Photosynthesis

Photosynthesiscan be defined as a redox reaction driven by light energy, in which carbon dioxide and water are converted into metabolits and oxygen. Photosynthesis is traditionally divided into two stages, the so-calledlight reactionsand thedark reactions. The first process is thelight-dependent process (light reaction), which occurs in the grana and requires the direct energy of light to make energy carrier molecules that are used in the second process. The light-independent process (or dark reaction) occurs in the stroma of the chloroplasts, where the products accumulated in the products of the light reaction are used to form C-C covalent bonds of carbohydrates. The dark reactions can usually occur if the energy carriers from the light process are present.

In the light reactions, light strikes chlorophyllain such a way as to excite electrons to a higher energy state. In a series of reactions, the energy is converted (along an electron transport process) into ATP and NADPH. Water is split in the process, releasing oxygen as a byproduct of the reaction. The ATP and NADPH are used to make C-C bonds in the dark reactions.

In the dark reactions, carbon dioxide from the atmosphere (or water for aquatic and marine organisms) is captured and reduced by the addition of hydrogen to form carbohydrates ([CH2O]_n). The incorporation of carbon dioxide into organic compounds is known ascarbon fixation. The energy comes from the first phase of the photosynthetic process. Living systems 69

4.1 INTRODUCTION

cannot directly utilize light energy, but they can, through a complicated series of reactions, convert it into C-C bond energy that can be released by glycolysis and other metabolic processes. So, the main role of the light reactions is to provide the biochemical reducing agent NADPH2and the chemical energy carrier (ATP) for the assimilation of inorganic carbon, as presented in Equation4.1:

2NADPþ3H2Oþ2ADPþ2Pi$2NADPH2þ3ATPþO2 ð4:1Þ The fixation of carbon dioxide happens in the dark (in the stroma of chloroplasts) using the NADPH2and ATP produced in the light reaction of photosynthesis (Equation4.2):

CO2þ4Hþ2NADPHþ3ATP$ðCH2OÞ ð4:2Þ Carbon dioxide is available in water in three different forms: CO2, bicarbonate (HCO₃), or carbonate (HCO₃²) (Figure 4.1), the relative amounts of which are pH dependent. Although plants and algae are known to be dependent exclusively on the Calvin-Benson-Bassham cycle (also known as theCalvin cycle) (Atomi, 2002), six autotrophic carbon-fixation pathways are known. These are (1) the Calvin cycle, (2) the acetyl-CoA pathway, (3) the 3-hydroxypropionate cycle, (4) the reverse tricarboxylic acid cycle, (5)3-Hydroxypropionate/4-hydroxybutyratecycle, and (6) Dicarboxylate/4-hydroxybutyrate cycle (GeorgeFuchs, 2011). This section discusses the Calvin cycle, which is the most important in microalgae.

In the Calvin cycle there is only one enzyme responsible for CO2 fixation: ribulose 1,5-biphosphate carboxylase/oxygenase, also known asRubisco.Figure 4.2shows the Calvin

CO₂(g) CO₂(aq) H₂CO₃ HCO₃^- CO₃^2- FIGURE 4.1 Different forms in which carbon dioxide is available in water.

3 Ribulose-bis-P 3ADP + 3P

3ATP

6 Glycerate-P 3 Ribulose-P

5 Glyceraldehyde-P

6 Glyceraldehyde-P

Organic compounds production

6ATP 6ADP + 6P

6NADPH

6NADP⁺ + 6P 6 Glycerate bis-P CALVIN CYCLE

CO₂ FIGURE 4.2 The dark process of CO2 capture and transformation through metabolism of photosyn- tethic microalgae (modified from Masojı´dek et al., 2004).

70 4. RESPIROMETRIC BALANCE AND CARBON FIXATION OF INDUSTRIALLY IMPORTANT ALGAE

cycle, where one molecule of ribulose 1,5-biphosphate and a CO2 are converted into two glycerate phosphate. CO2diffuses through the cell and is captured by the enzyme ribulose biphosphate (Rubisco).

CO₂ g

$CO₂ð Þ $aq H₂CO₃$HCO₃ $CO₃² The fixation of CO2occurs in four distinct phases (Masojı´dek et al., 2004):

1. Carboxylation. A reaction whereby CO2is added to the five carbon sugar ribulose bisphosphate (Ribulose-bis-P) to form two molecules of phosphoglycerate (Glycerate-P).

This reaction is catalyzed by the enzyme ribulose biphosphate carboxylase/oxygenase (Rubisco).

2. Reduction. To convert Glycerate-P into 3-carbon sugars (Triose-P), energy must be added in the form of ATP and NADPH2in two steps, which are the phosphorylation of Glycerate-P to form diphosphoglycerate (Glycerate-bis-P) and the reduction of Glycerate-bis-P to phosphoglyceraldehyde (Glyceraldehyde-P) by NADPH2.

3. Regeneration.Ribulose-P is regenerated for further CO2fixation in a complex series of reactions combining 3-, 4-, 5-, 6-, and 7-carbon sugar phosphates, which are not explicitly shown in the diagram.

4. Production. The primary end products of photosynthesis are considered to be carbohydrates, fatty acids, amino acids, and organic acids.

Besides the carboxylase activity described here, all Rubiscos (there is more than one type) are known to display an additional oxygenase activity in which an oxygen molecule, com- peting with CO2 for the enzyme-bound eno-diolate of RuBP, reacts with RuBP to form 3-phosphoglycerate and phosphoglycolate (Atomi, 2002). The latter product is subse- quently oxidatively metabolized via photorespiration, leading to a net loss in carbon dioxide fixation. Photorespiration thus represents a competing process to carbon fixation, where the organic carbon is converted into CO2without any metabolic gain. Photorespiration depends on the relative concentrations of oxygen and CO2where a high O2/CO2ratio stimulates this process, whereas a low O2/CO2ratio favors carboxylation. Rubisco has low affinity by CO2; itsKm(half saturation) is approximately equal to the level of CO2in air. Thus, under high irradiance, high oxygen level, and reduced CO2, the reaction equilibrium is shifted toward photorespiration. For optimal yields in microalgal mass cultures, it is necessary to minimize the effects of photorespiration, achieved by an effective stripping of oxygen and by CO2

enrichment. For this reason, microalgal mass cultures are typically grown at a much higher CO2/O2 ratio than that found in air, which is in turn an opportunity to reuse industrial gas emissions.

The source of nitrogen in cultivation of microalgae seems to cause changes in oxygen production during photosynthesis. The ratio between O2evolution rate and CO2uptake rate (the photosynthetic quotient, PQ) depends on the composition of the produced biomass and the substrates that are used. Especially oxidized nitrogen sources, which must be reduced before they are incorporated into the biomass, affect the PQ. When nitrate is used, it is expected at an evolution of 1.3 mol O2per mol of CO2assimilated, whereas nitrite promotes a release of 1.2 mol O2and ammonia 1.0 mol O2(Eriksen et al., 2007). Approximately 20%

of O2evolution equivalents can be accounted for by NO₃ uptake and assimilation under N-replete conditions (Turpin, 1991).

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4.1 INTRODUCTION