Bioresource Technology 92 (2004) 133–141

Chlorophyll production from Spirulina platensis: cultivation withurea addition by fed-batch process

Carlota de Oliveira Rangel-Yagui, Eliane Dalva Godoy Danesi,Jo~aao Carlos Monteiro de Carvalho *, Sunao Sato

Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of S~aao Paulo,

Av. Prof. Lineu Prestes, 580, B-16, 05508-900, S~aao Paulo-SP, Brazil

Received 1 February 2002; received in revised form 24 July 2003; accepted 7 September 2003

Abstract

The cyanobacterium Spirulina platensis is an attractive alternative source of the pigment chlorophyll, which is used as a natural

color in food, cosmetic, and pharmaceutical products. In this work, the influence of the light intensity and urea supplementation as a

nitrogen source using fed-batch cultivation for S. platensis growth and chlorophyll content was examined. Cultivations were carried

out in 5 l open tanks, at 30± 1 �C. Response surface methodology was utilized for analysis of the results, and models were obtained

for biomass productivity, nitrogen-cell conversion factor and chlorophyll productivity. The best cellular growth was observed with

500 mg/l of urea at a light intensity of 5600 lx, whereas the highest concentration of chlorophyll in the biomass was observed with

500 mg/l of urea at a light intensity of 1400 lx. Overall, the best chlorophyll productivity was observed with 500 mg/l of urea at

a light intensity of 3500 lx, providing the optimal balance between the cellular growth and the biomass chlorophyll content.

� 2003 Elsevier Ltd. All rights reserved.

Keywords: Spirulina platensis; Biomass production; Chlorophyll; Fed-batch process; Urea; Light intensity

1. Introduction

Chlorophyll is a naturally occurring pigment present

in photosynthetic plants, including algae, and in some

photosynthetic bacteria, known as cyanobacteria. The

greater part of industrially prepared chlorophyll-deriv-

atives is destined for the increasing demand for natural

colorants for food and beverages. Some of the industrial

production is also destined for the cosmetic and toiletrymarket, and to the pharmaceutical market (Hendry,

1996). Currently, most of the commercially produced

chlorophyll is obtained from vegetable sources (Gross,

1991). Nevertheless, there is a growing interest in the

biotechnology field for obtaining non-vegetable sources

of colors. The use of fermentation processes possess a

number of advantages when compared to vegetable

sources, including the possibility of continuous cultiva-tion, and the rapid multiplication of microorganisms

(Taylor, 1984).

*Corresponding author. Fax: +55-11-38156386.

E-mail address: jcmdcarv@usp.br (J.C.M. de Carvalho).

0960-8524/$ - see front matter � 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2003.09.002

Spirulina platensis is a cyanobacterium that has beenlargely studied due to its commercial importance as a

source of protein, vitamins, essential amino acids, and

fatty acids (Ciferri and Tiboni, 1985; Zhang et al., 1999).

More recently, special attention has been given to S.

platensis as a potential source of pharmaceuticals, and

other high value products such as chlorophyll (Boro-

witzka, 1995; Chen and Zhang, 1996). The utilization of

chlorophyll from S. platensis is an attractive alternativethat should be considered due to its high content of this

pigment, and ease of cultivation. The cyanobacterium S.

platensis possesses a high tolerance to alkaline pH, for

ease of cultivation; a large size for its cell aggregates for

ease of harvest; and an easily digestible cell wall (Jensen

and Knutsen, 1993).

It has been shown that the composition of the cultiva-

tion medium, cellular age, and light intensity are the mainfactors influencing chlorophyll content in S. platensis

biomass. Cultivations carried out under poor illumination

conditions present higher biomass chlorophyll content

than cultivations carried out under high illumination

conditions, suggesting an inverse proportional relation-

ship between light intensity and chlorophyll content

Nomenclature

[Cla] concentration of chlorophyll in the S. platensis

biomass (mg/g)

I light intensity (lx)

Ic codified value for the light intensity

K time constant (d�1)Mo initial mass of urea per unit volume (mg/l)

MuðtÞ accumulated mass of urea per unit volume

added at the time t (mg/l)

MuadðtÞ mass of urea per unit volume added at the

time t (mg/l)

Mut total mass of urea per unit volume added

(mg/l)

