پاسخ اسپیرولینا پلاتنسیس به رشد در سطوح افزایش یافته دی‏اکسید‏کربن و گاز خروجی

Introduction

Considerable anthropogenic emissions caused atmospheric CO₂ (carbon dioxide) concentrations to get higher from an interglacial level of 280 ppm to presently 385 ppm (1). Currently, main sources of energy are fossil fuels which combustion of them is responsible for increased CO₂ levels (2, 3, 4). More than 170 countries have ratified the Kyoto protocol of the United Nations to make efforts for reducing greenhouse gases by 5.2% on the basis of the emissions in 1990 (5). One of the most understudied methods of CO₂ reduction is the use of microalgae that convert CO₂ from a point source into biomass (6, 7). These microorganisms have some advantages to CO₂ mitigation such as their ability to capture CO₂ from high CO₂ streams like flue gases (4, 8 & 9) and more efficiency for converting solar energy into biomass rather than terrestrial plants (10). Numerous studies have suggested that marine cyanobacteria are good choice to achieve this purpose (11). The anthropogenic emission of CO₂ from coal-fired thermoelectric plants is responsible for up to 7% of global CO₂ (12). Industrial exhaust gases contain up to 15% CO₂ providing a CO₂-rich source for microalgae cultivation and a potentially more efficient route for CO₂ biofixation (13). Although earlier it was assumed that these microorganisms are susceptible to high CO₂ concentration, some microalgae are now reported to grow rapidly at this condition (14, 15). Utilization of this source faced to some challenges such as high temperature, decreasing growth of some microalgae in elevated CO₂ and the effect of toxic gases included flue gas such as SO_X, NO_X and CO on the growth of these organisms (8, 9).

Microalgae can fix carbon dioxide from different sources including CO₂ from the atmosphere, from industrial exhaust gases and in form of soluble carbonates (16). As microalgae react to concentrations of CO₂ differently, a bench-scale experiment is necessary for investigating response of selected microalgae to elevated CO₂ concentration. The aims of this study were evaluation of growth response of a cyanobacterium, Spirulina platensis PCC9108 to different CO₂ concentrations and emissive flue gas from a stove. The response was monitored in terms of changes in number of cells, dry weight of biomass and chlorophyll content. We investigated the effect of CO₂ and flue gas on total lipid and protein content, too.

Materials and Methods

Microorganisms and cultivation media

The microalgae Spirulina platensis PCC9108, was provided by the Pasteur Institute, France. Cells were cultivated in the modified culture medium including BG-11 and ASN-III with ratio 1:1 (v/v) (17). As the aim was investigation of different CO₂ concentrations effect on S. platensis growth, modified medium without carbon source, NaHCO₃, was used (18).

Cultivation conditions and aeration system

All runs were carried out using a working volume of 400 ml in 500ml Erlenmeyer flasks and initial biomass concentration was 0.1 gL^-1 with OD ca. 0.13 at 670 nm (19, 20). Discontinuous light with 16h light:8h dark photoperiod was provided by means of four 40W-fluorescent lamps and periodically confirmed by an illuminance meter, model LEYBOLD 666 230, providing a light intensity of 3200 lux (lx) at the vessel’s surface (21). In order to reduce the long lag phase, cultures cultivated in enriched air with 1% CO₂ for a week before inoculation (12, 21).

Preparation of different concentrations of CO₂ and injection into Erlenmeyer flasks performed by a system including air compressor, CO₂ capsule, pressure gauge and flow meter. The pressure of injected air was formerly adjusted to 100 mbar by microregulator -composed of several pressure regulators stabilizing the pressure step by step- and lastly flowmeter set up flow rate of the input air to culture medium (Fig. 1). Finally, projected CO₂-enriched air was monitored by CO₂ analyzer (model Guardian D-600). Using above system, growth of S.platensis investigated under five concentrations of CO₂ (v/v) (ambient air as control (0.036%), 4%, 6%, 8% and 10%). In order to study flue gas effect on S. platensis, effluent way of a working stove connected to suction gate of air compressor (Fig. 2). Flue gas components was determined by flue gas analyzer (Testo model t350 XL). Table 1 lists some average characteristics of the flue gas during the sampling interval. All runs were aerated at a rate of 0.25 volume of intended air per volume of media per minute (v/vm) (7).

Fig. 1, 2- Schematic diagram of aeration system, (1) for preparing different concentrations of CO₂ and injects it into microalgal culture media, (2) for injection of flue gas from stove to microalgal culture media.

Table1-data from analysis of flue gas injected into S. platensis media.

