Microbiological Research 197 (2017) 49–55

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Acclimation strategy of Rhodopseudomonas palustris to high light

irradiance
Dayana Muzziotti a , Alessandra Adessi a,b , Cecilia Faraloni c , Giuseppe Torzillo c ,
Roberto De Philippis a,b,∗
a
Department of Agrifood Production and Environmental Sciences, University of Florence, via Maragliano 77, 50144, Florence, Italy
b
Institute of Chemistry of Organometallic Compounds (ICCOM), CNR, Via Madonna del Piano, 10-50019 Sesto Fiorentino, Florence, Italy
c
Institute of Ecosystem Study (ISE), CNR, Via Madonna del Piano, 10-50019 Sesto Fiorentino, Florence, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The ability of Rhodopseudomonas palustris cells to rapidly acclimate to high light irradiance is an essential
Received 12 October 2016 issue when cells are grown under sunlight. The aim of this study was to investigate the photo-acclimation
Received in revised form 2 January 2017 process in Rhodopseudomonas palustris 42OL under different culturing conditions: (i) anaerobic (AnG), (ii)
Accepted 23 January 2017
aerobic (AG), and (iii) under H2 -producing (HP) conditions both at low (LL) and high light (HL) irradiances.
Available online 28 January 2017
The results obtained clearly showed that the photosynthetic unit was significantly affected by the light
irradiance at which Rp. palustris 42OL was grown. The synthesis of carotenoids was affected by both
Keywords:
illumination and culturing conditions. At LL, lycopene was the main carotenoid synthetized under all
Anaerobic growing conditions
Aerobic growing conditions
conditions tested, while at HL under HP conditions, it resulted the predominant carotenoid. Oppositely,
H2 -producing conditions under AnG and AG at HL, rhodovibrin was the major carotenoid detected. The increase in light intensity
Photo-acclimation produced a deeper variation in light-harvesting complexes (LHC) ratio. These findings are important for
Photo-protection understanding the ecological distribution of PNSB in natural environments, mostly characterized by high
light intensities, and for its growth outdoors.
© 2017 Elsevier GmbH. All rights reserved.

1. Introduction are responsible of transferring the electronic excitation to bacte-

riochlorophylls and then, charge separation occurs (Adessi and De
Photosynthetic bacteria contain pigments such as bacteri- Philippis, 2014; Feng et al., 2004). In purple bacteria, a total of five
ochlorophylls (BChls) and carotenoids mainly located in the (5) carotenoids have been found: lycopene, rhodopin, rhodovib-
light-harvesting complexes. In general, pigments not only absorb rin, anhydrorhodovibrin and spirilloxanthin (Feng et al., 2004).
light at different wavelengths but they also play important role in These pigments differ in the number of conjugated double C C
photoprotection of the photosynthetic apparatus (Feng et al., 2004; bonds present in their molecular structure. Lycopene and rhodopin
Kuo et al., 2012). In purple photosynthetic bacteria, carotenoids have eleven (11), rhodovibrin and anhydrorhodovibrin twelve (12)
trap light in the blue-green spectral region of visible light (Adessi and spirilloxanthin thirteen (13) double C C bonds (Feng et al.,
and De Philippis, 2014; Feng et al., 2004) while BChls have absorp- 2004; Li et al., 2014). Moreover, these carotenoids also differ in
tion peaks in the near UV region (390 nm) and in the visible region the presence of functional groups in their molecular structure (Chi
of the spectrum (Q band: Qx and Qy ). The absorption peaks of BChls et al., 2015; Georgakopoulou et al., 2006; Kushwaha et al., 2014;
in the near infrared region, i.e. 800, 850 and 880 nm, are due to the Mizoguchi et al., 2008). Carotenoids act as quenchers dissipating
Qy shift. However, the maximum absorption peak of this pigment is the excess of excitation energy via an efficient bacteriochlorophyll-
found at 875 nm. On the other hand, the Qx absorption band of BChls to- carotenoids energy transfer mechanism avoiding the formation
absorbs at 590 nm (Adessi and De Philippis, 2014). Carotenoids of Reactive Oxygen Species (ROS) or hunting the latter directly.
This last function of carotenoids is considered as a photo-protection
mechanism used by purple bacteria (Cogdell et al., 2000; Mizoguchi
et al., 2008).
∗ Corresponding author at: Department of Agrifood Production and Environmen-
Purple bacteria are capable of regulating their photosynthetic
tal Sciences, University of Florence, via Maragliano 77, 50144, Florence, Italy.
E-mail addresses: dayanaisabel.muzziottigil@unifi.it (D. Muzziotti),
unit according to environmental factors, for instance, light intensity
alessandra.adessi@unifi.it (A. Adessi), [email protected] (C. Faraloni), (Harada et al., 2008). The photosynthetic unit is composed by two
[email protected] (G. Torzillo), roberto.dephilippis@unifi.it (R. De Philippis). light-harvesting complexes (LHCs), named LH1 and LH2. Light har-

