biomass yield in flat panel

Optimization of Outdoor Cultivation in Flat PanelAirlift Reactors for Lipid Production by Chlorellavulgaris

Ronja M€unkel,1 Ulrike Schmid-Staiger,2 Achim Werner,1 Thomas Hirth1,2

1University of Stuttgart/Institute of Interfacial Process Engineering and Plasma Technology,

Nobelstrabe 12, 70569 Stuttgart, Germany; telephone: þ49-711-970-4069;

fax: þ49-711-970-4200; e-mail: [email protected] Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany

ABSTRACT:Microalgae are discussed as a potential renewablefeedstock for biofuel production. The production of highlyconcentrated algae biomass with a high fatty acid content,accompanied by high productivity with the use of naturalsunlight is therefore of great interest. In the current study anoutdoor pilot plant with five 30 L Flat Panel Airlift reactors(FPA) installed southwards were operated in 2011 inStuttgart, Germany. The patented FPA reactor works onthe basis of an airlift loop reactor and offers efficientintermixing for homogeneous light distribution. A lipidproduction process with themicroalgae Chlorella vulgaris (SAG211-12), under nitrogen and phosphorous deprivation, wasestablished and evaluated in regard to the fatty acid content,fatty acid productivity and light yield. In the first set ofexperiments limitations caused by restricted CO2 availabilitywere excluded by enriching the media with NaOH. Thehigher alkalinity allows a higher CO2 content of supplied airand leads to doubling of fatty acid productivity. The secondset of experiments focused on how the ratio of light intensityto biomass concentration in the reactor impacts fatty acidcontent, productivity and light yield. The specific lightavailability was specified as mol photons on the reactorsurface per gram biomass in the reactor. This is the firstpublication based on experimental data showing thequantitative correlation between specific light availability,fatty acid content and biomass light yield for a lipidproduction process under nutrient deprivation and outdoorconditions. High specific light availability leads to high fattyacid contents. Lower specific light availability increases fattyacid productivity and biomass light yield. An average fattyacid productivity of 0.39 g L�1 day�1 for a 12 days batchprocess with a final fatty acid content of 44.6% [w/w] wasachieved. Light yield of 0.4 gmol photons�1 was obtained forthe first 6 days of cultivation.

Biotechnol. Bioeng. 2013;xxx: xxx–xxx.

� 2013 Wiley Periodicals, Inc.

KEYWORDS: Chlorella vulgaris; outdoor cultivation; Flat PanelAirlift reactor; lipid production; carbon dioxide; lightavailability

Introduction

“Algal biofuels have the potential to contribute to improvingthe sustainability of the transportation sector, but innova-tions and R&D are needed to realize their full potential.”(“Sustainable Development of Algal Biofuels”, The NationalAcademies Press, Washington, US, 2012.) Discussed in thescientific literature, as well as in the world press, microalgaeare a promising alternative biomass source for biofuelproduction. However, there are still challenges that need to beaddressed to ensure stable large-scale production withpositive net energy balance (Chisti, 2007; Mata et al.,2010). Thereby special attention has to be paid to increasingthe productivity of the production systems, minimizingenergy demand and cost requirements (Griffiths et al., 2011),as well as increasing product quality to ensure an efficientdownstream process (Stephenson et al., 2010). Whethermicroalgae production will be feasible for energy purposescannot be answered based on data currently available.Especially robust data from field experiments are necessary tostudy the potential in detail (Wilhelm and Jakob, 2011).Although several data sets from outdoor experiments areavailable for biomass production (Eriksen, 2008; Lee, 2001)only very few have been published concerning the lipidproduction process under nutrient deprivation (Bondioliet al., 2012; Rodolfi et al., 2009). The same applies tophotosynthetic efficiency and biomass light yield. Thesevalues are available for biomass production for bothlaboratory experiments (Cuaresma et al., 2011; Meiseret al., 2004) and outdoor cultivations (Acien Fernandezet al., 2003) as well as for different strains. As mentioned bySchlagermann et al. (2012) no quantitative measurements

Correspondence to: R. M€unkel

Received 7 January 2013; Revision received 28 March 2013; Accepted 19 April 2013

Accepted manuscript online xx Month 2013;

Article first published online in Wiley Online Library

(wileyonlinelibrary.com).

DOI 10.1002/bit.24948

ARTICLE

� 2013 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2013 1

have yet been released for photosynthetic efficiency duringlipid production.

As shown in laboratory experiments and outdoorcultivations, deprivation of nutrients like nitrogen andphosphorus may lead to an increase in lipid content (Illmanet al., 2000; Rodolfi et al., 2009). This is caused by theaccumulation of triacylglycerides (TAG), which generallyserve as storage molecules in microalgae cells. Along with theaccumulation of TAG, the degree of fatty acid saturation isshifted towards saturated and monounsaturated fatty acids.When focusing on biodiesel production such a compositionis closer to the requirements for biodiesel, according tostandards such as EN 14214 (Griffiths et al., 2011). Recentstudies show that overall lipid productivity may increase withlimited nutrient supply (Griffiths et al., 2011; Lv et al., 2010;Stephenson et al., 2010). Another parameter influencing thelipid content along with the lipid productivity is the specificlight availability or effective irradiance (Su et al., 2011).Hereafter, specific light availability describes the ratio of lightstriking the reactor surface, to biomass concentration in thereactor. It is specified as mol photons per gram of biomassand refers to a defined time interval. Under natural lightconditions, light availability varies and depends uponweather conditions and the diurnal cycle. During the outdoorproduction process, specific light availability can beinfluenced solely by either changing the light path of thereactor or by adjusting the biomass concentration.

