होम Chinese Science Bulletin Hydrogen production bydraTGB hupLdouble mutant ofRhodospirillum rubrumunder different light...

Hydrogen production bydraTGB hupLdouble mutant ofRhodospirillum rubrumunder different light conditions

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10.1007/s11434-006-2171-4
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ARTICLES
Chinese Science Bulletin 2006 Vol. 51 No. 21 2611—2618

DOI: 10.1007/s11434-006-2171-4

Hydrogen production
by draTGB hupL double
mutant of Rhodospirillum
rubrum under different
light conditions
ZHU Ruiyan1, WANG Di1, ZHANG Yaoping2
& LI Jilun1
1. Department of Microbiology and Immunology, College of Biological
Science, China Agricultural University, Beijing 100094, China;
2. Department of Bacteriology, University of Wisconsin, Madison
WI53706, USA
Correspondence should be addressed to Li Jilun (email: lijilun@
cau.edu.cn)
Received April 24, 2006; accepted July 28, 2006

Abstract To increase H2 yield of Rhodospirillum
rubrum in two-stage hydrogen production process,
two deletion mutants were constructed. One is single
mutant designated R. rubrum UR801 that deleted
hupL gene encoding the large subunit of uptake hydrogenase, and the other is a double mutant designated R. rubrum UR805 lacked both draTGB encoding regulators for the activity of nitrogenase and hupL.
Comparing H2 yields of two mutants with R. rubrum
UR2 (wild type) and UR472 (ΔdraTGB) under different light conditions, the results showed that the H2
yield of R. rubrum UR801 under continuous light is
the highest (5700 mL of H2 per liter culture), and it is
1.56, 2.24 and 2.32-fold that of R. rubrum UR2,
UR472 and UR805, respectively. However, the total
H2 yield of R. rubrum UR805 in two-stage hydrogen
production process is the highest (4303 mL/L), and it
is 1.35, 1.21 and 1.04-fold that of R. rubrum UR2,
UR801 and UR472, respectively. Thus, R. rubrum
UR805 might be a valuable strain to produce a large
amount of hydrogen in two-stage hydrogen production process.
Keywords: Rhodospirillum rubrum, hydrogen photoproduction, twostage hydrogen production process, nitrogenase, uptake hydrogenase.