http://dx.doi.org/10.1016/j.micres.2017.01.007
0944-5013/© 2017 Elsevier GmbH. All rights reserved.
50 D. Muzziotti et al. / Microbiological Research 197 (2017) 49–55

vesting complex 1 (LH1) is located in between the reaction center of the devices. Under aerobic conditions, 0.25 L Erlenmeyer flasks
(RC) and light harvesting complex 2 (LH2), the latter being located stoppered with sterile cotton were used. Cultures were mixed using
at the periphery of the photosynthetic unit. LH1 and LH2 consist of a magnetic stirrer, and maintained at 30 ◦ C. All tests were run for
photosynthetic pigments, i.e. carotenoids and bacteriochlorophyll 48 h and the working volume of the cultures was 0.1 L except for H2 -
a, non-covalently bound to membrane proteins. producing conditions illuminated at 1500 ␮mol photons m−2 s−1 in
When purple bacteria are grown under low light intensities, the which, the working volume was 0.25 L. Under H2 -producing con-
number of LHCs increases in order to efficiently harvest the limited ditions, the production of H2 by Rp. palustris 42OL was used as an
number of available photons (Kuo et al., 2012; Li et al., 2014). On the indicator to start the sampling at 0 h, and the rubber-stoppered
opposite, when purple bacteria are grown at high light intensities vessels were connected with a tygon tube to a water-displacement
the level of LHCs declines to avoid photo-damage (Brotosudarmo gas collection system for measuring the volume of H2 produced. In
et al., 2015). In the process of light-harvesting, the peripheral LHCs, these vessels, prior to the start of the experiments the headspace
i.e. LH2 (Kotecha et al., 2013; Li et al., 2014), are the first structures to was flushed for 15 min with Ar in order to remove air. The light was
trap light (Olsen et al., 2008; Qian et al., 2008) and then, this energy supplied by a halogen lamp giving a light intensity of either 250 (LL)
is channeled to the LH1-RC core complex, where the primary charge or 1500 (HL) ␮mol photons m−2 s−1 . Henceforward, the low light
separation occurs (Adessi and De Philippis, 2014; Li et al., 2014; intensity and high light intensity will be referred to as LL and HL,
Šlouf et al., 2012). The synthesis of photosynthetic pigments and respectively. The time indicated as t-1 corresponded to the start-
RC/LH1 peptides is carried out by a large number of genes located ing point of the experiments at which cultures were illuminated at
within a structure known as super-operon (SO), and the expression 100 ␮mol photons m−2 s−1 for 1 h. The experiments were carried
of these genes depends on redox and light conditions (Kotecha et al., out in batch mode. All experiments were carried out in triplicate
2013). and the data are reported as the mean value ± standard deviation.
Rhodopseudomonas palustris is a very versatile organism, also in
its utilization of light. The regulation of light acclimation is a very 2.3. Analytical methods
complex process that allows to finely tuning the response to dif-
ferent environmental conditions in terms of light wavelength and The photon flux density was measured with a quan-
intensity (Evans et al., 2008). Rp. palustris strain 42OL, in particu- tum/radiometer/photometer model DO9721 equipped with a
lar, has previously shown a great photo-acclimation capacity when quantum sensor model LP9021 (Delta Ohm, Italy). Cell concen-
growing under H2 -producing conditions at high light intensities tration was determined spectrophotometrically as Optical Density
either natural or artificial (Adessi et al., 2012; Muzziotti et al., 2016). (OD) and the experimentally determined equation y = 0.4504 × OD
2
Under such conditions, Rp. palustris 42OL showed the capability to 660 – 0.0623 (r = 0.991) derived from an OD (␭ = 660 nm) versus cell
dissipate the excess of reducing power through the production of dry weight (g/L) calibration curve, was used to convert OD to
H2 (Muzziotti et al., 2016). cell dry weight. The in vivo absorption spectra were recorded
The aim of this study was to investigate the effects of high light on cells suspensions in 95% glycerol (Selyanin et al., 2016). The
irradiances on pigment composition and photosynthetic unit in cells were harvested by centrifugation for 2 min at maximum
the purple non sulfur bacterium (PNSB) Rp. palustris 42OL grown speed. The same volume of glycerol (95%) was added to the pel-
under three different conditions: Anaerobic and Aerobic growth let. The mixture was homogenized manually and the absorption
conditions as well as H2 -producing conditions. spectrum (350–900 nm) was recorded using a UV–vis Varian spec-
trophotometer. Molar ratios of light-harvesting complexes were
calculated by deconvoluting near-IR in vivo absorption spectra
2. Materials and methods
(Koblížek et al., 2010).

