01. Studies on the Growth OfChlorella Vulgarisin Culture Media

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StudiesontheGrowthofChlorellavulgaris inCultureMediawithDifferentCarbonSources

M. otari,a,* J. Golob,a M. Bricelj,b D. Klinar,c and A. Pivecca University of Ljubljana, Faculty of Chemistry and Chemical Technology,

Department of Chemical Technology,Akereva cesta 5, 1000 Ljubljana, Slovenia

bNational Institute of Biology, Vena pot 111, 1000 Ljubljana, Sloveniac Scientific Research Centre Bistra Ptuj, Slovenski trg 6, 2250 Ptuj, Slovenia

Dedicated to the memory of Professor Dr. Valentin Koloini

Diminishing oil reserves, rising oil prices and a significant increase in atmosphericcarbon dioxide levels have led to an increasing demand for alternative fuels. Microalgaehave been suggested as a suitable means for fuel production because of their advantages

related to higher growth rates, higher photosynthetic efficiency and higher biomass pro-duction, compared to other terrestrial energy crops. During photosynthesis, microalgaecan fix carbon dioxide from different sources, including the atmosphere, industrial ex-haust gases and soluble carbonate salts. To determine the most optimal conditions for thegrowth of Chlorella vulgaris in order to produce lipids that can be transformed into

biodiesel fuel, different nutritional conditions were investigated. For this purpose, threemedia, namely Jaworskis medium, an enriched solution from modified Dual Solvay pro-cess and natural mineral water, were prepared and analyzed for biomass production,chlorophyll content and lipid content. The best growth resulted in an enriched solutionfrom the modified Solvay process. This medium was diluted in different dilution ratios(1:100, 1:50, 1:10) and the best results were obtained in a medium diluted in a 1:10 ratioon the fifth day of culturing (3.72 106 cells mL1; 4.98 mg mL1 chlorophyll a).

Key words:

Carbon dioxide, microalgae, Chlorella vulgaris, batch culture, lipid content

Introduction

There is a growing consensus among govern-ments, scientists, and industrial organizations of de-veloped countries, about the threat of climatechanges due to the greenhouse effect caused byenormous emissions of anthropogenic carbon diox-ide (CO2) into the atmosphere.1

A variety of strategies can be adopted to limitand reduce CO2 emissions. These include improv-ing the efficiency of energy production, substitutingcarbon-rich fossil fuels such as coal and oil, withnatural gas and other energy sources that containless carbon or are carbon-free, and developing tech-nologies to capture CO2 in view of reutilizationand/or sequestration. Concerning the latter ap-proach, there is a range of potentially attractivetechnologies for CO2 capture based on physical andchemical absorption methods, cryogenic and mem-brane separation processes, and biological fixation.2

Biological CO2 approaches have drawn much

attention because they lead to production of bio-

mass energy in the process of CO2 fixation throughphotosynthesis.3 Plants and photosynthetic microor-ganisms are capable of performing this process.They use solar energy to convert CO2 and waterinto biomass.

Microalgae, a group of fast-growing unicellularor simple multicellular microorganisms, offer sev-eral advantages, including higher photosynthetic

efficiency, higher growth rates and higher biomassproduction compared to other energy crops.2,4,5,6

Microalgae can fix CO2 from different sources,which can be categorized as CO2 from the atmo-sphere, CO2 from industrial exhaust gases, andfixed CO2 in the form of soluble carbonates(NaHCO3 and Na2CO3).

2 Many microalgal strainshave been reported to have the ability to accumu-late large quantities of lipids. Allard and Templier(2000) have extracted lipids from a variety of fresh-water and marine microalgae and reported that thelipid content varied from 1 to 26 % (C. vulgaris1422 %).7 Nitrogen limitations were observed toinduce the increase of lipid content in somechlorella strains, including C. vulgaris (57.9 %).8,9

Traditional analysis of lipid content in biological

M. OTARI et al., Studies on the Growth of Chlorella vulgaris in , Chem. Biochem. Eng. Q. 23 (4) 471477 (2009) 471

Original scientific paperReceived: May 15, 2009

Accepted: October 12, 2009

* Corresponding author: E-mail: [email protected]; Phone: +386 2 7480256


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samples was conducted by solvent extraction andgravimetric determination. This method has someadvantages because of the simplicity of procedures,inexpensiveness as regards equipment and chemi-cals, but on the other hand, organic solvents are re-quired. Lately, supercritical fluid extraction as analternative extraction method is being introduced.10

Its main disadvantages are high investment costsfor the required equipment.

