V Riassunto Scopo: l'obiettivo della presente tesi è stato quello di investigare l'effetto di alti valori di intensità luminose in Rhodopseudomonas palustris ceppo 42OL. In particolare, l’acclimatazione ad alti valori di intensità luminose e la produzione di idrogeno come meccanismo primario per l’eliminazione dell’eccesso di potere riducente.
Metodi e Risultati: La sperimentazione condotta su Rhodopseudomonas palustris 42OL ad alti valori di intensità luminose e sotto diverse condizioni di crescita, dimostra che la produzione di idrogeno, rappresenta una via per smaltire l'eccesso di potere riducente e consente alle cellule il mantenimento di un buon stato fisiologico. Inoltre, l’ampia capacità di acclimatazione di Rp. palustris 42OL ad alti valori di intensità luminose e sotto diversi condizioni di crescita è stata confermata. A questo proposito, i cambiamenti nella composizione dei pigmenti è stato fondamentale nel processo di acclimatazione. È importante sottolineare che l’acclimatazione in Rp. palustris 42OL non è stata limitata ad un sistema d’illuminazione poli-cromatico; il batterio è stato capace di acclimatarsi ad un sistema d’illuminazione mono-cromatico. In questa tesi, vie alternative sono state studiate per smaltire l’eccesso di potere riducente in Rp. palustris 42OL. Quando la produzione di idrogeno in Rp. palustris 42OL è inibita, il batterio canalizza l’eccesso di potere riducente alla sintesi di biomassa che consente il mantenimento di un buon stato fisiologico, manifestato da un elevato e costante valore di Fv/Fm.
Conclusioni: I risultati ottenuti sostengono la fattibilità della produzione di idrogeno all’aperto utilizzando i batteri rossi non sulfurei e luce solare come prima fonte di energia.
Impatto del lavoro: con la presente tesi è stato compiuto un importante passo avanti nella comprensione del ruolo del processo di produzione di idrogeno per il mantenimento di una buona condizione fisiologica dei batteri rossi non sulfurei in condizione di elevata illuminazione. In laboratorio, è stata dimostrata la correlazione positiva tra alti valori di intensità luminose e produzione d’H2. I passi successivi dovrebbero essere quindi la progettazione di un adeguato fotobioreattore per sfruttare al meglio la luce solare nella produzione di idrogeno all’aperto.
VI Manoscritto sottomesso a pubblicazione il 2/11/2015
Dayana Muzziotti, Alessandra Adessi, CeciliaFaraloni, Giuseppe Torzillo, Roberto De Philippis (2015) H2 production in Rhodopseudomonas palustris as a way to
cope with high light intensities. Research in Microbiology (numero di registrazione da parte della rivista: RESMIC- S- 15- 0043).
Manoscritto in corso di preparazione e da sottomettere a pubblicazione Dayana Muzziotti, Alessandra Adessi, CeciliaFaraloni, Giuseppe Torzillo, Roberto De Philippis (2015/16) Photoacclimation process in Rhodopseudomonas palustris 42OL exposed to high light intensities. Research in Microbiology. ABSTRACTS
Dayana Muzziotti, Alessandra Adessi, Giuseppe Torzillo, Roberto De Philippis (2015) “H2 Production as a way to overcome the Damaging Effects of High Light
Intensities in Purple Non Sulfur Bacteria” 15th International Symposium on Phototrophic Prokaryotes, Tubingen (2- 6 Agosto, 2015- Germany) (Presentazione di un Poster).
Dayana Muzziotti Gil, Alessandra Adessi, Andrea Sanchini, Laura Dipasquale, Giuliana d’Ippolito, Angelo Fontana, Roberto De Philippis (2015) “ Production of Biohydrogen by Combining a Two Stage Fermentation Process Using Salt-Rich Substrates ”, Global Biotechnology Congress, Boston (22- 25 Luglio, 2015- USA) (Presentazione di un Poster).
Dayana Muzziotti Gil, Alessandra Adessi, Andrea Sanchini, Laura Dipasquale, Giuliana d’Ippolito, Angelo Fontana, Roberto De Philippis (2014) “Production of Biohydrogen through the Combination of Two Stage Fermentation Processes Using Salt-Rich Substrates”, European Hydrogen Energy Conference (EHEC) 2014, Seville (12-14 Marzo,2014- Spain) (Presentazione orale).
VIII Abstract Aim: the aim of this thesis was to investigate the effect of high light intensities on
Rhodopseudomonas palustris strain 42OL. Particularly, the acclimation to high light
irradiances and the production of H2 as a primary way to get rid of the excess of reductants.
Methods and Results: The experiments carried out on Rhodopseudomonas palustris 42OL under anaerobic and aerobic growing conditions as well as H2 producing conditions at high light intensities demonstrated that the production of H2 by this strain was a way to get rid of the excess of reductants and allowed the cells to maintain a good physiological status. Moreover, the huge capacity of Rp. palustris 42OL to acclimate to high light irradiances under different growing conditions was confirmed. In this connection, changes in pigments composition showed to be crucial during the acclimation process. Interestingly, the acclimation of Rp. palustris 42OL was not restricted to a poly-chromatic illumination system but also to mono-chromatic light. In this thesis, alternative ways to disposal the excess of reducing –power by Rp. palustris 42OL were also investigated. When the production of H2 in Rp. palustris 42OL was inhibited, this strain showed to deviate the excess of reductants to the synthesis of biomass which allowed maintaining a good physiological state, reflected by a constant and high Fv/Fm value.
Conclusions: all results obtained supported the feasibility of H2 production outdoor by purple non sulfur bacteria and the use of sunlight as energy source.
Impact of the study: In this thesis, an important step forward was made on the understanding of the role of the process of H2 production in giving to purple non sulfur bacteria the capability of maintaining a good physiological status under high light intensity. At laboratory scale, it was demonstrated the positive correlation between high light intensities and H2 production. Hence, the next step in the scaling up of the process should be the design of suitable photobioreactors shaped for optimizing the adsorption of the solar light for outdoor hydrogen production.
IX Manuscript submitted for publication on 02/11/2015
Dayana Muzziotti, Alessandra Adessi, CeciliaFaraloni, Giuseppe Torzillo, Roberto De Philippis (2015) H2 production in Rhodopseudomonas palustris as a way to
cope with high light intensities. Research in Microbiology (number given by the Editor RESMIC- S- 15- 0043).
Manuscript in preparation, to be submitted to publication
Dayana Muzziotti, Alessandra Adessi, CeciliaFaraloni, Giuseppe Torzillo, Roberto De Philippis (2015/16) Photoacclimation process in Rhodopseudomonas palustris 42OL exposed to high light intensities. Research in Microbiology. ABSTRACTS
Dayana Muzziotti, Alessandra Adessi, Giuseppe Torzillo, Roberto De Philippis (2015) “H2 Production as a way to overcome the Damaging Effects of High Light
Intensities in Purple Non Sulfur Bacteria” 15th International Symposium on Phototrophic Prokaryotes, Tubingen (2-6 August, 2015- Germany) (Poster exhibition).