With increasing burning of fossil fuels and consequent changes in global climate, much attention has
been paid to the developments of clean and reproducible energy sources. Hydrogen is regarded as one of the
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cl; eanest energy vectors. It possesses high combustion
heat and the final product is water after combustion.
Hydrogen production, found in many bacteria and microalgae[1,2], is catalyzed by hydrogenase or nitro―
genase[3 6]. Hydrogen production by photosynthetic
bacteria was first observed in Rhodospirillum rubrum[7,8] which has ability to produce hydrogen anaerobically from various organic acids under continu―
ous light and in absence of ammonia[9 11]. Hydrogen
photoproduction in R. rubrum, mainly catalyzed by
nitrogenase[7,8,12], occurs when cells produce excessive
reducing potential, so nitrogenase activity and reducing
potential are crucial to a large amount of hydrogen
production in R. rubrum.
The nitrogenase complex is composed of two components: MoFe protein (dinitrogenase) and Fe protein
(dinitrogenase reductase). MoFe protein, an α2β2
tetramer encoded by nifD and nifK, contains the active
site (FeMo-cofactor) of nitrogenase; Fe protein, an γ2
dimer encoded by nifH, functions as the direct electron
donor to MoFe protein. Nitrogen fixation in R. rubrum
is regulated at the transcriptional level and at posttranslational level. The genes encoding nitrogenase are expressed with sodium glutamate as nitrogen source, but
they are repressed by other fixed nitrogen sources[8].
The activity of nitrogenase can be inhibited by glutamine[13], darkness[14], ammonia[15] or phenazine methosulfate (PMS)[14,16]. The regulation of nitrogenase activity at posttranslational level, termed as “switch-off/
switch-on”[17] to distinguish it from biosynthetic regulation of nitrogenase components, is more rapid than
that of nitrogenase synthesis.
Posttranslational regulation of nitrogenase activity
has been well-characterized in R. rubrum[18]. Two enzymes are involved in the regulation of nitrogenase
activity in R. rubrum: dinitrogenase reductase ADPribosyltransferase (DRAT, encoded by draT)[19] and
dinitrogenase reductase-activating glycohydrolase
(DRAG, encoded by draG)[20]. DRAT transfers ADPribose group from NAD to Arg101 on one subunit of Fe
protein to inactivate nitrogenase in response to darkness
or introduction of fixed nitrogen source due to disrupting electron flow from Fe protein to MoFe protein.
DRAG removes ADP-ribose group from Fe protein
restoring nitrogenase activity when R. rubrum is reilluminated or when the fixed nitrogen source is exhausted. The draT and draG genes in R. rubrum, cloned
and sequenced, formed one transcriptional unit, which
are adjacent to nifHDK but transcribed in the opposite
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direction[21]. R. rubrum draT mutant is incapable of
modifying Fe protein by ADP-ribose group even in the
dark[22].
Uptake hydrogenase, found in many nitrogen-fixation bacteria, catalyzes hydrogen to proton and electron;
its physiological function is to recycle energy to nitrogenase to reduce nitrogen to ammonia. Activity of uptake hydrogenase in R. rubrum is rhythmic, which is
related to energy compensation; rhythmic period of
uptake hydrogenase activity varies with cultivation
conditions[23]. Uptake hydrogenase, composed of a
small subunit (encoded by hupS) and a large subunit
(encoded by hupL), has been purified in R. rubrum[24].
R. rubrum lacking uptake hydrogenase activity isolated
by Tn5 mutagenesis increases nitrogenase-dependent
hydrogen photoproduction, but hydrogen yields are
related to Tn5 insertion regions[12]. Comparison of hydrogen production rates of Rhodobacter capsulatus
hup− strain with wild type B10S reveals that hup− strain
could increase hydrogen production rate 1.3-fold under
argon atmosphere[25].
Inactivation of nitrogenase activity in the dark is
limiting in the case of hydrogen production continuously under light/dark cycle in R. rubrum; therefore,
draTGB hupL double mutant of R. rubrum was constructed in this work and its hydrogen production was
investigated under different light conditions.
1

Materials and methods

1.1

Bacterial strains and growth conditions

Strains and plasmids used in this study are listed in
Table 1.
Table 1 Strains and plasmids
Strains and plasmids

Relevant characterization

Ref.

R. rubrum
UR2

wild-type, Smr

UR801

ΔhupL::aaaC1, Smr Gmr

UR472

ΔdraTGB::kan, Smr Kmr
[27]
ΔdraTGB::kan ΔhupL::aaaC1, Smr
this study
KmrGmr

UR805

[21]
this study

Plasmids
pSUP202
pGEM T-easy
pRYZ
pUCGm
pRYZ1

2612

suicide vector for R. rubrum,
Apr Tcr Cmr
Apr

lab collection
Promega

1.8 kb-BamH I and Sph I of △hupL
this study
was cloned into pSUP202, Apr Cmr
r
r
aaaC1 source, Ap Gm
lab collection
2.7-kb BamH I and Sph I
this study
of △hupL::aaaC1 was cloned
into pSUP202, Apr Cmr Gmr