Microalgae Chlorella vulgaris was chosen as asubject for this research due to its easy growth in arelatively low-priced media without the necessity ofutilizing very specific compounds and its signifi-cant lipid content, as lipids are the most desirablecomponent from the energy point of view. Manydifferent studies have been conducted with thisstrain.1114

The Solvay process, employing a dual alkaliapproach, uses ammonia as catalyst to aid the reac-tion of CO2 with sodium chloride for the productionof sodium carbonate. The reaction of the Solvayprocess can be represented with the following equa-tion:

NaCl + H2O + NH3 + CO2 NaHCO3 + NH4Cl

After the reaction, sodium bicarbonate, whichis fairly insoluble, is separated by filtration. Sodiumcarbonate is subsequently obtained by heating so-dium bicarbonate. The ammonia is recovered by re-acting ammonium chloride with lime, Ca(OH)2,where limestone serves as the source of lime.

2NH4Cl + Ca(OH)2 2NH3 + CaCl2 + 2H2O

Among others, the use of limestone for the re-generation of ammonia renders the process ineffec-tive, mainly because of the consumption of lime-stone, the production of CO2, and extensive energyrequirement during calcination. In the Solvay pro-cess, for every two moles of CO2 that is capturedfrom power plants, one mole of CO2 from the calci-nation of limestone is released.15

In this work, we focused merely on the firststep of the Solvay process in which bicarbonate andammonium ions are formed. Both these ions areused as a source of carbon and nitrogen for the nu-trition of microalgae.

The purpose of this work was to establish thegrowth ofC. vulgaris in a medium where bicarbon-ate ions serve as a source of CO2 for photosynthe-sis. Besides carbon, other nutrients essential for al-gal growth were added.16 An investigation wasbased on batch experiments carried out in a lab inorder to optimize the medium composition for thebest microalgal biomass yield, and consequently forthe economic efficiency of the process if used in anup-scale for biofuel production.

Materials and methods

Microalgal culture and preparationof inoculum

The culture of Chlorella vulgaris (SAG211-12) was purchased from the Collection of AlgalCultures at the University of Gttingen (Germany).The culture ofC. vulgaris from agar slants was asepti-cally inoculated into several 200 mL Erlenmeyerflasks containing 150 mL of liquid Jaworski me-dium. The medium consisted of the following com-ponents (per litre of distilled water): 36 mgNa2HPO4 12H2O, 80 mg NaNO3, 12.4 mg KH2PO4,20 mg Ca(NO3)2 4H2O, 50 mg MgSO4 7H2O,15.9 mg NaHCO3, 2.25 mg EDTAFeNa, 2.25 mgEDTANa2, 2480 mg H3BO3, 1390 mg MnCl2 4H2O,

1000 mg (NH4)6Mo7O24 4H2O, 40 mg cyanocobala-min (B12), 40 mg thiamin HCl (B1) and 40 mg biotin.The flasks were exposed to direct sunlight and roomtemperature until they reached sufficient density ofmicroalgae for inoculation in further experiments.

Culture media

The first set of experiments was carried out inthree different media: the aforementioned Jaworskimedium, the solution from the modified Solvayprocess, and natural mineral water DonatMg

(Rogaka Slatina, Slovenia).

Preparation of solution from the modifiedSolvay process

During the preparation of this culture medium,we used only the first step of the Solvay process;therefore we named it the pure solution from themodified Solvay process. In a weak solution of so-dium chloride (2 g L1) in distilled water, the am-monia was blown in until pH value reached 10.7.Then, carbon dioxide was blown in the alkaline so-lution until the desired pH value of 6.8 wasreached.

An enriched solution from the modified Solvayprocess differed from the pure solution in the sup-plements added. These supplements were all con-stituents of Jaworskis medium, with the exceptionof NaHCO3. The effect of bicarbonate concentrationon growth, lipid content and chlorophyll contentwas investigated by dilution of the enriched solu-tion from the modified Solvay process. Dilutions atratios 1:10, 1:50 and 1:100 were applied in the sec-ond set of experiments.

Analysis of the solution from the modified

Solvay process

According to the ISO 5664 (1984)17 method,the initial concentration of NH4

+ was 1282 mg L1.