The objective of the current investigations was to set up alipid production process under outdoor conditions, generat-ing highly concentrated biomass with a high lipid contentaccompanied by a high productivity. The set-up was a lipidproduction process with Chlorella vulgaris in an outdoor pilotplant with 30 L photobioreactors under phosphorous andnitrogen deprivation. The established batch process wassolely limited by the parameters light and temperature, whichcannot be influenced. Process limitations caused by restrictedCO2 or nutrient availability were avoided. In pre-experiments

the CO2 content of supplied air was determined, warranting anon-CO2-limited lipid production process. Following this, ina second set of experiments the specific light availability wasvaried by setting different initial biomass concentrations. Theinvestigations resulted in a quantitative correlation betweenspecific light availability, lipid content and biomass lightyield.

Materials and Methods

Reactor Design and Plant Construction

All experiments were performed in an outdoor pilot plantlocated in Stuttgart, Germany (N48�44024″ E009�05056″) insummer and autumn of 2011. The plant consisted of five FlatPanel Airlift reactors facing south with a light path of 3 cm,total reactor volume of 30 L and a single-edge surface area of1.3m2 (Fig. 1). The Flat Panel Airlift reactor (FPA) (Schmid-Staiger et al., 2009) was originally developed at theFraunhofer IGB and is now commercially produced bySubitec GmbH (Stuttgart, Germany). The reactor works onthe principle of an airlift reactor and thus allows efficientintermixing based solely on aeration. Figure 2 shows a cross-section of the reactor. Staticmixers cause a circulating currentin every chamber of the reactor, leading to uniform lightdistribution. Every reactor was equipped as shown inFigure 3. The CO2 flow rate was continuously adjusted bya mass flow controller (Bronkhorst, Kamen, Germany, FIC inFig. 3) and added to the air flow. The total gas flow rate was setby an electropneumatic control valve (Bosch Rexroth, Lohr,Germany, PIC in Fig. 3) and supplied through a perforatedsilicone hose at the bottom of the reactor. Temperatureand pH value were measured by a combined electrode(Endress und Hauser, Stuttgart, Germany, TIC/QI in Fig. 3)and transmitted to the process control unit (Siemens CPU1214, München, Germany). All process parameters weremeasured continuously and recorded every minute.

Figure 1. Outdoor pilot plant with five 30 L Flat Panel Airlift Reactors for a two-stage lipid production process.

2 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2013

Process Conditions

To evaluate the relevant process correlations of a lipidproduction process with C. vulgaris (SAG 211-12) a two-stageoutdoor process was established. In the first stage tworeactors were operated to produce biomass in a fed-batchmode with repeated nutrient feeding. In stage two threereactors were operated in parallel for lipid production, whichwas induced by ammonium and phosphate deprivation andoperated as a batch process. Considerations and results

concerning the biomass production stage are not subjects ofthis publication.Modified DSNmedium (Pohl et al., 1987) was used in both

process stages. The medium consists of 3.5 g L�1 sea salt;1.38 g L�1 MgSO4·H2O; 0.56 g L

�1 CaCl2; 3.2mg L�1 Fe(III)citrate and 40ml L�1 micronutrient solution (20mg L�1

MnCl2·4H2O; 5mg L�1 ZnSo4·7H2O; 5mg L�1 CoSO4·7H2O; 5mgL�1; Na2MoO4·2H2O; 0.5mgL�1 CuSO4·5H2O).When biomass was transferred from stage one reactors

for biomass production into stage two reactors for lipidproduction, the culture was diluted with N/P-free DSN-medium to achieve the desired biomass concentration. Afterdilution, the ammonium concentration in the lipid produc-tion reactors was <30mg L�1. The phosphate concentrationwas <20mg L�1. Both nutrients were assimilated by 24 hafter start of experiments, at the latest. According to Liu et al.(2008) lipid productivity may be improved by additional ironfeeding. Hence, during the lipid production stage, 1mg L�1

Fe was added three times a week by addition of dissolved ironcitrate.The reactor was kept at a constant volume of 24.5 L.

Evaporation losses were substituted regularly with deionizedwater. The aeration rate was set to 500 L h�1 corresponding to0.34 vvm. The pH value was kept constant at 6.9 by settingthe CO2 content of the supplied air. The upper reactortemperature was fixed at 29�C by periodical spray cooling.During all experiments the optical density in all reactors wasdetermined five times a week in the morning. Samples fromall reactors were taken to analyze the lipid content of thebiomass three times per week.

