Effect of light on Synechocystis sp. and modellingof its growth rate as a response to average irradiance

Lorena Martínez & Antonio Morán & Ana Isabel García

Received: 22 June 2010 /Revised and accepted: 14 January 2011 /Published online: 24 March 2011# Springer Science+Business Media B.V. 2011

Abstract The growth rate and CO2 biofixation rate of aphotosynthetic organism depend basically on the availabil-ity of light, all other factors being optimum. In densecultures of cyanobacteria or micro-algae intended forbiomass production, incident irradiance on the reactorsurface is not the same as the intensity which is receivedby cells, as irradiance is attenuated by cell absorption andthe self-shading effect. In a well-mixed, dense culture, onlythe average irradiance, Iav, can be considered responsiblefor the photosynthetic response. In this study, the photo-synthetic response of Synechocystis sp., estimated from itsspecific growth rate, was measured for each Iav in batchcultures irradiated with different levels of external irradi-ance, Iext. The specific growth rate of Synechocystis sp.depends on Iav, in accordance with the model proposed byMuller-Feuga (J Exp Mar Biol Ecol 236:1–13, 1999). Anon-linear regression analysis estimated a maximum spe-cific growth rate of 0.108 h−1, at an Iav of 930 μmolphotons·m−2·s−1. This reveals that Synechocystis sp. is ahighly light-tolerant strain, suitable for outdoor cultures.Higher Iav levels caused photoinhibition in batch cultures.Parameters obtained from the Muller-Feuga model showthat the minimum irradiance needed to start growthmechanisms becomes less as light availability decreases,i.e. cells become more efficient in the use of light when itis scarce. This observation suggests that choosing for low-light adaptation may be a good strategy to improveproductivity in dense cultures, where light is a limitingfactor.

Keywords Synechocystis . Specific growth rate . Averageirradiance . Photoinhibition . Light-acclimation

Introduction

The industrial application of large-scale photosynthetic micro-organisms cultures for biological CO2 fixation is widelystudied because of its considerable potential: photoautotro-phic micro-algae and cyanobacteria present higher photo-synthetic efficiency than land plants (Janssen et al. 2002);they can be cultured in controlled conditions as a suspensionand it is quite easy to provide the culture with various lowcost nutrient sources, as waste water (Harmelen and Oonk2006). It must be taken into account that the key factor of abiological CO2 fixation based on photosynthetic micro-organisms is the free carbon and energy sources: flue gasesand light. However, only the optimization of the growth anddownstream processes as well as the development of lowcost photobioreactors will allow the eventual implementationof this technology.

Irradiance, apart from being the main energy source, canbe also a limiting substrate in conditions of low irradianceand an inhibiting substrate in conditions of high irradiance.Thus, the growth of micro-algae and cyanobacteria dependsdirectly on the amount of light received by cells and soresponse to irradiance must be studied before the use of aphotoautotrophic microorganism in a biological CO2 bio-fixation system.

Providing light to photosynthetic cultures is the mainconstraint in mass cultures of micro-algae. During the lastfew years biologists and engineers have made a great effortto develop different photobioreactors designs trying tomake light as easily available as possible to the cells(Tredici and Chini-Zitelli 1998; Janssen et al. 2002; Ono

L. Martínez :A. Morán :A. I. García (*)Natural Resources Institute, University of León,Avda. Portugal, 41,24071 León, Spaine-mail: ana.garcia@unileon.es

J Appl Phycol (2012) 24:125–134DOI 10.1007/s10811-011-9658-3

and Cuello 2006; Molina-Grima et al. 2000). However,available light can be modulated only in a limited way bycontrolling the light–dark cycle, light path, cell density andincident irradiance (Molina-Grima et al. 1996). The light–dark cycle depends mainly on mixing rate and photo-bioreactor geometry (Janssen et al. 2002) and should be ashigh as shear stress will allow. The other three parameters,light path, cell density and incident light, can be enclosed inonly one: the averaged irradiance, Iav. According to Acién-Fernández et al. (1998), Iav represents the light available foreach cell moving randomly in a dense culture, where thereis a heterogeneous light distribution caused by the self-shading effect. This parameter can be calculated using theexpression published by Molina-Grima et al. (1994):

Iav ¼ IextK 0

a � p � Cb� 1� exp �K 0

a � p � Cb� �� � ð1Þ

where

Iext incident light on the photobioreactor surface(μmol·photon m−2·s−1)

K′a attenuation constant (kg m−3)Cb biomass concentration (here in kg m−3) andp light path (m)

K′a is determined according to Acién-Fernández et al.(1997). Once K′a is known, it is possible to determine theaverage irradiance inside the culture, Iav, with a length pathof p and at a biomass concentration of Cb when the cultureis illuminated with an incident irradiance of Iext (García-Malea et al. 2006). The importance of Iav is that it makes itpossible to study the photosynthetic response vs. the realintensity the cells receive in a dense culture, not vs.irradiance on the photobioreactor surface. Iav allows thedetermination of a real optimal light range in a denseculture. Hence, in order to optimize micro-algae or cyano-bacteria biomass productivity and CO2 biofixation, aninvestigation of the influence of average irradiance on thegrowth rate is a fundamental step.

