Reference : Conception et réalisation d'un photobioréacteur adapté à l'étude de la production d'h...
Dissertations and theses : Doctoral thesis
Life sciences : Phytobiology (plant sciences, forestry, mycology...)
Conception et réalisation d'un photobioréacteur adapté à l'étude de la production d'hydrogène chez les micro-algues. Application à l'étude de l'influence d'une NAD(P)H déshydrogénase-plastoquinone-réductase chloroplastique de type II sur le potentiel de photoproduction d'hydrogène chez Chlamydomonas reinhardtii.
[en] Conception and building of a photobioreactor adapted to the study of the production of hydrogen in microalgae. Application to the study of the influence of a Type II NAD(P)H dehydrogenase plastoquinone reduction chloroplast on the potential of hydrogen photo-production in Chlamydomonas reinhardtii.
Mignolet, Emmanuel [Université de Liège - ULiège > > Labo de Bioénergétique >]
Université de Liège, ​Liège, ​​Belgique
Docteur en Sciences
Franck, Fabrice mailto
[en] photobioreactor ; hydrogen ; Chlamydomonas reinhardtii
[en] Conception and building of a photobioreactor adapted to the study of the production of hydrogen in microalgae.
Application to the study of the influence of a Type II NAD(P)H dehydrogenase plastoquinone reduction chloroplast on the potential of hydrogen photo-production in Chlamydomonas reinhardtii.

Context and abstract of the thesis

This project is part of a research framework to better understand the process of hydrogen photobioproduction in microalgae.
Overall, we can say that hydrogen photobioproduction depends on the set of biochemical reactions that define cellular energy metabolism. In Chlamydomonas, H2 photobioproduction is ensured by two enzymes (Hyda1 and Hyda2). These two enzymes are located in the chloroplast stroma, they catalyze the reaction of hydrogen production (Fig. 1). The electrons required for the synthesis of H2 come from two different metabolic pathways, situated upstream of the PSI and CytB6f. The transport of electrons between these two paths and the CyB6f is via the plastoquinones (PQ) pool. The first pathway for the reduction of PQ involves the photolysis of water via photosystem II, known as the PSII-dependent pathway. The second reduction pathway is thought to involve an enzyme catalyzing the oxidation of NAD(P)H, and the reduction of PQ. Thought to be located on the stroma side of the membrane, it is known as the PSII-independent pathway. Succinctly, the PSII-dependent pathway could be considered as a contribution to the reductant supply from light energy and the PSII-independent pathway as a contribution to the reductant supply from starch, or more generally, from cellular carbon substrates.
The existing situation: the NAD(P H-type II (Ndh-II) are enzymes capable of oxidizing NAD (P) H and transferring electrons to a quinone molecule (plastoquinone or ubiquinone). They are known as type II by analogy with mitochondrial complex I. As regards the chain of mitochondrial electron transport, NDH-II proteins constitute an alternative pathway to complex I and II to bring electrons to the ubiquinone pool. This alternative pathway would enable an adaptation to the yield of the electron transport chain based on the metabolic state of the algae.
As regards the chloroplast electron transport chain, Ndh-II proteins are thought to participate in several mechanisms of adaptation to light intensity variations. Their function would be for the non-photochemical reduction of the PQ pool.
In 2005, seven ''open reading frames'' corresponding to hypothetical type II NAD(P)H dehydrogenase (NDA1 to NDA2) were identified in the nuclear genome of Chlamydomonas.
A study of Chlamydomonas NDA gene expression highlighted the most important expression of the NDA2 gene. In order to specifically study the role of the NDA2 enzyme, it was inactivated by RNA-interference. (Fred Jans thesis 2011).
From the wt84 strain (Chlamydomonas reinhardtii), two lines of mutant deficient Nda2 were obtained:
G4: Nda2-RNAi (1)
P11: Nda2-RNAi (2)
If we consider these lines from a phenotypic point of view, two main theoretical hypotheses can be made:
is there a significantly decreased reduction of the PQ pool by the PSII-independent pathway?
is there an affected, reduced or even completely inhibited hydrogen photobioproduction?

