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Influence of phosphorus concentration and temperature on growth and phosphorus uptake by the microalga Scenedesmus obliquus
blOi BOUI (I Kll LOOY ELSEVIER
BioresourceTechnology67 (1999) 233-240
Influence of phosphorus concentration and temperature on growth and phosphorus uptake by the microalga Scenedesmus obliquus M.E. Martinez a*, J.M. Jim6nez a, F. E1 Yousfi b ~lnstituto de Biotecnolog[a, Departamento de Ingenieria Quimica, Universidadde Granada, Granada, Spain hDepartamento de Ingenier[aQu[mica, Universidadde Granada, Granada, Spain Received 18 December 1997; revised 20 June 1998; accepted 1 July 1998
Abstract
The growth of the freshwater microalga Scenedesmus obliquus has been studied in a mineral medium with P concentrations of between 0 and 372/~M and temperatures of 20, 25, 30 and 35°C. At all the temperatures, growth was inhibited by the P. The inhibition by P was highly dependent on temperature, detected at concentrations on the order of 1/aM at 35°C, 2/zM at 20 and 25°C, and 350/zM at 300C. The greatest specific growth rate, 0.047 h -t, was registered at 30°C. The specific growth rates were adjusted to a semistructured model of inhibition by the substrate. The inhibition constants, KI (/~M), increased with rising temperatures from 20 to 25 and 30°C, and decreased at higher temperatures. At 35°C, the lowest value was recorded for Kj 1.20 #M, and thus the inhibitory effect was the most pronounced. At 30°C, within the interval of P concentrations of 0-300/~M, it was possible for the variation in l~/Soto be fitted to a semistructured Monod kinetic model of limitation by the substrate. Yield of biomass varied with temperature. The highest yields were reached at 20°C. The specific rates of P consumption increased with So, tending towards a constant value. The highest values, obtained at 30°C and 35°C, were practically the same, and the lowest were obtained at 20°C. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Microalgae; Scenedesmus obliquus; Phosphorus removal; Batch growth
YPm,,
Nomenclature
A560 C
C0 K
KI Ks P-F PB qs S So Smax
SSQ t
T
Y~
absorbance of the cell suspension at 560 nm biomass concentration, mg 1-1 initial biomass concentration, mg 1-t parameter of eqn (4),/zmol 2 1-1 mg-1 inhibition constant,/~M saturation constant,/~M pseudo-regression coefficient biomass productivity, mg 1-~ h-t specific rate of phosphorus consumption, /~mol mg- 1 h - 1 phosphorus concentration,/~M initial phosphorus concentration,/~M phosphorus concentration corresponding to the maximum,/~M residual sum of squares culture time, h temperature, °C biomass/phosphorus yield, mg/lmol-t
*Corresponding author.
YPm,o
maximum mg/~mol- t minimum mg/~mol -t
biomass/phosphorus
yield,
biomass/phosphorus
yield,
Greek symbols #
]'/rn3
specific specific (1) and specific (1) and specific
/[/max
maximum specific growth rate, h-t
~/mt Jim2
growth rate, h-t growth rate, h-t, parameters of eqn eqn (2) growth rate, h-t, parameters of eqn eqn (2) growth rate, h -t, parameter of eqn
(1)
1. I n t r o d u c t i o n
Cultured microalgae, in secondarily treated sewage, can help eliminate inorganic substances such as ammonium and phosphorus, and at the same time can
0960-8524/99/$ - - see front matter © 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 8 ) 0 0 1 2 0 - 5
234
S.M.E. Martinezet al./Bioresource Technology67 (1999) 233-240
provide a source of maketable biomass, pigments and chemical products (Oswald and Benemann, 1980; Lavoie and De la Noiie, 1985; De la Noiie and Pruix, 1988). Nitrogen and phosphorus are considered the principal nutrients to be eliminated in order to control eutrophy, although other elements, such as iron, molybdenum, zinc and manganese, can constitute important factors in phytoplankton growth in waters exposed to secondarily treated sewage (Koopman et al., 1980). For reducing inorganic P, the most widely used procedure up to now has been physico-chemical dephosphatization. For the reduction of inorganic N, the use of biological processes of nitrification and denitrification are common. The primary disadvantage of these two types of elimination processes concerns the high operational costs of adding chemical products in the case of P, and strong