R. rubrum UR2 as wild type is a spontaneous streptomycin-resistant mutant of R. rubrum ATCC11170[21].
R. rubrum was grown at 30℃ (100 r/min) in modified
SMN medium, in which 20 mmol/L potassium phosphate buffer (pH 6.8) replaced MOPS in SMN medium[26] to lower cost. Escherichia coli DH5α as host
strain for all plasmid-cloning experiments and E. coli
S17-1 as donor of mating were grown in LB medium at
37℃. R. rubrum for hydrogen production was cultivated in MG medium[26] containing 7 mmol/L sodium
glutamate and 20 mmol/L potassium phosphate buffer
(pH 6.8, autoclaved solely). The preculture was grown
in modified SMN but not older than 15 h. Antibiotics
were used at the following concentration (μg/mL): for
R. rubrum, streptomycin (Sm), 100; gentamicin (Gm),
10; kanamycin (Km), 12.5; nalidixic acid (Nx), 20;
chloramphenicol (Cm), 5; and for E. coli, ampicillin
(Ap), 100; chloramphenicol, 25; gentamicin, 5; kanamycin, 50; tetracycline (Tc), 12.5.
1.2 Construction of uptake hydrogenase-deficient
mutant
To delete hupL gene encoding large subunit of uptake hydrogenase, two pairs of primers were designed
to amplify 5′ flanking region and 3′ flanking region of
hupL, respectively (Fig. 1).
P1: (5′-CGGATCCGGTGTCACCGCCGCCGGGCTG-3′),
P2: (5′-CGAGCTCCCCGCCTACGATGA TGTCGGC-3′),
P3: (5′-CGAGCTCCGGCCAAAGGTGTGGGC-3′),
P4: (5′-GGCATGCGATCCTGGGTATTGGGAAAC-3′).

The underlined bases in primers indicated introduced
enzyme sites, in which BamH I and Sph I sites existed
on vector pSUP202 but Sac I site did not. Two fragments, 912-bp 5′ flanking region of hupL and 903-bp 3′
flanking region of hupL, were amplified by PCR using
genome DNA of R. rubrum UR2 as template and
cloned into pGEM-T easy vector to be sequenced, respectively; and then the two fragments were cloned into
pSUP202 at BamH I and Sph I sites, yielding vector
pRYZ. Finally, aaaC1 fragment (encoding Gmr) from
pUCGm was inserted into pRYZ at the Sac I site, generating suicide vector pRYZ1 to delete hupL. After being transformed into E. coli S17-1, pRYZ1 was conjugated into R. rubrum UR2 and UR472. NxrGmr colonies were isolated after 7―10 d and replica-printed to
identify Cms colonies on modified SMN containing
chloramphenicol. NxrGmrCms colonies resulting from
double-crossover recombination event were verified by
PCR (Cmr is encoded by pSUP202). The hupL and
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Vol. 51 No. 21 November 2006

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Fig. 1. Physical map of hup cluster in R. rubrum and designed primers to amplify 3′ flanking region and 5′ flanking region of hupL.

draTGB hupL mutants were designated R. rubrum
UR801 (ΔhupL::aaaC1) and R. rubrum UR805
(ΔdraTGB::kanΔhupL::aaaC1), respectively.
1.3 Nitrogenase activity and hydrogen content measurements
Nitrogenase activity was measured by the acetylene
reduction method[28]. R. rubrum was grown in 125 mL
glass bottle sealed with rubber stopper containing 120
mL modified MG medium at 30―33℃ illuminated by
twelve 60-W standard superlux lamps with light intensity approximately 2000―2500 lux. 1 mL sample was
withdrawn anaerobically from culture with airtight syringe and injected into 9 mL vial containing oxygenfree argon gas. The reaction was started by injecting
10% (v/v) acetylene. The assay was incubated at 30℃
for 5 min by shaking in the light (200 r/min) and then
was terminated by the addition of 0.2 mL of 30% trichloroacetic acid. Nitrogenase activity in the dark was
measured by the same method except changing the
light into in the dark. Ethylene formation was measured
by gas chromatography equipped with flame ionization
detector: column, GDX-502; column length, 2 m; column temperature, 70℃; detector, 120℃; carrier gas, N2;
samples, 100 μL. When measuring hydrogen content,
100 μL gas samples were withdrawn directly from the
same cultivation and assayed by gas chromatography
equipped with a thermal conductivity detector: column,
molecular sieve 5 Å; column length, 2 m; column temperature, 60℃; carrier gas, Ar gas.
1.4

