3.4 mol H2/mol substrate by water displacement method.

Conclusion: On the basis of the above results, it is concluded that our strain is able to pro-

duce hydrogen biologically.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction the use of biomass is scanty, it is very much essential to study

Pseudomonas stutzeri, similar to most accepted Pseudomonas

spp., can grow up in minimal, chemically defined media, the

optimum temperature for growth is approximately 35 �C and

requires no additional growth factors. None of the strains bear

acidic environment, they do not grow at pH 4.5. Lipopolysac-

charide is a key antigenic molecule on the cell surface, a vital

heat-stable O-antigen of the genus. A variety of bacterial

strains are able to produce hydrogen using carbohydrate as a

substrate.1 Hydrogen is also used as a reductant in numerous

industrial processes.2 Presently, 40% H2 is synthesised from

natural gas, 30% through heavy oils, naphtha, 18% from coal,

4% by electrolysis, about 1% is from biomass.3 Even though,

(mobile).M. Krishnaveni).2013, JPR Solutions; Publi

the outcome of research. Hence, the present study was per-

formed to know the production of hydrogen using P. stutzeri

isolated at country bricks chamber furnace located opposite to

Periyar University, Salem.

2. Materials and methods

2.1. Isolation and identification of Pseudomonas sp

The soil sample was collected near country bricks chamber

furnace located opposite to Periyar University campus, Salem.

One gram of soil was serially diluted, concentration in the

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Fig. 1 e 16S rDNA sequence based phylogenetic distance analysis dendrogram presenting the position of identified strain

and related species.

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 2 1 0e2 1 2 211

range of 10�6 was streaked in to nutrient agar plates and

incubated for 24 h at 37 �C and observed for yellow colonies,

indicating the presence of Pseudomonas sp. The bacterial iso-

lates were further sub-cultured to obtain pure culture. The

obtained pure culture was analysed biochemically and further

sequenced. The 16S rRNA sequence obtained was amplified

using universal primer. The nucleotide sequence of the isolate

was compared with other related sequences. The obtained

sequence was submitted to gene bank for accession number.

2.2. Hydrogen production using biomass

The identified strain P. stutzeri was initially inoculated in to

peptone broth containing peptone 0.5 g and yeast extract

0.75 g and incubated for 24 h. 100 ml of peptone broth culture

was used to inoculate MYG medium containing malt, yeast

extract 0.5 g each, glucose 1.0 g/100 ml. 500 ml of MYG culture

was then inoculated in to 250ml productionmedia containing

beef extract 0.5 g, yeast extract 1.25 g, peptone 1.25 g, sodium

chloride 5.12 g, sugarcane bagasse 1.5 g. Mechanical heat

treatmentwas done to remove the lignin present in sugarcane

bagasse. The inoculated culture was allowed to grow for a

period of 16 days in a setup which can collect gas in to the

empty space, thereby pushing the water up. The produced gas

was sent for gas chromatography analysis.

Fig. 2 e Hydrogen production by Pseudom

3. Results and discussion

3.1. 16srRNA gene sequencing

16s rRNA gene sequencing confirms the strain as P. stutzeri

having accession number KF532951. The identified strain was

named as MKVSSG 2013. Palleroni et al4 classified P. stutzeri

into two types: one clustered around 62% G þ C that does not

tolerate a temperature of 43 �C, and another 65e66%GþC that

grows at 43 �C or higher. But our strain was showing only

53.3% Gþ C content. After initial analysis at NCBI, the relevant

sequences were downloaded and phylogenetic analysis was

performed (Fig. 1). It supports the alignment of the 16S rRNA

gene sequence with accession no. NR_103934.1 present in the

public domain. Similar study was performed with P. stutzeri

JX442762.5

3.2. Hydrogen production

The hydrogen gas produced by water displacement method6

was analysed with gas chromatography thermal conductiv-

ity detector. Fig. 2 depicts the result of gas chromatography

analysis. These studies were performed under aseptic condi-

tions and showed H2 recoveries between 2.56 and 3.4 mol

onas stutzeri e gas chromatograph.

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 2 1 0e2 1 2212

H2/mol substrate. The main disadvantage of hydrogen pro-

duction bymeans of biological way is lower yield of hydrogen,

maximally 4 mol H2/mol glucose when compared with other

processes.7

4. Conclusion

The isolated strain P. stutzeri was able to degrade plant

biomass and produce hydrogen from 2.56 to 3.4 mol H2/mol

substrate.

Conflicts of interest

All authors have none to declare.

Acknowledgement

The author thank Honourable Vice Chancellor, Dr. K. Muthu-

chelian Avl, Registrar Dr. K. Angamuthu Avl, Periyar

University for their administrative support and also the

author thank Managing Director, Mr. D. Jagadeesh Kumar,

Chrompark Research Centre, Namakkal for providing lab

facilities to carry out the research and Managing Director,

Dr. Sankarapandian Selvaraj, Helini Biomolecules, Chennai

for helping us in doing bioinformatics work.

r e f e r e n c e s

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3. Suzuki Y. On hydrogen as fuel gas. Int J Hydrogen Energy.1982;7:227e230.

4. Palleroni NJ, Doudoroff M, Stanier RY, Solanes RE, Mandel M.Taxonomy of the aerobic pseudomonads: the properties of thePseudomonas stutzeri group. J Gen Microbiol. 1970;60:215e231.

5. Penislus Shiyan S, Krishnaveni M. Hydrogen production byPseudomonas stutzeri JX442762 isolated from thermal soil atMettur power station, Salem District, Tamil Nadu, India. JPharm Res. 2013;6:112e116.

6. Kosourov S. Sustained hydrogen production by Chlamydomonasreinhardtii: effects of culture parameters. Biotechnol Bioeng.2002;78:731e740.

7. Kumar N, Das D. Continuous hydrogen production byimmobilized Enterobacter cloacae IIT-BT 08 using lignocellulosicmaterials as solid matrices. Enzyme Microb Technol. 2001;29:280e287.