Mutc codified value for the total mass of urea perunit volume added

p significance level

PCla (chlorophyll productivity) ratio between chloro-

phyll concentration (½Cla�Xmax) and cultiva-

tion time (mg/l/d)

PX (biomass productivity) ratio between formed cel-

lular concentration (dry mass) and the culti-vation time (mg/l/d)

t time (d)

T total feeding time (d)

X cellular concentration––dry matter (mg/l)

V volume of the tank (5 l)

Xmax maximum cellular concentration (mg/l)

YX=N (nitrogen-cell conversion factor) ratio between

the cellular concentration and the nitrogenadded in the tank (g/g)

134 C.de.O. Rangel-Yagui et al. / Bioresource Technology 92 (2004) 133–141

(Bogorad, 1962; Eloranta, 1986). Moreover, the use of

high light intensity in S. platensis cultivation can lead to

two main effects: (i) photoinhibition, decreasing the cel-

lular growth rate, and (ii) photoxidation, with severe cell

damage and, in extreme cases, total loss of the cultivation

(Jensen and Knutsen, 1993; Vonshak et al., 1994). Al-

though photoinhibition usually occurs at light intensities

above the saturation of the photosynthetic rate, this phe-nomenon can be observed at light intensities below the

saturation of the photosynthetic rate in cultivations under

stress conditions, such as low temperatures (Samuelson

et al., 1985). Nevertheless, it should be pointed out that

S. platensis requires more light for photosynthesis and

cellular growth than other cyanobacteria, since it grows

under high salinity and pH conditions (Kebede and

Ahlgren, 1996).According to Piorreck et al. (1984), the concentration

of chlorophyll in S. platensis biomass increases with an

increase of nitrogen concentration in the cultivation

medium. The conventional S. platensis nitrogen sources

are nitrates. However, Stanca and Popovici (1996)

demonstrated that the utilization of urea as a nitrogen

source in S. platensis cultivation leads to an increase in

both the total biomass and the biomass chlorophyllcontent. Urea is easily assimilated by S. platensis, prob-

ably due to its spontaneous hydrolysis to ammonia under

alkaline cultivation (Danesi et al., 2002) and/or urease

action (Carvajal et al., 1980). According to Boussiba

(1989), when both ammonia and nitrate are present

in the medium, ammonia is assimilated preferentially.

However, ammonia can be toxic to S. platensis, when

present at high concentrations. The utilization of a fed-batch process for urea addition, with an exponentially

increasing mass flow, may be a way to avoid this prob-

lem, by tuning the rate of urea addition to the rate of

urea utilization by the microorganism (Pirt, 1975).

In this work, the influence of light intensity and urea

utilization on S. platensis growth and its chlorophyll

content was investigated. S. platensis cultivations using

urea as the nitrogen source and fed-batch process, with

an exponentially increasing mass flow, were compared

to the standard cultivation of S. platensis using potas-

sium nitrate as the nitrogen source and a batch process,

at different light intensities.

2. Methods

2.1. Microorganism and growth conditions

S. platensis was grown in geometrically lengthened

open tanks (Richmond, 1983), with a total area of 1250

cm2, 5 l operation volume, and equipped with paddlewheels providing an agitation of 18 rpm. A standard

cultivation medium containing KNO3 as the nitrogen

source (Paoletti et al., 1975) was utilized in the standard

cultivations (Section 2.3.1), and the same medium with

urea replacing KNO3 was utilized for all other cultiva-

tions (Section 2.3.2). The carbon sources present in the

standard medium were 8.89 g/l Na2CO3 and 15.15 g/l

NaHCO3. The initial pH of the medium was set at9.5 ± 0.2, and no adjustments were made throughout the

cultivations. The temperature was set at 30± 1 �C.Different light intensity values (I) were examined by

adjusting the distance of fluorescent lamps from the

tank surface, and measuring the light intensity value in a

Sekonik luximeter. Since the cultivations were carried

out in open tanks, water loss due to evaporation oc-

curred. Therefore, the total volume of each tank wascorrected daily, with the addition of deionized water

until the initial volume level observed for the tank was

reached.