2.1. Bacterial strain and culture media

2.4. Pigment analyses

Rp. palustris strain 42OL (Adessi et al., 2016) used in this study
For pigment analyses, up to 20 mL of culture were harvested
(collection number CSMA73/42) was originally isolated from a
and centrifuged at 3900 × g for 15 . The supernatant was discharged
pond containing wastewaters of a sugar refinery and is deposited
and the pellet was preserved for the extraction. Between 2–3 mL of
at the CSMA Culture Collection (WDCM number 147). The cultures
Acetone: Methanol (7:2 v/v) solution was added to the cells. The
run under anaerobic and aerobic conditions were carried out in
suspension was vortexed for 10 , centrifuged again under the con-
RPN medium (Bianchi et al., 2010) containing malate 2.0 g L−1 and
ditions mentioned above and the extract was kept in vials. The
NH4 Cl 0.5 g L−1 ; cultures run under H2 -producing conditions were
extraction was repeated until cells were colorless. Following clar-
carried out in RPP medium (Bianchi et al., 2010) containing malate
ification by centrifugation, the absorption spectrum of the extract
4.0 g L−1 . For hydrogen production experiments, the cultures were
was measured in the range 400–900 nm. The extracts were then
pre-grown in RPP medium containing 1 g L−1 glutamate.
analyzed by HPLC, at room temperature (25 ◦ C), using the Agilent
1100 Series system. The instrument was equipped with the UV–vis
2.2. Culture conditions diode-array detector (Agilent DAD 61315B). Pigments were sep-
arated on the Phenomenex Luna 3 ␮ C8(2) 100 Å column with a
The inocula were prepared using the same medium for the binary solvent system (0 min 100% A, 20 min 100% B, 25 min 100%
experiments and exposed to 100 ␮mol photons m−2 s−1 . The tem- B, 27 min 100% A, 30 min 100% A [A: 80% methanol + 20% ammo-
perature was maintained at 30◦ C. After 5–6 days, the pre-cultures nium acetate (28 mM), B: methanol)]. The solvent flow rate was
were centrifuged (15 at 3900 × g) and the pellets re-suspended 0.8 mL min−1 . Pigments were identified according to their spectrum
in RPN or RPP medium for the tests. The initial O.D.(␭ = 660nm) was absorption and compared with literature (Brotosudarmo et al.,
adjusted to 0.8. The experiments were carried out under anaerobic 2015; Chi et al., 2015). No other pigments were detected. The quan-
and aerobic conditions as well as under H2 -producing conditions. tification of pigments was determined using their molar extinction
The devices used for anaerobic and H2 -producing conditions were, coefficient. Total carotenoids content was determined according
respectively, 0.1 L rubber-stoppered vessels and 0.25 L rubber- to Soon et al., 2014. The BChl a was assayed spectrophotometri-
stoppered vessels. Prior to the start of the experiments, the cultures cally using the extinction coefficient value of 75 mol−1 m3 cm−1 at
were flushed with Ar in order to remove the air from the headspace 775 nm.
 D. Muzziotti et al. / Microbiological Research 197 (2017) 49–55 51