Experimental Design

The aim of the first experiment was to identify the properCO2 content of supplied air to avoid CO2-limitation duringthe lipid production process. During the lipid productionstage, no buffering nutrients like phosphate are present and aconstant CO2 content in the supplied air would result insevere variations of the pH value. To avoid those variations aPID-controller continuously set the CO2 content of suppliedair to compensate the pH shift caused by changing CO2

solubility and a variable CO2 uptake rate.In order to evaluate the proper CO2 content of the supplied

air, the culture medium of the three reactors was enrichedwith different amounts of NaOH. Cultivation was conse-quently possible in all reactors at identical pH values butdifferent CO2 contents of supplied air. NaOH neutralized thedifferent amounts of dissociated carbon dioxide in the culturemedium. During preliminary tests with algae-free medium,it was shown that a NaOH concentration of 3.9mMcorresponds to a CO2 content of supplied air of 2.0% underequilibrium conditions at a pH value of 6.9 and a reactortemperature of 28�C. Under equivalent conditions, aconcentration of 7.7mM NaOH corresponds to 4.0% CO2

in supplied air. During cultivation the first reactor was set to aNaOH concentration of 3.9mM, while the second reactorwas set to 7.7mM NaOH. The third reactor was operated

Figure 3. Flow diagram of an outdoor FPA reactor for the lipid production stage.

All three reactors were equipped identically.

Figure 2. A: Cross-section of a Flat Panel Airlift reactor with 3 cm light path.

Arrows show the circular current generated by ascending gas bubbles. B: Schematic

representation of the different light zones in the reactor and the flow profile leading to a

homogenous light distribution.

Münkel et al.: Outdoor Cultivation of C. vulgaris for Lipid Production 3

Biotechnology and Bioengineering

without NaOH addition as a reference. The CO2 content ofsupplied air was constantly regulated to keep the pH value at6.9. The CO2 content of supplied air varied according to theNaOH content in the medium, the photosynthetic activityand the culture temperature. The reactors were inoculatedsimultaneously with an identical initial biomassconcentration.

The aim of the second set of experiments was to evaluatethe influence of the specific light availability by settingdifferent initial biomass concentrations. Three reactors wereinoculated simultaneously with biomass concentrationsbetween 1.3 and 4.1 g L�1. All reactors were set to a NaOHconcentration of 3.9mM. The experiment was performedtwice, in order to achieve a sufficient quantity of data (run 1and 2). The time-dependent courses of biomass concentra-tion, lipid content and lipid concentration of run 1 arepresented in detail. Time-dependent courses for run 2 areshown in the Appendix. However, results of run 1 and 2 weretaken into account for the evaluation of the influence ofspecific light availability on biomass light yield and final lipidcontent.

Determination of Irradiance

The radiation was measured by three vertically installed lightsensors aligned to the west, south and east. Additionally, onesensor measured the diffuse light. The dataset was recordedevery tenth minute in Wm�2. The position of the reactorswas rotated by 4� on the east–west axis. Taking into accounttheir trigonometric relationships, the three sensors alignedwest, south and east were used to calculate the light intensityon the reactor surface facing the sun. The data from thediffuse light sensor were taken into account when theradiation on the reactor surface facing away from the sun wasdetermined. The total radiation on both reactor surfaces wascalculated as the sum of the radiation on the sun-facing andthe shaded reactor surfaces. The photosynthetic activeradiation (PAR) was estimated as 45% of the total radiation.By integrating the photosynthetic active radiation for thetime interval of 24 h, the irradiance IPAR in mol photons perday and reactor surface (front and rear) was determined.Another sensor recorded the horizontal radiation in Wm�2.These data were used to calculate the horizontal radiation inMJm�2 day�1.

Determination of Optical Density and Dry WeightConcentration

The optical density was determined at 750 nm in aspectrophotometer (Hitachi 150-20, Japan). The followingcorrelationwas used to convert the optical density OD750 intobiomass concentration:

DW ¼ 0:1907� OD750; r2 ¼ 0:98

The dry weight concentration was determined to confirmthe correlation. Sample with a volume of 10mL were

centrifuged and washed twice (20min per step at 5,000 rpm).The samples were dried for 24 h at 100�C and weighed.

Determination of Fatty Acid Profile and Fatty Acid Content

The fatty acid profile was analyzed by a method based on thatdescribed by Lepage and Roy (1984). The modified methodused in this work is described by Meiser et al. (2004). Prior totransesterification the samples were centrifuged and washedtwice as described in the section above. The samples wereanalyzed in a gas chromatograph (Agilent 7890A, column:SupelcoSPBTm-PUFA Fused Silicia Capillary Column). Asum of all fatty acids provided an estimate of the total fattyacid content.

Calculations

Biomass productivity and fatty acid productivity describe thechange in biomass concentration respective fatty acidconcentration according to a defined time interval. The totalfatty acid content refers to the total biomass and the contentof a single fatty acid refers to the total amount of fatty acids inthe biomass. To evaluate the influence of irradiance alongwith biomass concentration on the lipid production process,the specific light availability IPAR,spec is defined according toEquation (1):

IPAR;spec ¼RIPARdt

1DWVð1Þ

IPAR,spec gives the ratio of irradiance on reactor surface tobiomass in the reactor and refers to a defined time interval.IPAR describes the irradiance in mol photons on the reactorsurface (front and back) and is integrated over the definedtime interval. Ø DWdescribes the mean biomass concentra-tion during the considered time interval, V is the total culturevolume and t is the cultivation time. The photosyntheticefficiency PEPAR describes the ratio of chemically fixed energyto radiation energy impinging the reactor surface and iscalculated according to Equation (2):

PEPAR ¼ ðDFAW� 37; 4kJg�1 þ ðDDW� DFAWÞ � 16; 7 kJg�1ÞV217kJ mol photons�1 � R

IPARdt

� 100

ð2Þ

DFAW corresponds to the change in total fatty acidconcentration in the culture broth. DDW describes thechange in biomass concentration. The mean energy contentof the algae biomass was assumed to be 37.4 kJ g�1 for fattyacids and 16.7 kJ g�1 for the residual biomass (Atwater andBenedict, 1902). IPAR describes the irradiance in mol photonson the reactor surface (front and back) and is integrated overthe defined time interval. The mean energy content of thephotosynthetic active radiation is 217 kJmol photons�1

(Tredici, 2010).