Hydrogen production experiments

Hydrogen production was performed in one liter
fermentor illuminated by two 60-W standard superlux
lamps. The light intensity on the surface of the fermentor was approximately 40000―60000 lux. 20 mL inocula were added into fermentor containing 980 mL
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modified MG medium (10 mL potassium phosphate
buffer was autoclaved solely and then added into the
fermentor). After inoculation, oxygen-free Ar gas (0.3
L/min) was flowed into fermentor for 10 min to obtain
anaerobic condition. The temperature and agitation
speeds were 30℃ and 250 r/min, respectively. The pH
was not adjusted during cultivation.
Water displacement was used for hydrogen collection. A 600 mL bottle filled with a CO2 trap (1% NaOH,
2% NaCl) was equipped on the gas exit of fermentor.
2
2.1

Results
Verification of hupL deletion mutant

The NxrGmrCms colonies were identified by colony
PCR. Two pairs of primers: PA (5′-CCAATGCCGACATCGTAG-3′), PB (5′-TCGGTAAAGCCCACACC-3′)
and PC (5′-CTCTCTATACAAAGTTGGGC-3′), PD (5′ACAGGTGGCGTGCATCTC-3′) were designed to
verify the double-crossover mutants (Fig. 2(a)).
PA and PB existed on 3′ end of 5′ flanking region and
on 5′ end of 3′ flanking region of hupL, respectively.
Because wild type possesses of intact hupL gene,
1.7-kb fragment should be amplified, whereas 970-bp
fragment should be amplified in hupL mutant due to
replacement of hupL by Gm fragment. PC existed on 3′
end of Gm fragment, and PD was located in chromosome (downstream of 3′ flanking region of hupL). No
band should be amplified in wild type because of no
Gm fragment on its chromosome, whereas 1.3-kb
fragment should be amplified in hupL deletion mutant.
According to the results of Fig. 2(b), the NxrGmrCms
colony was the right hupL deletion mutant.
R. rubrum draTGB hupL double mutant was derived
from R. rubrum UR472. The verification of draTGB
hupL mutant was the same as that of R. rubrum UR801,
so the data were not shown.
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ARTICLES
brum UR472 and UR805 were 347 and 325 nmol
C2H4·mL−1·h−1·A600−1 in the light, and 31 and 32 nmol
C2H4·mL−1·h−1·A600−1 in the dark (8.9% and 9.8% of
that in the light), respectively. These results showed
that draTGB deletion leads to decreased nitrogenase
activity.

Fig. 2. Primers to verify hupL deletion mutant (a) and verification of R.
rubrum UR801 (b). Lanes 1 and 3, Products of PCPD and PAPB using
genome DNA of R. rubrum UR2 as template, respectively; lanes 2 and 4,
products of PCPD and PAPB using genome DNA of R. rubrum UR801 as
template, respectively.

2.2 Nitrogenase activities of R. rubrum UR2 and its
derivatives
Nitrogenase activities of R. rubrum UR2 and its derivatives were measured both in the light and in the
dark when cultures were grown to A600 of 2.5―3.0
(Table 2). Nitrogenase activities of R. rubrum UR2 and
UR801 were 749 and 790 nmol C2H4·mL−1·h−1·A600−1 in
the light, and 31 and 42 nmol C2H4·mL−1·h−1·A600−1 in
the dark (4.1% and 5.3% of that in the light), respectively. These results demonstrated that hupL deletion
has no significant effects on acetylene reduction catalyzed by nitrogenase. Nitrogenase activities of R. ru-

Table 2 Comparison of nitrogenase activities of R. rubrum UR2 with
its derivatives in the light and in the darka)
Nitrogenase activityb)
Strains
Genotype
ND/NLc)
in the light in the dark
UR2
WT
749
31
4.1%
UR801
ΔhupL
790
42
5.3%
UR472
ΔdraTGB
347
31
8.9%
UR805 ΔdraTGBΔhupL
325
32
9.8%
a) Each activity is from at least triplicate assays from different individually grown cultures.b) Specific nitrogenase activity unit: nmol
C2H4·mL−1·h−1·A600−1; c) ND/NL indicates the ratio of nitrogenase of each
strain in the dark to that in the light.

