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

نویسندگان

1 کارشناس ارشد فیزیولوژی گیاهی، دانشگاه اصفهان، ایران

2 دانشیار فیزیولوژی گیاهی، دانشگاه اصفهان، ایران

3 استادیار سیستماتیک گیاهی، دانشگاه اصفهان، ایران

4 دانشیار مهندسی شیمی، دانشگاه اصفهان، ایران

5 کارشناس ارشد زیست شناسی سلولی مولکولی، دانشگاه اصفهان، ایران

چکیده

  مقدمه: میکروارگانیسم‏های فتوسنتز کننده به دلیل توانایی‏شان در به دام انداختن دی اکسید کربن، پتانسیل خوبی در تثبیت دی اکسید کربن حاصل از منابع مختلف، از جمله گاز خروجی کارخانه‏های صنعتی، به عنوان منبع کربن دارند. در سال‏های اخیر با افزایش روز افزون گازهای گلخانه ای به ویژه دی اکسید کربن تلاش‏های زیادی برای بررسی امکان کاهش تولید دی اکسید کربن از کارخانجات صنعتی با استفاده از میکروارگانیسم‏ها انجام شده است .   مواد و روش ‏‏ ها: به منظور دستیابی به این هدف، پاسخ رشد سیانوباکتری اسپیرولینا تحت تیمار با غلظت‏های مختلف دی اکسید کربن (036/0 تا 10 درصد) و نیز گاز خروجی از دودکش در مقیاس آزمایشگاهی مطالعه شد. برای ایجاد مقادیر مورد نظر دی اکسید کربن از کمپرسور هوا و کپسول دی اکسید کربن استفاده شد .   نتایج: بالاترین غلظت تولید بیومس در غلظت 8 درصد گزارش شد. همچنین، رشد این سیانوباکتری تحت تیمار با گاز خروجی از دودکش نیز مشاهده شد، اگرچه میزان تولید بیومس در این تیمار تا حدی کاهش یافت. شایان ذکر است محتوی لیپید تام آن با افزایش غلظت دی اکسید کربن از میزان موجود در جو تا 4 و6 درصد به ترتیب 5/14، 8/15 و 16 درصد وزن خشک محاسبه شد. در مقابل محتوی پروتئین تام آن با وجودی که مقدار قابل توجهی به دست آمد، حدود 46 درصد وزن خشک، ولی در تیمارهای مختلف تغییر قابل ملاحظه‌ای مشاهده نشد .   بحث و نتیجه ‏ گیری: هدف از انجام این مطالعه، بررسی امکان رشد این میکروارگانیسم با استفاده از دی­اکسید کربن موجود در گاز خروجی کارخانجات صنعتی بود و نتایج بدست آمده از آن نشان داد که ارگانیسم تحت بررسی پتانسیل خوبی برای تثبیت زیستی دی اکسید کربن از منابع مختلف، به ویژه گاز حاصل از احتراق، داشته و نیز به واسطه محتوی پروتئینی بالا به عنوان کاندید خوبی برای تولید پروتئین تک یاخته مطرح می‌باشد .  

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Growth response of Spirulina platensis PCC9108 to elevated CO2 levels and flue gas

نویسندگان [English]

  • Seyedmahdi Hoseini 1
  • Abas Almodares 2
  • Saeid Afsharzadeh 3
  • Mohamadsadegh Hatami pour 4
  • Fatemah Montazeri 5
1 M.Sc. of Plant Physiology, University of Isfahan, Iran
2 Associated Professor of Plant Physiology, University of Isfahan, Iran
3 Assistant Professor of Plant Systematic, University of Isfahan, Iran
4 Associated Professor of Chemical Engineering, University of Isfahan, Iran
5 M.Sc. of Cell & Molecular Biology, University of Isfahan, Iran
چکیده [English]

