2. Material And Methods
2.1. Strain and inoculum maintenance
The strain of Chlamydomonas reinhardtii CC425 (cw15 arg2 sr-u-2-60 mt+) was from the National Renewable Energy Laboratory. The strain was maintained in axenic culture Tris-acetate-phosphate, TAP medium [27], with arginine supplementation (100 mg L− 1), at 24 ± 0.5 ºC, pH 7.2, 12 h light/dark cycle, and light intensity of 60 µmol m− 2 s− 1 obtained with cool white fluorescent tubular lamps in an air-conditioned room. These conditions were used as the control in the biomass optimization experiment for the first experimental step.
2.2. Experimental design and hydrogen production
The experiments were carried out in triplicate simultaneously in an incubator with photoperiod and temperature control (411 FPD Ethiktechnology). The strain was cultivated under two experimental steps in TAP medium [27] with arginine supplementation (100 mg L− 1). The first step was utilized to obtain the biomass needed for the second step.
In order to improve the biomass yield in the first step, a factorial design with a central condition (24 + 1) was used to generate a matrix with 17 experiments. Experiments were carried out at two levels of temperature (24 and 32°C), pH (6.2 and 8.2), light intensities (200 and 500 µmol m− 2 s− 1), and with or without glucose addition (0 and 1 g L− 1), as well as a central condition in the intermediate level of these 4 parameters (28°C, pH 7.2, 350 µmol m− 2 s− 1 and 0.5 g L− 1 of glucose). The experiment was performed with batch cultures in triplicate using glass tubes closed with screw lids (20 mL) the stationary phase was stabilized.
According to the results of the first step for the biomass optimization, the culture was maintained under aerobic growth conditions (continuous illumination of 200 µmol m− 2 s− 1 with a cool white lamp; pH 6.2 and 32 ± 0.1 ºC) in 500 mL Erlenmeyer flasks with 300 mL of culture, until the middle of the exponential phase.
The second step was performed to induce hydrogen production through the anaerobic phototrophic process in batch cultures under either sulfur deprivation or sulfur limitation. The biomass of Erlenmeyer flasks with 300 mL of culture from the first step was centrifuged and suspended in TAP medium in 500 mL Duran glass bottles with 300 mL of culture and sealed with a butyl cover (photobioreactor). The temperature and pH from the first step were maintained, and there was a variation of the concentration of the sulfur (0 or 50 µmol L− 1) and light intensity (60 or 200 µmol m− 2 s− 1), as shown in Table 1. The biomass at the end of the first step was centrifuged in 33.3 Hz for 10 min, washed twice with culture medium selected for each condition, and suspended in the respective photobioreactors. The atmospheric conditions of the photobioreactors were changed by adding nitrogen gas for 10 min, previously sterilized using a filter with 0.2 µm of porosity. Experiments were carried out until stabilization of H2 production.
Aliquots of 500 µL of gas of headspace were collected every 12 h using a syringe and valve (push button valve 22285) to measure the gas composition by gas chromatography (Shimadzu GC-2010) using a thermal conductivity detector (TCD) and argon as a carrier gas. The results obtained are shown in moles of hydrogen and were calculated based on the regression method with calibration curves (r2 = 0.996). The hydrogen production curves are shown in µmol L− 1 of hydrogen/mg L− 1 of chlorophyll a.
|
Culture |
Temperature |
pH |
Sulfur |
Light |
Condition |
|
Medium |
(ºC) |
(µmol L− 1) |
(µmol m− 2 s− 1) |
||
|
TAP |
32 |
6.2 |
0 |
60 |
1 |
|
organic carbon |
200 |
2 |
|||
|
source: |
50 |
60 |
3 |
||
|
acetic acid |
200 |
4 |
Furthermore, other parameters were calculated from the data, such as the maximum production of hydrogen per chlorophyll a (in µmol L− 1 of H2 / chlorophyll a in mg L− 1, represented by Pmax) and per mol (in mmol of H2 / liter of culture, represented by H), hydrogen volume (milliliters of H2 per liter of culture, represented by VolH2), which were calculated using the ideal gas equation, and hydrogen production rate (µmax) by Gompertz mathematical model [28] (Eq. 1). Equation H represents the accumulated production of hydrogen (µmol H2 L− 1), t is the hydrogen production time (hours), and e is Euler's number (2.71828). The equation results in parameters A (hydrogen production potential in µmol L− 1), µmax (hydrogen production rate in µmol H2 L− 1 h− 1) and K (lag phase time). The specific hydrogen production rate, µspecific (µmol H2 mg Chl a− 1 h− 1), was calculated from the µmax divided by the initial biomass in chlorophyll a (mg L− 1) of the photobioreactor.
(1)
2.3. By-product production and nutrient uptake
The ethanol and organic acid concentrations, such as acetic acid, were detected at the end of the second step using gas chromatography in a Shimadzu GC-2010/FID with an HP-INNOWAX capillary column [29]. The ammoniacal nitrogen and phosphate concentrations, as well as chemical oxygen demand (COD) were analyzed from samples taken from the initial photobioreactor and at the end of the hydrogen production process. Nutrient uptake was calculated based on the percentage of the difference between the initial and final concentrations of these compounds. The nutrient analyses were carried out according to APHA [30].
2.4. Biomass and biochemical analysis
The biomass of factorial design in the first step was measured by weighing the suspended solids [30] and the yield was calculated based on the difference between the final and initial biomass values. Biomass of the photobioreactors was estimated at the point when the algae were transferred from the 1st to the 2nd experimental step. The biomass, in terms of cell density was measured by cell counting using a Fuchs Rosenthal chamber in an Olympus BX5 microscope [30], and by the chlorophyll a amount, using the extraction method with 80% ethanol [31, 32]. Samples of 3 mL were taken from each photobioreactor for the analyses, and 0.5 mL of the samples were fixed with Lugol's acetic solution and stored in the dark until cell count.
The biochemical characterization of dry biomass was performed with the samples from the initial photobioreactor (sacrifice reactor) and at the end of the hydrogen production phase. The dry biomass was obtained by centrifuging the cultures followed by heat treatment in an oven at 60 ºC for 24 h of the pelletized biomass until a constant weight was achieved. Carbohydrates were determined using the phenol colorimetric method [33] and the total protein was estimated by the total nitrogen analysis TKN [30] with a protein conversion factor of 4.71 [34].
2.5. Analysis of results
The Protimiza Experimental Design software was used to analyze the experimental design of optimization of C. reinhardtii biomass in the first step and to find the parameters that have had positive effects on the biomass yield through the Pareto Chart with a significance level of 5%. Furthermore, the Statistica 7.0 software was used for the Analysis of Variance (ANOVA) and Tukey post-hoc tests to differences among the biomass yields. The best biomass yield was selected combining the higher biomass yield averages with Pareto Chart.
The results of the hydrogen production parameters were processed using the Gompertz mathematical model according to Zwietering et al. [28] in the statistical software Origin Pro 8.0. Analysis of Variance (ANOVA) and Tukey post-hoc tests were used to assess the differences among the hydrogen production parameters, such as the hydrogen production rate, maximum production of hydrogen, as well as biomass yields by chlorophyll a and cell density. Furthermore, the same statistical comparisons were made of the amount of nutrient uptake (ammoniacal nitrogen, phosphate, COD, and acetic acid) and ethanol produced. The T-Student test was used to compare the initial and final carbohydrate and protein amounts of the biomass in the photobioreactors. We adopted p < 0.05 to indicate the significant differences.