and then, under anaerobic and H2 -producing conditions, remained

stable until the end of the experiment. Oppositely, under aerobic
conditions, LH2/LH1 ratio dropped through the time. However, it
has to be stressed that under H2 -producing conditions, LH2/LH1
ratio was always higher than under anaerobic and aerobic con-
ditions, indicating the presence of a higher number of peripheral
light-harvesting complexes (i.e. LH2) under this condition. At HL
under anaerobic and H2 -producing conditions, LH2/LH1 ratio dra-
matically decreased during the first hours of exposure to light.
Thereafter, this ratio remained stable under both conditions. On the
other hand, when Rp. palustris 42OL was grown in HL under aero-
bic conditions, LH2/LH1 ratio constantly declined along time with
a higher rate than under LL, showing that always under aerobic
conditions peripheral light-harvesting complexes (i.e. LH2), were
sensitive to the presence of oxygen, in particular under HL condi-
tions. In any case, under all culture conditions the drop in LH2/LH1
ratio in the first hours was significantly deeper at HL than under LL
(Fig. 1).

3.2. Absorption spectra of pigments and carotenoid composition

in Rp. palustris 42OL grown under anaerobic, aerobic and
H2 -producing conditions and illuminated at LL and HL

The absorption spectra (400–900 nm) of extracted pigments

for Rp. palustris 42OL cultivated under anaerobic, aerobic and H2 -
producing conditions and illuminated at LL (a) or HL (b) intensity
are depicted in Fig. 2. When Rp. palustris 42OL was grown under
anaerobic and aerobic conditions at LL, pigment content decreased
between 0 and 24 h. Thereafter, it remained substantially stable
until the end of the experiment. At HL under anaerobic and aer-
obic conditions, the trend of decrease of carotenoids along the
time of the experiments was comparable to that observed at LL.
Fig. 1. Molar ratio of light- harvesting complexes (LH2/LH1) of Rp. palustris 42OL However, at HL BChl a peaks completely disappeared under anaer-
cultivated under AnG (䊉), AG () and HP (䊏) conditions, at LL (a) and HL (b). Molar
obic and aerobic conditions. Under H2 -producing conditions at LL,
levels of light- harvesting complexes (LH1, LH2) were determined after deconvolu-
tion of absorption spectra (see Materials and Methods). Empty symbols indicate the
the absorption peaks of carotenoids and BChl a slightly decreased
starting point (t-1) at which cells were exposed to 100 ␮mol photons m−2 s−1 for between 0 and 24 h. Afterwards, they remained stable until the end
1 h. t0 (grey square and grey arrow) corresponds to the beginning of H2 production of the experiment. It is worth mentioning that a change in culture’s
under H2 -producing conditions. color from dark red to light red was observed during the first hour
of light exposure (data not shown). When Rp. palustris 42OL was
grown under H2 -producing conditions at HL, carotenoids increased
2.5. Statistical analysis between 0 and 24 h and then, they dropped at 48 h. On the other
hand, only slight changes in the absorption of BChl a were observed
The statistical analysis of the data was carried out with Graph- through the time.
Pad, using One-Way ANOVA analysis followed by a post-test Fig. 3 shows the cellular concentrations of BChl a and total
(Tukey’s multiple comparisons test). The statistical significant dif- carotenoids in Rp. palustris 42OL grown 48 h under anaerobic, aer-