Since the kinetic theories published by Michaelis andMenten (1913), many models have been proposed toestimate growth rate, μ, as a function of average irradiance,Iav, also taking into account photoinhibition (Molina-Grimaet al. 1994; Yun and Park 2003; Muller-Feuga 1999; Bensonand Rusch 2006; García-Malea et al. 2006). Of these, twomodels were considered in this study because they have beenused to study light response of micro-algae in dense cultureswhere irradiance varied with time. However, they predictvery different responses. The first model, published byGarcía-Malea et al. (2006; Eq. 2), which is an improvedversion of the model proposed by Molina-Grima et al.(1994), shows a hyperbolic relation between μ and Iav: thegrowth rate increases with average irradiance until a

maximum growth rate is reached. Thereafter, an increase inirradiance does not lead to any increase in the growth ratebecause of photoinhibition.

m ¼ mmax � Iav aþb�Iexpð Þcþ dð Þ aþb�Iexpð Þ þ Iav

aþb�Iexpð Þ ð2Þ

where

μmax is the maximum growth rate, characteristic for eachmicroorganism, in s−1 constant values a, b and c areparameters given by non-linear regression, whichrelate growth rate with average irradiance, takinginto account photoinhibition

The second model, proposed by Muller-Feuga (1999), incontrast, predicts a maximum growth rate, μs, at a givenaverage irradiance value, Is, Eq. 3. At this irradiance, Is,photosystem II has reached its saturation point and it isreceiving all the energy it can process. At any higheraverage irradiance, over the saturation point, the photosys-tem collapses by reason of the excess of light and thegrowth rate falls.

m ¼2 � ms � 1� Ie

Is

� �� Iav

Is� Ie

Is

� �

1� IeIs

� �2þ Iav

Is� Ie

Is

� �2 ð3Þ

where

Ie average irradiance at the energy compensation point.Lower intensity causes cell weight loss, μmolphotons·m−2·s−1

Is saturation point average irradiance, μmolphotons·m−2·s−1

μs saturation point growth rate, s−1

Iav average irradiance in the culture, μmolphotons·m−2·s−1

Another question to bear in mind is the ability of micro-algae and cyanobacteria to adapt their photosynthetic rate tochanges on the increase or lowering of available light, thesechanges not being so intense so as to incur in a photo-inhibition situation. This ability was called light–shadeadaptation and means that cells that are previously adaptedto low irradiance shows a higher photosynthetic rate whenthey are exposed to favourable light levels than cells thathave not been shaded. In the same way, those cells are moresusceptible to photoinhibition (Gallegos et al. 1983). Thisprocess is known as photoacclimation and basicallyinvolves changes in pigment contents and other protectivemechanisms which tend to reduce the amount of lightenergy received by the reaction centre PSII (Richmond

126 J Appl Phycol (2012) 24:125–134

1986). A practical consequence is that if it were possible toavoid high light intensities, by means of a high-densityculture and proper mixing rates, growing cells adapted tolow-light intensities would show a higher photosyntheticrate and thus, a higher productivity and CO2 biofixation.

The aim of the present study was to determine theinfluence of available irradiance, Iav, on the growth rate ofSynechocystis sp., a freshwater cyanobacterium investigatedfor the establishment of local biofixation systems. Thestrain was cultured under different irradiances, Iext, goingso far as to reach maximum noonday irradiances. At thesame time, the best mathematical models for explaining thegrowth response of Synechocystis sp. as a function ofexternal irradiances, Iext, and average irradiance, Iav, were tobe studied. A kinetic model able to estimate growth responseas a function of culture conditions would be a useful tool fordetermining the productivity and viability of processes.

Materials and methods

The strain studied is a freshwater Synechocystis sp., isolatedand identified at genus level at the Natural ResourcesInstitute of Leon, Spain (Martínez et al. 2005). This nativeSynechocystis sp. strain was considered a good photosyn-thetic organism for CO2 biofixation, on the basis of itsspecific growth rate, as high as 0.073 h−1, and on its goodlight utilization efficiency (Martínez 2009). The inoculumwas grown under laboratory conditions, in a 100-mL bubbledconical flask, with controlled injection of 5% CO2 in air,under a 16/8 photoperiod of 200 μmol photons·m−2·s−1, at atemperature of 25°C, and in Mann and Myers medium(Mann and Myers 1968).

Photobioreactor and culture conditions Synechocystis sp.was cultured under different incident irradiances, Iext, from400 to 2,400 μmol photons·m−2·s−1, in a 1 L bubble-column photobioreactor (García-Malea et al. 2006), with adiameter of 80 mm and a height of 400 mm. The culturewas run, in batch mode, for 6 days. pH and CO2 additionwere controlled on demand. Air was bubbled at0.4 L·min−1·L−1, and 10% CO2 (v/v) in air was spargedinto the medium The reactor surface was illuminated by aset of fluorescent lamps (33W, 2150 lumen, Philips,France), installed on a vertical semi-cylindrical lightreflector, the lighting unit. Each of the irradiances on thereactor surface, Iext, was achieved by adjusting the distanceand number of such lighting units.