472 M. OTARI et al., Studies on the Growth of Chlorella vulgaris in , Chem. Biochem. Eng. Q. 23 (4) 471477 (2009)


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The ratio of bicarbonate and carbonate in thereaction solutions and the concentrations of bicar-bonate and carbonate were determined from the13C NMR spectra of the reaction solutions. 13C NMRspectra were recorded on a Brucker DPX 300 spec-trometer operating at 75.475 MHz with 17480scans. First, the standard solution was prepared from13.10 mL H2O, 0.79 mL D2O, 71.4 mg NaHCO3and 44.9 mg acetone. The f = 1.78 resulted fromthe 13C NMR spectrum of the standard solution.The known amount of the acetone was added to thereaction solution before measuring of the 13C NMRspectra. About 10 % of D2O was added for theNMR lock. Since the resonance of pure bicarbonateis at 160.82 ppm and the carbonate at 168.24 ppm,1

the percentage of the bicarbonate in the bicarbo-nate/carbonate mixture was calculated as:

% HCO 3-

= 100(168.24-d(sample))/(168.24-160.82),

where d(sample) is the chemical shift of the bicar-bonate-carbonate 13C NMR resonance of the reac-tion solution.

The concentrations of carbonate and bicarbon-ate C(HCO3/CO32) in the reaction solutions werecalculated using the following equation:18

C(HCO3/CO3

2) =

= 1.78 C(acetone) I(HCO3/CO32)/0.5 I(acetone),

where C(acetone) is the concentration of theacetone added to the reaction solution, andI(HCO3/CO32)/I(acetone) is the ratio of intensitiesof the resonance of bicarbonate-carbonate and themethyl resonance of the acetone.

Preparation of culture mediumwith natural mineral water

The natural mineral water DonatMg contains thefollowing cations, anions and non- disintegratablecompounds (per litre of DonatMg): 1.05 mg NH4

+,

3.3 mg Li+

, 1500 mg Na+

, 13 mg K+

, 1030 mg Mg+

,380 mg Ca2+, 6.8 mg Sr 2+, < 0 . 1 m g F e2+,0.17 mg Mn2+, 0.23 mg F, 59 mg Cl, 0.29 mg Br,0.08 mg I, < 2 m g N O3, < 0.007 mg NO2,2400 mg SO42, < 0.02 mg HPO4,, 7700 mg HCO3,16.6 mg metaboric acid, 156 mg metasilicic acid,approximately 3800 mg dissolved carbon dioxide.This medium was prepared according to Golob(1998).19 By mixing in a vessel with a large enoughsurface, the dissolved carbon dioxide was elimi-nated. The natural mineral water was diluted withdistilled water at a 1:2 ratio. Then, 6 g CaCl2 6H2Oand 0.5 g NH

4Cl per litre of natural mineral water

were added. This medium was diluted with distilledwater at a 1:50 ratio, and 80 mg NaNO3 and 36 mgNa2HPO4 12H2O per litre were added.

Culture conditions

Experiments were carried out in 3000 mLErlenmeyer flasks containing 1750 mL of each indi-vidual medium with initial inoculum of approxi-

mately 105

cells mL1

. The alga was grown in batchculture in a growth chamber at 25 C under 12/12hour light/dark cycles. Illumination was providedusing PAR fluorescent lights. The light intensitywas between 100120 mE m2 s1. The flask con-tents were continuously stirred with a magnetic stir-rer at 150 rpm. Samples were collected every 24, 48or 72 hours, depending on the sets of experiments.The cell number, chlorophyll content, and lipidcontent were monitored.

Microalgal cell counting and dry weight

A direct microscopic count (cells mL1) wasperformed on a sample of microalgal suspensionusing a Brker-Trk counting chamber (Brand,Germany) and a Nikon Eclipse 80i microscope(Nikon Corporation, Japan). Optical density of themicroalgal suspension was measured by absorbanceat 550 nm (A550) in an HP 8452 UV/VisibleSpectrophotometer. The spectrophotometer wasblanked with each medium, respectively. At the endof the experiments, 100 mL of the culture broth wasremoved from every flask, respectively. The sam-ples were filtered through glass microfibre discs

(Sartorius stedim biotech, Gttingen, Germany) andthe dry weights of pellets were measured after dry-ing at 105 C for 2 hours.

pH and light measurement

The sample pH was directly determined using apH meter (model 211, Hanna Instruments). The pHmeter was calibrated daily. Light intensity was mea-sured with the Li-Cor measuring device.