ferences of the analysis follow this scale: (*) significant, P < 0.05; obic and H2 -producing conditions at LL and HL. When Rp. palustris
(**) significant, P < 0.01; (***) significant, P < 0.001; (****) significant, 42OL was grown at LL, the final cellular concentration of BChl a
P < 0.0001; (ns) not significant, P > 0.05. (␮g mg dryweight −1 ) was significantly higher under H2 -producing
conditions than under the two other conditions tested, namely
3. Results anaerobic and aerobic (P < 0.05 and P < 0.01, respectively). At HL,
BChl a was lower than at LL under all conditions. In anaerobic condi-
The effect of light irradiance on the photosynthetic unit of Rp. tions, it was very low (0.60 ± 0.12 ␮g mg drywieight −1 ) and in aerobic
palustris 42OL was assessed by comparing cells grown under anaer- conditions almost no detectable. Under H2 -producing conditions,
obic, aerobic and H2 -producing conditions with either LL or HL BChl a concentration was 7.32 ± 0.64 ␮g mg drywieight −1 , statistically
intensity. significantly higher (P < 0.001). Concerning the total carotenoid
content of the cells, under LL it was different among the three
3.1. LH2/LH1 ratio in Rp. palustris 42OL under anaerobic, aerobic conditions (P < 0.05), with the lowest value observed under aerobic
and H2 -producing conditions and illuminated at LL and HL conditions (0.52 ± 0.05 mg g drywieight −1 ) and the highest under H2 -
intensities producing conditions (1.68 ± 0.23 mg g drywieight −1 ). Under HL, the
total amounts of carotenoids were globally lower compared to LL,
Fig. 1 shows modifications in LH2/LH1 molar ratio in Rp. palus- being under H2 -producing conditions significantly (P < 0.01) higher
tris 42OL cultivated under anaerobic, aerobic and H2 -producing (0.56 ± 0.06 mg g drywieight −1 ) than under anaerobic and aerobic
conditions illuminated both at LL (a) and HL (b) intensity. At LL, conditions (0.26 ± 0.03 and 0.28 ± 0.01 mg g drywieight −1 , respec-
under all conditions tested, LH2/LH1 ratio declined between 0–4 h tively).
52 D. Muzziotti et al. / Microbiological Research 197 (2017) 49–55

Fig. 2. Absorption spectra of pigments in Rp. palustris 42OL cultivated under AnG, AG and HP conditions and illuminated at LL (a) and HL (b) intensity.
Absorption spectra of pigments were measured at 0 (Black, ), 24 (Black, ) and 48 (Grey, ) hours. 䊉䊉 refer to carotenoids; 䊉 refers to BChl a peaks.

3.3. Carotenoid composition under different culture conditions ysis showed that Rp. palustris 42OL synthetized five carotenoids:
lycopene, anhydrorhodovibrin, rhodovibrin, rhodopin and spiril-
Fig. 4 shows the percentage distribution of carotenoids after loxanthin. At LL, under anaerobic and aerobic conditions, lycopene
48 h of incubation under anaerobic, aerobic and H2 -producing was the main carotenoid synthetized and its percentage was
conditions and illuminated at LL (a) and HL (b) intensity. HPLC anal- rather similar between these two conditions, 46.6 and 54% of total

Fig. 3. Cell concentration of BChl a and total carotenoids in Rp. palustris 42OL grown 48 h under AnG, AG and HP conditions and illuminated at LL (a) and HL (b) intensity.
Concentrations are expressed as ␮g BChl a (mg dry weight)−1 and mg total carotenoids (g dry weight)−1 , respectively.
 D. Muzziotti et al. / Microbiological Research 197 (2017) 49–55 53