C.de.O. Rangel-Yagui et al. / Bioresource Technology 92 (2004) 133–141 135

2.2. Preparation of the inoculum

A 200 ml suspension of S. platensis grown in standard

culture medium was used as the inoculum (Paoletti et al.,

1975). Cultures were cultured in a 500 ml Erlenmeyer

flask on a rotary shaker (agitation of 100 rpm), at a

temperature of 30 �C and light intensity of 3500 lx. The

inoculum was recovered during the exponential-growthphase, filtered, and washed with a 0.9% NaCl solution to

completely remove the KNO3. The cells were re-sus-

pended in the same standard medium in the absence of

KNO3 and used to inoculate the cultivation tanks. For

the standard cultivations (Section 2.3.1), the KNO3

concentration was 2.57 g/l, whereas for the experiments

with urea as the nitrogen source (Section 2.3.2) the urea

concentration was 80 mg/l and urea was added on al-ternate days to reach the desired final concentration

(Section 2.3.2.2). For all experiments, the starting cel-

lular concentration was 50 mg/l (dry weight).

2.3. Cultivation of S. platensis

2.3.1. Standard cultivations

The standard cultivations correspond to ones carried

out utilizing the standard culture medium (Paoletti et al.,1975). At least one standard cultivation was carried out

at each light intensity studied, in order to compare the

utilization of urea as a nitrogen source with the well

established utilization of KNO3 (Paoletti et al., 1975).

The standard experiments were not considered in the

Table 1

Experimental design for the full factorial search and general results for maxim

([Cla]), biomass productivity (PX ), chlorophyll productivity (PCla), and nitrog

per unit volume (Mut) and the light intensity (I)

Experiment Mut (mg/l) I (lx) Xmax (mg/l) [

A 380 2000 661

B 620 2000 671

SA;Ba – 2000 623

C 380 5000 1553

D 620 5000 1632

SC;Db – 5000 1423

E 500 3500 1266

SEc – 3500 1081

F 500 3500 1222

G 500 3500 1290

H 332 3500 1214

I 668 3500 1185

SF;G;H;Id – 3500 1193

J 500 5600 1945

SJe – 5600 1878

K 500 1400 408

SKf – 1400 455

a Standard cultivation (using 2.57 g/l KNO3) carried out at the same lightb Standard cultivation (using 2.57 g/l KNO3) carried out at the same lightc Standard cultivation (using 2.57 g/l KNO3) carried out at the same lightd Standard cultivation (using 2.57 g/l KNO3) carried out at the same lighte Standard cultivation (using 2.57 g/l KNO3) carried out at the same lightf Standard cultivation (using 2.57 g/l KNO3) carried out at the same light

regression analysis, but were considered for comparing

the two processes by multifactor analysis of variance

(MANOVA).

2.3.2. Experiments with urea as the nitrogen source

2.3.2.1. Experimental design. A central composition de-

sign for two independent variables (total mass of ureaadded per unit volume and light intensity) at five levels

each (Box et al., 1978) was carried out to evaluate

the effect of these variables on biomass productivity,

chlorophyll productivity and nitrogen-cell conversion

factor. The central value for the total mass of urea per

unit volume, Mut ¼ 500 mg/l was chosen based on pre-

liminary studies (Rangel, 2000), and the addition was

according to Eq. (1) (Section 2.3.2.2). The central valuefor light intensity (I) was chosen to be 3500 lx (Ferraz

et al., 1985). The values of these two independent vari-

ables are given in Table 1. The experiments E, F, and G

are repetitions of the central composition (central values

for I and Mut), carried out in order to verify the exper-

imental variability, taken into account in the multifac-

torial regression analysis.

2.3.2.2. Urea feeding regime. A fed-batch process with

exponentially increasing mass flow was used, according

to the following equation:

MuðtÞ ¼ MoeKt ð1Þ

where MuðtÞ is the accumulated mass of urea per unit

volume added at the time t, Mo is the initial mass of urea

um cellular growth (Xmax), concentration of chlorophyll in the biomass

en-cell conversion factor (YX=N) as a function of the total mass of urea

Cla] (mg/g) PX (mg/l/d) PCla (mg/l/d) YX=N (g/g)

13.1 36.7 0.48 3.2

15.5 37.2 0.58 2.1

14.2 34.6 0.49 1.5

6.2 97.1 0.60 8.5

9.2 113.6 1.05 5.5

14.9 88.9 1.32 3.5

13.9 74.5 1.04 5.0

13.1 60.1 0.79 2.6

10.1 71.9 0.73 5.0

12.5 80.6 1.01 5.3

7.5 71.4 0.54 7.5

13.1 69.7 0.91 3.6

12.1 70.2 0.85 2.9

6.0 114.4 0.69 8.1

11.6 110.5 1.28 4.6

19.9 22.7 0.45 1.5

19.0 23.9 0.45 1.0

intensity used in experiments A and B.

intensity used in experiments C and D.

intensity used in experiment E.

intensity used in experiments F, G, H, I.

intensity used in experiment J.

intensity used in experiment K.