All growth curves started from an initial DO of 0.2, in a16/8 photoperiod. The temperature of the cultures wasmaintained at 30±2°C during the light period and at 22°Cduring the dark period, trying to reproduce a near realsituation for outdoor mass cultivation. pH was maintained

at 8. Homogeneous culture samples were collected everyday, and simultaneously, the irradiance in the centre of thereactor, Iint, was measured. The irradiance at the centre ofthe reactor when Cb=0, I0, was measured just beforeinoculation.

Analytical methods The monitoring of cultures at differentirradiances was achieved by daily measurement of thebiomass concentration, total organic carbon (TOC) andaverage irradiance at the centre of the reactor. Eachmeasurement was carried out in triplicate. Biomass concen-tration was determined relative to dry weight by filtering10 mL of culture through a 0.45 μm nitrocellulose Whatmanfilter, washed with 20 mL 0.5 N HCl to dissolve precipitatedsalts. The filter was dried at 105°C for 24 h. For TOCmeasurements, 1 mL 0.1 HCl was added to 10 mL of culturesample to bring pH down to pH 4, ensuring that all dissolvedinorganic carbon was converted into CO2 (Eaton et al. 1995).After that, samples were sparged with O2 for 2 min in orderto desorb CO2 and measured by an automatic infraredanalyzer TOC-5000 (Simadzu Corporation, Japan). Irradi-ance (PAR), both Iext and Iav at the centre of the reactor, weremeasured with a spherical radiometer QSL-2100 (Biospher-ical Instruments Inc., USA). The irradiance measured insidethe culture with the spherical radiometer is a real averageirradiance because it detects light as cells receive it. The lightpath, p, at the centre of photobioreactor, is calculated as:diameter of the photobioreactor/2 − diameter of the sphericalradiometer/2, which equals to 0.04 m.

Culture parameters Daily growth rate (μi, h−1) and pro-

ductivity (Pi, in terms of g biomass·L−1·d−1) were calculat-ed from changes in the biomass concentration, as shown inEqs. 4 and 5. CO2 biofixation was determined from themeasured TOC (Eq. 4).

▪ Growth rate (μi, h−1):

m ¼ Ln Cbið Þ � Ln Cbi�1ð Þti � ti�1

ð4Þ

▪ Productivity (Pi, g biomass·L−1·d−1):

Pi ¼ Cbi � Cbi�1ð Þti � ti�1ð Þ ð5Þ

▪ CO2 biofixation (Fi, g CO2·L−1·d−1):

Fi ¼ TOCi � TOCi�1

ti � ti�1ð Þ � 4412

ð6Þ

where

Cbi biomass concentration measured at time ti, in g L−1

TOCi total organic carbon concentration measured at timeti, in g C·L−1

J Appl Phycol (2012) 24:125–134 127

ti amount of time when daily sample was measured,in h

In batch cultures, biomass concentration and irradianceinside the culture, Iint,i, change over time. Before inocula-tion (Cb = 0), average irradiance I0, was measured at thecentre of photobioreactor (p=0.04 m). Interior irradiance (atp=0.04 m) related to the growth rate calculated for eachtime i, μi, was determined as the average value:

Iint;m;i ¼ Iint;i þ Iint;i�1

2ð7Þ

where,

Iint,m,i average irradiance, in μmol photons·m−2·s−1,measured at p=0.04 m for culture time ti

In the same way, the average irradiance, Iav,i, related to aspecific growth rate, μi, was calculated as the average valuebetween Iav,i−1 and Iav,i.

Statistical analysis

Non-linear regression of experimental variables for themathematical models described and statistical parameterswere determined by Origin 6.1 software.

Results

Influence of incident irradiance (Iext) on the growth rate

Growth curves during batch culturing, obtained at differ-ent incident light intensities, can be found in Fig. 1a. InFig. 1b, it can be seen how the irradiance measured at thecentre of the reactor, Iint, declined as the biomassconcentration increased. The highest accumulated biomassconcentration was observed for Iext=1,200 μmol pho-tons·m−2·s−1 and 1,600 μmol photons·m−2·s−1, with 4.42 gbiomass·L−1 and 4.53 g biomass·L−1, respectively. Theaccumulated biomass concentration was lower for Iext=2,000 μmol photons·m−2·s−1 cultures and was almost thesame for Iext=2,400 μmol photons·m−2·s−1 and 800 μmolphotons·m−2·s−1. In the batch culture at 400 μmol pho-tons·m−2·s−1, the biomass concentration on day 6 was thelowest, 1.67 g·L−1.