In order to study the effect of the deficiency of the enzyme (Nda2) on the potential for hydrogen production, a set of tools and specific experimental protocols had to be developed to meet the second hypothesis (above).
The aim of this project was mainly be to develop the study of modifications in hydrogen production.

Purpose and specificity of the thesis:
The purpose and specificity of this project was to break away from complex technical difficulties:
knowing that the two mutant lines of Chlamydomonas reinhardtii were wall-less cells and therefore, highly sensitive to hydrodynamic stress;
knowing that the study of hydrogen photobioproduction by microalgae was based on the capacity of degassing the culture medium, i.e. the need to obtain optimal hydrodynamic agitation favoring gas exchange.

The main challenge was therefore to develop a system to find a consensus technique in order to resolve the contradiction between preserving the fragile algae while causing sufficient hydrodynamic agitation to degas the culture medium.

In 2005, experimental tools for the study of hydrogen production by microalgae were poorly developed.

The majority of H2 photoproduction experiments were conducted in glass bottles according to the Melis protocol.

The Melis protocol is based on a temporal separation of two phases:

the first is a phase of growth of microalgae in a culture medium that is optimal for photosynthesis and the accumulation of reserves (starch);

the second phase begins after the transfer of the microalgae into a sulfur depleted medium. This procedure consists of causing biochemical stress, inducing reorganization and metabolic adaptation which will eventually lead to hydrogen photobioproduction.

Although it is sufficient for a basic approach, the experimental procedure of bottle culture has several major drawbacks when it comes to studying the processes of hydrogen production over long periods (100 to 200 hours).

In the bottles, the homogenization of the culture medium is conducted using magnetic stirrers moved by rotary shakers. To obtain good homogenization, a minimum speed of agitation is necessary. This process induces a relatively large hydrodynamic stress, resulting in significant cell death.
On the other hand, microscope observation of Chlamydomonas cells grown in sulfur depleted medium deficient shows that the majority of algae lose their flagella and thus their natural mobility. This phenomenon reinforces the idea that magnetic stirrers severely limit the quality of experimental results: the agitation necessary for good homogenization must be more vigorous in sulfur depleted medium than in an optimal medium.

Another major problem encountered in experiments using bottles is the quality of degassing. Generally, the analysis of gas products is done by gas chromatography; samples are taken in the gas phase, and then analysed.

This method is unsuitable for this type of experiment.

Is the gas phase in equilibrium with the liquid phase?
Is there a risk of over or under-saturation of hydrogen?
Is there an influence from pressure?
Is there a risk of inhibiting the production of hydrogen by its own concentration in the medium?
Does periodic and repeated sampling induce disturbances in how the reaction takes place?

In order to solve these uncertainties and technical difficulties, a new and original concept of algal culture has been developed.

The basic principle of this system involves placing a suspension of algae (culture medium) in a photobioreactor made of two transparent tubes connected at their ends. The photobioreactor is fixed on an oscillating table. A glass ball placed in one of the tubes slides because of the force of gravity, and this enables the homogenization of the medium.

In addition to obtaining optimal homogenization without hydrodynamic stress (by three different types of mixing), a geometric feature of the whole has enabled active degassing of the hydrogen produced in the culture medium.

Parallel and in annex to this photobioreactor (PBR), a system enabling the measurement and analysis in real time of the concentration of hydrogen in the gas phase has been developed. This system is based on the principle of polarographic analysis of oxygen concentration developed by Leland C. Clark. The O2 and H2 concentrations can be measured by a periodic reversal of the voltage across the electrodes.

Finally, a theoretical model of evolution and equilibrium of hydrogen concentration in the PBR has been developed to monitor and compare the experimental results.

The findings:

The new concept of microalgae culture developed in this project has solved several problems.
The homogenization of the culture medium by mixing the phases and stirring with a glass ball is a particularly effective alternative compared to mixing with the magnetic stirrers commonly used in bottles. Cell death by hydrodynamic stress can be reduced to virtually zero, whereas experience has shown that in this field, mixing using magnetic stirrers is a relatively harmful process.
The original system of mixed phases with active degassing (at atmospheric pressure) has resulted in superior yields of hydrogen being produced than in other systems.
The real-time analysis of changes in concentrations of oxygen and hydrogen via the probes has increased the accuracy of the results.

