Hydrogen (H 2 ) production from experiments with Spirulina maxima 2342 is reported in this work. The performance of this photosynthetic microorganism for producing H 2 was evaluated for the first time under specific experimental conditions (e.g., a biomass concentration of 0.34 ± 0.02 g, a light intensity of 150 μE . s-1 . m-2 and reaction times of 19.3 ± 1.2 h). The performance of this photosynthetic microorganism for producing hydrogen was successfully improved by the addition of sodium dithionite (a reducing agent) as an innovative method for increasing the gas production, and as a main contribution of this work. Quantitative gas chromatography (GC) analyses of H2 to verify the production performance were successfully carried out at low concentration levels. GC analyses were performed by means of a conventional thermal conductivity detector coupled to a separation system of a Molecular Sieve column 500 mm × 3175 mm (L × ID). Low detection limits were consistently obtained with the GC system used. The separation of H2 in culture samples was efficiently achieved in average retention times of 1.47 min. The H2 produced in this process was subsequently used for power generation using a Proton Exchange Membrane Fuel Cell (PEMFC).
The limited supply of fossil fuels and their strong environmental impact prompt the use of unconventional energy sources to face the future energy demand of the world [
Photo-biological production of H2 has advanced significantly in recent years, and it is on the way to becoming a mature technology as an energy vector. A variety of photosynthetic and non-photosynthetic microorganisms, including unicellular green algae, cyanobacteria, anoxygenic photosynthetic bacteria, obligate anaerobic, and nitrogen-fixing bacteria have been used for the H2 production [
Solar energy can be converted into chemical energy in the form of H2 gas by using oxygenic and anoxygenic photosynthetic microbes. Major challenges, such as inhibitory amounts of oxygen produced, during oxygenic photosynthesis, and inhibition of H2-producing nitrogenase by ammonia, are being studied in genetic engineering experiments [
Cyanobacteria are photosynthetic prokaryotes that are promising “low-cost” microbial cell factories due to their simple nutritional requirements, metabolic plasticity, and availability for their genetic manipulation [
Some species of cyanobacteria produce hydrogen in a natural way as a byproduct from either a dark anaerobic fermentation or a photosynthetic process using fixed-carbon compounds which are produced in daylight under aerobic conditions [
On the other hand, analytical methodologies for detecting/quantifying the hydrogen production in photo- biological reactions using Spirulina maxima 2342 have been rarely reported in the literature (e.g., [
In this paper, a new experimental application of the Spirulina maxima 2342 for the production of H2 is reported. The performance of this photosynthetic microorganism for producing H2 was successfully improved under some particular experimental conditions. The main objective of this work is to report quantitatively the production of hydrogen from the use of Spirulina maxima 2342 by using controlled experimental conditions of the following key processes: biomass production, anaerobiosis/dark process and the detection/quantification of hydrogenproduction at low concentrations by GC coupled with TCD. Details of the experimental work methodology and the results are outlined.
The experimental work methodology used for the production of hydrogen from Spirulina maxima 2342 microorganisms is schematically shown in
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 [
Duplicate experiments at a laboratory temperature of 25˚C were systematically carried out by using home- made glass biomass photobioreactors (
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 [
According to the work methodology described in
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 [
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
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.
The performance of the Spirulina maxima 2342 as aphotosynthetic microorganism for producing hydrogen was successfully improved by the addition of sodium dithionite (a reducing agent) as an innovative method to increase the gas production. Such an improvement was achieved by means of the following experimental stages: biomass production: the systematic culture of photosynthetic microorganisms where an oxygenic photosynthesis to CO2 fixation in the photobioreactor was promoted; anaerobiosis/dark process, where the microorganisms are exposed to a synthesis of hydrogenase enzyme (to start the hydrogen production from carbon hydrates stored under dark conditions), followed by an illumination process to complete the H2 production; and the detection/ quantification of hydrogenproduction at low concentrations by TCD-GC.
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 [
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 [
Detection/quantification of hydrogen at low concentrations by TCD-GC system used in this experimental study is shown in
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.
Chromatography Column | Mol Sieve GC column (5 m × 3.175 mm) |
---|---|
Carrier gas Carrier gas flow Detector temperature Filament temperature Column temperature | Argon Ar Chromatographic grade: 99.998% purity (INFRA) 30 mL∙min−1 110˚C 140˚C 60˚C |
Total injection pressure (mm Hg) | Partial injection pressure (mm Hg) | Retention time (min) | Peak area (counts) | Precision error (%) |
---|---|---|---|---|
2 5 10 20 40 | 0.2 0.5 1.0 2.0 4.0 | 1.48 ± 0.01 1.48 ± 0.01 1.48 ± 0.01 1.47 ± 0.00 1.44 ± 0.01 | 4827 ± 469 11,766 ± 236 17,079 ± 2177 35,657 ± 1133 68,533 ± 2597 | 9.7 2.0 12.7 3.2 3.8 |
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
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
Ptotal (N∙m−2) | Vtotal (m3) | PH2 (N∙m−2) | VH2 (m3) | Xi | nH2 (mol) | nH2 (mol∙h−1) |
---|---|---|---|---|---|---|
87017.91 | 4.4 × 10−5 | 22.7 | 6.6 × 10−8 | 0.0015 | 6.1 × 10−10 | 21 × 10−10 |
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 [
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 [
Borodin et al. in 2002 [
Dutta et al. in 2005 [
It demonstrated the H2 production from Spirulina maxima 2342. The production involves two different metabolic processes which are linked: the light-dependent production(photosynthetic) and the anaerobiosis/darkness (fermentative) and the hydrogen gas was detected under established experimental conditions in a gas chromatograph with TCD obtaining 21 × 10−10 mol of H2∙h−1/2.5 mg Chlorophyll a and with a reaction time of 19.3 ± 1.2 h.
The authors wish to thank Dr. Portugal, E., Sánchez, D. and Betancourt, V. of the Electrical Research Institute, Cuernavaca, Morelos, Mexico, for the technical help. This work was carried out with financial support through the project CONACYT INF224765.
A. U. Juantorena,E. Santoyo,O. Lastres,G. Hernández,A. Bustos,S. A. Gamboa,P. J. Sebastian, (2016) Gas Chromatography as an Analytical Monitoring Technique for Hydrogen Production from Spirulina maxima 2342. Green and Sustainable Chemistry,06,78-87. doi: 10.4236/gsc.2016.62007
nH2: mole number of hydrogen
PH2: hydrogen partial pressure [N・m−2]
VH2: hydrogenpartial volume [m3]
T: absolute temperature [K]
R: universal gas constant [N・m・mol−1・K−1]
Xi: gas molar fraction
Ptotal: total pressure of the gas mixture [N・m−2]
Vtotal: total volume of the gas mixture [m3]