0

100

200

300

400

500

600

700

0 2 4 6 8 10 12 14 16

t (days)

Mu (

t) (m

g/l)

Fig. 1. Mass of urea accumulated in function of time for the experi-

ment H––Mut ¼ 332 mg/l (�), experiment G––Mut ¼ 500 mg (j), and

experiment I––Mut ¼ 668 mg/l (M), calculated according to Eq. (1).

136 C.de.O. Rangel-Yagui et al. / Bioresource Technology 92 (2004) 133–141

per unit volume added to the cultivation, and K is the

time constant. The total feeding time (T ) was 14 d,

therefore t6 T . The cultivations were fed on alternatingdays, in a way that the urea was added at times t equalto 0, 2, 4, 6, 8, 10, 12 and 14 d. The initial mass of urea

was fixed at 80 mg/l, and according to Eq. (1), when

t ¼ T ,MuðtÞ ¼ Mut, whereMut is the total mass of urea per

unit volume added. The variation of the mass of urea

added with time, according to Eq. (1), for three different

experiments, G, H, and I is shown in Fig. 1 (Table 1).

The mass of urea per unit volume added at the time t(MuadðtÞ), was calculated as follows:

MuadðtÞ ¼ MuðtÞ �Muðt�hÞ ð2Þ

where Muðt�hÞ corresponds to the accumulated mass of

urea per unit volume added at the time t � h, calculatedutilizing Eq. (1), where h is the interval of feeding (in this

case, 2 d). The urea feeding was carried out by means of

the addition of 20 ml of urea stock solutions at knownconcentrations.

2.4. Analytical methods

The S. platensis cellular concentration (X ) was de-

termined by the turbidimetric method (Leduy and

Therien, 1977), at 560 nm, on a Celm E-225-D spectro-

photometer. The concentration of chlorophyll a in the S.

platensis biomass ([Cla]) was determined spectrophoto-

metrically at 661.7 nm (Myers and Kratz, 1955), also ona Celm E-225-D spectrophotometer. Briefly, knowing

the cellular concentration, a sample containing 5 mg of

S. platensis (dry weight) was harvested by filtration

through Millipore cellulose acetate membranes (pore

diameter of 0.45 lm). Subsequently, the membrane

containing the biomass was placed in a tube with 10 ml

of acetone, immersed in an ice bath, and then chloro-

phyll was extracted for 2 min using a Tecnal TE-039homogenizer. The chlorophyll extract obtained was fil-

tered through a Millipore polytetrafluorethylene mem-

brane (pore diameter of 1 lm), and the optical density

was measured at 661.7 nm. The optical density was

correlated with the chlorophyll content according to a

calibration curve obtained utilizing pure chlorophyll

(Sigma).

The ammonia concentration in the cultivations was

determined in alternate days, using an Orion 95-12

ammonia ion selective electrode, connected to an Orion

710-A potentiometer. The conductivity values obtainedwere correlated to the actual ammonia concentrations in

the cultivations through calibration curves (Leduy and

Samson, 1982), prepared on each analysis day, using

solutions of known concentrations of ammonia.

The total carbonate concentration present on the

cultivations was also determined on alternate days, by

titration with hydrochloric acid (Pierce and Haenisch,

1948). Before the analysis, sodium hydroxide was addedto the samples in order to convert all the bicarbonate

into carbonate. The dissociated carbonate was then

titrated with hydrochloric acid, in two steps: the first one

characterized by the phenolphthalein indicator change

of color from pink to colorless, and the second one

characterized by the methyl orange indicator change of

color from orange to pink. The two endpoints are re-

lated to the carbonic acid formation by the HCl addi-tion, according to the following reaction:

CO2�3 þHþ $ HCO�

3 ðFirst stepÞ

HCO�3 þHþ $ H2CO3 ðSecond stepÞ

Since the first step of the titration (phenolphthaleinendpoint) will neutralize not just the carbonate but also

the excess of hydroxide added, the total carbonate

concentration (g Na2CO3/l) was calculated from the

volume of HCl used on the second step of the titration

multiplied by two. All analytical determinations de-

scribed above were repeated at least two times to verify

reproducibility.