Specific growth rates with different exterior irradiancesand on the various days of culturing are shown in Fig. 2.Each batch culture showed similar behaviour: high growthrates during the early days of culturing, when biomassconcentrations were low and, as biomass concentrationsrose, the growth rate slowed, being reduced to similarly lowvalues. The highest specific growth rate of 0.095 h−1

occurred at low biomass concentrations. This result wasobserved for the 1,600 μmol photons·m−2·s−1 culture, atCb=0.95 g·L−1. In contrast, the lowest growth rates at lowbiomass concentrations, that is, during the earliest days ofbatch culturing, were found at the highest (2,000 and

a

b

0.00

1.00

2.00

3.00

4.00

5.00

0 1 2 3 4 5 6 7time (d)

Cb

(g

L-1

)

400 800 1200 1600 2000 2400

Fig. 1 a Growth curves and b interior irradiance, Iint, in μmolphotons·m−2·s−1, measured simultaneously for the cultures grown atdifferent irradiances

0.00

0.02

0.04

0.06

0.08

0.10

0 1 2 3 4 5 6 7time (d)

spec

ific

gro

wth

rat

e (h

-1)

400 800 1200 1600 2000 2400

Fig. 2 Specific growth rates for batch cultures of Synechocystis sp. atdiffering irradiances on the photobioreactor surface, Iext, expressed inμmol photons·m−2·s−1

128 J Appl Phycol (2012) 24:125–134

2,400 μmol photons·m−2·s−1) and the lowest (400 μmolphotons·m−2·s−1) irradiances on the reactor surface.

The maximum daily productivity and biofixation ofCO2 found for each culture at different external irradianceswere studied. Unlike the maximum specific growth rates,μmax, maximum productivity and biofixation of CO2 tookplace at high biomass concentrations, up to 0.9 g·L−1. Theresults presented in Fig. 3 show that productivity andbiofixation of CO2 increased with irradiance up to1,600 μmol photons·m−2·s−1, where maximum values of1.56 g biomass·L−1·d−1 or 1.96 g fixed CO2·L

−1·d−1 wereattained.

In order to compare these results with other culturesystems and organisms, it is crucial to know whatirradiance really reaches the cell walls of photosyntheticcells, as presented below.

Modelling of specific growth rates and average irradiance

Figure 4a presents specific growth rates and their respectiveaverage light intensities, μi−Iint,m,i, measured at the centre ofthe cylindrical photobioreactor (p=0.04 m). Each Iint,m,i isstated for each daily growth slope. As observed for incidentirradiances, the specific growth rate of Synechocystis sp.increases with available irradiance, Iint,m,i, up to a maximumvalue of around 930 μmol photons·m−2·s−1. At higher Iint,m,i,the specific growth rate slows. It should be noted that there isa slight deviation from these experimental results due to thetemperature range (±2°C).

Interior irradiance values, Iint,i, shown in Fig. 1b andmeasured at each sampling time, are the result ofattenuation caused by different biomass concentrations.Using the data from Fig. 1b and according to Acién-

Fernández et al. (1997), Iav,i for each growth slope where aspecific growth rate of μi was observed, was determinedusing the Eq. 1. The appropriateness to both proposedmodels (Eqs. 2 and 3) by non-linear regression analysis, ofexperimentally observed specific growth rates μi, with Iav,ivalues, was determined by means of their non-linearregression coefficient, R2, which were in all cases accept-able. The experimental specific growth rates vs. averageirradiances and the best fitting curves to the García-Maleaet al. (2006) and Muller-Feuga (1999) models are shown inFig. 5. As can be seen from Table 1, non-regressioncoefficients R2 do not indicate a clear preference for eitherof the models; since when the regression coefficient is goodfor one model, it is also good for the other. Hence, on thebasis of R2 values, both models could be used to calculategrowth rates for batch cultures at different irradiances.However, the García-Malea et al. (2006) model does notexplain the decrease in growth rates at high averageirradiances, once a given maximum value has been reached.This effect was clearly observed for cultures receiving 400,1,600 and 2,400 μmol photons·m−2·s−1 (Fig. 5) and can beattributed to photoinhibition. Thus, the Muller-Feuga(1999) model, which includes a photoinhibition term,better reproduces the observed responses of Synechocystissp. to irradiance for batch cultures at different averageirradiances Iav.

Parameters obtained after non-linear regression of theexperimental data to the Muller-Feuga model are shown inTable 2. According to parameters calculated, the modelestimates there will be increasing maximum specific growthrates with average irradiances from 423±11 μmol pho-tons·m−2·s−1 up to 930 ±22 μmol photons·m−2·s−1, where-upon the maximum growth rate of 0.108 h−1 will bereached.