Dayana Muzziotti Gil, Alessandra Adessi, Andrea Sanchini, Laura Dipasquale, Giuliana d’Ippolito, Angelo Fontana, Roberto De Philippis (2015) “ Production of Biohydrogen by Combining a Two Stage Fermentation Process Using Salt-Rich Substrates ”, Global Biotechnology Congress, Boston (22-25 July, 2015- USA) (Poster exhibition).
Dayana Muzziotti Gil, Alessandra Adessi, Andrea Sanchini, Laura Dipasquale, Giuliana d’Ippolito, Angelo Fontana, Roberto De Philippis (2014) “Production of Biohydrogen through the Combination of Two Stage Fermentation Processes Using Salt-Rich Substrates”, European Hydrogen Energy Conference (EHEC) 2014, Seville (12-14 March, 2014- Spain) (Oral presentation).
XI
Preface
This thesis is constituted of an introductive section given detailed information about the metabolic versatility of purple non sulfur bacteria. An exhaustive description of the photo-fermentation process in purple non sulfur bacteria is also offered, with particular consideration to the conditions needed to produce hydrogen. The acclimation (chromo-acclimation) to high light intensities in purple non sulfur bacteria is detailed described, and the role of photo-pigments into this process is highly considered. Moreover, a short description of all ways to dissipate the excess of energy by photosynthetic organisms, is included in this introduction. The use of different techniques to understand the molecular/ energetic status of the photosynthetic unit is presented, with particular attention to Pulse- Amplitude- modulation (PAM) fluorescence and Saturation Pulse Method of Quenching Analysis. In this section, a general view regarding the inhomogeneity problems of light distribution during the photo- fermentation process using purple non sulfur bacteria is offered. Furthermore, short statements about one topic with a few references in literature is described, i.e. production of hydrogen as a way to discard the excess of reducing power generated as a result of high light intensities exposure. The main aim of this thesis was to study the behavior of the purple non sulfur bacterium Rhodopseudomonas palustris strain 42OL to different culturing conditions illuminated at high light intensities, with particular interest to the production of hydrogen as a way to dispose the excess of reductants and as a mechanism to preserve a well physiological status. Besides, the acclimation to high light intensities in this strain was also one of the main objectives to be studied, particularly the trend of photo- pigments.
The results obtained during this PhD program are presented in chapters, each of them considering different aspects in regards to the aims to be reached. Chapter I is divided in two sections. Paragraph I.1 contains the experiments carried out under different culturing conditions and illumination patterns. From
XII this research came out that Rp. palustris 42OL is capable of acclimating to high light irradiances and underlines the production of hydrogen as a way to discard the excess of reductants allowing the strain to maintain a good physiological status. Paragraph I.2 includes the studies related to extra ways to discard the excess of reducing –power when Rp. palustris 42OL, illuminated at high light intensities, is unable to produce hydrogen. From this research arose that under such conditions, this strain really has other ways to dispose the high intracellular content of reducing power which allow it to keep an equilibrated redox balance. However, an initial period of production of H2 seems to be needed to allow such
acclimation. Chapter II, constitutes of two sections, comprises the investigations related to the acclimation of purple non sulfur bacteria under different growing conditions to high light intensities using different illumination patterns. Paragraph II.1 encloses the investigations related to chromo- acclimation to high light intensities in Rp. palustris cultures, growing under aerobic and anaerobic growing conditions as well as H2 producing conditions. From these experiments
arose that pigments play a crucial role during the acclimation process, and their behavior depends on light intensity conditions. Paragraph II.2 comprises the investigations related to the acclimation of purple non sulfur bacteria under aerobic and anaerobic growing conditions to high light intensities, using a mono-chromatic illumination system. From this work came out that Rp. palustris 42OL cultivated under anaerobic growing conditions is able to acclimate to high light intensities and to maintain a good physiological state. Moreover, the metabolic versatility of this bacterium is confirmed. It has to be stressed that some of the investigations carried out in this thesis are preliminary, and constitute the base for further researches. Little information about the topics explored in this thesis was found in literature, especially with regard to the production of hydrogen by purple non sulfur bacteria to overcome the excess of excitation energy owing to high light intensities, showing the novelty of this study.
XIII The last part of this thesis consists of the main and general conclusions derived from the set of experiments. The main general conclusion is that the production of H2 outdoors using purple non sulfur bacteria requires the
improvement in the design of photobioreactors with particular attention to achieving an adequate light distribution to the cells in the lower layers of the culture, this issue representing the biggest challenge to be achieved. In this thesis, a key step in the knowledge needed for the scaling- up of H2 production
process outdoors was made, because it was demonstrated that purple non sulfur bacteria are able to tolerate high light irradiances while producing H2.
Moreover, it was demonstrated the positive correlation between high light intensities and high H2 production rates. Hence, the next step in the scaling up
of the process should be the design of suitable photobioreactors, shaped for optimizing the adsorption of the solar light for outdoor hydrogen production.
1
Table of content
Introduction 4
1. Purple Non Sulfur Bacteria 5
1.1. Taxonomy 5
1.2. Ecology 6
2. Metabolism of Purple Non Sulfur Bacteria 7
2.1. Anaerobic metabolism 7
2.1.1. Anoxygenic photosynthesis 8
2.2. Aerobic metabolism 9
3. Photofermentation and H2 production 10
3.1. Mechanism of H2 production 11
4. The photosynthetic unit (PSU) 13
4.1. Structure of the photosynthetic unit (PSU) 14 4.1.1. Energy transfer in the photosynthetic unit (PSU) 17 4.2. Pigments present in Purple Non Sulfur Bacteria 17 4.2.1. Function of carotenoids in photosynthetic organisms 19
5. Characteristics of Light 21
5.1. Light intensity and quality in H2 production 23
5.2. Physiological effect of light intensity on
photosynthetic microorganisms 25
6. Chlorophyll fluorescence 29
6.1. Discovery of Chlorophyll fluorescence 29
6.2. Pulse- Amplitude- Modulation (PAM) Fluorescence 30 6.2.1. Principle of Pulse- Amplitude- Modulation Fluorescence 31 6.2.2. What does Chlorophyll fluorescence yield proof? 34 6.2.3. Saturation pulse method of quenching analysis 35
2 6.2.5. Relative electron transport rate and light response
curve 39
6.3. Fast Repetition Rate Fluorometry 41
6.3.1. Theory of fast repetition rate fluorometry 42
6.3.2. Excitation protocols 43
7. Ways to dissipate the excitation energy 44
Aim of the thesis 56
Results 58
Chapter I 60
I.1 H2 production in Rhodopseudomonas palustris as a way to cope
with high light intensities 62
I.1.1 Introduction 64
I.1.2 Materials and Methods 66
I.1.3 Results 69
I.1.4 Discussion and conclusions 76
I.2. Photo-acclimation studies on inhibiting H2 producing
cultures of Rp. palustris 42OL using the fast repetition
rate technique 86
I.2.1 Introduction 86
I.2.2 Materials and Methods 88
I.2.3 Results 91
I.2.4 Discussion 95
3
Chapter II 102
II.1 Effects of high light intensities on Rp. palustris 42OL grown under aerobic and anaerobic conditions as well as H2 producing
conditions 104
II.1.1 Introduction 104
II.1.2 Materials and Methods 106
II.1.3 Results 109
II.1.4 Discussion 132
II.1.5 Conclusions 136
II.2. Effects of high light intensities on Rp. palustris 42OL grown under
monochromatic light in aerobic and anaerobic conditions 141
II.2.1 Introduction 141
II.2.2 Materials and Methods 143
II.2.3 Results 147
II.2.4 Discussion 160
II.2.5 Conclusions 163
General Conclusions 168
4
Introduction
5
1.