  Introduction : Because their ability to capture CO2, photosynthetical microorganisms have some advantages to CO2 mitigation from high CO2 streams such as flue gases and they can use CO2 as carbon source. Recently, experts have made efforts to exploit microorganisms intended for recovering CO2 from power plants.   Materials and method s: To achieve this purpose, we studied the growth response of the cyanobacterium Spirulina platensis PCC9108 under different concentrations of carbon dioxide (ranging from 0.036% to 10%) and flue gas in a bench-scale system. Preparation of different concentrations of CO2 and injection into Erlenmeyer flasks was performed by a system including air compressor, CO2 capsule, pressure gauge and flow meter.   Results : The main goal of studying this paper is a survey of organism's potential to grow by generated CO2 from flue gas of power plant. It already had the potential and highest biomass production recorded at 8% CO2 (v/v). Also we proved that S.platensis PCC9108 can be grown under flue gas, although biomass production decreased fairly. Total lipid content of algae interestingly enhanced with elevated CO2 levels from ambient air to 4% and 6% which ranged from 14.5 to 15.8 and 16 dry weight (wt.) % respectively. In contrast, total protein content illustrated no difference between all treatment and its value was about 46 wt.%.   Discussion and conclusion : The results of present study suggested that understudied S.platensis PCC9108 is appropriate for mitigating CO2 because of its carbon ï‌xation ability. Also due to its high protein content, this cyanobacterium is a good candidate to produce SCP (single cell protein).  

کلیدواژه‌ها [English]

  • Biofixation of CO2
  • Biomass production
  • Flue gas
  • Total lipid content
  • Total protein content
  • Spirulina platensis

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 SOX, NOX 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).

 

 

 

 

CO2 capsule

 

Air compressor

 

heater

 

Pressure gauge

 

microregulator

 

flowmeter

 

Sparging air to erlene mayer flask contained medium

 

Monitor CO2 concentration by CO2 Analyzer

 

(1)

 

filter

 

 

 

Air compressor

 

Pressure gauge

 

microregulator

 

flowmeter

 

Sparging air to erlene mayer flask contained medium

 

Monitor gas components by flue gas Analyzer

 

engine

 

stove

 

(2)

 

filter

 

 

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.

 

Temperature

O2

CO2

CO

NO2

NO

Flue gas component

140.5°C

14.4%

4%

7 ppm

4 ppm

24 ppm

Values

 


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):

Cb (gL−1) = 0.460× OD670 + 0.050 R2= 0.991, where Cb, 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 (μmax, d−1) during the logarithmic phase (17):

µmax = (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 = (Xt − X0) / (t − t0)

where Xt 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 Pmax (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 (Xmax) and maximum productivity (P) values for S. platensis observed at 8% CO2, but about highest maximum specific growth rate (µmax), belonged to 6% CO2. Values for Xmax, µmax 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 Xmax, µmax 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.

CO2 Level (%,v/v)

Xmax (mg/ml)

µmax (day-1)

P (gl-1day-1)

Ambient air

0.55 b

0.27 a

0.073 a

4% CO2

1.16 d

0.35 b

0.123 b

6% CO2

1.30 e

0.41 c

0.151 c

8% CO2

1.75 f

0.27 a

0.163 c

10% CO2

0.22 a

-----

-----

Flue gas

0.98 c

0.28 a

0.086 a

 

3

 


Each value is the mean of three samples. For each parameter, different lowercase letters across a row indicate significant difference between means, while the same letters show insignificant difference (ANOVA, Duncan’s test, p<0.05).

 

Effect of increasing concentrations of CO2 and flue gas on total lipid and protein content

 

From the data obtained in this work, total lipid content in the S. platensis ranged from 13.8 to 16 wt.% and reached to highest amount at 6% CO2. Generally, total lipid content of understudied S. platensis was not changed with increasing CO2 concentration or under flue gas (Fig. 6). Also, as shown in Fig. 6, there was not any difference in total protein content between treatments. However, results have shown that this cyanobacterium has high total protein content ranging from 45.8 to 47.5 wt.%.

 

 

4

 

 

5

Fig. 3-5-Chlorophyll a content (3), Dry weight (4) and Number of cells (5) of S. platensis under different concentrations of CO2 (v/v) (ambient air as control (0.036%), 4%, 6%, 8% and 10%) and flue gas. Each value is the mean of three repeats. Different letters in front of symbols indicate significant difference between means, while the same letters show insignificant difference (ANOVA, Duncan’s test, p<0.05).

 

 

Fig. 6- Effect of elevated CO2 and flue gas on total lipid and protein of S. platensis. Each value is the mean of three repeats. Different letters indicate significant difference between means (ANOVA, Duncan’s test, p<0.05).