Equation (3) gives the biomass light yield YPAR. Thebiomass light yield describes the amount of biomassproduced per mol photons impinging the reactor surface.

YPAR ¼ DDW� VRIPARdt

ð3Þ

Results

Influence of CO2 Content of Supplied Air on Lipid Production

Enriching the reactors with different amounts of NaOHpermitted us to evaluate the effect of different dissolved CO2

concentrations without changing the pH value of the system.The identical pH values ensured the comparability of thesecultivations and kept the ratio of dissolved CO2 to HCO3

�

and CO3�2 constant.

Figure 4 shows the oscillating course of the CO2 content ofsupplied air during the lipid production stage of all threereactors. The time-dependent course of the CO2 content ofsupplied air can be described with Equation (4):

dcCO2;l

dt¼ kLaðc�CO2;l

� cCO2;lÞ � ˙mCO2 ð4ÞThe saturation concentration c� corresponds to the CO2

content of supplied air, where cCO2;l is the actual concentra-tion in the culture media and ˙mCO2 the CO2 uptake ratecaused by CO2-assimilation. The volumetric mass transfercoefficient kLa is a system-specific variable and identical forall reactors during the experiment. Assuming a constantpH value, the change in cCO2;l is insignificant. During thenight the CO2 uptake rate ˙mCO2 was zero, c

� equaled cCO2;l

disregarding CO2 production caused by respiration. Thesystem was close to equilibrium. Depending on the NaOHconcentration, CO2 contents of supplied air of 0%, 1.6%, and3.5% were sufficient to keep the pH value at 6.9. During

daytime there was CO2 uptake by the algal cells along withphotosynthetic CO2-assimilation and therefore ˙mCO2 in-creased. The gas–liquid equilibrium could not be sustained.Initiated by the PID-controllers that kept the pH valueconstant, CO2 contents of supplied air increased to 0.5%,3.0%, and 5.5% at noontime. The increase in supplied CO2

was additionally caused by the higher reactor temperature,which resulted in lower solubility of CO2 in the liquid phase.According to Figure 5A the biomass concentration

increased during cultivation under nitrogen and phosphorusdeprivation from 1.9 to 6.2 g L�1 in 8 days in both reactorsenriched with NaOH.However, the biomass concentration inthe reference reactor remained at 3.0 g L�1 after Day 4. Thefinal fatty acid content in all three reactors did not differsignificantly and reached 35–40% of the biomass. The finalfatty acid concentration was mostly influenced by thedifferent biomass concentrations. The lipid concentrationreached 2.5 and 2.6 g L�1 in the two NaOH-enriched reactorscompared with 1.2 g L�1 in the reference reactor. A summaryof biomass and lipid productivities for all three reactors isshown in the Appendix (Table AI).

Influence of Biomass Concentration on Lipid Production

In order to quantify the influence of biomass concentrationon lipid production and hence light availability per cell, thethree reactors were inoculated with different initial biomassconcentrations. The experiment was carried out twice (run 1und 2). For both runs the three reactors were inoculatedsimultaneously and operated in parallel under nitrogen andphosphorus deprivation. Figure 6 shows the time-dependentcourses of biomass concentration, fatty acid content and fattyacid concentration of run 1. Time-dependent courses for run2 are shown in the Appendix (Fig. A1). The initial biomassconcentrations of run 1 were 1.3, 2.5, and 3.8 g L�1.According to Figure 6A, the initial biomass concentrationincreasedmore than threefold during cultivation. The highestincrease was observed for the culture with the highest initialbiomass concentration, where the biomass concentrationincreased from 3.8 to 12.4 g L�1 in 14 days. This correspondsto a mean biomass productivity of 0.67 g L�1 day�1.The fatty acid content of the biomass for each reactor was

analyzed eight times during the experiment. In all cultivationsthe fatty acid content increased continuously and reached thehighest fatty acid content at Day 12. The fatty acid contentincreased from an initial figure of 10% to 53.7% for the reactorwith the lowest initial biomass concentration, and up to 44.6%for the reactor with the highest initial biomass concentration.The fatty acid concentration was calculated according

to these data (Fig. 6C). This highlighted the influence ofbiomass concentration on the lipid concentration. The reactorwith an initial biomass concentration of 1.3 g L�1 reached afatty acid concentration of 2.4 g L�1 at Day 14. The reactorwith the highest biomass concentration showed the highestfatty acid concentration of 5.2 g L�1 after the same period oftime. A summary of biomass and fatty acid productivities forall three reactors is shown in the Appendix (Table AII). In a

0 1 2 3 4 5 6 7 80

1

2

3

4

5

6

cultivation time [d]

CO

2 con

tent

in s

uppl

ied

air

[% v

v −

1 ]

Figure 4. Time dependent course of the CO2 content of supplied air during the

lipid production stage in three FPA reactors with increasing NaOH concentrations

added: 0 mM (~), 3.9 mM (&), and 7.7 mM (�). A PID controller kept the pH value

constant at 6.9 by varying the CO2 content of supplied air.