Purple Non Sulfur Bacteria
Purple Non Sulfur Bacteria (PNSB) are anoxygenic phototrophic bacteria which are capable of performing the anoxygenic photosynthesis in the absence of oxygen (Adessi & De Philippis, 2014). Furthermore, these bacteria possess photosynthetic pigments such as bacteriochlorophyll a (BChl a) and carotenoids (Cars), which display an important role in photosynthesis. Purple bacteria are capable of conserving energy by photophosphorylation and, in fact, it constitutes one of their most interesting issues (Madigan & Jung, 2008). These micro-organisms are important in biotechnology because many products and sub- products, derived from their metabolism, can be used for the development of environmentally sustainable bio-products. Nowadays, H2 produced by these
bacteria is considered one of the most important products of their metabolism.
1.1. Taxonomy
From the taxonomical point of view, PNSB belong to Alpha (α)-proteobacteria or Beta (β) – (α)-proteobacteria, but most of them pertain to the first group. Alpha (α)-proteobacteria and Beta (β) – proteobacteria differ in morphology and physiology. Moreover, differences in the number of chemotaxonomic characters (cytochrome c structures, lipid and quinone compositions, lipopolysaccharides structures), have also been observed. The differences between these two sub-groups have been genetically confirmed through DNA- rRNA hybridization. Furthermore, from 16S rRNA analysis came out that most of PNSB (the genera Rhodospirillum (R.), Rhodobacter (Rb.), Rhodopila (Rp.), Rhodomicrobium (Rm.) and Rhodopseudomonas (Rps.)) belong to the α- group of the proteobacteria (Imhoff, 2006).
6 The Alpha (α)-proteobacteria group is composed of the following sub-groups:
1) α-1 for Rhodospirillum and relatives; 2) α-2 for Rhodopseudomonas and relatives; 3) α-3 for Rhodobacter and relatives.
On the other hand, the species of PNSB that belong to the genus Rhodocyclus (Rc.) are found in the β group of proteobacteria (Imhoff, 2006).
1.2. Ecology
In nature, PNSB live in the anoxic regions of water bodies and sediments. These zones must receive an adequate quantity of light in order to guarantee their bacterial growth. It has to be stressed that, even though, these bacteria need a sufficient quantity of light to grow, the quality of it is so important, too. The proper combination of quantity and quality of light allows the bacteria to grow under phototrophic conditions. Aquatic environments with a notable concentration of organic matter and low oxygen tension constitute the preferred habitat of these bacteria. Many species of PNSB have been found in eutrophic waters and lakes. One of the most well studied Purple Non Sulfur (PNS) bacterium, Rps. palustris, has been found in wet rotting leaves and is usually found in soils (Imhoff, 2006).
7
2. Metabolism of Purple Non Sulfur Bacteria
PNSB are photosynthetic microorganisms with a wide metabolic versatility which represents an advantage for them, because such versatility has allowed them to thrive in different environments. The most representative PNS bacterium is Rhodopseudomonas palustris. It belongs to Alfa (α)-proteobacteria and is widely found in nature owing to its metabolic versatility. Many studies have been conducted on Rp. palustris, and they have made in evidence not only that this bacterium has the four modes of metabolism (i.e. photoautotrophic or photosynthetic, photoheterotrophic, chemoheterotrophic and chemoautotrophic), but also that there is an exceptional flexibility among them. Rp. palustris has become a model organism to study the metabolism and changes on it due to variations in, for instances, light and electron sources (Larimer et al., 2004).
Some genetic studies have shown that Rp. palustris has many energy- metabolic- associated- genes and it explains its metabolic versatility. The regulation of these genes by Rp. palustris under environmentally stressing conditions allows it to grow (Larimer et al., 2004).
2.1. Anaerobic metabolism
Under anaerobic conditions and light, PNSB are able to grow phototrophically. In one hand, they can grow photoheterotrophically using organic substrates as both electrons donors and carbon source. On the other hand, the photoautotrophic metabolism in which reduced sulfur compounds or hydrogen are used as electron donors, also allows them to grow (Imhoff, 2006). These bacteria might also grow under anoxic conditions by either fermentation or anaerobic respiration (Madigan & Jung, 2008). Moreover, PNSB are capable of growing under anaerobic dark conditions using either organic or inorganic
8 compounds as electron acceptors (Imhoff, 2006). It has to be stressed that anaerobic respiration is very important in PNSB either under dark or light. Under dark conditions, it allows a slow bacterial growth while under light, the bacteria are capable of dissipating the reducing power through the development of the photosynthetic electron transport and, in that way, to maintain an optimal redox balance (McEwan, 1994).
Genetic studies have shown that under anaerobic conditions, Rp. palustris is able to use light as energy source by cyclic photophosphorylation. This ability is due to the presence of a number of photosynthetic genes in its genome (Larimer et al., 2004).
2.1.1. Anoxygenic photosynthesis
Anoxygenic photosynthesis (Fig.1) is a process performed by PNSB. When these bacteria grow under photoheterotrophic conditions, the anoxygenic photosynthesis is very active. This process has fascinated many scientists during many decades because of its intrinsic properties: absence of air and no O2
evolution. Moreover, the use of organic acids as electron donors instead of water has also attracted the scientific world (Ghirardi et al., 2009). Another aspect which contributes to the interest in this process is the ability to generate ATP in the absence of O2 but, oppositely, using light as energy source (Adessi &
De Philippis, 2014).
The anoxygenic photosynthesis is a cyclic mechanism in which two BChl molecules are used as both the primary electron donors and the final electron acceptors. At the reaction center (RC) of any photosynthetic organism, the separation of charge takes place, and the excitation energy is kept into an energy- rich chemical bond. Charge separation at the RC is the result of
9 absorption of light by BChl molecules and the transfer of one electron from a chemical compound (donor) towards another compound (acceptor).
The mechanism of the anoxygenic photosynthesis is well understood. Initially, light harvesting complexes (LHs) absorb a photon and the excitation energy is directed to two BChls in the RC. Thereafter, charge separation occurs and this energy is utilized for the release of an electron which reduces the quinone into a semiquinone. After the absorption of a second photon, the quinone is doubly reduced and then, capable of picking up protons from the cytoplasmic space and translocating them through the membrane to reach the cytochrome bc1 complex. Afterwards, electrons are funneled to the cytochrome
c2 (Cyt c2) and protons are released into the periplasmic space. Under this
condition, the Cyt c2 is able to reduce the previously oxidized BChls localized at
the RC. Subsequently, this cycle electron transport is closed (Adessi & De Philippis, 2014).
2.2. Aerobic metabolism
In the presence of O2, PNSB evolve cellular respiration to grow and to
produce ATP for their metabolic functions. When these bacteria respire aerobically, carbon compounds are metabolized but a little part of them is assimilated. PNSB are capable of fixing CO2 under autotrophic and heterotrophic
conditions (Adessi & De Philippis, 2012). The widely metabolic versatility in PNSB is observed also under aerobic dark conditions, because they are able to grow using the aerobic respiration (McKindlay, 2014). Genetic studies in Rp. palustris indicate that it possess four sets of genes for terminal oxidases that work in the presence of oxygen (Larimer et al., 2004).