2.3 Hydrogen content in biogas produced by R. rubrum
Hydrogen content in biogas produced by R. rubrum
cultivated in modified MG medium under continuous
light was assayed. Although hydrogen production by R.
rubrum UR2 and its derivatives did not start at the
same time, hydrogen had been produced in four R. rubrum strains at 30th h. Gas samples were withdrawn
from glass bottle and analyzed during the period of
30―120 h of cultivation. The results showed that hydrogen content of R. rubrum UR2 and its derivatives
exceeded 85% from 30 to 80 h, indicating that hydrogen produced by R. rubrum in modified MG medium
was pure (Fig. 3). Hydrogen content of hup+ strains (R.
rubrum UR2 and UR472) decreased significantly to

Fig. 3. Hydrogen content in biogas produced by R. rubrum UR2, UR472 (a) and UR801, UR805 (b) under continuous light. At the time points indicated, gas samples were taken from three different individually grown cultures and measured. The data were mean of triplicate measurements.

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70%―80% at 120th h (Fig. 3(a)), whereas hup− strains
(R. rubrum UR801 and UR805) did not decline obviously at the same time (>90%) (Fig. 3(b)). These results indicated that hydrogen produced by R. rubrum
hup– strains was purer than hup+ strains. Therefore, it
was beneficial for hydrogen purification using R. rubrum hup– strains for hydrogen production.
2.4 Comparison of hydrogen production of R. rubrum
UR2 with its derivatives under continuous light
In order to compare hydrogen yields of four strains
under continuous light with those in two-stage hydrogen production process, hydrogen production of R. rubrum under this condition was investigated first. Hydrogen photoproduction of R. rubrum UR2 and its derivatives are shown in Fig. 4.
Hydrogen production rate in R. rubrum UR2 appeared about 18―20 h after inoculation, attained highest (59 mL·L−1·h−1) at 45―50 h, and then declined
markedly when the cultivation time was over 85 h and
disappeared after 100 h. Hydrogen production by R.
rubrum UR801, lasting a longer time than by R. rubrum
UR2, began at 18―20 h; hydrogen production rates
attained the highest of 70―80 mL·L−1·h−1 and de-

creased obviously in cultures older than 150 h and disappeared after 160 h (Fig. 4(a)).
Hydrogen production of R. rubrum UR805 under
continuous light, similar to R. rubrum UR472, started
about 30―36 h which was 12―14 h later than R. rubrum UR2; hydrogen production rates attained the
highest (47―54 mL·L−1·h−1) at about 60 h and decreased obviously after 90th h (Fig. 4(b)).
Besides hydrogen, there was CO2 (data not shown)
in the biogas produced by R. rubrum, so solution containing 1% NaOH and 2% NaCl was used for hydrogen
collection to remove CO2. As shown in Table 3, hydrogen yields of R. rubrum UR2 and UR801 were 3647
and 5700 mL/L under continuous light, respectively.
Productivities were 45 mL·L−1·h−1 for R. rubrum UR2
and 47 mL·L−1·h−1 for R. rubrum UR801. Under continuous light, hydrogen yield of R. rubrum UR801 was
1.56-fold that of R. rubrum UR2.
Total hydrogen yield of R. rubrum hup mutant was
7.3 L/L over a period of 16 d[12] (productivity was approximately 20 mL·L−1·h−1); However, total hydrogen
yield of hupL deletion mutant R. rubrum UR801 in this
study was 5700 mL/L within 6.5 d. Productivity of R.
rubrum UR801 was 47 mL·L−1·h−1, which was 2.35-

Fig. 4. Hydrogen production rates of R. rubrum UR2, UR801 (a) and UR472, UR805 (b) under continuous light.
Table 3 Comparison of hydrogen yields of R. rubrum UR2 with its derivatives under different light conditionsa)
Strains

H2 yield (mL/L)b)

H2 yields after 72 h

Productivity (mL/L/h)
Two-stage

Yx/Ync)