Fig. 3 Maximum daily biomass productivity and CO2 biofixationobserved for Synechocystis sp. Batches cultured at incident irradianceson the reactor surface from 400 to 2,400 μmol photons·m−2·s−1 for6 days

Fig. 4 Synechocystis sp. specific growth rates, μi, in h−1, at averagelight intensities observed during daily growth, Iint,m,i. Batches culturedat incident light intensities on the reactor surface from 400 to2,400 μmol photons·m−2·s−1 for 6 days

J Appl Phycol (2012) 24:125–134 129

Discussion

Influence of incident irradiance (Iext) on the growth rate Arecently isolated Synechocystis strain, with a high potential forits application in CO2 biofixation systems, was studied in thepresent work. In order to asses this potential, its response toirradiance was studied. On the basis of accumulated biomass,this strain shows an apparent growth limitation by lowirradiance. The growth limitation appears also at the highestirradiances and then, the best growth rates are expressed at

medium irradiances. This behaviour seems to indicate thateven at the lowest biomass densities, an irradiance of400 μmol photons·m−2·s−1 on the reactor surface is notsufficient to saturate the photosynthetic system of Synecho-cystis sp. Similar results were found by Spektorova et al.(1986) who reported growth inhibition from low irradiance at300 μmol photons·m−2·s−1 (75 Wm−2, sodium lamps) forChlorella sp. f. marina cultures, with Cb≤0.5 g L−1. Thus,the photosynthetic activity expressed by cells externallyirradiated at 400 μmol photons·m−2·s−1 lies in the light

Fig. 5 Specific growth rate (μ, h−1) vs. average irradiance (Iav, μmolphotons·m−2·s−1). The curves best fitting the García-Malea et al.(2006) and Muller-Feuga (1999) models for Synechocystis sp. culturesirradiances on the reactor surface (Iext) from 400 to 2,400 μmol

photons·m−2·s−1 are also shown. Batches cultured at irradiances on thereactor surface from 400 to 2,400 μmol photons·m−2·s−1 for 6 days.The cells were not previously adapted to each irradiance

Iext Equation 2, García-Malea et al. (2006) Equation 3, Muller-Feuga (1999)μmol·m−2·s−1 R2 R2

400 0.906 0.911

800 0.981 0.980

1,200 0.968 0.958

1,600 0.898 0.927

2,000 0.884 0.782

2,400 0.952 0.945

Table 1 Non-linear regressioncoefficients determined by non-linear regression analysis of ex-perimental data to the twogrowth models considered

130 J Appl Phycol (2012) 24:125–134

limitation zone of the response curve. The opposite effect wascaused by the strongest irradiances where specific growthrates were 25% lower than the highest. This decrease can berelated to photoinhibition. Once high biomass concentrationsconsiderably attenuated the irradiance inside the photobio-reactor, photoinhibition began to be less severe or did nottake place at all and growth rates were similar to cultures thatwere not inhibited.

When the maximum daily productivity and biofixationof CO2 found for each culture at different externalirradiances were compared, both increased with irradianceup to 1,600 μmol photons·m−2·s−1. At higher irradiances,Synechocystis sp. reduced its productivity and CO2 bio-fixation. These outcomes agree with previous observations(Fig. 2). According to those results, an irradiance below1,600 μmol photons·m−2·s−1 limits Synechocystis sp.growth, while irradiances higher than this cause a certaindegree of photoinhibition.

It must be pointed out, however, that this photoinhibitiondid not cause irreversible deactivation of the photosynthesisreaction centres, since irreversible photoinhibition leads toculture collapse and the cessation of growth, an effect notobserved here. It therefore seems that Synechocystis sp. maysuffer photoinhibition at irradiances above 1,600 μmolphotons·m−2·s−1, but when cells move to the centre of thereactor, where the available irradiance is reduced, light mayeven limit growth. This is the point at which cells are able toregenerate their photosynthetic systems, continuing photo-synthetic processes. Then both photoinhibition and photo-limitation take place simultaneously in the cells, slowinggrowth, but not stopping it. This phenomenon is character-istic of dense cultures, and it was previously observed byMolina-Grima et al. (1996) in continuous cultures ofIsochrysis galbana. From these results, it may be concludedthat the cyanobacterium Synechocystis sp., under theconditions described in this study, shows maximum produc-tivity when Iext=1,600 μmol photons·m−2·s−1.

Modelling of specific growth rates and average irradiance Thefact that the Muller-Feuga (1999) model successfullyconverges when the García-Malea et al. (2006) model is

unable to do so might partly be an outcome of the largenumber of parameters involved in the latter. The equationproposed by Muller-Feuga (1999) offers the advantage ofmodelling the relationship between light and growth with asmaller number of parameters. Another advantage of thismodel is the easier identification of growth states by meansof the parameters calculated by the model. For the García-Malea et al. (2006) model, only parameter c gives anyimmediate information relating growth with light because itequals the average irradiance needed to reach half themaximum growth. The remaining parameters adjust theshape of the curve and their physiological interpretation isdifficult. However, the Muller-Feuga (1999) model wasdeveloped to explain the three fundamental rules forsubstrate metabolism of an organism, which are (1) theneed for a maintenance ration to avoid weight loss, Ie, whengrowth is nil; (2) the existence of a saturation ration, Is, atwhich specific growth rate is at its maximum, μs; and (3)the existence of an optimum ration, lying at some pointbetween the saturation ration, Is, and the maintenanceration, Ie.