10
3. Photofermentation and H
2production
When PNSB grow under photoheterotrophic anaerobic conditions and in the absence of nitrogen, they are able to carry out a biological process of H2
production known as photo- fermentation. In this process, the reducing power derived from the oxidation of organic compounds and the energy coming from the light are used to reduce protons to H2. The production of this biogas by
PNSB implicates several sub- processes: Anoxygenic photosynthesis (previously described), ATP synthesis, TCA cycle, nitrogenase activities (Fig. 1) (Adessi & De Philippis, 2012). Under photofermentative conditions, Nitrogenase is the key enzyme involves in H2 production and it develops an activity similar to an ATP-
powered hydrogenase because of H2 is the only product (McKindlay & Harwood,
2010). From the biochemical point of view, this enzyme is a two protein complex constitutes of one subunit with a molecular weight of 250 kDa and another subunit of about 70 kDa. The major subunit consists of a dinitrogenase containing Fe and Mo as cofactors while the minor is a dinitrogenase reductase (containing Fe). Currently, three isozymes of nitrogenase (Mo- nitrogenase, V- nitrogenase and Fe- nitrogenase) have been found and each of them produces different quantities of H2 (Adessi & De Philippis, 2012).
Nitrogenase activity is inhibited by NH4+. This cation acts at 3 distinctive
levels. In the first level, the transcription of nifA (a gene which encodes a RNA polymerase sigma 54- dependent transcriptional activator) is blocked due to fixed nitrogen signals (e.g., NH+4). At second level, conformational and structural
changes on NifA happens owing to the presence of NH4+. In this way, the
transcriptional activator is unable to bind to its binding site and as a consequence, the transcription of nitrogenase (nif) gene does not occur. At the third and last level, NH4+acts directly on nitrogenase generating a “switch off”
on it or the inactivation of the enzyme through ADP -ribosylation mediated by DraT (Masephol et al., 2002).
11 3.1. Mechanism of H2 production
Nitrogenase, in the absence of nitrogen source, catalyzes the production of H2 through this reaction:
8H+ + 8e- + 16ATP → 4H2 + 16ADP (1)
In order to produce H2 through photofermentation, a source of ATP and
electrons is needed. Nature has resolved this important issue coupling the production of H2 with anoxygenic photosynthesis. The latter provides both the
ATP and electrons required during the photofermentation process. In the anoxygenic photosynthesis, electrons are accepted by ferrodoxins (which have been previously reduced in an ATP- consuming reaction). Thereafter, they are transferred to nitrogenase which reduces H+ to H2 in an ATP dependent reaction
(Adessi & De Philippis, 2012).
It has to be mentioned that under some circumstances, i.e. environmental stressing conditions, the production of H2 by the cell is considered as a way to
deviate the excess of reducing power deriving from other metabolic processes (Scoma et al., 2014).
12 Fig.1. Main steps involved in H2 production through photo-fermentation carried out by PNSB (Source: Adessi & De Philippis, 2012).
13
4. The photosynthetic unit (PSU)
Photosynthetic organisms have an active structure to drive photosynthesis, and it is known as the photosynthetic unit (PSU). Historically, Gaffron and Wohl were the first to establish that light energy absorbed by this structure is converted in excitation energy and transferred to the “photoenzyme”, for initiating photosynthesis. Later, G. Wilse Robison (1967) created two models to explain how the excitation energy travels. In the lake model, the exciton may travel freely to all RC. Oppositely, in the puddles model, the exciton moves only to its own RC. Nevertheless, further studies on this field have demonstrated that the real picture is “in-between”, because energy exchange among the different puddles may occur. Nowadays, it is thought that due to the presence of LHCs, the pebble- mosaic model is the real situation (Govindjee, 2010).
Photosynthesis, a process carried out by photosynthetic organisms, is a light- dependent process in which light is absorbed and transformed in chemical energy. Photo-pigments are responsible of absorbing light and of transferring the excitation energy to the RC. In purple bacteria, the absorption spectrum includes the two ends of the visible spectrum and the absorption of light by Cars and BChls happens at specific points of it. In regards to Cars, they absorb in the range 450- 550 nm while BChls absorb at 800 and 875 nm in the near infrared region. This photo- pigment has also other absorption peak at 590 nm owing to the Qx shift of BChla. During Photosynthesis, LHs absorb light and transform it
into excitation energy which is transferred to the RC. Thereafter, primary charge separation in the RC occurs. It has to be stressed that just some BChls localized in the RC are involved in photo- chemical reactions. Indeed, most of them harvest light and channel the excitation energy to the RC (Adessi & De Philippis, 2014).
14 Fig.2. Absorption spectrum of PNSB. Peaks slightly vary among species (Source: Adessi & De Philippis, 2014).
4.1. Structure of the photosynthetic unit (PSU)
The molecular structure responsible of carrying out the photosynthetic process is known as the PSU. This structure is located in the photosynthetic membrane and it is composed of the LHCs and the RC (Law et al., 2004). There is a strong interaction between the RC and the Light Harvesting 1 Complex (LH1) to form the RC- LH1 core complex, and around this core complex, peripheral LHs, such as Light Harvesting 2 Complex (LH2), are located (Harada et al., 2008). It has to be stressed that Light- Harvesting Complexes (LHs) absorb light with a high efficiency and channel it to the RC, following a gradient. In order to harvest light, purple bacteria must periodically synthetize the RCs and LHs. Both structures are constituted of proteins which bind photo-pigments: BChls and Cars. It has to be mentioned that the different spectral characteristics between
15 these photo- pigments are not only due to different chemical structures, but also based on differences in the chemical environments within the proteins that bind them (Law et al., 2004). It has recently been reported the presence of 300 molecules of BChls and 200 molecules of Cars per PSU (BChl: Cars ratio is 3:2) (Adessi & De Philippis, 2014). In purple bacteria, LHs absorb in different part of the spectrum. The LH1 has its maximum absorption peak at 875 nm (B875 band) which corresponds to the near- IR of the spectrum. On the other hand, the LH2 absorbs at 800 and 850 nm (B800 and B850 bands) (Koblízek et al., 2005).
Some studies on the RC of Rb. sphaeroides has shown that it is constituted of three polypeptides known as L, M and H. Furthermore, ten co- factors (four BChl a, two bacteriopheophytin (Bphe) a, two ubiquinone molecules, an ion of non-heme Fe 2+, and a Cars molecule) are part of the RC. The L and M subunits of the RC each possess 5 transmembrane α- helices. Moreover, they have a pseudo- twofold rotational structural symmetry and are non- covalently bound to the co- factors present in the RC. A pair of BChls, known as P dimer, is localized on the periplasmic side of the membrane and the BChl macrocycles in such dimer are parallel to each other and perpendicular to the membrane surface (Leonova et al., 2011). In regards to LHs, they are formed of repetitions of an elementary unit constitute by α and β hydrophobic apo-proteins and such subunits (which are organized in rings of different sizes), bind two BChl molecules and one or two Cars molecules (Adessi & De Philippis, 2014). The LH1 possess a circular configuration and have 16 αβ- dimer units. Moreover, this ring has a hole at the center in which the RC is localized (Law et al., 2004). Concerning the LH2, it has the same confirmation as LH1; however, it is composed of different number of subunits and its cycles are smaller (Adessi & De Philippis, 2014).