L

Two-stage

L/D

L

UR2

3647 (80 h)

3193 (101 h)

480

45

31

1.35

UR801

5700 (121 h)

3558 (141 h)

1158

47

25

1.21

UR472

2540 (60 h)

4120 (114 h)

2210

42

36

1.04

UR805

2455 (75 h)

4303 (121 h)

2513

33

36

1.00

a) L, Under continuous light; L/D, under light/dark cycle condition; Two-stage, R. rubrum was cultivated for 72 h under continuous light and then
transferred to light/dark cycle condition. b) Each yield is from at least replicated assays from different individually fermentation, Times in brackets
indicates that hydrogen production lasts; c) Yx/Yn indicates ratio of hydrogen yield of UR805 to different strains in two-stage hydrogen production
process.

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ARTICLES
fold that of R. rubrum hup mutant strain constructed by
Kern[12].
2540 mL/L hydrogen were produced by R. rubrum
UR472 and 2455 mL/L by UR805 under continuous
light, and productivity was 42 mL·L−1·h−1 in the case of
R. rubrum UR472 and 33 mL·L−1·h−1 in the case of R.
rubrum UR805 (Table 3). Hydrogen yields of R. rubrum UR472 and UR805 under continuous light decreased respectively to 69.6% and 67.3% of that of R.
rubrum UR2, resulting from lower nitrogenase activities in draTGB deletion mutants than that in wild type
R. rubrum strain (Table 2).
In a word, H2 yield of R. rubrum UR801 under continuous light is the highest, and it is 1.56, 2.24 and
2.32-fold that of R. rubrum UR2, UR472 and UR805,
respectively.
2.5 Comparison of hydrogen production of R. rubrum
UR2 with its derivatives in two-stage hydrogen production process
To improve hydrogen yield of R. rubrum under
light/dark cycle, draTGB hupL double mutant was constructed and its hydrogen production was investigated.
Because R. rubrum grew not very well in modified MG
medium under light/dark cycle condition from the beginning of cultivation, and hydrogen yields of R. rubrum were low under this condition (data not shown), R.
rubrum was cultivated under continuous light for 72 h
and then shifted to light/dark cycle condition (this
process was termed as “two-stage hydrogen production” process). Hydrogen production of R. rubrum UR2
and its derivatives in this two-stage hydrogen production process were shown in Fig. 5. Within 72 h, hydrogen production of R. rubrum UR2 and its derivatives

were similar to those under continuous light. When
cultures were shifted to darkness, hydrogen production
rates of draT+ strains (R. rubrum UR2 and UR801) or
draT– strains (R. rubrum UR472 and UR805) decreased
significantly. However, R. rubrum UR2 and UR801
recovered hydrogen production rates to relatively low
level of 38―44 mL·L−1·h−1 when the cultures were
reilluminated (Fig. 5(a)), whereas R. rubrum UR472
and UR805 rapidly restored to high level of 51―58
mL·L−1·h−1 (Fig. 5(b)).
As shown in Table 3, total hydrogen yields of R. rubrum UR2 and UR801 in two-stage process were 3193
and 3558 mL/L, respectively. They were 87.5% and
62.4% of those under continuous light, respectively.
Under this condition, productivities were 31 mL·L−1·h−1
for R. rubrum UR2 and 25 mL·L−1·h−1 for UR801. Total
hydrogen yields of R. rubrum UR472 and UR805 were
4120 and 4303 mL/L in this two-stage process, respectively. They were 1.62 and 1.75-fold that of under continuous light, respectively, and the productivities of R.
rubrum UR472 and UR805 were 36 mL·L−1·h−1. Hydrogen yield of R. rubrum UR805 was the highest
among the four strains in two-stage hydrogen production process, which was 1.35-fold higher than that of R.
rubrum wild type under the same condition.
In comparison of hydrogen yields of R. rubrum UR2
with its derivatives under light/dark cycle condition (12
h/12 h) in two-stage hydrogen production process (Table 3), the results showed that hydrogen yield of UR2
under this condition was 480 mL/L; R. rubrum UR805
exhibited the highest hydrogen yield of 2513 mL/L,
which was 5.2-fold that of R. rubrum UR2 under
light/dark cycle condition. Thus, R. rubrum UR805

Fig. 5. Hydrogen production rates of R. rubrum UR2, UR801 (a) and UR472, UR805 (b) in two-stage hydrogen production process. v indicates that R.
rubrum was cultivated in the dark for 12 h.