Fig. 4. Relative percentages of carotenoids in Rp. palustris 42OL cells grown under AnG, AG and HP and illuminated at LL (a) and HL (b) intensity.

carotenoids, respectively. However, under anaerobic conditions observation on the sensitivity of LH2 to the presence of oxygen
the relative amounts of carotenoids were differently represented, (Magis et al., 2010; Magis et al., 2011; Rinalducci et al., 2008).
lycopene being the most abundant, while the second most rep- This phenomenon was previously ascribed to the damages caused
resented carotenoid was rhodopin, followed by rhodovibrin and by light-induced reactive oxygen species (ROS) on photosynthetic
spirilloxantin, with a negligible amount of anhydrorhodovibrin. unit’s proteins (Tandori et al., 2001). From the results obtained
Differently, in aerobic conditions, after lycopene, the other four in the present study, it is possible to conclude that under aer-
were almost all equally distributed. Under the same growth con- obic conditions the rate of protein degradation due to ROS was
ditions (anaerobic and aerobic) after 48 h of growth under HL, faster under high than under low light conditions, pointing out
a completely different pattern of distribution was observed in the presence of an additional stress to the photosynthetic unit,
comparison with LL. In this case, rhodovibrin was the dominant which under aerobic conditions is neither newly synthesized nor
carotenoid, being 42.2 and 37.6% of total carotenoids, respectively, recovered. Actually, it was previously reported that in PNSB the
followed by rhodopin, spirilloxantin, anhydrorhodovibrin and only synthesis of photosynthetic unit only occurs under anaerobic con-
at last lycopene. The cultures grown under H2 -producing con- ditions (Bauer et al., 2003). Under H2 -producing conditions at LL
ditions showed a drastically different percentage composition of Rp. palustris 42OL did not modify its Light-Harvesting Complexes
carotenoids. Lycopene was always the most abundant, with a very ratio during the short lag phase of H2 production. Then, once Rp.
significant prevalence representing the 79% at LL and the 65% at palustris 42OL started to produce H2 , a slight initial decrease in
HL. The other four carotenoids were present in much lower per- Light-Harvesting Complexes ratio was observed, followed by a sta-
centages, almost equivalent than under LL, and with a prevalence bilization of the LH2/LH1 ratio. At HL, Light-Harvesting Complexes
of anhydrorhodovibrin and rhodopin under HL conditions. ratio decreased in correspondence with the lag phase of H2 produc-
tion and then remained substantially constant. These findings point
out that under H2 -producing conditions, similarly to anaerobic con-
4. Discussion
ditions, the Light-Harvesting Complexes ratio was only affected in
the first few hours after the increase of the light intensity from
The results gathered in this study clearly show that light inten-
pre-culture conditions (100 ␮mol photons m−2 s−1 ) to LL or HL con-
sity, together with culturing conditions, strongly affect LHCs ratio
ditions, but that after this period of acclimation LH2/LH1 ratio was
as well as pigment concentration and composition. In particular,
not affected anymore by the light intensity.
when Rp. palustris 42OL was grown at LL under aerobic condi-
The results obtained also showed that the synthesis of pigments
tions, a slow decrease in Light-Harvesting Complexes (LH2/LH1)
in Rp. palustris 42OL is dependent on both light intensity and culture
ratio was observed while under anaerobic conditions this value,
conditions. Under anaerobic and aerobic conditions at LL, BChl a
after a decrease in the first four hours, remained constant until
decreased during the first 24 h and then remained constant until the
the end of the experiments. At HL, a faster and linear decrease
end of the experiment. Oppositely, under the same growing condi-
along the time of LH2/LH1 ratio was observed under aerobic con-
tions but at HL, BChl a resulted almost not detectable already since
ditions, but not under anaerobic conditions, confirming previous
54 D. Muzziotti et al. / Microbiological Research 197 (2017) 49–55