0

1

2

3

4

5

6

7bi

omas

s co

ncen

trat

ion

[g L

−1]

0

10

20

30

40

50

60

fatty

aci

d co

nten

t [%

w w

−1]

A

B

0 2 4 6 8 100

0.5

1

1.5

2

2.5

3


fatty

aci

d co

ncet

ratio

n [g

L−1

]

C

Figure 5. Parallel outdoor cultivation of C. vulgaris in three parallel 30-L-FPA

reactors under nitrogen and phosphorus deprivation at pH 6.9. Time-dependent

courses of the biomass concentration (A), the fatty acid content (B) and the fatty acid

concentration (C) for different NaOH concentrations in the media: 0 mM (5), 3.9 mM

(&) and 7.7 mM (�).

0

2

4

6

8

10

12

14

biom

ass

conc

entr

atio

n [g

L−1

]

0

10

20

30

40

50

60

fatty

aci

d co

nten

t [%

w w

−1]

A

B

0 2 4 6 8 10 12 14 16 180

1

2

3

4

5

6


fatty

aci

d co

nten

trat

ion

[g L

−1]

C

Figure 6. Parallel outdoor cultivation of C. vulgaris in three parallel 30-L-FPA



concentration (C) for different initial biomass concentrations: 1.3 g L�1 (5), 2.5 g L�1

(&) and 3.8 g L�1 (�). Run 1.


12 days batch process with an initial biomass concentration of3.8 g L�1, an average fatty acid productivity of 0.39 g L�1

day�1 with a final fatty acid content of 44.6% was achieved.The fatty acid profiles of three samples taken from the

reactor with the highest final fatty acid content of 53.7% atDays 1, 6, and 12 are shown in Figure 7. In comparison withDay 1 only the fatty acid C18:1 increased from 2.3% to 48.5%of the total amount of fatty acids at Day 6. Regarding thecontent of fatty acids related to the total biomass, the amountof C16:0 and C18:3 increased as well. C16:0 increased from2.7% to 7.1% DW, C18:1 from 0.3% to 20.5% DWand C18:3from 4.7% to 9.5% DWat Day 6 (data not shown in figures).From Day 6 to Day 12, the fatty acid profile did not changesignificantly but the total fatty acid content further increasedfrom 42.2% to 53.7%.

Effect of Specific Light Availability on Fatty Acid Contentand Biomass Light Yield

The initial biomass concentrations of all reactors of run 1 and2 varied between 1.3 and 4.1 gDWL�1. The horizontalradiation during the two cultivation periods is shown inFigure 8. For run 1 the daily mean radiation was nearlyconstant at 16MJm�2 day�1. However, during run 2 the dailymean radiation varied between 5.2 and 20.5MJm�2 day�1.These two datasets were used to compare the biomass light

yields and final fatty acid contents achieved for variousbiomass concentrations and time intervals of lipid produc-tion stage. In Figure 9 biomass light yield is plotted overspecific light availability calculated according to Equation (3).The considered time intervals cover the cultivation time fromDay 0 to 6, fromDay 0 to 8, fromDay 0 to 10 days, fromDay 0to 11 days and from Day 0 to 12. The mean biomassconcentration of the corresponding time interval was takeninto account for the calculation. Evidently, the biomass lightyield decreases with increasing specific light availability.Maximum biomass light yield for the lipid production stagewas achieved in the reactor with the highest initial biomass

concentration of 3.8 g L�1 during the first run, which resultedin a specific light availability of 1.9mol photons g�1. This lowspecific light availability resulted in a biomass light yield of0.4 gmol photons�1 and refers to the time interval fromDay 0 to 6. The fatty acid light yield for the identical timeinterval was 0.18 g fatty acids per mol photons andcorresponds to the highest value achieved during theexperiments. Biomass light yield decreased to 0.1 gmol

Figure 7. Fatty acid profile of C. vulgaris cultivated outdoor in a 30-L-FPA reactor

under nitrogen and phosphorus deprivation with an initial biomass concentration of

1.3 g L�1 and a final fatty acid content of 53.7% of the biomass.

0

10

20

0 2 4 6 8 10 120

10

20


horiz

onta

l rad

iatio

n [M

J m

−2 d

−1]

run 1

run 2

Figure 8. Daily horizontal radiation during run 1 and run 2 of the experiments

concerning the specific light availability. Test runs took place from September to

October 2011.