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Vol. 51 No. 21 November 2006

ARTICLES
would be a valuable strain for hydrogen production
under light/dark cycle.
3

Discussion

To improve hydrogen yield in two-stage hydrogen
production process, R. rubrum draTGB hupL double
mutant UR805 was constructed. Nitrogenase activities
of R. rubrum UR805 and UR472 were measured both
in the light and in the dark. Nitrogenase activity of R.
rubrum UR472 or UR805 in the dark decreases to approximately 10% of that in the light, and it is not consistent with the result of draT mutant R. rubrum UR213
(draT::kan) constructed by Liang et al.[22] presumably
due to different measurements (Nitrogenase activities
of R. rubrum UR472 and UR805 used in this study
were measured in the dark but that of UR213 was
measured in the light after cultivation in the dark for 40
min). Although nitrogenase of draT mutant could not
be modified, its activity and hydrogen production rates
are low in the dark probably as a result of low energy
level.
Uptake hydrogenase activity in R. rubrum wild type
is rhythmic, which is related to energy compensation[23].
Resulting from deletion of uptake hydrogenase, R. rubrum UR801 lacks energy compensation; therefore, its
hydrogen production is more easily affected by energy
level than R. rubrum UR2. Although hydrogen production of R. rubrum is illuminated by two 60-W standard
superlux lamps, light intensity of sunlight affects energy level in vivo to lead to unstable hydrogen production rates of R. rubrum UR801.
Under continuous light, hydrogen yield of R. rubrum
UR801 is 1.56-fold that of R. rubrum UR2. This means
that hupL deletion could increase hydrogen yield of R.
rubrum significantly. Hydrogen yield of R. rubrum
UR805 is the highest in two-stage hydrogen production
process, but it is interesting that there is no significant
difference in hydrogen yields between R. rubrum
UR472 and UR805 either under continuous light or in
two-stage hydrogen production process. Why does
hupL deletion have obvious effects on hydrogen yields
in draT+ strains but not in draT - strains and why are
hydrogen yields of draT– strains in two-stage hydrogen
production process higher than those under continuous
light? The mechanism needs to be clarified further.
With 30 mmol/L malate as carbon source, the theoretical hydrogen yield of photosynthetic bacteria is 4
L[29], but hydrogen yields of both R. rubrum UR801
under continuous light and UR472 as well as UR805 in
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two-stage hydrogen production process exceed the
theoretical maximum yield based on malate consumption. These results are similar to the data described by
Kern that hydrogen production by R. rubrum hup mutant continues at low rates after lactate was exhausted[12]. Two possible explanations for this observation are: (1) Sodium glutamate could be converted into
hydrogen besides as nitrogen source[12]; (2) CO2 Fixation to carbon source supplies energy and protons for
hydrogen formation.
Hydrogen yield of R. rubrum UR805 is 4303 mL/L
in two-stage hydrogen production process within 6.5 d
cultivation. The productivity of R. rubrum UR805 (36
mL·L−1·h−1) increases by 44% compared to that of
UR801 (25 mL·L−1·h−1) in two-stage process and by
80% to that of R. rubrum hup mutant (20 mL·L−1·h−1)
constructed by Kern et al.[12] under continuous light.
Thus, draTGB hupL double mutant R. rubrum UR805
might be valuable for hydrogen production in two-stage
hydrogen production process.
Acknowledgements This work was supported by the National High Technology Research and Development Program
of China (Grant No. 2003AA214010).

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Chinese Science Bulletin

Vol. 51 No. 21 November 2006