The effects of light intensity and wavelength on hydrogen evolution by Rhodopseudomonas rutila, a nonsulfur purple photosynthetic bacteria newly isolated from ...
Agric.
Biol.
Chem.,
49 (1),
35-38,
1985
35
Wavelength Dependence of Photoproduction of Hydrogen
by Rhodopseudomonasrutila Yuichi Nogi, Teruhiko
Akiba* and Koki Horikoshi*
Department of Agricultural Chemistry, College of Agriculture Setagaya-ku, and Veterinary Medicine, Nihon University, Tokyo 154, Japan * The Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan Received May 9, 1984
The effects of light intensity and wavelength on hydrogen evolution by Rhodopseudomonas rutila, a nonsulfur purple photosynthetic bacteria newly isolated from paddy soil, were investigated. The maximal specific rate of hydrogen evolution was achieved at a light intensity of about 12 klux with an incandescent light. As to wavelength dependence, four major peaks of hydrogen evolution were observed at 900, 860, 810 and 590nm. Light of longer wavelengths above 590nm that is absorbed by bacteriochlorophyll a was more effective for hydrogen evolution than light of shorter wavelengths below 540 nm that is absorbed by carotenoids.
Hydrogen production
by photosynthetic
light intensity on the growth rate of photo-
bacteria has received muchattention because synthetic hydrogen promising
gas is considered fuels obtainable
as one of the from renewable
bacteria was earlier reported by several authors.10~13) The effect of light intensity on hydrogen evolution has also been re-
resources. The photoproduction of hydrogen in photosynthetic bacteria is catalyzed by nitrogenase or hydrogenase. The hydrogen pro-
ported for Rhodospirillum rubrum,14) Rhodopseudomonas capsulatal l fl 5 ) and Rhodopseudomonas sphaeroides.13) It has been
sphaeroides^ R. palustris^ and Rhodospirillum rubrurn5'6) is known to be mediated by the
evolution of these photosynthetic bacteria increase proportionally with an increase in light intensity until they are saturated at 6.5 to 12.0
duction of Rhodopseudomonas capsulata,1
>2) R.
function of nitrogenase. The relationship between nitrogenase and H2photoproduction in photosynthetic bacteria has been well established
through
the many studies
so far re-
demonstrated that both growth and hydrogen klux. Recently, the response to sunlight of hydrogen evolution by Rhodopseudomonas
sphaeroides has been demonstrated by Kim et
ported, some of which were reviewed earlier by al.16) Photosynthetic bacteria contain a variety several authors.7 ~9) The hydrogen production of pigments and bacteriochlorophylls in their catalyzed by nitrogenase proceeds during photoheterotrophic growth under anaerobic conditions in the light when reducing power
cells as intermediates for transferring light energy. Some authors have suggested that the efficiency of light absorption varies with the
and ATPare available. Both reducing power wavelength.17>18) Few reports have appeared to and ATPare generated on cyclic photophos- date, however, concerning the effect of the phorylation with light as a primary source of wavelength of light on hydrogen evolution. energy and substrates as sources of electrons. Thus illumination is one of the major factors
affecting both growth and hydrogen produc-
tion of photosynthetic bacteria. The effect of
In the previous paper,19) we described the isolation and characterization of a new nonsulfur purple photosynthetic bacterium, Rhodopseudomonas rutila. Photosynthetically
36
Y. Nogi, T. Akiba and K. Horikoshi
grown cells of this strain showed nitrogenase
activity and produced hydrogen under anaerobic conditions
in the light.