the very first minutes of exposure. As it was previously reported carotenoids, namely rhodopin, rhodovibrin, spirilloxantin and
(Kuo et al., 2012; Soon et al., 2014; Zhou et al., 2014), under LL the anhydrorhodovibrin were present at lower percentages. The high
cells need an amount of BChl a rather high in order to efficiently relative concentration of lycopene, a non-polar carotenoid with the
capture the light needed for their photoheterotrophic metabolism. lowest number of conjugated C C bonds among the carotenoids
On the other hand, the absence of BChl a at HL under both anaerobic found in Rp. palustris 42OL, probably contributed to maintain an
and aerobic conditions seems to point out that the light intensity efficient transfer of the excitation energy to BChl a under this con-
was so high to damage this pigment, in particular in the pres- dition of low light availability. Indeed, according to the literature,
ence of oxygen. However, the presence of significant amounts of the excitation energy transfer increases with the decrease of the
BChl a under aerobic conditions at LL suggests that the rate of pig- number of conjugated double bonds in the carotenoid molecule
ment degradation under this condition was relatively slow. When (Chi et al., 2015; Šlouf et al., 2012). Moreover, lycopene, due to
the cells were grown under H2 -producing conditions, the nega- its chemical structure, easily embed into the plasma membrane,
tive effect of HL on the concentration of BChl a was much less thus possibly contributing to the stability of the photosynthetic unit
dramatic suggesting that the capability to synthesize hydrogen par- (Brotosudarmo et al., 2015; Šlouf et al., 2012).
tially prevented the degradation of this pigment. This finding seems A completely different picture was observed when anaerobic
to confirm that the synthesis of H2 plays a role of scavenger of the and aerobic cultures were exposed for 48 h at HL conditions. Under
excess of reducing power generated under HL conditions (Muzziotti these conditions, polar carotenoids became predominant, in par-
et al., 2016), reducing in this way the damage on the photosynthetic ticular owing to the presence of large amounts of rhodovibrin and
unit and its pigments. rhodopin. The predominant presence of this kind of carotenoids
When Rp. palustris 42OL was grown under anaerobic and aer- suggests that they were synthesized as a protection of the cells
obic conditions, under both LL and HL conditions, the amount of growing under HL. Indeed, polar carotenoids, owing to their chem-
carotenoids in the cells significantly decreased in the first 24 h and ical structure, were reported to be capable to act as efficient
then remained constant untill the end of the experiment. These quenchers of the excess of excitation energy (Brotosudarmo et al.,
findings suggest that Rp. palustris 42OL, under growth conditions, 2015; Chi et al., 2015; Cogdell et al., 2000; Feng et al., 2004; Šlouf
activated in the first 24 h a light-acclimation process protecting et al., 2012). Moreover, it was reported that polar carotenoids
the cells from the damage due to the increase in light intensity by aggregate more effectively than non-polar ones within phospho-
degrading carotenoids. However, contrary to what has frequently lipid bilayers (Gruszecki and Sielewiesiuk, 1990), suggesting that
been observed in phototrophs, no de novo synthesis of carotenoids they may act more efficiently in quenching ROS being embedded in
was observed in the 48 h of the experiment under light-stress the plasma membrane, closer to the sites where ROS are produced.
conditions (Erickson et al., 2015; Leverenz et al., 2015). In this con- When cells were grown for 48 h under H2 -producing conditions,
nection, it has to be stressed that recently the green microalga lycopene was always the most abundant carotenoid, both under
Chlorella ohadii under high light irradiances was reported not to LL and HL conditions, while the other carotenoids showed rather
increase its intracellular carotenoid content but to activate non- similar percentages under the two light conditions. Thus, the capa-
radiative routes for dissipating the excess of energy under HL bility of Rp. palustris 42OL cells to withstand light intensities as high
conditions (Treves et al., 2016). In the case of the purple bacterium as 1500 ␮mol photons m−2 s−1 without significantly changing the