16 Fig.3. PSU of purple bacteria. On the left, it is indicated the supramolecular structure. On the right side, individual BChls and the electronic couplings are shown (Source: Adessi & De Philippis, 2014).
17 4.1.1. Energy transfer in the photosynthetic unit (PSU)
In the PSU of purple bacteria, the energy absorbed from the light is transferred from the LHs to the RC following the Föster mechanism. The efficiency of energy transfer depends on several parameters such as the distance among pigments and the spectral overlap between the fluorescence emission of the donor pigment and the absorption of the acceptor pigment. In purple bacteria, the excitation energy is transferred thanks to the creation of an energy gradient and the direction of this transfer is LH2 to LH1 and finally, to the RC. Some studies have suggested that anoxygenic photosynthesis occurs as a consequence of the directionality of excitation energy transfer, which seems to be very efficient (Law et al., 2004).
4.2. Pigments present in Purple Non Sulfur Bacteria
Photosynthetic organisms have photo- pigments such as chlorophyll (Chl) and Cars, to harvest light energy. These pigments are organic compounds and, chemically speaking, they are lipoliphic and associated to protein complexes (Masojídek et al. 2004). In purple bacteria, the evolution of BChls is as follow: BChl a → BChl c → BChl g, and this pigment is constituted of a tetrapyrrole ring containing a central magnesium atom and a long- chain terpenoid alcohol (Papageorgiou, 2010).
18 PNSB have around 50 different kinds of Cars and their synthesis includes two main pathways:
a) The spirilloxantin pathway which contain normal spirilloxantin, unusual spirilloxantin, sphaeroidene and carotenal pathways;
b) b) The okenone pathway which comprises okenone and R.g.-keto carotenoids pathways.
(Adessi & De Philippis, 2014)
It has been recently reported that the biosynthesis of Cars in some species of PNSB, such as Rhodopseudomonas (Rps.) and Rhodospirillum (Rsp.) species, follows the normal spirilloxanthin pathway in which several reactions, proceeding in a sequence mode, allow the synthesis of mainly spirilloxanthin and, in small quantity, of the other five to seven (all) Cars (Mizoguchi et al., 2008). In PNSB, i.e. in Rp. palustris, 5 Cars have been found: lycopene, rhodopin, rhodovibrin, anhydrorhodovibrin and spirilloxanthin. The main difference among them concerns the numbers of conjugated C=C bonds (Feng et al., 2004).
19 Fig.4. Chemical structure of some photosynthetic pigments in purple bacteria: a) BChl a, and b) Cars (Source: Brotosudarmo et al., 2015).
4.2.1. Function of Carotenoids in photosynthetic organisms
It is well known that Cars play a crucial role in Photosynthesis. They are involved in the transfer of excitation energy and are used by photosynthetic organisms as protection. Specifically, (1) Cars harvest light in the LHs, and transfer the excitation energy towards the RC; precisely to Chl. (2) Cars also play a crucial role as structural entities in the PSU. (3) A third function concerns the photo-protection these photo-pigments give to the cells exposed to high light intensities (Masojídek et al., 2004).
20 Roles (1) and (2) have been explained previously. So, the (3) function of Cars will be explicated in the following lines. When BChls change their energetic levels and become excited, they may react with O2 and as a result, highly
reactive singlet oxygen species may be produced. Cars can prevent the formation of such toxic O2 species deviating the excitation energy from BChls to
heat. In that way, the excited molecule comes back to its ground state. Moreover, Cars can act as “quencher” of the dangerous and toxic singlet oxygen species due to the reactivity of their conjugated double bonds. It has to be stressed that the performance of Cars as a “quencher” occurs once the deviation of the excess of excitation energy to BChl becomes limiting (Hellingwerf et al., 1994). Tandori et al. (2001) reported that PNSB are capable of tolerating high light irradiances because of the protective role of Cars. Moreover, they explained that this protection is due to the rapid triplet- state energy transfer from excited BChl to Cars, which contribute to the dissipation of excitation energy to heat and the quenching of singlet oxygen species. From their studies, they concluded that in the absence of Cars, the RC can be subjected to photo-damage.
21 5. Characteristics of light
Solar light is the main source of energy on the Earth. It allows the development of many biological processes such as photosynthesis. Light, is defined as electromagnetic radiation and it has its intrinsic properties. Among its characteristics, the most important and amazing concerns its speed of traveling. Light travels at ~ 3 x 10 8 m s-1. Moreover, the electromagnetic radiation is divided in different wavelengths with a range between 10-3 and 10
-8
m. Gamma and X-rays have shorter wavelengths. On the other hand, the visible part of the spectrum ranges from 380 to 750 nm, which corresponds from the violet to the far red. It has to be stressed that the wavelengths of the visible part of the spectrum are used to drive photosynthesis, and thus are defined as the photosynthetically active radiation (PAR). According to the quantum theory, photon (quantum) is the base unit of light, and its energy is the product of its frequency and the Planck’s constant. During photosynthesis, photons are absorbed by photo-pigments; however, they must have enough energy to trigger the photosynthetic process (Masojídek et al., 2004).
Photobiology studies the relationship between light and living matter. Light is vital in the life of a microorganism, but when it is in excess it can really produce damage at cellular levels. Once a photon is absorbed by the cell, heat is generated and cell may reach temperature up to 200 °C in short time. As a result of it, proteins are injured (Hellingwerf et al., 1994). Light can either be measured as radiant flux energy or irradiance, in units of power per area (W m-2 or J m-2 s
-1
) or as the photosynthetic photon flux density (mostly used in photosynthesis) measured in μmol quanta m-2 s-1 or μE m-2 s-1(Hellingwerf et al., 1994; Masojídek et al., 2004).
22 Fig.5. Spectra of electromagnetic radiation and spectral pattern of visible light (Authors: Averill & Eldredge). (Source:
http://catalog.flatworldknowledge.com/bookhub/4309?e=averill_1.0-ch06_s01).
23 5.1. Light intensity and quality in H2 production
In the last years, a large number of H2 production studies by PNSB have
been focused on many aspects related to light, specifically the optimization of H2 production under optimal light irradiance. In a H2 production process, light
can be supplied artificially or naturally (sunlight). Moreover, a combination of both sources is also possible (Basak & Das, 2006). In regards to artificial light, incandescent (tungsten lamp) and halogen lamps are the most used in studies with PNSB, because they include the absorption areas of both Cars and BChl a (Kuo et al., 2012). Basak and Das (2006) have recently reported that the artificial illumination for a study with PNSB can come from different sources: fluorescence lamps, optical fibers, neon tubes, halogen lamps and light emitting diodes (LEDs). In regards to LEDs system, it has widely been used across the world due to its low- cost energy consumption and long life expectancy. One of the most important and relevant characteristic of this system, is the combination of a narrow wavelength band with an intensification of light energy. LED lamps can be built for a specific wavelength band in order to match the photosynthetic absorption spectra of PNSB. Therefore, this system allows the enhancement of the light conversion efficiency (Kuo et al., 2012). In order to optimize the H2 production process in PNSB, some studies have been conducted
on determining the effect of specific wavelength on both H2 production and
bacterial growth. It has been reported that the red- infrared emission region of light plays a crucial role on both H2 production and bacterial growth (Uyar et al.,
2007). Hellingwerf et al. (1982) have also reported the effect of specific wavelengths on bacterial growth in these bacteria. Kuo and co-workers (2012) showed the effect of different light sources (fluorescence light; halogen lamp; incandescent light; LED: blue; green; red; white and yellow) on bacterial growth and Cars content in PNSB.
