Download GENERATION OF HYDROGEN BY Rhodobacter sphaeroides

GENERATION OF HYDROGEN BY Rhodobacter sphaeroides –
EFFECT OF WAVELENGTH AND LIGHT INTENSITY
K. Górecki, M. Waligórska, K. Seifert, M. Łaniecki
Department of Chemistry, A. Mickiewicz University, Poznań, Poland
Introduction
Purple nonsulphur bacteria Rhodobacter sphaeroides are capable of photoheterotrophic growth when they
are supplied with energy by light, with organic compounds being used as carbon and nitrogen sources. The
energy in the form of quanta of light absorbed by photosynthetic complexes is converted into proton
gradient used in a production of adenosinotriphosphate (ATP), a cell’s ‘energy currency’. The ATP
is a direct substrate for nitrogenase complex, which fixes dinitrogen. When no dinitrogen is present in the
environment, only dihydrogen is produced. Photoheterotrophic hydrogen production, known also under
the name of photofermentation, is directly regulated by light intensity. At low light wavelenghts, the more
intense light is available for bacteria, the more hydrogen is produced.
This paper focuses on qualitative and quantitative light influence on hydrogen production. Two filters
were used: RG780, which is opaque for light of wavelength lower than 700 nm; and BG7, which generates
a band of 350 – 600 nm.
Figure 1. Scheme of hydrogen generation in photofermentation
process.
Experimental procedures
Figure 2. Comparison of Rhodobacter sphaeroides absorption
spectrum (solid line) and transmittance spectra of two filters used
(dashed for RG780, dotted for BG7). Absorption maxima are indicated
with regard to photosynthetic dyes (Bchl – bacteriochlorophyll, Crt –
carothenoids). AppA absorption maximum is also shown.
Results
Light intensity experiment
To estimate the effect of light intensity on hydrogen
production under RG780 filter, samples were cultivated
under different light intensity.
Inoculum
 Rhodobacter sphaeroides O.U. 001 (ATCC
4919) bacteria were cultivated on Van Niel’s
medium.
Medium and procedures
 All experiments were done on modified Bielb
and Pfenning medium. The pH value of the
medium was brought to 6.8 before inoculation.
Experiments were performed in glass reactors
(25 cm3) with working capacity of 12.5 cm3.
Medium was inoculated with 30% v/v bacteria
(equivalent in dry weight = 0.365 g) and
cultivated for 12 hours. All samples were
deaerated with argon before starting the
illumination.
Light source
 300 W Ultra-Vitalux lamp by Osram (Munich,
Germany). For filter experiments, an opaque box
had been build with desired filters built in at light
pathway. The RG780 and BG7 filters were
obtained from Schott (3 – 4 mm thick) and were
positioned perpendicularly to light pathway.
Analytical methods
 The amount of produced hydrogen was estimated
by gas chromatography (GC-3800 from Varian,
TCD detector, Carboplot P7 capillary column).
 Nitrogenase activity was estimated by acetylene
reduction assay.
 Protein concentration was estimated by Lowry’s
protein assay.
RG780 filter in 50 W/m2
Two simultaneous cultures of R. sphaeroides
were inoculated with the same amount of
bacteria and placed under light of 50 W/m2
intensity.
BG7 filter in 30 W/m2
BG7 filter by Schott overlaps the Soret band of
bacteriochlorophyll of 375 nm and the AppA
absorption maximum of 420 nm. Due to filter
high absorption all experiments were performed
in light with intensity of 30 W/m2 only.
Figure 3. Total amount of produced hydrogen and light conversion
efficiency (■ — hydrogen produced by RG780 cultures;  —light
conversion efficiency of RG780 cultures;  — hydrogen produced by
control cultures;  — light conversion efficiency of control cultures).
Conclusions
 At low light intensities blue light decreases hydrogen
production significantly and biomass growth moderately.
 Studies performed at infrared region (800 – 1000 nm)
indicated much better hydrogen production by
Rhodobacter sphaeroides whereas biomass growth was
only delayed.
 An increase of light intensity within the infrared region
resulted in significant increase of photobiologically
generated hydrogen.
 Light conversion efficiency was decreased by RG780 filter
at low light intensities but increased at high light
intensities.
 An
application
of
RG780 filters in outdoor
photobioreactors operating under solar irradiation can
lead towards higher amounts of hydrogen produced and
as well as protection of bacteria from photoinhibition or
damage by strong ultraviolet radiation.
Acknowledgements This work was supported by Polish Ministry of Science and Higher
Education – project N N204 185440.
Figure 4. A: hydrogen production; B: protein
Figure 6. A: hydrogen production; B: protein
concentration ( •, ■ — RG780 cultures; ,  — control
cultures).
concentration ( •, ■ — BG7 cultures; ,  — control
cultures).
Figure 5. Nitrogenase specific activity ( — RG780
cultures,  — control cultures).
Figure 7. Nitrogenase specific activity ( —BG7 cultures,
 —control cultures).