The main objec-
lengths. Rubber-stoppered 20-ml test tubes (1.7 x 13cm) containing 10ml of cell suspension (=ca. 5mg dry cells) were incubated anaerobically at 30°C for 4hr under illumination with monochromatic light of various
tive of the present study was to investigate the wavelengths (450 to 950nm) that was obtained with the effect of the wavelength of light on hydrogen light from a 1.5kW xenon lamp by passing it through evolution (action spectrum of hydrogen evolu- a diffraction grating and collimated with appropriate tion). The discussed.
effect
of light
intensity
is also
lenses. A slit (1.7x6cm) was placed just in front of each test tube to ensure that the light reached a definite area. The incident light intensity at the surface of test
tubes was measured with a bolometer equipped with a MATERIALS AND METHODS Organism and culture conditions. Rhodopseudomonas rutila (=JCM* 2524; ATCC 33872), a new strain isolated in our laboratory,19) was grown anaerobically under illumination at 30°C in a 1-liter flask containing 900ml of medium in a thermostatic chamber. The medium contained (g per liter) 4.0g sodium L-malate, 1.0g yeast
extract (Difco), 1.0g KH2PO4 and 0.2g MgSO4-7H2O (pH 7.0, with NaOH). The culture flask was illuminated from all sides with fluorescent lamps at an intensity of
thermopile (Ohkura Electric Co., AM-1001), and regulated at approximately 1,000juW/cm2 with a suitable filter over the range of wavelengths tested. Anaerobiosis and agitation in the test tubes were achieved by de-
gassing and flushing with argon and by stirring magnetically. The reaction was started by the addition of 1.0ml of 4.0% sodium L-malate and H2 evolved after 4hr incubation was determined by gas chromatography as
described below.
Assay of hydrogen gas. Quantitative measurements of H2 gas were performed by gas chromatography with a ditions, the organism produced molecular hydrogen from thermal conductivity detector and a column (4 m by 3 mm) L-malate through the nitrogenase activity. packed with molecular sieve 13X (60 to 80 mesh); the column was kept at 50°C. The carrier gas was N2at a flow Preparation of resting cell suspensions. Bacterial cells at rate of 40 ml/min. the late-log phase were harvested by centrifugation at 4°C, washed three times with 50mMsodium phosphate buffer Assay of L-malate. The concentrations of sodium l(pH 7.0), and resuspended in the same buffer. These cell malate were analyzed by high performance liquid chrosuspensions were used throughout the present study. matographywith a differential refractometer and a column (Shodex Ionpack KC-811, 30cm by 8mm) kept at Measurements of hydrogen evolution at different light 50°C. The eluent was 0.1% H3PO4 at a flow rate of intensities. Rubber-stoppered 30-ml flasks containing 10 ml 1.0 ml/min. of cell suspension {=ca. 19mg dry cells) were incubated approximately
anaerobically
7.5 klux. When grown under these con-
at 30°C for 6hr under illumination
at dif-
RESULTS AND DISCUSSION
ferent intensities. Anaerobiosis and agitation in the flasks were achieved by degassing and flushing with argon and Light dependence of hydrogen evolution by stirring magnetically. The reaction was started by the The rates of hydrogen evolution were obaddition of 1.0ml of 4.0% sodium L-malate as the substrate.
The flasks were illuminated from both sides with two 125-W metal halide lamps (Toshiba Sunlight DR125/T) which emitted a continuous spectrum of light similar to that of sunlight. The light intensity was controlled by means of the distance from the light source and measured with a photometric sensor (Li-Cor Inc., Model
served to becomeconstant after an 1 hr lag in the incubations at any light intensity used. The specific rate of hydrogen evolution and the yields of hydrogen on L-malate actually
consumed increased with the increase of intensity, both becoming maximum at 12 syringes from the flasks at one hour intervals and assayed as shown in Fig. 1. At 24 klux, the highest for H2 by gas chromatography as described below. Liquid intensity used, the hydrogen evolution samples (1.0ml) for assaying of L-malate were taken with conversely decreased. A similar effect LI-185B).
Gas samples
(0.5ml)
were taken
in gas-tight
light klux, light rate with
syringes from the flasks at the beginning and the end of incubations to. determine the amountof the substrate consumed during the incubations.
respect to light intensity was earlier demon-
Measurements of hydrogen evolution at various wave-
green photosynthetic
* Japan Collection of Microorganisms, The Institute
strated
in other photosynthetic
bacteria:
The
growth of Chloropseudomonas ethylicum, 10) a bacterium,
and Rhodo-
of Physical and Chemical Research, Wako, Saitama 351-01.