 

Discussion and conclusion

 

Algae play a vital role in the biogeochemical cycling of carbon. Although many algae consume different sources of organic carbon, CO2 is the main source of carbon for the majority of them. Growth response of Spirulina platensis PCC9108 in terms of dry weight, number of cells and chlorophyll a showed similar pattern and all suggested best growth observed at 8% CO2 treatment. Morais and colleagues reported best growth of S. platensis at level of 6% CO2 in air, while Andrews and coworkers observed best growth of S. platensis at levels of 6% and 12% CO2 (21, 25). Repeated measures analysis showed that growth rate of alga at first day for control are more than other treatments. However, from second days its growth rate at 8% and 6% CO2 were uppermost. These results suggested that growth of alga inhibited under effect of CO2 – elevated levels at first 24h, but then adapted and grew progressively.

Previously, inhibition of microalgal growth at high CO2 levels reported, but it’s reason is not well illustrated. The general notion is that, the toxicity of a very high CO2 concentration is due to the lowering of pH and increase in the algal performance at higher levels of CO2 may be because of the enhanced availability of dissolved CO2 (5). Interestingly, some researchers have reported that there was no inhibition at elevated concentrations of CO2. For instance findings by Geoghegan reveal that the growth rates of C. vulgaris were similar whether the algae were grown in 5% CO2 or high CO2 concentrations such as 20% CO2 in air. Also, work by Morais and Costa (21) shows that C. vulgaris grown in vertical tubular photobioreactor actually grows better in 12% CO2 than in 6% CO2 in air.

As results shown, S. platensis used in this study grew well under flue gas. Partially inhibition in growth possibly is due to toxic effect of CO, SOx and NOx or high temperature. SOx and NOx can have a significant effect on the growth rates and health of the microalgae. Of greatest concern is the effect of them on the pH of the microalgal growth medium (26). For algae to utilize CO2 from power plants, it needs to tolerate concentrations of about 15 to 20% CO2 in air (27). Most authors found that at these CO2 concentrations the growth of microalgae is completely or partially inhibited (7). However it has been shown that the inhibition can be alleviated by adaptation to CO2. The inoculums of alga adapted to 5% CO2 resulted in enhanced algal growth in 15% CO2 compared with culture grown by ambient air. Also, algae can be adapted to high CO2 concentration by gradual increases of CO2 concentration (28).

Our results shown total lipid and protein content of understudied S. platensis, not changed so much under effect of diverse CO2 concentration and flue gas. Work by Chiu et al. (2009) shows an increase in the biomass production and lipid accumulation with a CO2 concentration increase in the aeration of Nannochloropsis oculata cultures. Likely, increase of lipid content affected by CO2 levels is a strains specific character (29). Richmond mentioned that, cellular contents of cyanobacteria (eg. Spirulina platensis) were not changed by varying environmental conditions, compared with eukaryotic microalgae (17). However, this alga is an excellent candidate for producing single cell protein (SCP) due to its high protein content. Also, understudied S. platensis due to potential of biomass production under high CO2 concentration and flue gas is a good candidate for CO2 biofixation and decreasing atmospheric CO2. Morovere, produced biomass could be utilized for the marketable production of several chemicals which are either unique or found at relatively high concentrations, such as phycocyanin, myxoxanthophyl and zeaxanthin (30).

 

Acknowledgments

 

This study was supported by grants of the University of Isfahan, Iran.

 

 

 

References

(1) Czerny J, Barcelos Ramos J, Riebesell U. Influence of elevated CO2 concentrations on cell division and nitrogen fixation rates in the bloom-forming cyanobacterium Nodularia spumigena. Biogeosciences. 2009; 6: 1865-75.

(2) Brennan L, Owende P. Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sust Energ Rev. 2010; 14(2): 557-77.

(3) Kadam KL. Power plant flue gas as a source of CO2 for Microalgae cultivation: economic impact of different process options. Energ Convers Manage. 1997; 38(6): 505-10.

(4) Hsueh HT, Chu H, Yu ST. A batch study on the bio-fixation of carbon dioxide in the absorbed solution from a chemical wet scrubber by hot spring and marine algae. Chemosphere. 2007; 66(5): 878-86.

(5) Chinnasamy S, Ramakrishnan B, Bhatnagar A, Das KC. Biomass Production Potential of a Wastewater Alga Chlorella vulgaris ARC 1 under Elevated Levels of CO2 and Temperature. Int J Mol Sci. 2009; 10(2): 518-32.

(6) Wang B, Li Y, Wu N, Lan CQ. CO2 bio-mitigation using microalgae. Appl Microbiol Biot. 2008; 79(5): 707-18.

(7) Chiu SY, Kaoa CY, Chen CH, Kuan TC, Ong SC, Lin CS. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresource Technol. 2008; 99 (9): 3389-96.