2.5. Statistical analysis

The response surface methodology (RSM) was used

to understand the overall effect of light intensity andurea fed-batch addition on S. platensis cultivation. RSM

is a group of techniques used to evaluate relationships

between one or more measured responses and a number

of quantitative independent variables that may have

important effects on the measured responses (Box et al.,

1978). In this paper, response equations with the cor-

respondent surface plots were generated in the following

form:

Yn ¼ b0 þ b1Mutc þ b2Ic þ b11M2utc þ b22I2c þ b12MutcIc

ð3Þwhere Yn is the predicted response for the dependent

variable (PX , PCla, and YX=N),Mutc is the codified value for

the total mass of urea added, Ic is the codified value for

0

400

800

1200

1600

2000

0 2 4 6 8 10 12 14 16 18

t (days)

X (m

g/l)

5

6

7

8

9

10

11

pH

Fig. 2. Experimentally determined growth curves (�) for the experi-

ment G (�) and for the corresponding standard cultivation with

KNO3––SF;G;H;I (�), and experimentally determined pH curves for the

experiment G (j), and for the corresponding standard cultivation with

KNO3––SF;G;H;I (�). The error bars correspond to the standard devi-

ations.

C.de.O. Rangel-Yagui et al. / Bioresource Technology 92 (2004) 133–141 137

the light intensity, and the b values are the estimated

polynomial coefficients. The codified variables Mutc and

Ic correspond to the fitted values obtained using ap-

propriate scaling procedures, according to the following

equations:

Mutc ¼Mut � 500

120ð4Þ

Ic ¼I � 3500

1500ð5Þ

The analysis of the data and mapping of the fitted re-

sponse surfaces were carried out using the software

S-PLUS 2000. For the independent variables (Mutc andIc), a significance level p < 0:05 was considered for the

coefficients b1 and b2. However, for the coefficients ob-

tained from combinations of the studied independent

variables (M2utc, I

2c , and the interaction MutcIc), b11, b22,

and b12, a significance level p < 0:15 was considered. Thecoefficients obtained from combinations of the studied

independent variables were taken into account in the

polynomials only when a better approximation wasobtained between the values estimated by the polyno-

mial and the experimental results. This could be ob-

served by an increase in the R2 value for the polynomial,

or by a decrease in the p value for the coefficients b1 andb2. Nevertheless, the analyses of variance for the re-

gressions were calculated considering an error of at most

5% (p < 0:05).

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16 18t (days)

Tota

l car

bona

te c

once

ntra

tion

(g N

a 2C

O3/l

)

Fig. 3. Experimentally determined total carbonate concentration

curves for the experiment G (r), and for the corresponding standard

cultivation with KNO3––SF;G;H;I (}). The error bars correspond to the

standard deviations.

3. Results and discussion

There have been numerous studies of the utilization

of diverse nitrogen sources in S. platensis cultivation,

and the best results for biomass production have been

attributed to the use of nitrates (Faintuch, 1989; Cornet

et al., 1998), thus confirming the wide utilization ofcultivation media containing KNO3 (Paoletti et al.,

1975) and NaNO3 (Stanca and Popovici, 1996) as the

nitrogen source. S. platensis cultivations generally have

nitrates added as the nitrogen source when grown as

batchcultures, and the typical initial nitrate source

concentration is �2.5 g/l (Paoletti et al., 1975; Stanca

and Popovici, 1996). No negative effect of this nitrate

concentration during S. platensis cultivation has beenreported. Faintuch (1989) demonstrated that the growth

rate obtained with the initial KNO3 concentration sug-

gested by Paoletti et al. (1975) is higher than the growth

rate obtained utilizing KNO3 concentrations of at most

1.0 g/l. This phenomenon was observed for cellular

concentrations of �200 mg/l, corresponding to the ini-

tial stages of cultivations done in the current study (Fig.