Chlorophyll content determination

To determine chlorophyll content of microalgalcells, two different methods were applied: the spec-trophotometric technique and the fluorometrictehnique.

The sample of microalgal suspension was cen-trifuged for 10 minutes at 13000 rpm. The super-natant was decanted and the pellet resuspended in90 % methanol. Chlorophyll was then extractedfrom the sample during one hour of incubation in awater bath at 50 C. The sample was again centri-fuged for 10 minutes at the same speed.

For the spectrophotometric determination ofchlorophyll, the absorbance of light green super-natant was measured at two wavelengths, 665 (A665)and 750 nm (A750), using the HP 8452 UV/Visiblespectrophotometer. The spectrophotometer was



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blanked with methanol. The chlorophyll content ofthe sample was calculated using the following for-mula:20

chlorophyll a

A A V

V l

MeOH

sample=

-

139 665 750. ( )

(mg mL1

)

where VMeOH is the methanol volume (mL), Vsample isthe sample volume (mL) and l is the width ofcuvette (cm).

For fluorometric determination of chlorophylla, the fluorescence at an excitation wavelength of430 nm and an emission wavelength of 663 nm wasmeasured. Fluorescence measurements were carriedout using a Jasco FP 750 spectrofluorometer. Theapparatus was blanked with methanol. The chloro-phyll a content of the sample was calculated using

the calibration curve that was made with chloro-phyll a from alga Anacystis nidulans (Sigma) asstandard.

Lipid extraction and measurementof lipid content by fluorescent spectrometry

For the determination of lipid content, a fluo-rescent spectrometric method was applied. Thesamples of 12 mL were taken from each culture, re-spectively, centrifuged for 10 minutes at 4000 rpm,then 8 mL of supernatants was removed and the restof the samples were stored at 20 C. Low tempera-tures and subsequent thawing caused a disruption ofmicroalgal cells. For extraction, 4 mL of hexanewere added, sealed up, and mixed well. Lipids wereextracted during 45 minutes of incubation in a wa-ter bath at 45 C. During and at the end of in-cubation, the samples were vigorously mixed on avortex mixer. After separation in two phases,1.5 mL of light fraction (lipids in hexane) was takenfor staining with 5 mL of 7.8 104 mol L1Nile Red(Sigma) dissolved in acetone according to Elsey(2007), who reported on using Nile Red, a lipid-sol-uble fluorescent probe revealing several character-

istics advantageous to in situ screening used forlipid measurement.21

The samples were then excited at 486 nm andthe emission was measured at 570 nm using a JascoFP 750 spectrofluorometer. Emission intensitieswere recorded over a period of 300 seconds. Foreasier comparison of lipid content in different me-dia, sample emission intensities were compared atdetermined time points (150 seconds).

Supercritical CO2 extractionof microalgal lipids

In order to acquire sufficient microalgal bio-mass for supercritical fluid extraction of lipids, thealgal growth was carried out in a 10 L vessel with a

ten times diluted and enriched solution from themodified Solvay process containing nutrients thatare present in Jaworskis medium, with the excep-tion of NaHCO3. The system was aerated at anair-flow rate of 270 L h1. Other culturing condi-tions were the same as in the previous sets of exper-iments.

After six days of growth, microalgal cells wereharvested by sedimentation in the dark for 3 days,when clear supernatants were decanted, and bycentrifugation (Rotanta 460 R, Hettich Zentrifugen)of the residues of supernatants and sediments to-gether at 4000 rpm for 10 minutes. The sedimentswere collected and dried using a freeze-drier(CHRIST, Alpha 24 LSC). We used lyophilisationas the drying method because of losses and alter-ation of lipid composition caused by conventional

drying at different temperatures.9Supercritical CO2 extraction of lipids from

microalgae Chlorella vulgaris was carried out usinga flow apparatus. The freeze-dried microalgal bio-mass (360 mg) was submitted to supercritical CO2at a pressure of 300 bar and temperature of 50 C.The extraction was carried out in consecutivebatches. The fluid amount (CO2) was measuredwith a gas meter. The lipids from supercritical ex-tracts were determined gravimetrically, weighingthe glass test tube before and after extraction.