37
Wavelength Dependence of Hydrogen Production c
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Light
1
1
1
1
10
15
20
25
intensity
( klux)
Fig. 1. Effect of Incident Light Intensity on Hydrogen Evolution by Rhodopseudomonas rutila Cells. R. rutila cells were incubated on L-malate at 30°C for 6hr under illumination with two metal halide lamps at different light intensities. The amounts of H2 evolved and lmalate consumed were determined after 6-hr incubation. -%-, specific rate of hydrogen evolution; of hydrogen from L-malate.
500
1
-O-, yield
Table I. Effect of Incident Light Intensity on Yield of Hydrogen
The amounts of hydrogen evolved during 6 hr-incubation and light intensities in photon units of Einstein (E) were determined as described in the text. L ig h t in t e n s ity (k l u x )
Yi el d o f h y dr o ge n (m l- H 2 / E / m 2 )
3 4.5 6 12 24
0 .60 0 .55 0.69 0.43 0.21
600
700
Wavelength
800
900
1000
( nm)
Fig. 2. Effect of Wavelength on Hydrogen Evolution Rhodopseudomonsrutila Cells.
by
R. rutila cells were incubated at 30°C for 4hr under illumination with monochromatic light of different wavelengths.
Wavelength dependence of hydrogen evolution Figure 2 shows the wavelength dependence
of hydrogen evolution (action spectrum of hydrogen evolution). Four main peaks appeared for the hydrogen evolution at wavelengths of900, 860, 810 and 590nm, and three minor
peaks
at 540,
500
and
460nm.
The
accuracy of measurementswas confirmed by experiments repeated three times. As has been
reported in a previous article,19) the absorption spectrum of a cell extract of R. rutila showed absorption maxima at 860, 805 and 590nm due to bacteriochlorophyll a, and at 524, 490 and 465nm due to carotenoids. The results shown in Fig. 2 indicate that light of longer
microbium vannielii,n) a nonsulfur purple photosynthetic saturatedevolu-at 1,000 ft-c (=ca. bacterium, ll klux). was Hydrogen
wavelengths absorbed by bacteriochlorophyll a has a more conspicuous effect on hydrogen evolution than that of shorter wavelengths
tensity can be interpreted as'15*effects on the rate of photophosphorylation.1
around 500nm absorbed by carotenoids. The results presented in Fig. 2 support the idea that
tion and growth of Rhodopseudomonascapsulatal) sphaeroides3) rated at and 1,000 R. ft-c. These effectswereofalso light satuin-
The yields of hydrogen based on the incident light intensity were calculated as shown in a tungsten incandescent lamp, that emits light Table I. The incident light intensity in photometric units ofklux was converted to a photon of longer wavelengths above 800nm, may be unit using the factor for ofa juE/m2/sec, metal halide lampconversion (1 klux=14 /iE/m2/sec).20) The yield was not significantly influenced by an increase in light intensity
below the saturation point.
appropriately used as a favorable light source for hydrogen evolution of photosynthetic bacteria rather than a fluorescent lamp that emits
light showing a main emission spectrum near 600 nm.
In future hydrogen production with cultures of photosynthetic bacteria will be performed outdoors in this respect, culturing of selection of
a natural sunlit a problem in photosynthetic a light source
environment. In experiments on bacteria is the which can be ap-
38
Y. Nogi, T. Akiba and K. Horikoshi
propriately used for illumination. We chose a metal halide lamp that emits light similar to sunlight in experiments on light intensity dependence.
The peak at 900nm in Fig. 2 seemed to be
not due to bacteriochlorophyll a since the latter showed no absorption maximumin this region.19)
This
result
suggested,
presence of a light-harvesting suggested
the existence
at 904nm.
teriochlorophyll synthetic towards
center. Morita21)
of a photochemical
On the other
hand, bac-
type pigment B890 in photo-
bacteria has been reported to shift a longer wavelength on illumi-
nation.22'2^ But no further information is available to indicate which of these is the case
in our organism. The action spectrum of growth of Rhodopseudomonas sphaeroides showed the occurrence of considerable
growth near 900nm.24) It has also been noted that light of wavelengths shorter than 540nm has almost the same effect on growth as light of wavelengths longer than 600nm. This fact is in contrast with the wavelength dependence of hydrogen evolution where light of shorter
seen in this study wavelengths around
500nmwas observed to be comparatively less effective
than
light
Acknowledgments.