2). Therefore, the utilization of the fed-batch process forthe cultivations having KNO3 as the nitrogen source

could lead to nitrate concentrations lower than 1.0 g/l

during most part of the cultivation, and thus lower the

productivity. To confirm this assumption on the utili-

zation of fed-batch process in cultivations having KNO3

as the nitrogen source, three experiments were carried

out, at 3500 lx, with the addition of KNO3 in the same

manner as the urea (fed-batch process with exponen-

tially increasing mass flow), and the cellular growth re-sults were similar to the cellular growth results obtained

for the standard cultivations (data not shown). Al-

though lower cellular concentrations were not observed

for the cultivations with fed-batch addition of KNO3,

there is no need to use this process as opposed to the

batch cultivation. If the concentration of KNO3 was

high enough to cause inhibition due to increased salinity

of the medium, as happens with increasing concentra-tions of NaCl (Vonshak et al., 1996), the fed-batch

would be a better option, but this is not the case in the

current study.

Typical results for cellular growth (X ), pH, and total

carbonate concentration throughout the cultivation, are

presented in Figs. 2 and 3, respectively. As can be seen

138 C.de.O. Rangel-Yagui et al. / Bioresource Technology 92 (2004) 133–141

from Fig. 2, an increase in pH occurred with cellular

growth for both nitrogen sources. This observation can

be correlated to the carbon source consumption. The

bicarbonate ions are assimilated by the cyanobacteria

and subsequently converted into carbon dioxide and

carbonate. During the first one is utilization in photo-

synthesis and excretion of ion carbonate into the me-

dium, an increase in the pH of the system is generateddue to the shift of the bicarbonate–carbonate equilib-

rium towards bicarbonate (Fig. 4).Moreover, S. platensis

incorporates nitrate and urea also leading to increases

in the medium pH.

As can also be seen from Fig. 2, the cultivation with

added urea exhibited better cellular growth curves than

with KNO3 addition. The utilization of nitrate as the

nitrogen source requires the S. platensis metabolism toreduce nitrate to nitrite, and then to ammonia, which is

further assimilated (Hattori and Myers, 1966). The

ammonia present in the cultivations could enter the cells

without energy expenditure, according to an extracel-

lular/intracellular gradient of pH, and further be as-

similated, with glutamine synthetase (Boussiba, 1989).

According to the results, the utilization of fed-batch

process indeed avoided inhibitory ammonia concentra-tions (Belkin and Boussiba, 1991) in the cultivations

carried out with urea. Maximum ammonia concentra-

tions in the order of 10�4 M were observed. However,

concentrations of ammonia below 10�6 M were ob-

served for experiment H, corresponding to the smallest

Mut utilized.

According to Fig. 3, exhaustion of the carbon source

did not occur during cultivation and the same patternwas observed for all experiments. Therefore, the or-

ganism must have reached stationary growth phase due

to other factors such as exhaustion of the nitrogen

source or shading effect.

Fed-batch processes may cause volume alteration

during the cultivation, with an increase in the volume

(Yamane and Shimizu, 1984). This is particularly im-

portant in the alcoholic fermentation by fed-batch pro-cess using mash from sugar cane, currently employed in

Brazil (Echegaray et al., 2000). In this work, although

fed-batch process was utilized for the urea addition, no

increase in the volume of the tanks was observed. In

CELL

2HCO3 + CO3 + H2O

photosynthesis CULTIVATION

HCO3 + OH CO3 + H2O

CO2 − −

− −

2

−2

Fig. 4. Schematic representation of the bicarbonate assimilation by

S. platensis.

fact, evaporation occurred in all the cultivations, and

the volume of the tanks had to be corrected daily, as

mentioned earlier.

The results of Xmax, [Cla], PX , PCla, and YX=N for the

cultivations with urea, as well as the standard cultiva-

tions, with KNO3, are presented in Table 1. The highest

cellular growth value was observed with 500 mg/l of urea

and at a light intensity of 5600 lx (experiment J), and thelowest cellular growth value was observed with 500 mg/l

of urea and a light intensity of 1400 lx (experiment K).