24 Uyar and co- workers (2007) while working with Rhodobacter sphaeroides O.U. 001 showed that an increase in light intensity up to 270 W m-2 promotes the maximum hydrogen production rate. Further increment in light intensity did not change the rate. From their experiments and according to their results, they stated that in order to obtain a high hydrogen production rate, the light intensity should reach at least 270 W m-2 at the darkest point of the photobioreactor. This operational problem is normally considered one of the major issues in this kind of processes, because low light intensities are the rate determining parameter. It has been reported that alteration of light intensity and light spectrum modify the conversion of light to H2. Experiments have
shown the inversely relationship between light conversion efficiency and light intensity (Nakada et al., 1998). It has to be mentioned that during the H2
production process by PNSB using artificial illumination, the most relevant point consists of selecting the right lamp which must include the near IR, because the absorption maximum of BChl a is positioned in this region (Adessi & De Philippis, 2014).
Sunlight can also be used to produce H2 using PNSB. It offers economic
advantages because it is free and contains the full spectrum of light. However, light intensity of sunlight change due to environmental conditions and period of the year, so that, such variations of light intensity truly affect the performance of H2 production outdoor (Chen et al., 2011). Recently, Adessi and De Philippis
(2014) proposed some ways to circumvent the problems of H2 production using
sunlight as illumination system. Furthermore, they stated that a combining system using sunlight and artificial light to produce H2 might be used.
25 5.2. Physiological effect of light intensity on photosynthetic organisms
Photosynthetic organisms convert light energy into chemical energy; however, it is a dangerous process in which these organisms must face multiple stresses. Fortunately, they have evolved some mechanisms to tolerate such kind of difficulties. Whether light absorption exceeds the capacity for the photosynthetic electron transport, cells can be damaged. So that, photosynthetic organisms must create a balance between the need of harvesting light under low light intensities and the accumulation of light when they are exposed to high light irradiances. For that reason, photosynthetic organisms have developed several ways to overcome this pressure and to dissipate the excess of light (Bruce &Vasil’ev, 2010).
In nature, photosynthetic organisms often face changes in light intensity and in order to cope with them, they have developed different kinds of acclimation mechanisms to adjust the photosynthetic activity in response to light conditions (Masojídek et al., 2004). The photo-acclimation capacity of photosynthetic organisms is determined by structural and functional adaptations to long- term light conditions (Belshe et al., 2007). The term Chromatic photo-acclimation is more appropriate because it refers to responses to light intensity and spectral distribution (Duxbury et al., 2009). Falkowski and Laroche (1991) established that photo- acclimation allows maximizing the PAR and prevents photo-damage.
The response to light intensity by photosynthetic organisms depends on the quantity of it they receive. Under low light conditions, photosynthetic organisms increase their pigmentation and number of PSU as well as the size of LHs. Oppositely, when photosynthetic organisms are under high light intensities, pigment contents decrease. The modification of pigments occurs at a timescale of days (Masojídek et al., 2004).
26 Photosynthetic organisms can also rapidly response to changes in light intensities through the development of down-regulation processes, for example, the decrement in the absorption of light and/or alternative energy sinks, when photosynthetic capacity is exceed. These protective mechanisms most likely limit damage to the PSU; however, a lower quantum yield is obtained. Down- regulation mechanisms are so important because they allow photosynthetic organisms to tolerate rapidly changes in light environment (Belshe et al., 2007). It has been reported that alteration in pigments composition due to ligh intensity conditions permits photosynthetic organisms to maintain an optimal photosynthetic activity. This type of acclimation is known as Chromo- acclimation and it plays a crucial role in ecology, distribution and evolution of photosynthetic organisms (Gloag et al., 2007).
It has been reported that PNSB are capable of controlling its photosynthetic activity according to light availability. Under low light intensities, they increase the number of LHs that allow light absorption (Kuo et al., 2012). Moreover, PNSB are able to shift the absorption spectrum of their peripheral LHs owing to light intensities. Among these bacteria, Rps. acidophila and Rps. cryptolactis, are capable of changing their LH2 to a more blue- shifted B800- 820 complex. On the other hand, Rps. palustris strain 2.6.1, when growing under low light intensities, synthetizes an intracytoplasmic membrane to acclimate to such conditions. In this bacterium, there is not a replacement of B800-B850 band by 820 nm absorbing LH2 (Gall & Robert, 1999).
Gall and Robert (1999) reported that once Rp. palustris strain 2.6.1 acclimate to a particular light intensity, the Cars composition in the LH2 changes according to that intensity of light. Therefore, under low light and high light intensities this complex has different Cars absorption characteristics. The absorption peaks at 468, 496 and 530 nm in the LH2 are detected under high
27 light intensities while the peaks observed at 466, 491 and 526 nm are found under low light intensities.
Mizoguchi et al. (2008) recently reported that Rps. sp. Strain Rits and Rps. palustris have similar photosynthetic apparatus and the synthesis of LH depends on light intensities. When these bacteria grow under normal and high light conditions, RC-LH1 and LH2 were synthetized as the core and peripheral components, respectively. On the other hand, when these bacteria grow under low light conditions light- harvesting 4 (LH4) is the major peripheral LH.
When running an experiment at 30 μE s-1 m-2 with Rps. sp. Strain Rits, Mizoguchi and co-workers (2008) found that due to light intensity, this bacterium synthetized 7 types of Cars: lycopene, anhydrorhodovibrin, spirilloxanthin, rhodopin, 3,4- didehydrorhodopin, Rhodovibrin and OH- spirilloxanthin. Moreover, this group of research studied the light- intensity dependence upon the composition of Cars in Rps. species. They found that, the relative amount of each Car in each species strongly depended on the light intensity. In the extracts of cells from Rps. sp. Strain Rits and Rps. palustris CGA009, 3, 4- didehydrorhodopin was a major Car under normal and high light conditions, while rhodopin was predominant under low- light conditions. 3, 4-didehydrorhodopin was found to be first accumulated at a remarkable amount in Rps. species including the Rits strain and Rps. palustris CGA009/DSM123 under three different light intensities: 3, 30 and 200 μE s-1 m-2.
Harada and co-workers (2008) have also reported the huge photo-acclimation capacity of PNSB. Under low- light intensities, the amount of RC- LH1 core and LH2 was higher than under high light irradiances. They also found that some of these bacteria synthetize distinct peripheral LH according to light intensities. Rhodopseudomonas (Rps.) palustris, for instances, synthetizes RC-LH1 and LH2 under high light intensity and the latter can be mainly substituted
28 by LH4 under low light intensities. This group of research also refers to changes in Cars composition in the LH owing to light intensity and oxygen concentration as well as growth phase. They concluded that such alterations are acclimation responses to environmental conditions which, a propos, are controlled by regulating the relevant gene expression level.
