The biomass production with the systematic culture of photosynthetic microorganisms used Spirulina maxima 2342 (UTEX collection). These microorganisms was cultivated under illumination conditions of 156 W, which

were provided by 4 fluorescent lamps of 39 W. Stirring and air bubbling conditions were also used for obtaining a total biomass volume of 20 L. A standard mineral was used as a culture medium [19] . In addition, general features or composition of Spirulina maxima 2342 is determined.

Duplicate experiments at a laboratory temperature of 25˚C were systematically carried out by using home- made glass biomass photobioreactors (Figure 2). After 25 days of incubation, a sample volume of biomass (250 mL) was collected and concentrated by a filtration process. The quality of the photosynthetic biomass samples was monitored in terms of the microorganism-biomass quality index parameters: dry-weight content of biomass and Chlorophyll a concentration, according to the methodology suggested by Arnon in 1949 [20] . Digital photographs of Spirulina maxima (UTEX-2342) cells are shown in Figure 2. Such images were recorded with a digital camera (NIKON) coupled to an optical microscope HITACHI KP-D50 and a scanning electron microscope (SEM).

Physicochemical properties of the photosynthetic biomass samples were also measured. The optical density (OD) of the biomass samples were measured by spectrometry using a DU 650 Beckman spectrophotometer calibrated at a wavelength of 750 nm, whereas the pH measurements were performed by a 430 Corning pH-meter. Dry weight of the biomass samples was determined after filtration through a filter paper Whatman (diameter 47 mm). The remaining residue collected on the filter paper was also dried in an oven at 75˚C for 24 h.

Chlorophyll-a [Chla] concentration (in mg) of biomass was spectrophotometrically determined by measuring the absorbance responses at 645 nm (A645) and 663 nm (A663) using 15 mL of methanol (as solvent) and 10 mL of sample (as aqueous solution). Chlorophyll-a [Chla] concentration (in mg) was subsequently calculated by means of the equation suggested by Arnon in 1949 [20] :

According to the work methodology described in Figure 1, the hydrogen production from Spirulina maxima 2342 was conducted by a mixed process of anaerobiosis and darkness. The experiment was carried out in two identical home-made glass biomass photobioreactors at a laboratory temperature of 25˚C. The biomass photobioreactors have a sample volume of 300 mL, which were equipped with a vacuum/pressure Teflon^® stopcock [17] both for facilitating the sample collection and to preserve the sample without air contamination for its later chemical analysis. These photobioreactors were initially set under vacuum conditions for filling the biomass samples. A volume of 250 mL of biomass culture was added to each photobioreactor, and 2 g of sodium dithionite were finally added to promote the oxygen removal process, which inhibits the hydrogenase enzyme that affect the production of hydrogen.

Sodium dithionite (also known as sodium hydrosulfite, Na₂S₂O₄) is a white crystalline powder with a weak sulphurous odour. It can be obtained from sodium bisulphite through the reaction: 2NaHSO₃ + Zn → Na₂S₂O₄ + Zn(OH)₂∙Na₂S₂O₄ is usually recommended as reducing agent for redox and solubility reactions [21] [22] .

To ensure a homogeneous mixing of the biomass sample, a stirring process for 5 min was additionally performed. The photobioreactors were then placed into a dark anaerobic process by passing an argon gas stream (industrial grade) of 3 ± 0.1 mL∙s⁻¹ for 1 h to increase the removal of dissolved oxygen.

After the anaerobiosis/dark process, a vacuum process was again applied to the photobioreactor sample during 5 min for trying to complete the oxygen removal, which was difficult to achieve due to the dynamics of the photosynthesis process.

To have a photoproduction of hydrogen (at low concentrations by gas chromatography coupled with TCD), with high performance, a stirring-illumination process with a 100 W lamp, a light intensity of 150 µE∙s⁻¹∙m⁻², and a reaction time of 19.3 ± 1.2 h were needed. A picture showing some details of the experimental system used for the photoproduction of hydrogen gas is presented in Figure 2.

To have a statistical reproducibility of the hydrogen photoproduction process, three experimental runs with 2 replicates for each one were carried out. To verify the hydrogen gas photoproduction, the biomass reactors were connected to a gas chromatograph (GC) system equipped with a thermal conductivity detector (TCD) for a reliable detection and quantification of the hydrogen gas produced with the biomass samples. The GC system was previously calibrated with hydrogen standards of high purity. Calibration curves were specifically prepared, which were subsequently used to quantify the amount of hydrogen produced with the experimental system.

In this stage, the production of the photosynthetic biomass of Spirulina maxima 2342 as a fundamental raw material of the process was obtained. After applying the photosynthetic experimental conditions (previously described), the Spirulina maxima 2342 biomass samples were efficiently and systematically produced. According to the microorganism-biomass quality index measurements, the Spirulina maxima 2342 samples presented the following average properties: a dried biomass of 0.34 ± 0.02 g; a pH of 10.19 ± 0.05; and Chlorophyll a (mg) and Chlorophyll a/biomass concentrations of 2.5 ± 0.3 mg and 7 ± 1 mg∙g⁻¹, respectively. According to the quality index parameters reported by Oswald in 1977 [23] , the Chlorophyll a/biomass concentrations measured in the samples falls within the expected values for such photosynthetic microorganisms (between 3 and 10 mg∙g⁻¹).

In this stage, it was found that the photosynthetic microorganisms simultaneously co-produced hydrogen and oxygen gases in a very similar way over the experimental time. This co-production of oxygen affected the amount of hydrogen produced in the photobioreactor because it inhibited the performance of the hydrogenase enzyme [24] . To remove the oxygen produced by the microorganisms, and therefore to solve this co-production problem, sodium dithionite were added as reducing agent. This optimum dose of sodium dithionite enabled the oxygen removal from the anaerobiosis/dark process to be successfully achieved.