Figure 9. Outdoor cultivation of C. vulgaris in 30-L-FPA reactors under nitrogen

and phosphorus deprivation at pH 6.9. Two runs with three reactors each operated in

parallel were set up. The initial biomass concentration varied between 1.3 and

4.1 g L�1. Biomass light yield (& run 1, & run 2) describes the change in biomass

concentration per irradiance on the reactor surface for different time intervals (Day 0–

6, Day 0–8, Day 0–10, Day 0–11, and Day 0–12) and is plotted over the corresponding

specific light availability. The specific light availability describes the ratio of the sum of

irradiance on the reactor surface to mean biomass in the reactor. Final fatty acid

contents of all reactors are also shown (�).



photons�1 under experimental conditions with high specificlight availability of 8.5mol photons g�1. The final fatty acidcontent for all six reactors is also shown in Figure 9. Specificlight availabilities lower than 4mol photons g�1 increases thefatty acid content from 10% in the biomass production stageto a final lipid content of between 35% and 45% within12 days. For a final fatty acid content of more than 50% ahigher specific light availability is needed.

The photosynthetic efficiency was calculated according toEquation (2).Maximal PEPAR was achieved during the secondrun with the highest initial biomass concentration. For a timeinterval of 6 days in the beginning of the lipid productionstage, the mean PEPAR was 4.9% corresponding to a biomasslight yield of 0.4 gmol photons�1. However, when aiming toachieve a final fatty acid content of more than 50% a meanPEPAR lower than 2% is inevitable.

Discussion

The aim of the first experiment was to identify the properCO2 content of supplied air to avoid CO2-limitation duringthe lipid production process. To evaluate the proper CO2

content of supplied air, the culture medium of the threereactors was enriched with different amounts of NaOH.Consequently, cultivation was possible in all reactors atidentical pH values but different CO2 content of supplied air.As shown in Figure 5A, the course of biomass concentrationis affected by the amount of NaOH in the media and thus bythe concentration of dissolved CO2. These results permit theassumption that, in the lipid production stage, biomassproductivity was limited due to a low CO2 concentrationwhen no NaOH was added and the CO2 content of suppliedair was around 0.5% during noontime. Neither a minimumvalue of NaOH nor a minimum value of CO2 of supplied aircan be derived from the results. An increase of NaOH from3.85 to 7.70mM showed no effect on biomass productivity.Therefore, running the process with a NaOH concentrationof 3.85mM stands to reason.

In regard to Figure 5B, the fatty acid content of the biomassis not affected by different amounts of NaOH in the culturemedium and thus by the concentration of dissolved CO2

in the liquid phase. However, there is a clear differenceconcerning the fatty acid concentration due to the influenceof biomass productivity (Fig. 5C).

The performed experiments show the importance ofoptimizing the production process with regard to the CO2

supply. This corresponds to the results gained by Chiu et al.(2009) and Lv et al. (2010). Both reported dependence of thebiomass and lipid productivity on the CO2 content of suppliedair. Due to the differences in the employed production systemsand algae strains, their quantitative results are not comparableto the present experiments. To describe the influence of theCO2 content of supplied air on the production process indetail, all process conditions such as temperature, reactor type,gassing conditions, algae strain and media composition haveto be taken into account. According to Equation (4), thedynamics of CO2 transport from gas phase to liquid phase can

be described by the volumetric mass transfer coefficient kLaand depends on the fluid dynamics of the system, as well as thegassing conditions, including bubble size, their distributionand retention time. Various influences on the kLa value ofphotobioreactors and its identification are already described indetail in the literature (Acien Fernandez et al., 2003; Talbotet al., 1990; Zhang et al., 2002).

On the other hand, the saturation concentration of CO2 inthe culture media influences the dynamics of CO2 transportand depends on the temperature and salinity of the liquidphase. Further, the CO2 uptakemechanism of the speciesmayinfluence the optimal CO2 concentration for a non-limitedproduction process. The dynamics of CO2 uptake by algaecells and the impact on the production system is described indetail by Nedbal et al. (2010).

Based on the knowledge obtained, CO2 limitation could beprecluded in a second set of experiments. The initial biomassconcentrations were varied to investigate the influence ofdifferent specific light availabilities. The specific lightavailability was specified as mol photons per gram ofbiomass in the reactor and refers to a defined time interval.According to Figure 6A and B, both the biomass concentra-tion and the lipid content was influenced by the specific lightavailability. The reactor with the highest specific lightavailability showed the highest final lipid content butobtained the lowest biomass productivity. Vice versa, thehighest initial biomass concentration accompanied with thelowest specific light availability led to the highest biomassproductivity along with the lowest final lipid content. Here,the biomass concentration increased 3.4-fold in 14 days. Evenif the increase is not only due to a rise in lipid concentration, astrong increase in biomass concentration under N-depletedconditions is well documented. Liu et al. (2008) reported a1.4- to 7.8-fold increase in biomass concentration fordifferent strains during N-depleted cultivation. Biomassproductivity under N-depleted conditions may be verydifferent depending on algae strain and cultivation con-ditions. However, it is a significant parameter for designing aneconomically feasible production process.