aeration in the case of N. Algae can use a wide variety of nitrogenous compounds, both inorganic and organic, as a nitrogen source to synthesize amino acids. Phosphorus, used in the cellular processes related to energy transfer and nucleic-acid synthesis, is taken preferentially from inorganic phosphates in the forms of H2PO4 and HPO 2-. The consumption rate of P depends on the concentration of P in the medium, the intracellular P content, pH, the concentrations of the ions Na ÷, K + and Mg2+, and temperature (Kaplan et al., 1986). Ambient temperatures outside the cells substantially affects the growth of all microorganisms, not only directly acting on the chemistry of cell growth and death but also indirectly influencing the solubility of CO2 and 02 in the nutrient medium. Most microalgae, including Scenedesmus obliquus, are mesophilic and can grow at temperatures between 15 and 40°C. There are few studies available which focus on the influence of temperature on the microalgal growth and substrate consumption. Nevertheless, the optimal temperatures for growth, substrate conversion and product synthesis differ from each other, and thus the optimal temperature for the culture depends on the aim to be achieved (Soeder and Hegewald, 1988). Phosphorus in cells is used for the formation both of organic and inorganic compounds. Algae use three different processes to transform P into high energy organic compounds: phosphorylation at the substrate level, oxidative phosphorylation, and photophosphorylation. The general reaction can be represented by: A_n Energy l.Jr+ri • ATP n
In the first two processes, the energy comes either from the oxidation of the respiratory substrates or from the electron transport system of the mitochondria. In the
third process, light energy is transformed and incorporated into ATP. In the present work, we examined the growth and P consumption of the miroalga Scenedesmus obliquus under conditions of excess mineral nutrients and with P and temperature within the ranges 0 to 372 ktM and 20 to 35°C.
2. M e t h o d s
2.1. Microalga and culture media
The freshwater microalga Scenedesmus obliquus 276-3a was used (from the algal collection of the University of G6ttingen, Germany). The culture medium composition in gl -~ was: 1.011 KNO3, 0.247 MgSO47H20, 0.015 CaCI22H/O, 0.007 FeSO4 7HzO (complexed with EDTA). The micronutrients added in mg 1-1 were: 0.06 Mn, 0.07 Zn, 0.06 Cu, 0.01 B, 0.001 Mo. A total of 73 different initial concentrations of P (So), determined by the analysis of the culture media after sterilization by filtration, were used. The phosphorus was added in the form of NaH2PO42H20, and the pH of the culture medium was adjusted to an initial value of 6.5 2.2. Experimental conditions
All experiments were carried out in a batch-culture system consisting of four photobioreactors, each of 1 1 working capacity, jacketed for the circulation of water, equipped with a thermostat, stirred magnetically and having a continuous supply of air. The working temperatures were 20, 25, 30 and 35°C. All cultures were supplied with air, sterilized by filtration, at a specific rate of 0.16v/vmin -~. Continuous illumination was provided by OSRAM L40W/10 daylight fluorescent lamps. The intensity of the illumination, measured in the interior of the bioreactors by a Gossen-Mavolux luxometer, was 11.334 Klux. The inoculum was a culture in mineral medium, 5.06 mM in P, solidified with agar and incubated for 4 days under continuous illumination at room temperature. The cells were transferred to the culture medium by a platinum loop to obtain an initial biomass concentration (Co) of approximately 8 mg 1-1. 2.3. Analytical methods
Cell concentrations (C), mgl -~, were indirectly measured by the absorbance of the cell suspension at 2 = 5 6 0 n m . A straight-line calibration of A560-dry weight, mg 1-I, was obtained. The P concentration in the culture medium was determined by a colorimetric method of reduction with
S.M.E. Martinez et aL/Bioresource Technology 67 (1999) 233-240
ascorbic acid in samples from which the cells had been previously separated by centrifugation (Healey, 1978). The intracellular P concentration was determined by the same method after digesting the cells with persulphate (Okada et al., 1982).