Analytic determinations

Triplicate samples of culture media were collected aseptically every 24h and pH was also controlled by digital pH meter (CONSORT) at the same time. The biomass concentration (X, gL⁻¹) was estimated spectrophotometrically by measuring the optical density at 670 nm using a spectrophotometer (model SECOMAM XS5 Uvi light), and converted using a calibration curve to the dry weight (22):

C_b (gL⁻¹) = 0.460× OD₆₇₀ + 0.050 R²= 0.991, where C_b, is the biomass concentration. A same calibration curve was also provided to measuring number of cells as another growth parameter to verify biomass concentration that was (22):

N = [71.67 × OD₆₇₀ - 1.329] × 10³ R² = 0.99, where N is the number of cells per ml of culture media.

Biomass analysis

For extracting chlorophyll, 1ml of sample centrifuged for 5min at 13000 rpm and pellet were washed two times. One ml ethanol was added and incubated at 70°C in water bath for 20 min. Samples were centrifuged again (13000 rpm) and finally measured absorbance of supernatant at 650 and 665 nm (5). Chlorophyll content was measured with under equation(17):

Chlorophyll a (mg L^-1) = 16.5 (A₆₆₅) – 8.3 (A₆₅₀)

For lipid extraction, the microalgal cells were collected by centrifugation of a 30-mL sample of culture at 3000 g for 15 min. The cells were washed twice by dissolving pellet in deionized water and centrifuging again. Sample was precipitated in methanol/chloroform (2/1, v/v) and homogenized for 15 min to disrupt cells. Chloroform and 1% NaCl were added to mixture to obtain a ratio of methanol, chloroform, and water of 2:2:1. Chloroform phase was collected by centrifuging. Chloroform was evaporated under vacuum in a rotary evaporator to remove organic solvent. The residual substances from evaporation was weighted as lipid and divided to dry weight of sample (23).

Total protein was determined according to Bradford assay (1976) using bovine serum albumen standards. For cell disruption, 5 ml lysis buffer (including 2% v/v Triton x-100, 1% w/v SDS, 100mM NaCl, 10mM Tris-HCl and 1mM EDTA) was added to the sample, vortex with glass bead for 10 min and at last incubated at 90°C in water bath for 10 min. Protein precipitation was followed using two proportions of cold 25% and 10% trichloroacetic acid (TCA). Precipitated protein was suspended in 0.5 mL 1.0 N NaOH for the Bradford assays (24). All samples for lipid and protein content analysis harvested at the end of linear phase.

Kinetic parameters

The number of cells per ml was used to calculate the maximum specific growth rate (μ_max, d⁻¹) during the logarithmic phase (17):

µ_max = (ln N₂ – ln N₁) / t₂ – t₁

where N is the number of cells and t represents the time in which samples were taken.

Productivity (P, g L⁻¹ d⁻¹) was obtained using the below equation (17):

P = (X_t − X₀₎ / (t − t₀)

where X_t is the biomass concentration (g L⁻¹) at t (d) and X₀ is the biomass concentration at inoculation (t₀). Maximum productivity during cultivation was designated P_max (g L⁻¹ d⁻¹).

Statistical analysis

The experimental results were evaluated by comparison of the growth curves and analysis of variance (ANOVA) of the kinetic parameters, significance being tested by the Duncan test at Sig<0.05. Repeated measures analysis of variance tests were used for comparison of growth parameters in different treatments during 7 days.

Results

Growth response to elevated CO2 levels

The highest chlorophyll a content (21.86 µg mL^-1), dry weight (1.75 mg mL^-1) and number of cells (294×10³ per ml) of S. platensis, which were 8, 4 and 3 times more than that of alga under ambient CO₂, respectively. Maximum chlorophyll a content was recorded at 6% CO2, although maximum cell number and dry weight were observed at 8% CO₂ level (Fig. 3-5). Interestingly, understudied S. platensis showed a sharp increase in chlorophyll synthesis and biomass production when the CO₂ concentration was raised from ambient concentration to 8%. However, growth of algae and chlorophyll synthesis inhibited under treatment by 10% CO₂.The highest maximum biomass concentration (X_max) and maximum productivity (P) values for S. platensis observed at 8% CO₂, but about highest maximum specific growth rate (µ_max), belonged to 6% CO₂. Values for X_max, µ_max and P of algae in the presence of different concentrations of carbon dioxide and flue gas are shown in Table 2.

Growth response to flue gas

Although, growth of Spirulina platensis PCC9108 under flue gas decreased somewhat, but in the highest chlorophyll a content was ca. 7 times more than that of alga under ambient CO₂ (Fig. 3). Also, number of algal cells for treatment with 4%, 6% and 8% CO₂ and flue gas were in same statistical level (Fig. 5). However, obtained biomass (dry weight) is lower than that recorded for 8% CO₂ (Fig. 4). Productivity and specific growth rate of algae were lower than that gained for all runs. Values for X_max, µ_max and P of algae under treatment with flue gas are listed in Table 2.

Table 2- Effect of elevated CO₂ and flue gas on growth parameters of S. platensis. All parameters calculated for logarithmic growth phase.