There is a very interesting point related to BChl a synthesis in PNSB. Interestingly, Harada and co-workers (2008) found that the presence of BChl a intermediates vary from one strain to the other and depends greatly on light intensity conditions. It is so interesting to note that due to the complexity of light utilization by PNSB, evolution has focused on the development of regulatory bacteriophytochromes to regulate such mechanism. Moreover, the presence of these structures in these bacteria allows PNSB to acclimate to different environmental conditions, specifically to light wavelength and light intensity (Adessi et al., 2012).
29
6. Chlorophyll fluorescence
Nowadays, Chl fluorescence is used to obtain information about several aspects related to photosynthesis. In the past, this technique was used by selective groups of research; however, it has changed in the last years. Among the aspects that can be studied applying Chl fluorescence there are: identity and organization of various pigments and pigment complexes, excitation energy transfer among them, and electron- transfer reactions (Govindjee, 2010).
In PNSB is possible to apply Chl fluorescence in order to understand the condition of the photosynthetic apparatus. However, it must be underlined that PNSB contain BChl a instead of Chl, and it has to be clearly identified the differences concern to the absorption maximum of each molecule. In regards to BChl a, it has its absorption maximum in the near IR (Adessi & De Philippis, 2014). It has been recently reported in literature the use of BChl a fluorescence to study photosynthesis in PNSB (Cleland et al., 1992; Adessi et al., 2012; Ritchie & Runcie, 2013; Ritchie, 2013).
6.1. Discovery of Chlorophyll fluorescence
The discovery of Chl fluorescence started in the nineteenth century when E.N. Harvey observed the luminescence which was defined by him as “light emission”. From that period until now, many studies and discoveries have been made in this field. However, the real discovery of this phenomenon was attributed to Sir John Herschel. He recognized the existence of this phenomenon in a solution of quinine sulfate. Nevertheless, he named it as epiploic dispersion instead of fluorescence. After some years of studies, Sir G.G Stokes acknowledged this phenomenon and called it as fluorescence. Moreover, Sir G.G Stokes established that fluorescence was concerned with light emission. In 1874, N.J.C Müller observed that the red Chl fluorescence was weaker in green living
30 leaf than in a diluted Chl solution, and from his studies he concluded the inverse relationship between Chl fluorescence and photosynthesis. Later, Kaustsky and Hirsch (1931) bowed to a short report whose title was “New experiments on carbon dioxide assimilation”. They observed that when dark- adapted leaves are illuminated, the time of Chl fluorescence was correlated with the time of CO2
assimilation. This correlation was previously reported by Otto Warburg in 1920 (Govindjee, 2010).
6.2. Pulse- amplitude- modulation (PAM) fluorescence
Chl fluorescence has become an excellent tool to study photosynthesis because it gives a lot of information about what happens in the PSU. Precisely, this technique gives information about the efficiency of primary energy conversion (Koblížek et al., 2005; Adessi et al., 2012) which is fundamental to understand the development of photosynthesis. Hans Kaustsky and co-workers were the first to consider the potential of this technique to understand how photosynthesis develops. They discovered the dark/ light induction phenomenon (Kautsky effect; Kaustsky & Hirsch, 1931).
PAM fluorescence has been successfully applied in PNSB (Adessi et al., 2012; Ritchie & Runcie, 2013; Ritchie, 2013) owing to structural and functional similarities between the RC of purple bacteria and the RC of photosystem II (PSII) of oxygenic photosynthetic organisms (Adessi et. al, 2012).
31 6.2.1. Principle of pulse- amplitude- modulation (PAM) fluorescence
In order to understand the principles of this technique, differences between fluorescence intensity and fluorescence yield must be established. The former may vary in several orders of magnitude according to light conditions whereas fluorescence yield does vary in less than a factor of 5-6. Moreover, fluorescence yield gives direct information about photosynthesis. Chl fluorometers are capable of measuring fluorescence yield under all situations and one of their most relevant advantages is the capacity to measure fluorescence yield without damaging the cells. The PAM fluorescence has the following characteristics: a very low measuring light to measure the fluorescence yield of a dark- adapted sample; a selective detection system to differentiate between the fluorescence excited by the measuring light and the much stronger signals caused by ambient and actinic light; a very fast time response in order to resolve the changes in fluorescence yield upon dark- light and light-dark transitions (Schreiber, 2010).
In order to properly develop the PAM fluorescence, the instrument must have all conditions cited previously, and it is mandatory to efficiently separate the fluorescence from the much stronger excitation light. It can be achieved using optical filters (Schreiber, 1983; Renger & Schreiber, 1986; Horton & Bowyer, 1990). Moreover, excitation energy is responsible of carrying out photosynthetic reactions (actinic light) in conventional Chl fluorometers. Due to the need of differentiating between fluorescence and ambient light, fluorescence excitation can be modulated either mechanically or electronically and a selective fluorescence amplifier is required to read the modulated signal. In the past, modulation fluorometers with mechanical choppers and lock- in amplifiers had been extensively used in photosynthesis studies (Duysens & Sweers, 1963; Bonaventura & Myers, 1969), but the first fluorometer fulfilling
32 the 3 requirements referred above is the PAM-101 Chl Fluorometer (Schreiber, 1986; Schreiber et al., 1986).
The PAM technique has the following characteristics:
a) Fluorescence measuring light involves very short ( μs- range) pulses; b) At low frequency, the intensity of each pulse can be quite high without
producing an important increase of fluorescence yield;
c) the resulting μs- fluorescence pulses are detected by a photodiode detector characterized by fast response and a great linearity range; d) A system to reject background signals;
e) the pre-amplified signal is further processed by a selective –window- amplifier; which amplifies the difference between the signal during the excitation pulse and the signal a few μs after it; in principle, the difference signal can be “disturbed” only by very rapid changes between the two μs- window periods;
f) the switching on/off of the actinic light and the triggering of discharge flashes is synchronized to be in middle of the dark periods between measuring light pulses;
g) the measuring light frequency can increase automatically upon triggering of actinic illumination and, in that way, rapid induction and relaxation kinetics can be followed.