We thank
Professor
B. Maruo,
Nihon University, for the valuable advice and discussion. This study was supported by a Solar Energy Science Grant from The Institute of Physical and Chemical Research. REFERENCES
however, the
reaction center in Chromatiumthat responded to light
been constant in our experiments.
of longer
above 600nm. These differences
wavelengths
in wave-
length dependence between growth and hydrogen evolution are of interest from the
1) P. Hillmerand 2)
H. Gest, /. Bacterial,
A. Colbeau, B. C. Kelley, Bacteriol, 144, 141 (1980).
and
129, 732 (1977).
P. M. Vignais,
J.
3) B. A. Macler, R. A. Pelroy, and A. Bassham, J. Bacteriol, 138, 446 (1979). 4) J. S. Kim, K. Ito, and H. Takahashi, Agric. Biol Chem.,
44,
827 (1980).
5) R. P. Carithers, D. C. York, and D. I. Arnon, /. Bacteriol, 137, 779 (1979). 6) J. Miyake, N. Tomizuka, and A. Kamibayashi, /. Ferment. Technol, 60, 199 (1982). 7) H. Gest, Adv. Microbiol Physiol, 7, 243 (1972). .8) J. R. Benemann and R. C. Valentine, Adv. Microbiol Physiol,
8, 59 (1972).
9) D. C. York, "The Photosynthetic Bacteria," ed. by R. K. Clayton and W. R. Sistrom, Plenum Press, New York, N.Y., 1978, p. 657. 10)
S.
C.
Holt,
S.
F.
Conti,
Bacteriol, 91, 349 (1966). ll) W. C. Trentiniand M. P. Starr, (1967).
and
R.
C.
J. Bacteriol,
Fuller,
J.
93, 1699
12) O. Hirayama, E. Ando, K. Wamori, and N. Hara, Nippon Nogeikagaku Kaishi, 48, 97 (1974). 13) H. Sawada and P. L. Rogers, J. Ferment. Technol, 55,
297
(1973).
14) H. T. Stiffler and H. Gest, Science, 120, 1024 (1954). 15) P. Hillmer and H. Gest, /. Bacteriol, 129, 724 (1977). 16) J. S. Kim, K. Ito, and H. Takahashi, Agric. Biol. Chem.,
46,
937 (1982).
point of view of the efficiency in the electron
17) C. A. Wraight and R. K. Clayton, Biochim. Biophys.
One of the technical difficulties in measurement of wavelength dependence is the shadowing of cells. The degree of shadowing depends on
18) F. Gobel, "The Photosynthetic Bacteria," ed. by R. K, Clayton and W. R. Sistrom, Plenum Press, New
transfer
chain.
Acta,
York,
333,
N.Y.,
246 (1973).
1978,
p. 907.
19) T. Akiba, R. Usami, and K. Horikoshi,
Int. J. Syst.
Bacteriol, 33, 551 (1983). the number of cells. Because of this, a suspenL. Lange, P. Nobel, B. Osmond, and H. Ziegler, sion of resting cells instead of growing cells 20) O."Physiological Plant Ecology," Vol. 12A, was used in the present study and the cell Encyclopedia of Plant Physiology, Springer-Verlag, concentration of the suspension was supBerlin, 1981. pressed at a certain concentration (=ca. 21) S. Morita, Biochim. Biophys. Acta, 153, 241 (1968). 0.5mg/ml; see Materials and Methods) 22) W. J. Vrendenberg and J. Amesz, Biochim. Biophys. Acta, 126, 244 (1966). which at least allows enough hydrogen evolu23) D. C. Fork and J. Amesz, Annu. Rev. Plant Physiol, tion to assay by gas chromatography within 20, 305 (1969). the limited incubation time. Although the 24) K. J. Hellingwerf, W. D. Vrij, and W. N. Konings, /. Bacteriol, 151, 534 (1982). shadowing effects were not apparent at this cell density, the degree of shadowing might have