Although the highest cellular growth was observed at

the highest light intensity studied, it is well known that

there is a light saturation point from which photoinhi-

bition of the cellular growth is observed, and the use

of higher light intensities should lead to this effect

(Samuelson et al., 1985; Vonshak et al., 1994).The cultivations carried out at 5000 and 5600 lx, with

urea as the nitrogen source, exhibited less color (yellow–

green) than the standard cultivations (dark green). This

phenomenon usually occurs when nitrogen is limiting

growth, suggesting that utilization of higher masses of

urea may be possible at higher light intensities. How-

ever, the difference observed in maximum cellular con-

centration (Xmax) was not significant enough to justifythe use of higher masses of urea only for biomass pro-

duction. Moreover, as can be seen from the results in

Table 1, the nitrogen-cell conversion factor obtained in

experiment D (I ¼ 5000 lx, Mut ¼ 620 mg/l), YX=N ¼ 5:5,is lower than the nitrogen-cell conversion factors ob-

tained in experiment C (I ¼ 5000 lx, Mut ¼ 380 mg/l),

YX=N ¼ 8:5, and in experiment J (I ¼ 5600 lx, Mut ¼ 500

mg/l), YX=N ¼ 8:1. Generally, as a limiting substratecomes close to excess, the efficiency of utilization of that

nutrient decreases.

The MANOVA applied to the results in Table 1 in-

dicates that better results were obtained with urea as

nitrogen source instead of KNO3 for the variables Xmax

(p ¼ 0:0167), PX (p ¼ 0:0425) and YX=N (p ¼ 0:0043). Forthe PCla, the results were not different (p ¼ 0:2180).

As can be see from the concentration of chlorophyllin the biomass values ([Cla]) presented in Table 1, there

is an inverse relationship between light intensity and the

concentration of chlorophyll in the biomass. The highest

value of [Cla] was observed with 500 mg/l of urea at

1400 lx, and the lowest value was observed with 500 mg/l

of urea at 5600 lx. Thus, it can be inferred that the

cultivation of S. platensis under poor illumination con-

ditions generates cells with higher chlorophyll contents,in order to optimize the light capturing (Eloranta, 1986).

However, the highest [Cla] values obtained at poor il-

lumination conditions did not compensate for the low

cellular growth observed, since there was no increase in

total chlorophyll productivity (PCla) for the cultivations

carried out at poor illumination conditions. In fact, the

highest chlorophyll productivity values were obtained

with intermediate light intensity, in agreement with

Fig. 5. Response surface showing the variation in biomass pro-

ductivity (PX ) with the light intensity (Ic) and the total mass of urea

per unit volume added (Mutc) to S. platensis cultivations.

Fig. 6. Response surface showing the variation in nitrogen-cell con-

version factor (YX=N) with the light intensity (Ic) and the total mass of

urea per unit volume added (Mutc) to S. platensis cultivations.

Fig. 7. Response surface showing the variation in chlorophyll pro-

ductivity (PCla) with the light intensity (Ic) and the total mass of urea

per unit volume added (Mutc) to S. platensis cultivations.

C.de.O. Rangel-Yagui et al. / Bioresource Technology 92 (2004) 133–141 139

Kebede and Ahlgren (1996). Therefore, at intermediate

light intensities there is a better balance between the

energetic gain with chlorophyll molecules biosynthesis,

expressed in cellular growth, and the energy spent in the

chlorophyll biosynthesis.

Among the cultivations carried out at the same light

intensity, the ones with higher total mass of urea added

presented higher concentrations of chlorophyll in thebiomass ([Cla]). For example, experiments H, G, and I

with total masses of urea added per unit volume (Mut) of

332, 500, and 668 mg/l, respectively, resulted in [Cla]values varying from 7.5 to 13.1 mg/g (Table 1). This

observation is in agreement with the results reported by

Piorreck et al. (1984) that also observed lower concen-

trations of chlorophyll in the biomass obtained from

cultivations carried out with limiting nitrogen concen-trations. There is an apparent relationship between ni-

trogen addition and the production of biomass with

higher chlorophyll contents, once that nitrogen appears

in the chlorophyll molecule composition (Bogorad,

1962). Moreover, the shading effect may have also

contributed to the higher concentrations of chlorophyll

observed in the biomass obtained from cultivations with

higher total mass of urea added, when cultivations werecarried out at the same light intensities. The cultivations