(Schreiber, 2010)
Light emitting diode (LED, in most PAM fluorometers) and flash discharge lamp (e.g., Xe- PAM) are used to produce the pulse- modulated measuring light. Moreover, LEDs can be modulated at high frequencies (MHz range) and they are economic and easy to handle. On the other hand, flash lamps provide extremely strong measuring light, which is useful when analyzing low Chl samples. Regarding the optical set-up, it is important that all lights, i.e. measuring, actinic,
33 saturation pulse, far- red background light and flash light, are equally distributed over the sample. In order to obtain a homogeneity distribution of light, most PAM fluorometers used multibranched fiber optics. Usually, one branch of the fiber optics is used for transporting the fluorescence signal to the detector whereas the other branches are utilized for controlling the light beams to the sample. It has to be stressed that the distance and angle between the end of the optic fibers and the sample may be modified in order to avoid disturbed ambient light but, at the same time, to contribute in the production of a high signal (Bilger et al., 1995). Most PAM fluorometers have fluorescence detectors constituted of photodiodes. It is an advantage because this kind of detectors has a larger linearity with light intensities. Moreover, they control the background of the signal maintaining it at low level even when the signal level is extremely high. PAM technique is not only useful to determine the fluorescence of Chl, but also to measure other photosynthetic parameters. For instances, changes in P700 can be measured with the same PAM-101 system as Chl fluorescence using special emitter- detector units (Schreiber et al., 1988; Klughammer & Schreiber, 1994, 1998). The same is true for P515 absorbance changes (Klughammer et al., 1998) and NADPH fluorescence changes (Mi et al., 2000). The absorbance changes of cytochromes (Cyt f, Cyt b-563 and Cyt b-559) has also been measured through the creation of a 16- channel LED-array spectrophotometer working in the range of 530- 600 nm (Klughammer et al., 1990). Furthermore, a computer –controlled pulse modulation system for the analysis of photoacoustic signals (Klughammer et al., 1990) has been applied for studying the pulse modulated heat release, O2 –evolution and CO2 –uptake associated with stroma
34 6.2.2. What does Chlorophyll fluorescence yield proof?
As explained previously, PAM fluorometers measure the quantum yield of Chl fluorescence, and each pulse sending to the sample analyses the probability to emit the energy absorbed as fluorescence. During this kind of analysis, it is mandatory to consider the competition between fluorescence emission and photo-chemical energy conversion and non- photochemical energy conversion (heat) (Schreiber U., 2010) (For more details about it, see section 7).
When fluorescence emission competes with photo-chemical energy conversion, two situations are possible: 1) under dark- adapted samples, RCs are opened and fluorescence yield is minimal (Fo) whereas 2) under saturating light,
RCs are closed and fluorescence yield is maximal (Fm). The difference between
Fm and Fo is known as “variable fluorescence”, denoted as Fv. In order to excite
the Chl molecule, it must be illuminated with sufficiently strong continuous light and few seconds later, fluorescence yield rises from Fo to Fm. The resulting
kinetic of fluorescence gives information about one of the most important photosynthetic parameters: electron transport rates (Schreiber, 2010).
As mentioned previously, Chl excitation occurs as a result of light absorption by pigments which channel this energy to the RC, where charge separation takes places. It has to be stressed that under normal physiological conditions, energy conversion at the RC is acceptor side limited (Duysens & Sweers, 1963; Schreiber, 2010). Moreover, there is not a direct proportionality or linearity between fluorescence yield and non- photochemical quenching (NPQ), because the energy received by a partially closed RC may be send to neighboring open centers. The magnitude of non-linearity rises with the degree of connectivity between PSU (Schreiber, 2010).
35 6.2.3. Saturation pulse method of quenching analysis
Fluorescence quenching refers to the obtaining of a maximum fluorescence yield when alternative pathways, i.e. photochemistry and nonradiative energy dissipation into heat, are reduced. In literature, these alternative pathways are known as photochemical quenching (pQ) and NPQ (Schreiber, 2010; Ritchie & Runcie, 2013; Ritchie, 2013), and they refer to the utilization of excitation energy to charge separation at the RC and the dissipation of excess excitation energy as heat, respectively (Schreiber, 2010).
The photosynthetic apparatus of most photosynthetic organisms normally reaches a stable status after dark- adaptation, under regular physiological conditions. This status is characterized by a fully oxidized state of the acceptor side in the PSU and, as a consequence, pQ is maximal and NPQ minimal. In order to determine fluorescence quenching during illumination, two important parameters must be calculated after dark adaption: Fo and Fm. Moreover, the
determination of Fo and Fm in the dark state are fundamental for assessing both
pQ and NPQ, in an illuminated sample by the saturation pulse method (Schreiber, 2010).
In the saturation pulse method, the acceptor side in the photosynthetic apparatus is completely reduced by a saturating pulse and, as a result, pQ is totally repressed. For each saturating pulse a maximum fluorescence yield (Fm’)
and a stable fluorescence yield (F), are reached. It has to be stressed that, using the saturation pulse method, the lowering of Fm is selectively used for evaluating
36 Historically, the quantification of pQ and NPQ has been made through the application of particular coefficients: qQ (control by redox state of acceptor) and
qE (control by energy state) (Dietz et al., 1985; Schreiber et al., 1986). These
coefficients can be quantified in the range between 0 and 1 (Bilger & Schreiber, 1986).
When evaluating Fo’(minimal fluorescence yield obtained after darkened of illuminated sample), it is fundamental the complete oxidation of the primary electron acceptor in order to determine the NPQ. It has to be stressed that under some situations, the determination of Fo’ can be difficult, so that, the
dissipation of energy as nonradiative (heat) can be indicated as the NPQ parameter created by Bilger and Björkman .In this case, there is no need to calculate Fo’ (Schreiber, 2010). The assessment of NPQ considers the presence
of traps for nonradiative energy dissipation (Lavorel & Etienne, 1977; Butler, 1978). It has to be clearly said that the utilization of the saturation pulse method in photosynthesis offers many advantages because both the pQ and NPQ, can be simultaneously assessed. Moreover, it is possible to obtain the optimal quantum yield of energy conversion (which is well ascribed by fluorescence parameters (Fm-Fo)/Fm=Fv/Fm)) and the effective quantum yield (which
correspond to fluorescence parameter (Fm’-F)/Fm’= ΔF/Fm’ (Genty et. at, 1989))
37 6.2.4. Determination of quantum yield
As it is well known, there is a relationship between fluorescence yield and quantum yield. The mathematical equations include the competition between fluorescence, pQ and NPQ, and establish the relation between these 3 parameters. Historically, math derivations have been made in order to understand how these 3 parameters are connected. It has to be mentioned that these equations are based on the “law of energy conservation”.
1) The sum of quantum yields (Ф) of photochemistry (P), fluorescence (F) and NPQ (D) is unity:
ФP+ ФF + ФD= 1 (2)
2) After a saturating pulse, the photochemical quantum yield, ФP , reaches
zero, while the remaining yields assume maximum values:
ФFm + ФDm= 1 (3)
3) As an assumption, the ratio between fluorescence and NPQ remains stable during a short saturating pulse:
ФDm/ ФFm = ФD/ ФF (4)
4) By combining equations 3 and 4, ФD can be expressed in terms of
fluorescence yield:
38 5) By combining equations 2 and 5, ФP can be expressed in terms of
fluorescence yield:
ФP= 1- ФF-ФF/ ФFm + ФF= (ФFm – ФF)/ ФFm (6)
It is possible to calculate the photochemical quantum yield both after dark- adaptation and during illumination. In the first case, the fluorescence yield obtained after a saturating pulse in known as Fm whereas, during illumination,
the fluorescence yield is defined as F, and the maximum yield generated by a saturating pulse is Fm’. The photochemical quantum yield obtained after dark
adaptation, corresponds to the optimal quantum yield. On the other hand, during illumination the quantum yield refers to an effective quantum yield. For the assessment of both quantum yields, the following math equations are used: Optimal quantum yield:
(ФP) max = (Fm-Fo)/ Fm = Fv/Fm (7)
Effective quantum yield:
ФP= (Fm’-F)/ Fm’ = ΔF/Fm’ (8)