Results and discussion

Effect of different culture media on growthof Chlorella vulgaris

The growth of microalgae Chlorella vulgaris indifferent culture media was primarily followed bycounting algal cells under the microscope (Fig. 1).


F i g . 1 Comparison of growth curves of microalgaeChlorella vulgaris cultured in different culture media: Jawor-

skis medium (p), an enriched solution from modified Solvay

process with dilution in a 1:10 ratio (), a pure solution frommodified Solvay process with dilution in a 1:10 ratio (), anatural mineral water medium (), a diluted natural mineralwater medium with added supplements ()


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The Jaworski medium was chosen due to itswell-defined composition favourable for the culturegreen algae, and on the basis of preliminary experi-ments that showed relatively good growth of differ-ent algal species in this medium. The growth wastracked for 29 days due to slow increasing ofmicroalgal biomass, and because the maximum cellnumber had not yet been determined until day 26 ofculturing. The fastest growth was obtained in an en-riched solution from the modified Solvay process.The maximum cell number (7.8 106 cell mL1) wasreached on the seventh day. After that day, the cellnumber decreased rapidly. In comparison with thepure solution from the modified Solvay process,which contained only NaHCO3 and NH4Cl as nutri-ents necessary for algal growth, the maximum cellnumber in the enriched solution was more than ten

times higher. No evident growth was present in theculture medium with natural mineral water. Due tohigh turbidity resulting from precipitation, micro-algal growth did not even start. When diluted andadjusted to proper ion strength with an addition ofsodium nitrate and phosphate, the growth in thefirst few days was comparable with that inJaworskis medium, but after day 7 the cell numberdecreased. The cell number was six-times lowercompared to the cell number in the enriched solu-tion from the modified Solvay process; thereforewe did not followed it any further, as media with

poor biomass yield was not of interest to us.

Effect of bicarbonate concentration on growth,chlorophyll content, and lipid content

Bicarbonate concentration of 1.05 g L1 in thepure solution from the modified Solvay process wasdetermined by the 13C NMR analysis. Compared tothe result (1.15 g L1) obtained by titration of a so-lution with 0.1 mol L1 HCl in the presence ofmethyl orange as indicator, both methods showedrelatively good mutual agreement. Also, by meansof13C NMR analysis, a 99.8 % portion of bicarbon-

ate was determined.Fig. 2 shows the growth of algae in batch cul-

tures under different concentrations of an enrichedsolution from the modified Solvay process, as themaximum cell number in this medium was out-standing and had been reached within a short periodof five days. The best growth resulted in the leastdiluted medium (1:10), and the poorest growth re-sulted in the most diluted medium (1:100). Themaximum cell number in all three media wasreached around the fifth day of culturing.

Every time a sample was taken, the pH valueof the medium was determined. Numeric results re-garding pH values in different diluted media on thefirst and ninth day of cultivation of microalgae

Chlorella vulgaris are presented in Table 1. Afterdiluting the medium, the pH value slightly de-creased due to more added water. Gradually, the pHvalue increased until day nine of cultivation, proba-bly due to consumption of bicarbonate ion in themedium as a sole source of carbon for algal growthand photosynthesis. This is in compliance withSorensen et al. (1996), who maintained that, sincealgae use CO2(aq) from bicarbonate to compensatethe lack of CO2 from gas supply, this results in anincrease of pH.22

Chlorophyll a content (Fig. 3) was measuredfrom the second day on, since a slight increase incell number was observed due to the lag phase ofmicroalgal cells. As in the case of cell number, thehighest chlorophyll a content was obtained in themedium with a 1:10 dilution ratio, and a peak inchlorophyll a content was observed on day five ofthe experiment. Chlorophyll measurements did notdirectly coincide with direct cell counts. The dis-crepancy in chlorophyll concentration is likely dueto the variability of levels within individual cells,and not as a result of changes in the overall bio-mass. With regard to the growth curve of a sample


F i g . 2 Comparison of growth curves of microalgaeChlorella vulgaris cultured in an enriched solution from mo-dified Solvay process at different dilution ratios: 1:10 (p),1:50 (), 1:100 ()