In the lipid productivity the influence of the specific lightavailability on the biomass productivity was crucial.Obviously, a low specific light availability led to the highlipid productivity. Hsieh and Wu (2009) described a strongimpact of the correlation between biomass concentration andirradiance on productivity in photobioreactors with differentdepths. Furthermore, Su et al. (2011) focused on a lipidproduction process under N-deficient conditions in labora-tory with varying initial biomass concentrations. Theyreported an increase of inoculum concentration leading toan increase of volumetric lipid productivity, accompanied bya decrease of lipid content when operating the process at thesame level of irradiance. Although the work was performedwith Nannochloropsis, their conclusions are consistent with thecurrent work. Similar experiments were performed in flatpanel reactors under outdoor conditions by Feng et al. (2011)with Chlorella zofingiensis. For the inoculation of photo-bioreactors with different optical densities under nitrogen


deprivation, an increase in lipid content has been reportedwhen initial optical density decreases. The increase of lipidcontent is accompanied by a decrease in lipid productivity. Ithas to be pointed out that the initial biomass concentration inthe current work was around 50� higher compared with theexperiments performed by Feng et al. (2011).Quantifying the influence of specific light availability on

the lipid production process, Figure 9 shows the decrease ofbiomass light yield with increasing specific light availability.The correlation shown is applicable for different timeintervals of the experiment, for different levels of radiationand for varying initial biomass concentrations. The increasein final lipid content over the specific light availability is alsoplotted. The combination of both correlations allows thesettings for a production process solely based on the targetedproduct quality and on the local radiation to be defined.When explaining the strong decrease in biomass light yield

with increasing specific light availability, various differentfacts must be considered. The literature contains evidencethat low cell densities especially in thin layer reactors can leadto light inhibition that reduces the photosynthetic efficiencyalong with biomass light yield (Richmond et al., 2003). Thisauthor suggests the application of the highest cell densitypossible. Furthermore, the decrease of biomass light yield isenforced by the increase in final lipid content. The higher thelipid content, the higher is the amount of energy bound pergram of biomass. According to the energy content of lipidsand residual biomass used in Equation (6), the heating valueof the biomass increases from 18.8 to 27.1MJ kg�1 when thelipid content increases from 10% to 50% of the biomass.Therefore, the light energy demand for the synthesis of lipid-rich biomass is higher. Concurrently, during lipid synthesisthe photon demand increases compared with the demand forprimary sugar synthesis, due to additional reduction steps(Wilhelm and Jakob, 2011). Zemke et al. (2010) describe alower efficiency of the biochemical synthesis of lipids basedon a photon balance.The photosynthetic efficiency depends on specific light

intensity in the same way as biomass light yield. With amaximum PEPAR of 4.9% for the first 6 days observed in thereactor with the highest initial biomass concentration and anoverall PEPAR lower than 2% for a final lipid content of morethan 50%, the values were in the lower range for outdoorcultivation. Tredici (2010) estimated a realistic PE in relationto the total spectrum of 5.4% for a biomass productionprocess under outdoor conditions with sufficient nutrientsupply. However, it has also been noted that the PE will besignificantly lower for a lipid production process undernitrogen deprivation. Described in Zemke et al. (2010), thesevalues are far below the theoretical maximum PE for lipidsynthesis where thermodynamics becomes the limitingfactor. Concerning the cell-internal conversion from photonsto lipid molecules, an efficiency of 26% is theoreticallypossible (Zemke et al., 2010).According to Schlagermann et al. (2012), no quantitative

measurements or assessments of the photosynthetic efficien-cy for a lipid production process under nitrogen deprivation

can be found in the literature. Prior to this work the PE ofsuch a process had not been published either for laboratory orfor outdoor experiments.Comparing the maximum lipid productivity observed in

this workwith data from the literature, only a small number ofdata sets are available. In an acclaimed work by Rodolfi et al.(2009) a lipid productivity of 0.204 g L�1 day�1 was reportedfor Nannocloropsis cultivated in N- and P-free media in 110-Lphotobioreactors under outdoor conditions in Florence(Italy). The experiments were set up with a daily harvestedculture volume of 40%, which resulted in lower biomassconcentrations compared with the current study. In the workby Rodolfi et al. (2009) the specific light availability was higherand therefore the final lipid content reached more than 60%.Bondioli et al. (2012), working in the same group, reported alipid productivity of 6.5 gm�2 day�1 for a similar productionsystem (grams per square meter of illuminated reactor surfaceper day) with a final lipid content of 68.5% for Nannochloropsisunder nitrogen deprivation. In comparison, the highest fattyacid productivity of 0.39 g L�1 day�1 shown in the currentwork can be recalculated on the basis of the illuminatedreactor surface and is hence 7.4 gm�2 day�1. The area for thebiomass production stage has to be considered as well whencalculating the areal fatty acid productivity. Realistically, atleast one third of the total area is needed for the first processstage. Assuming that the floor space equals the illuminatedreactor surface, the areal fatty acid productivity would be4.9 gm�2 day�1. Even compared with lipid productivitiesobtained in laboratory experiments, the current results areamong the highest for photoautotrophic processes. Only Palet al. (2011) reported a lipid productivity of 0.41 g L�1 day�1

forNannochloropsis under nitrogen deprivation and continuousillumination in the laboratory.Aside from lipid productivity, the composition of the fatty