Detection/quantification of hydrogen at low concentrations by TCD-GC system used in this experimental study is shown in Figure 2. This analytical system was calibrated for measuring the hydrogen concentrations with precision and accuracy. Praxair^® gas standards of high purity (99.9999%) were used for the calibration of the TCD-GC. A standard gas mixture containing 5% He, 10% H₂, 10% Ar, 45 % N₂ and 30% CH₄ was used. The operating conditions of the GC system are summarized in Table 1.

Typical chromatograms obtained from the injection of the hydrogen standards are shown in Figures 3(a)-(e). A similar separation pattern was consistently achieved during the hydrogen analysis of samples obtained from the three production experiments. From these chromatograms, it can be observed that the hydrogen gas was acceptably eluted from the rest of the gases with average retention times of 1.47 min and the same are increasing in size in increasing the injection pressure observed.

Gas standards at total pressures of 2, 5, 10, 20 and 40 mm Hg were injected into the TCD-GC with some replicates (n = 3). According to the composition of the standard mixture, five calibration points with partial pressures of hydrogen (0.2, 0.5, 1.0, 2.0 and 4.0 mm Hg) were processed together with their respective detection responses (or peak areas). Calibration curves between TCD responses (in peak area units) and partial pressure data were then prepared for the analysis of the gas samples obtained from the three production experiments (Figures 4(a)-(c)). The linear regression equations are also included in such figures.

Table 2 summarizes both the values of the independent (partial pressure measurements) and dependent (TCD responses) variables of the TCD-GC analyses, together with the precision data. The calibration curves were then used to quantify the hydrogen generated by the photosynthetic microorganisms from the peak areas obtained from the gas samples. These partial pressure values, molar fractions and concentration units were calculated by

using the equation of the ideal gas law. This quantitative methodology was valid due to the low work pressures, restricted condition for such a general equation. Hydrogen detections in culture samples Spirulina maxima 2342 were for total injection pressures with minimum of 91 mm Hg and up to 367 mm Hg.

Example of a typical chromatogram obtained from the hydrogen production experiments is shown in Figure 2. Hydrogen detection is confirmed by means of the peak observed at a retention time of 1.44 min. The typical chromatogram of hydrogen detection is obtained during the injection of gas samples at an injection pressure of 258 mm Hg (Experiment 3).

A summary of the experimental results obtained at H₂ injection pressures in the standard gas between 0 and 40 mm of Hg is shown in Table 3. It was obtained a ratio of 21 × 10⁻¹⁰ mol of H₂ h⁻¹/2.5 mg of Chlorophyll a in our experiments with biomass of Spirulina maxima 2342, in the experimental conditions established and in two phases of experimentation. The first one, production of photosynthetic biomass and then inducing the H₂ production by anaerobiosis/darkness and the second the hydrogen photoproduction with a reaction time of 19.3 ± 1.2 h.

This method of obtaining photosynthetic hydrogen in 2 phases has been demonstrated in this work and has also been reported by other authors. For example, Nath and Das in 2004 [25] , confirmed that the integration of a fermentation process with photosynthesis is a way to improve the hydrogen productivity. Skizim, et al. in 2012 [26] used Cyanothece sp. Miami BG 043511 cultivated in ASP2 medium at 30˚C without inorganic nitrogen combining 12 h light, 12 h darkness and with a light intensity of 30 µE s⁻¹∙m⁻². The maximum sustained rate of hydrogen production in Cyanothece sp. Miami BG 043511 in 2.5 h reported by Skizim et al. in 2012 [26] was 16.4 µmol of H₂ per g dry weight⁻¹∙h⁻¹ (15.8 mL of H₂ liter⁻¹∙h⁻¹). Rocheleau in 2000 [27] obtained 0.11 μmol∙g⁻¹ with Spirulinaplatensis darkness/anaerobiosis fermentation in laboratory work, confirming that Cyanobacteria produces hydrogen by dark fermentations, but not in the light phase.

Environmental and nutritional conditions that optimize the yield of hydrogen (H₂) from water using a two- step photosynthesis/fermentation (P/F) process was reported for the hypercarbonate-requiring cyanobacterium “Arthrospira maxima” by Ananyev et al. in 2008 [28] , in relation to fermentative H₂ production by bacteria. Anaerobic H₂ production in the dark from whole cells catalyzed by a bidirectional [NiFe] hydrogenase is demonstrated to occur in two temporal phases involving two distinct metabolic processes that are linked to prior light-dependent production of NADPH (photosynthetic) and dark/anaerobic production of NADH (fermentative), respectively.

Borodin et al. in 2002 [29] reported that higher rates for the same strain grown in a photobioreactor where actively growing cells were held at a chlorophyll concentration of 4 µg/mL (6.3 mL of H₂ liter⁻¹∙h⁻¹ or 70 µmol of H₂ mg of Chl⁻¹∙h⁻¹). In Cyanothece sp. ATCC 51142, when supplemented with 50 mM glycerol extremely high rates of 373 and 465 µmol of H₂ per mg of Chl⁻¹∙h⁻¹ have been reported [30] .

Dutta et al. in 2005 [31] reported 167.6 µmol of H₂ per mg of Chl⁻¹∙h⁻¹ for Anabaena variabilis sp. ATCC 29413 PK84 mutant, that they are significantly higher than the maximum rates published to date for both heterocystous and diazotrophs. The hydrogen production in Arthrospira maxima via bidirectional hydrogenase was only 3.1 µmol of H₂ per mg of Chl⁻¹∙h⁻¹ reported by Carrieri et al. in 2008 [32] .