Ta b l e 1 pH values of different diluted media during theexperimental period

Dilution ratiopH value

1st day 9th day

1:10 7.10 9.56

1:50 7.07 9.38

1:100 7.03 9.07


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with a 1:50 dilution ratio, the cell number peakedon day five, but an increase in chlorophyll a contentoccurred on days three and six. Both methods,spectrophotometry and fluorometry, which were ap-plied for chlorophyll determination, showed a simi-lar pattern (Fig. 4). The chlorophyll concentrationsmeasured fluorometrically were higher as thismethod is more sensitive. These optical methodscan significantly under- or overestimate chlorophylla concentration, partly because of the overlap of ab-

sorption and fluorescence bands of co-occurring ac-cessory pigments, and partly due to chlorophylldegradation products.23

Fig. 5 shows the relative lipid content in cells ofmicroalgae Chlorella vulgaris cultured in an en-riched solution from the modified Solvay process atdifferent dilution ratios. The highest intensities weredetected in a sample of medium diluted at a 1:10 ra-

tio. Peak emission intensity was obtained on the fifthday of cultivation as reported for cell number andchlorophyll a content. In the medium with the dilu-tion ratio of 1:50, all measurements were in a narrowrange with a slight increase on day five. In the me-dium with the dilution ratio of 1:100, the curve ofemission intensity peaked during days six and sevenas previously reported for cell number. For absolutelipid measurements per unit cell, one needs to de-

velop the requisite calibration curve that correlatesfluorescence to lipid content, whether determinedgravimetrically or by means of lipid standards.

Supercritical extraction of microalgal lipids

Supercritical extraction with CO2 was per-formed in 360 mg of freeze-dried microalgal bio-mass. Extraction was carried out at 300 bar(30 MPa) and 50 C (323.1 K) in nine batches in acumulative time of 240 minutes. In this time, thecumulative amount of CO2 used was 295.68 L. Theyield of supercritical extraction of lipids was only

1.69 %. Mendes et al. (2003) obtained from about20 % of lipids in Chlorella vulgaris at severe condi-tions (35 MPa, 328.1 K), and establishing that theconcentration of lipids was higher when crushedcells were used in the supercritical extraction. Thecrushing is important, because the cell wall is struc-turally a polymer (sporopollenin) derived fromcarotenoid polymerisation,24 which makes the ex-traction of the internal compounds difficult.

Conclusion

Despite several reports discussing different me-dia for cultivation of microalgae Chlorella vulgaris,as far as the authors know, none of them discusses


F i g . 3 Comparison of chlorophyll a content of microalgaeChlorella vulgaris cultured in an enriched solution from mo-dified Solvay process at different dilution ratios: 1:10 (p),1:50 (), 1:100 (). Results shown here were obtained by

fluorometric technique.

F i g . 4 Comparison of chlorophyll a content determinedby spectrophotometric and fluorometric techniques inmicroalgal cells of Chlorella vulgaris cultured in an enriched

solution from the Solvay process diluted in a 1:10 ratio

F i g . 5 Comparison of emission intensities of Nile Red ex-cited at 486 nm and recorded at 570 nm at determined time

point 150 seconds after addition to a light fraction of hexaneextracts of lipids from microalgal cells (Chlorella vulgaris) cul-

tured in an enriched solution from modified Solvay process atdifferent dilution ratios: 1:10 (p), 1:50 (), 1:100 ()


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the application of the principles of the Solvay pro-cess for CO2 fixation through photosynthesis car-ried out by microalgae. If compared with thegrowth of microalgae in Jaworskis medium, the re-sults showed a faster growth with a two timeshigher biomass yield in a solution from the modi-fied Solvay process enriched with essential ele-ments required for microalgal nutrition and are thesame as in Jaworskis medium. The maximum cellnumber was reached already on the fifth day. Thebest results were obtained in the medium diluted ina 1:10 ratio. It was found that the chlorophyll a andthe lipid contents also peaked around day five.

The yield of lipids from freeze-dried micro-algal biomass extracted with supercritical CO2 at30 MPa and 50 C was low. With this method, we

only qualitatively proved the presence of lipids inmicroalgal cells of Chlorella vulgaris cultured un-der normal nutrition. However, an improvement inthe efficiency of extraction and consequently quan-tification of lipids will be the aim of further re-search, along with the production of microalgal bio-mass with higher lipid content as the result of expo-sure to nitrogen starvation. These parameters are ofvital importance for the application of the system ina larger scale.

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01. Studies on the Growth OfChlorella Vulgarisin Culture Media