It was observed that the variation of several experimental factors could lead to significant distortions between the theoretical model of evolution of the concentration of hydrogen (depending on the gas collected in the gasometer) and the measurements made using gaseous chromatography (GC). Those factors are the following:

the quantity of culture medium in the reactor;
he geometry of the interface between gas phase and liquid phase;
he speeds of the mixing cycles;
the pressure of the PBR;
dynamic permeability and porosity;
changes in production rates of hydrogen in different strains of microalgae.

Because of these factors, changes in the experimental protocol were required:

fixing an interval of time (within each experiment) to obtain the speed ​​of stable production;
a study on the influence of pH, which helped validate the experimental range usually used;
a study on the influence of hydrogen concentration on the rate of production.

These changes have not only increased measurement accuracy, but have also confirmed certain hypotheses concerning a probable inhibition of hydrogen production by its own concentration in the culture medium.

Confirmation of the inhibition of hydrogen production by its own concentration in the culture medium is undoubtedly the most important piece of information provided by the latter optimization of the protocol.

Parallel to this, the observation of significant discrepancies between the theoretical model of the evolution of hydrogen concentration (depending on the gas collected in the gasometer) and the measurements made by gas chromatography (GC), result in the latter (GC) seeming to be unreliable in this type of experiment.

The contribution of hydrogen / oxygen probes in the field of hydrogen photobioproduction by microalgae

Developing of the system of measuring O2/H2 probes by time multiplexing (periodic alternation of the polarity probe) has provided new insights into the possibilities of monitoring the evolution of hydrogen concentration in real time. Although these probes were made by hand and used without the real purpose of gaining precise quantification, their potential in this area of ​​research is clearly visible. It is obvious that developing a similar system, but more accurate in terms of electrode geometry, would allow the calibration and enhancement of measurements (from a quantitative point of view). Besides this, an optimization of surface characteristics of the electrodes would also improve performance and increase capacity of use. A perfectly sealed probe head could work in the liquid medium and directly provide the values ​​of hydrogen concentration in the liquid phase.

It is likely that this method of measuring hydrogen concentration by polarography, may in future replace the current method of gas chromatography.

Taking all these elements into account:

In the semi-closed PBR, the main problem for obtaining the values ​​of metabolic fluxes to study is keeping the characteristics of the gas exchange in well-defined operating range (pressure, H2 concentration and mixing dynamics). It is demonstrated in this paper that this procedure is relatively easy to carry out: the only constraint is to precisely define the characteristics of the PBR and the theoretical models.
In the second part of the project, the photobioreactor is used to develop and complete a protocol analysis to characterize the relative contributions of the two metabolic pathways involved in hydrogen photobioproduction (the chloroplast electron transport chain) in the microalgae Chlamydomonas reinhardtii.
Two publications have been written regarding the biochemical approach of the second part; interested readers can find there all the results and comments that this project has brought to the field.

Follow-up to, and the future of, this project:

Thanks to the PBR and its multi-adaptability, an original approach to the theoretical modeling of the metabolism of hydrogen production is possible if the following are known:

the various flux values ​​of the metabolic pathways;
the volumes of hydrogen produced;
production rates based on metabolic configurations;
the speed and quantity of starch consumed;
the speeds of mitochondrial respiration and their involvement in hydrogen production (via PEDO2).

By standardizing and unifying the various units of measurement used to characterize the flow and the amounts of material as well as the electrochemical potentials and the gradients, through which the matter passes, it should be possible to construct a matrix model according to Kirchhoff's laws.

From this model, it should be possible to deduce other values and other metabolic constants without having to necessarily pass through experimentation.
This model would also, by successive iterative approaches and extrapolation, determine new optimal operating ranges for the PBR.
Laboratoire de bioénergétique
Production d'hydrogène par les micro-algues
Researchers ; Professionals ; Students ; General public

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