with higher total mass of urea added, at a fixed light

intensity, yielded higher values of cellular concentra-

tions (X ), which can generate higher shading effect

among cells, leading to a higher chlorophyll biosynthesis

rate in order to increase the efficacy of photons caption

and thus compensate for the lack of light intensity for

the cells not located on the surface (Hendry, 1996).Through the multivariable regression analysis applied

to the results obtained with urea as nitrogen source

(Table 1), the following equations were obtained for the

biomass productivity (PX ), nitrogen-cell conversion fac-

tor (YX=N), and chlorophyll productivity (PCla) as a

function of the codified variables (Mutc and Ic):

PX ¼ 71:80þ 33:31Ic þ 4MutcIc

ðR2 ¼ 0:97; p < 0:0001Þ ð6Þ

YX=N ¼ 5:03þ 2:25Ic � 1:20Mutc � 0:48MutcIc

ðR2 ¼ 0:97; p < 0:0001Þ ð7Þ

PCla ¼ 0:927þ 0:116Ic þ 0:134Mutc � 0:171I2 � 0:093M2utc

ðR2 ¼ 0:69; p ¼ 0:0227Þ ð8Þ

The surface responses corresponding to the multivari-

able analysis regressions equations for PX , YX=N, and PClaare presented in Figs. 5–7, respectively. According to

Fig. 5, the biomass productivity (PX ) shows a direct re-

lationship with the light intensity. A decrease in the

biomass productivity was observed with high urea ad-dition, at low light intensities, whereas at high light in-

tensity the inverse occurred. As can be seen from Fig. 5,

and also from the coefficient associated to the variable Ic

in Eq. (6), because the regression was done with codified

values, the light intensity is the dominant factor.

140 C.de.O. Rangel-Yagui et al. / Bioresource Technology 92 (2004) 133–141

The results presented in Fig. 6 indicate that high ni-

trogen-cell conversion factors (YX=N) occur with lower

total mass of urea added per unit volume (Mut) values

and at high light intensities. In other words, the nitro-

gen-cell conversion factor increased with the increase in

light intensity and decreased with the increase in the

total mass of urea added. At low light intensities, the

increase in Mut leads to a decrease in YX=N becausethe cellular growth is limited by the light intensity.

Moreover, as mentioned previously, the excess of urea

decreases the efficiency of utilization of this nutrient,

and therefore the YX=N values.

The response surface obtained for chlorophyll pro-

ductivity, presented in Fig. 7, shows that the chlorophyll

productivity for S. platensis cultivations reaches a max-

imum value with intermediate light intensity and inter-mediate total mass of urea values. The best chlorophyll

productivity was observed with a total mass of urea per

unit volume of 500 mg/l and at 3500 lx, resulting in a

better balance between the S. platensis cellular growth

and the biosynthesis of chlorophyll molecules, as stated

before.

4. Conclusions

S. platensis was cultivated at different light intensities,

utilizing urea as the nitrogen source. The utilization of

fed-batch process for urea addition, with an exponen-

tially increasing mass flow, avoided high concentrations

of ammonia (from urea hydrolysis) in the cultivations,

which could lead to toxicity to S. platensis. The results

show that better biomass productivity values are ob-

served with the use of urea as the nitrogen source, whencompared to KNO3, the traditional nitrogen source for

S. platensis cultivation. The best chlorophyll produc-

tivity, which reflected a balance between cellular growth

and chlorophyll concentration in the biomass, was ob-

served with 500 mg/l of urea at a light intensity of 3500

lx. Therefore, the use of urea as a nitrogen source uti-

lizing fed-batch process can be considered a promising

alternative for S. platensis cultivation for achievingoptimal biomass and chlorophyll production.

Acknowledgements

C.O. Rangel-Yagui is grateful for the financial sup-

port from CAPES––Coordenac�~aao de Aperfeic�oamento

de Pessoal de N�ııvel Superior (Brazil), in the form of a

Master of Sciences fellowship. E.D.G. Danesi is grateful

for the financial support from FAPESP––Fundac�~aao deAmparo �aa Pesquisa do Estado de S~aao Paulo (Brazil), in

the form of a Ph.D. fellowship. The authors are grateful

to Kleber Albuquerque Almeida for the help with the

experiments.

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