The reason that in some studies a decrease in growth rateafter reaching a saturating irradiance was not reported(Molina-Grima et al. 1994, 1996; Acién-Fernández et al.1998; Jeon et al. 2005), and results showed a hyperbolictendency similar to that described by García-Malea et al.(2006) can be found in the fact of specific growth rates andtheir respective average irradiances were measured after aperiod of adaptation of the cultures to each irradiance. Thecultures analysed in the present study were produced in adiscontinuous mode, starting with a biomass concentrationof 0.01 g·L−1. Thus, average irradiances during the earlydays of culturing were so high that they may well havesaturated the reaction centres, leading to the slowdown ingrowth rates observed here.

The modelling of growth rates and irradiance has let us tofind the maximum growth rate of 0.108 h−1 at 930±22 μmolphotons·m−2·s−1. This optimum average irradiance is ratherhigher than those reported for other strains of micro-algae:391 μmol photons·m−2·s−1 for Selenastrum tricornutum(Benson and Rusch 2006), 485 μmol photons·m−2·s−1 for

Non-linear regression parameters, Muller-Feuga (1999)

Iext μs Ie Is R2

μmol photons·m−2·s−1 h−1 μmol photons·m−2·s−1 μmol photons·m−2·s−1

400 0.068±0.01 24±4 423±11 0.911

800 0.072±0.08 65±2 577±8 0.980

1,200 0.075±0.02 180±10 584±16 0.958

1,600 0.108±0.03 248±22 930±22 0.927

2,000 0.047±0.01 299±20 947±93 0.882

2,400 0.036±0.04 319±30 1,328±25 0.945

Table 2 Non-linear regressionparameters after fitting experi-mental data μi−Iav,i to the modelproposed by Muller-Feuga(1999)

J Appl Phycol (2012) 24:125–134 131

Porphyridium cruentum (Muller-Feuga 1999) or 90 to150 μmol photons·m−2·s−1 for Haematococcus pluvialis(García-Malea et al. 2006).

In the cultures grown for this study, average irradiancevalues as high as the saturating irradiance of 930±22 μmolphotons·m−2·s−1 were reached only at the beginning of thediscontinuous cultures, when biomass concentrations werelow. In high-density cultures (>2 g L−1), extremely strongexternal irradiances would be required for this, greater than2,400 μmol photons·m−2·s−1, to achieve an irradiance insidethe photobioreactor of around 930±22 μmol pho-tons·m−2·s−1 or higher. This may be observed in Fig. 2a,where Iint values lower than 200 μmol photons·m−2·s−1 arefound for Cb>2 g L−1 in all the growth curves. Thus, in adense Synechocystis sp. culture, light absorption will makeit difficult to find any photoinhibition, on the assumption,of course, that culture mixing is adequate. This resultallows the conclusion to be reached that the cyanobacteri-um Synechocystis sp. shows strong resistance to highexternal irradiances, a quality desirable in outdoor cultures.

Modelling of growth rates not only shows the responseof these microorganisms to received irradiance, but rather italso gives additional useful physiological information, as Ie.The parameter Ie, by definition, is the minimum averageirradiance that a photoautotrophic cell needs to begingrowing and gaining weight by harvesting energy fromlight. Muller-Feuga (1999) deduced this parameter forPhorphyridium cruentum at 25°C from the experimentalresults of Dermound et al. (1992), as 15 μmol pho-tons·m−2·s−1. According to the Ie values estimated by thegrowth model (see Table 2), Ie is not constant as may beexpected, but increases with increases in irradiance on thereactor surface. The explanation for this may be thephotoacclimation effect. For the batch cultures presentedhere, minimum average irradiances, Ie, occurred when therewere high concentrations of biomass, after the cultures hadbeen running for some days. During this period of time,cells had been adapting to strikingly different irradiances. Itcan be observed that Synechocystis sp. cells in the2,400 μmol photons·m−2·s−1 cultures had to adapt theirphotosynthetic mechanisms to be able manage to grow inthe wide range from 2,000 μmol photons·m−2·s−1 tovirtually 0 μmol photons·m−2·s−1, doing this in less than6 days. However, for the 400 μmol photons·m−2·s−1

cultures, the adaptations were only from 400 down toalmost 0 μmol photons·m−2·s−1.

As observed, the cultures exposed to the highestirradiances, which reached average irradiances as high asnormal noonday light (2,000 to 2,400 μmol pho-tons·m−2·s−1) did show some degree of photoinhibitionbecause the photosynthetic centres were not able to processall the energy received at the reaction centres (Falkowskiand Owens 1980; Molina-Grima et al. 1996; Vonshak and