acid profile plays an important role for future applications.The ratio of chain lengths as well as the degree of saturationmay change, depending on cultivation conditions. In general,when algae species are able to accumulate storage lipidsduring nitrogen-depleted cultivation the amount of TAGincreases. TAGs produced mainly consist of saturated andmonounsaturated fatty acids. Polyunsaturated fatty acids aretypical components of membrane lipids and production isnot enhanced by nitrogen deprivation. High amounts ofsaturated and monounsaturated fatty acids are the basis foran economically feasible production of biofuels frommicroalgae. The importance of such acids in accordancewith the biodiesel standard EN 14214 is described in Griffithset al. (2011). Among others, C. vulgaris is one of the mostpromising candidates for the accumulation of triglyceridesand therefore interesting for biodiesel production (Stephen-son et al., 2010).In Figure 7 the amount of the four main fatty acids is

shown for Days 1, 6 and 12 of the lipid production stage. FromDay 1 to Day 6 the ratio of saturated and monounsaturatedfatty acids to the total amount of fatty acids increases from aninitial level of 26.2% to 66.7% at Day 6. From Day 6 to 12 theratio does not show a further increase. For the design of an



industrial process, the temporal course of fatty acid concen-tration together with the temporal course of the fatty acidprofile is the basis for determining the optimal harvesting time.

Conclusions

When focusing on a two-stage lipid production process underoutdoor conditions, process parameters such as CO2

availability and specific light availability must be chosencarefully. Depending on the reactor type and algae strain, thetarget pH value, the CO2 concentration of supplied air andthe alkalinity of themedia have to be considered to avoid CO2

limitations. The current investigation showed highly im-proved lipid productivity with enhanced CO2 concentrationin the media. For a non-limited production process theproduct quality can be set by adjusting the specific lightavailability. Here, the specific light availability was specifiedas mol photons per gram of biomass in the reactor and refersto a defined time interval. High specific light availability leadsto high fatty acid contents. Lower specific light availabilityincreases fatty acid productivity and biomass light yield. If allthis is taken into account, a production process undernitrogen and phosphorus deprivation allows high fatty acidproductivity along with a high final fatty acid concentration.Such a process may lead to a final fatty acid content of around45% of the harvested biomass, even under the outdoorconditions common in central Europe.

Nomenclature

DW biomass concentration (g L�1)– CO2 content in the supplied air (% vv�1)˙mCO2 CO2 uptake rate (g L

�1 day�1)cCO2;l concentration of CO2 in the culture media (g L�1)t cultivation time (day)FAW fatty acid concentration (g L�1)Ihor horizontal radiation (MJm�2)YPAR light yield (gmol photons�1)IPAR Irradiance on reactor surface (mol photons day�1)OD750 optical densityPEPAR photosynthetic efficiency (%)A reactor surface (front and rear) (m2)V reactor volume (L)c� saturation concentration of CO2 in culture media

(g L�1)IPAR,spec specific light availability (mol photons g�1)kLa volumetric mass transfer coefficient (s�1)

The work was carried out within the project “EtaMax: Mehr Biogasaus lignozellulosearmen Abfall- und Mikroalgenreststoffen durchkombinierte Bio-/Hydrothermalvergasung” (Bundesministerium fürBildung und Forschung FKZ-Nr 03SF0350A).

Appendix

0

2

4

6

8

10

biom

ass

conc

entr

atio

n [g

L−1

]

0

10

20

30

40

50

60

fatty

aci

d co

nten

t [%

w w

−1]

A

B

cultivation time [d]0 2 4 6 8 10 12 14

0

1

2

3

4

5

fatty

aci

d co

ncen

trat

ion

[g L

−1]

C

Figure A1. Parallel outdoor cultivation of C. vulgaris in three parallel 30-L-FPA



concentration (C) for different initial biomass concentrations: 1.3 g L�1 (5), 2.7 g L�1

(&) and 4.1 g L�1 (�). Run 2.


References

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Table AII. Parallel outdoor cultivation of C. vulgaris in three parallel 30-L-FPA reactors under nitrogen and phosphorus deprivation at pH 6.9 with

different initial biomass concentrations.

Day 0–7 Day 0–14

Initial biomassconcentration (g L�1)

Biomassproductivity(g L�1 day�1)

Fatty acidproductivity(g L�1 day�1)

Fatty acidcontent at

Day 7 (% DW)

Biomassproductivity(g L�1 day�1)

Fatty acidproductivity(g L�1 day�1)

Fatty acidcontent at

Day 14 (% DW)

Run 1 1.3 0.43 0.24 42.6 0.28 0.18 49.52.5 0.68 0.42 43.8 0.49 0.33 51.73.8 0.95 0.44 32.1 0.67 0.38 41.8

Run 2 1.4 0.41 0.29 50.1 0.28 0.18 49.62.7 0.55 0.29 35.0 0.41 0.26 45.74.1 0.58 0.27 26.7 0.41 0.27 40.1

Table AI. Parallel outdoor cultivation of C. vulgaris in three parallel 30-L-FPA reactors under nitrogen and phosphorus deprivation at pH 6.9 with

different NaOH concentrations in the media.

NaOHconcentration(mM)

CO2 content in suppliedair at noon (%)

Days 0–8

Biomass productivity(g L�1 day�1)

Fatty acid productivity(g L�1 day�1)

Fatty acid content atDay 8 (% DW)

0 0.5 0.16 0.13 36.83.9 3.0 0.55 0.29 39.77.7 5.5 0.54 0.31 41.5



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biomass yield in flat panel

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