(8) Maeda K, Owada M, Kimura N, Omata K, Karube I. CO2 Fixation from the Flue Gas on Coal-Fired Thermal Power Plant by Microalgae. Energ Convers Manage. 1995; 36(6/9): 717–20.

(9) Brown LM. Uptake of Carbon Dioxide from Flue Gas by Microalgae. Energ Convers Manage. 1996; 37(6/8): 1363-67.

(10) Patil V, Tran K, Giselrad HR. Towards sustainable production of biofuels from microalgae. Int J Mol Sci. 2008; 9(1): 1188-95.

(11) Jacob-Lopes E, Scoparo CHG, Franco TT. Rates of CO2 removal by Aphanothece microscopica N¨ageli in tubular photobioreactors. Chem Eng Process. 2008; 47: 1365–73.

(12) Yoo C, Jun SY, Lee JY, Ahn CY, Oh HM. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresource Technol. 2010; 101: 71–4.

(13) Mata TM, Martins A, Caetano NS. Microalgae for biodiesel production and other applications: A review. Renew Sust Energ Rev. 2010; 14(1): 217–32.

(14) Kodama M, Ikemoto H, Miyachi S. A new species of highly CO2 -tolerant fast growing marine microalga suitable for high density culture. J Mar Biotechnol. 1993; 1: 21-5.

(15) Morais MG, Costa JAV. Carbon dioxide fixation by Chlorella kessleri, C vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors. Biotechnol Lett. 2007a; 29 (9): 1349-52.

(16) Sydney EB, Sturm W, de Carvalho JC, Thomaz-Soccol V, Larroche C, Pandey A. Potential carbon dioxide fixation by industrially important microalgae. Bioresource Technol. 2010; 101(15): 5892-96.

(17) Richmond A. Handbook of microalgal culture: biotechnology and applied phycology. 3rd ed. Oxford: Blackwell Science Ltd., 2004.

(18) Soletto D, Binaghi L, Ferrari L, Lodi A, Carvalho JCM, Zilli M, et al. Effects of carbon dioxide feeding rate and light intensity on the fed-batch pulse-feeding cultivation of Spirulina platensis in helical photobioreactor. Biochem Eng J. 2008; 39(2): 36975.

(19) Morais MG, Radmann EM, Andrade MR, Teixeira GG, Brusch LRF, Costa JAV. Pilot scale semicontinuous production of Spirulina biomass in southern Brazil. Aquaculture. 2009; 294(1): 60-4.

(20) Andrade MR, Costa JAV. Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate. Aquaculture. 2007; 264(1-4): 130-34.

(21) Morais MG, Costa JAV. Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor. J Biotechnol. 2007b; 129(3): 439-45.

(22) Li-Hai Fan, Ya-Tao Zhang, Lin Zhang, Huan-Lin Chen. Evaluation of a membrane-sparged helical tubular photobioreactor for carbon dioxide biofixation by Chlorella vulgaris. J Membrane Sci. 2008; 325(1): 336-45

(23) Takagi M, Karseno YT. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella Cells. J Biosci Bioeng. 2006; 101(3): 223-26.

(24) Barbarino E, Lourenco SO. An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae. J Appl Phycol. 2005; 17(5): 447-60.

(25) Andrews R, Liu K, Crocker M, Crofcheck C, Shea A. Feasibility of Capture & Utilization of CO2 from Kentucky Power Plants by Algae Systems. 2008; Final Report. Retrieved from http://www.docin.com/p-106293599.html

(26) Stepan DJ, Shockey RE, Moe TA, Dorn R. Carbon dioxide sequestering using microalgal systems. 2002; Retrieved from http://www.osti.gov/scitech/servlets/purl/882000

(27) Hanagata N, Takeuchi T, Fukuju Y, Barnes DJ, Karube I. Tolerance of microalgae to high CO2 and high temperature. Photochemistry. 1992; 31(10): 3345-48.

(28) Yun Y, Park JM, Yang J. Enhancement of CO2 tolerance of Chlorella vulgaris by gradual increase of CO2 concentration. Biotechnol Tech. 1996; 10(9): 713-16.

(29) Lv JM, Cheng LH, Xu XH, Zhang L, Chen HL. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresource Technol. 2010; 101(17): 6797-804.

(30) Vonshak A. Spirulina platensis (Arthrospira) Physiology, cell-biology and biotechnology. London: Taylor & Francis, 1997.