Guy 1992). In such a situation, photosynthetic cells start upcertain defensive mechanisms to dissipate the absorbedenergy, such as degradation of photosystem II. When theirradiance is reduced, and cells are able to use all thephotons absorbed, the photosynthetic defence mechanismsare blocked and photosystems can be reactivated oncemore. If average irradiances are severe, it is possible forsome photosystems to be permanently damaged andbecome inactive (Zou and Richmond 2000). These dam-aged photosystems will not be able to work again whenirradiance is reduced by the effects of cell shading. Thisindicates that the photosynthetic cells will require moreavailable light to absorb the minimum amount needed tostart to grow: they will need a higher Ie because only someof the photosystems remain active. In contrast, culturesreceiving a moderate average irradiance during the earlydays of culturing keep all their photosystems in workingorder and are ready to process more absorbed energy whenlight levels are lowered. Hence, in this case, cells are able toabsorb more energy when receiving the same quantity ofphotons than cells from the cultures exposed to the highestirradiances. The results presented here showed that Ieincreased with external irradiance, and then averageirradiance increased, taking values from 24±4 μmol pho-tons·m−2·s−1 to 248±15 μmol photons·m−2·s−1 for the non-photoinhibited cells (from 400 to 1,600 cultures). Forcultures with an incident irradiance of up to 1,600 μmolphotons·m−2·s−1, minimum average irradiance, Ie, seemedto remain constant, showing that once photosystems enteredthe zone of photoinhibition, they were permanently dam-aged and deactivated to the same extent, regardless of howmuch higher the inhibiting received light was.

If cultures with a high concentration of biomass aredesirable, as occurs in high-yield production systems, thislast point leads to consideration of the need to adapt theinoculum to low irradiances, rather than to high irradiances.However, in the present study, it has been observed thatphotoacclimation to low irradiances renders cells able touse less light energy for growth. This condition is highlydesirable in high-density cultures, where photosyntheticcells are exposed for most of the time to low irradiancesinside the reactor, owing to the self-shading effect. In awell-mixed outdoor reactor, cells will be exposed only forshort periods of time to high irradiances at midday onlywhen cells are within a few millimetres down the reactorsurface. However, they stay much longer within the culture,receiving low irradiances for most of the time. Furthermore,as observed here and by other authors (Molina-Grima et al.1999; Yun and Park 2003; García-Malea et al. 2006), indense cultures, where photoinhibition is found, only alimited part of the PSII photosystem is irreversiblydamaged, while the rest can be regenerated when irradianceis reduced. This seems to indicate that photoacclimatation

132 J Appl Phycol (2012) 24:125–134

to low light would be more useful than photoacclimation tohigh light intensities as a way of improving biomassproduction in outdoor systems. In that way, cells will uselight more efficiently when it is limited as occurs in innerzones of photobioreactors, thus improving their photosyn-thetic efficiency. To avoid long periods of exposure to highirradiances in regions near the surface of the culture,thorough mixing would be required.

Nonetheless, it must be stated that the present studyshould not be considered the last word in respect ofmaximum specific growth rates, but as an initial stepanalysing photosynthetic behaviour of a strain of thecyanobacterium Synechocystis over a wide range ofphotosynthetic states, biomass concentrations and averagelight intensities. Additional studies including a period ofadaptation and steady-state growth are also required.

Conclusions

In dense photoautotrophic cultures, the response to irradi-ance must be studied by considering not the incidentirradiance, but the average irradiance, which is the light thatactually reaches the cells. Batch cultures of the native strainSynechocystis sp. allowed the determination of a prelimi-nary optimum average irradiance range. Experimental datashowed a maximum specific growth rate and CO2 biofix-ation of 0.092 h−1 and 1.96 g L−1 day−1, respectively. Themaximum values were found for cultures irradiated with anincident light of 1,600 μmol photons·m−2·s−1. To determinehow the specific growth rate varied with available irradi-ance, the average irradiance was calculated using a simpleprocedure and two different referenced growth models wereanalysed. The main difference between these models is theshape of the growth curve in response to average irradiance:the Muller-Feuga (1999) model predicts a decrease in thegrowth rate after reaching μmax, while, in contrast, theGarcía-Malea et al. (2006) model estimates that the growthrate will follow a hyperbolic relationship with averageirradiance. It was found that the experimental behaviour ofSynechocystis sp. in batch cultures without previous lightadaptation was closer to the model proposed by Muller-Feuga (1999). Non-linear regression of the experimentaldata to this model resulted in a maximum specific growthrate of 0.108±0.03 h−1, observed at an average irradianceof 930±22 μmol photons·m−2·s−1, revealing that the nativecyanobacterium Synechocystis sp. is a strain tolerant of highirradiances. The results also indicate that there is areduction in the minimum irradiance needed to startgrowing, Ie, when the available irradiance is lower,indicating that cells which are adapted to low irradiancesare able to use light more efficiently. This work suggests a

new concept for improving productivities of high-densitycultures: their adaptation to low-light levels.

Acknowledgements This work was supported by IDOM Interna-tional, Spain. We wish also to thank Dr. Vernet for the helpfulcomments provided.