Rhodocyclus gelatinosus it was previously reported a decrease in the relative composition in carotenoids seems to be related with its
production of both carotenoids and bacteriochlorophyll with the capability to synthesize H2 . Indeed, as mentioned above the synthe-
increases in light intensity (Prasertsan et al., 1993). This acclima- sis of H2 in Rp. palustris 42OL under HL was suggested to be capable
tion mechanism to high light intensity seems to be connected with of scavenging the excess of electrons generated by photosynthesis
a reduction in the antenna size and consequently in the amount of under these conditions (Muzziotti et al., 2016). Consequently, it can
light absorbed, a phenomenon frequently observed in microalgae be hypothesized that an efficient way for getting rid of the excess
during the acclimation to high irradiance (Falkowski and Laroche, of electrons under HL was effective in avoiding the need to accu-
1991). mulate polar carotenoids for protecting the cells from the harmful
Under H2 -producing conditions a completely different pattern effects of high light intensities.
was observed for the pigment content of the cells. At LL, the absorp- In conclusion, this study showed that Rp. palustris 42OL pos-
tion spectrum of pigments showed that both BChl a and carotenoids sesses an efficient strategy of photoacclimation to high light
slightly dropped down in the first 24 h. Thereafter, they kept stable intensities based on Light-Harvesting Complexes ratio regulation
until 48 h, when in any case showed a higher concentration in com- and pigment changes, specifically carotenoids. Furthermore, it was
parison with anaerobic and aerobic conditions. On the other side, at also found that the excess of reducing power generated as a con-
HL, carotenoids increased during the first 24 h of exposure to light sequence of high light intensities can be actively discarded by the
and then declined at 48 h. Under H2 -producing conditions, the con- cells under conditions allowing the production of H2 , in this way
centration of BChl a and carotenoids at 48 h were the highest among maintaining unchanged the carotenoid composition.
all conditions tested at HL. These results suggest a tight connection
between the presence and stability of the photosynthetic pigments
Conflict of interests
in Rp. palustris 42OL and its capability to produce H2 , a metabolic
process that was previously suggested capable of dissipating the
The authors declare that they have no conflict of interest.
excess of electrons under HL conditions maintaining the bacterium
in a good physiological status (Muzziotti et al., 2016). Moreover,
it is worth stressing that carotenoids, under H2 -producing condi- Acknowledgments
tions, were newly synthetized in the first 24 h of exposition at HL
conditions as an acclimation response to high light. The authors gratefully acknowledge Prof. Ana Margarita Silva
Considering the composition of the carotenoids extracted from Benavides for her encouraging support in organizing experimen-
the cells, a significant difference was observed between the cells tal work carried out at CNR-ISE, and Mr. Bernardo Cicchi for his
grown under the various conditions tested. When cells were technical support. The authors also acknowledge the Italian Min-
grown under anaerobic and aerobic conditions at LL, lycopene istry of the Environment (MATTM; project PIRODE), CNR (Italian
was always the most abundant carotenoid detected (46.6 and National Research Council) (EFOR project), Ente Cassa di Risparmio
54.0% of total carotenoids, respectively), while the other four di Firenze (Project HYDROLAB2 ). RDP would also like to mention
 D. Muzziotti et al. / Microbiological Research 197 (2017) 49–55 55

the contribution given to his activities by the participation in the Kuo, F., Chien, Y., Chen, C., 2012. Effects of light sources on growth and carotenoid
IEA-HIA (International Energy Agency – Hydrogen Implementation content of photosynthetic bacteria Rhodopseudomonas palustris. Bioresour.
Technol. 113, 315–318.
Agreement), Annex 34. Kushwaha, K., Saini, A., Saraswat, P., Agarwal, M., Saxena, J., 2014. Colorful world of
microbes: carotenoids and their applications. Adv. Biol., http://dx.doi.org/10.
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