References

Acién-Fernández FG, García-Camacho F, Sánchez-Pérez JA,Fernández-Sevilla JM, Molina-Grima E (1997) A model forlight distribution and average solar irradiance inside outdoortubular photobioreactors for the microalgal mass culture.Biotechnol Bioeng 55:701–710

Acién-Fernández FG, García-Camacho F, Sánchez-Pérez JA, Fernández-Sevilla JM, Molina-Grima E (1998) Modeling of biomass produc-tivity in tubular photobioreactors for microalgal cultures: effects ofdilution rate, tube diameter and solar irradiance. Biotechnol Bioeng58:605–616

Benson BC, Rusch KA (2006) Investigation of the light dynamics andtheir impact on algal growth rate in a hydraulically integratedserial turbidostat algal reactor. Aquacultural Eng 35:122–134

Dermound D, Chaumont D, Thebault JM, Dauta A (1992) Modellingof growth of Porphyridium Cruentum in conncection with twoindependent factors; light and temperature. Bioresource Technol42:113–117

Eaton AD, Clesceri LS, Greenberg AE (eds) (1995) Standard methodsfor the examination of water and wastewater. American PublicHealth Association, Washington (1995)

Falkowski PG, Owens TG (1980) Light and shade adaptation: twostrategies in marine phytoplankton. Plant Physiol 66:592–595

Gallegos CL, Platt T, Harrison WG, Irwin B (1983) Photosyntheticparameters of arctic marine phytoplankton: vertical variationsand time scales adaptation. Limnol Oceanogr 28(4):698–708

García-Malea MC, Acién FG, Fernández JM, Cerón MC (2006)Continuous production of green cells of Haematococcus pluvia-lis: modeling of the irradiance effect. Enzyme Microb Tech38:981–989

Harmelen T, Oonk H (2006) Microalgae biofixation processes:applications and potential contributions to greenhouse gasmitigation options. http://www.ieaghg.org/index.php?/2009112028/biofixation-network.html. Accessed 22 November2007.

Janssen M, Tramper J, Mur LR, Wilffels RH (2002) Enclosed outdoorphotobioreactors: light regime, photosynthetic efficiency, scaleup and future prospects. Biotechnol Bioeng 81:193–210

Jeon YC, Cho CW, Yun YS (2005) Measurement of microalgalphotosynthetic activity depending on irradiance and quality.Biochem Eng J 27:127–131

Mann JE, Myers J (1968) On pigments, growth and photosynthesis ofPhaeodactylum tricornutum. J Phycol 4:349–355

Martínez L (2009) Eliminación de CO2 con microalgas autóctonas—CO2 biofixation with native microalgae. Dissertation. Universityof León

Martínez L, García AI, Morán A (2005) Isolation and selection ofmicroalgae species for CO2 biofixation. J Biotechnol 135(S):122–126

Michaelis L, Menten ML (1913) Die Kinetik der Invertinwirkung.Biochem Z 4:334–336

Molina-Grima E, García Camacho EF, Sánchez Pérez JA, FernándezSevilla JM, Acién Fernández FG, Contreras Gómez A (1994) Amathematical model of microalgal growth in light-limitedchemostat culture. J Chem Technol Biotechnol 61:167–173

J Appl Phycol (2012) 24:125–134 133

Molina-Grima, Fernández-Sevilla JM, Sánchez-Pérez JA, García-Camacho F (1996) A study on simultaneous photolimitation andphotoinhibition in dense microalgal cultures taking into accountincident and averaged irradiances. J Biotechnol 45:59–69

Molina-Grima, Acién-Fernández FG, García Camacho F, Chisti Y(1999) Photobioreactors: light regime, mass transfer and scale up.J Biotechnol 70:231–247

Molina-Grima E, Acién-Fernández FG, García-Camacho F, Camacho-Rubio F, Chisti Y (2000) Scale-up of tubular photobioreactors. JAppl Phycol 12:355–368

Muller-Feuga A (1999) Growth as a function of rationing: a modelapplicable to fish and microalgae. J Exp Mar Biol Ecol 236:1–13

Ono E, Cuello JL (2006) Feasibility assessment of microalgal carbondioxide sequestration. Technology with photobioreactor and solarcollector. Biosystems Eng 95:597–606

Richmond AE (1986) Microalgal culture. CRC Crit Rev Biotechnol4:369–488

Spektorova LV, Nosova LP, Goronkova OI, Albitskaya ON, FilippovskijYuN (1986) High-density culture of marine microalgae—promisingitems for mariculture. II. Determination of optimal light regime forChlorella sp. f. marina under high-density culture conditions.Aquaculture 55:221–229

Tredici MR, Chini-Zitelli G (1998) Efficiency of sunlight utilization:tubular versus flat photobioreactors. Biotechnol Bioeng 57:187–197

Vonshak A, Guy R (1992) Photoadaptation, photoinhibition andproductivity in the blue green alga Spirulina platensis, grownoutdoors. Plant Cell Environ 15:613–616

Yun YS, Park JM (2003) Kinetic modeling of the light-dependentphotosynthetic activity of the green microalga Chlorella vulgaris.Biotechnol Bioeng 83:303–311

Zou N, Richmond A (2000) Light-path length and population densityin photoacclimation of Nannochloropsis sp. (Eustigmatophy-ceae). J Appl Phycol 12:349–354

134 J Appl Phycol (2012) 24:125–134