microalgae, can grow at the expense of its internal P reserves. At each temperature, /~ rose with an increase in So, reached a maximum value and then decreased. This decrease indicated a certain inhibitory effect of the P. This effect, known for some time, enabled Rhode (1948) to differentiate 3 groups of freshwater microalgae according to their capacity to tolerate concentrations of P: So < 0.65/~M; So = 0.65 #M and So > 0.65/~M. The inhibition by P was highly dependent on temperature, occurring at concentrations of roughly 1/~M at 35°C, 2~M at 20 and 25°C and 350 #M at 30°C. In addition, # rose at temperatures of 20 to 25 and 30°C. At P concentrations lower than 0.5/~M, there were no significant differences between the values of/~ at 25, 30 and 35°C, these being greater than at 20°C. Nevertheless, inhibition by the substrate at 35°C appeared at a very low P concentration, compared with the other temperatures; thus, at So > 2.55 .uM, the specific rates at 35°C were lower than those at 20°C. Different modified models of inhibition by substrate and toxicity were tested at 20, 25, and 35°C. The one which best reproduced the experimental tz/So variations observed was the one of partial inhibition by the substrate, eqn (1):
3.Resuland tsdiscussion In the present work, the influence of temperature was analysed in relation to changes in the specific growth rate, biomass/substrate yield, specific rate of P consumption and biomass productivity, in experiments with different initial P concentrations. For all temperatures and initial P concentrations, growth curves without lag phases were obtained, the first phase being exponential growth followed by a phase of linear increase in biomass with time. The development of the cultures was tracked for a minimum time of 27 h and a maximum of 177 h.
3.1. Specific growth rate The specific growth rates, /~ = dlnC/dt, during the exponential-growth phases, were plotted against the initial phosphorus concentration (So) in /zM for the 4 temperatures tested (Fig. 1). In all cases, when the P concentration in the culture medium approached zero, the specific growth rate approached a finite value other than zero. This observation, confirmed experimentally by cultures with phosphorus-free nutrient medium and discussed by different authors (Rhee, 1973; Droop, 1974), showed that Scenedesrnus obliquus, like other
0.051
235
[dinIKIS+ldmzS2+tZm3KsKl lt =
(1)
KsKr+K1S+S z
At 30°C, in the interval of P concentrations of 0 to 300 pM, where no inhibition by the substrate was
0
/ // O
0
0
/
0
0 0
0
00
0
~J
0"02 0
~ tt"
2
4
6
8
10
12
50
~
!
120
190
~ !
i 0
260
330
400
So Fig. 1. Variation of the specific growth rate, p, with the initial phosphorus concentration, S~, at temperatures: 35°C.
<>
20°C, [] 25°C,
o
30°C, and []
S.M.E. Martinez et al./Bioresource Technology 67 (1999) 233-240
236
Table 1 Influence of temperature on kinetic parameters T, *C
Model
/zm~, h -l
Sin=, #M
#ml, h -I
#m2, h - l
,td,m3, h -I
Ks, #M
KI, #M
SSQ
P-r 2
20 25 30 35 30
Partial inh. Partial inh. Partial inh. Partial inh. Monod modif.
0.0355 0.0405 0.0466 0.0384
3.20 3.31 20.08 0.56
0.0438 0.0454 0.0471 0.0446 0.0466
0.0219 0.0287 0.0350 0.0317 0.0256
0.0255 0.0320 0.0272 0.0356
1.33 0.96 0.25 0.62 0.20
10.50 15.80 955.72 1.20
3.7701x 10-5 8.4140X 1 0 - 6 2.6025x 10 -5 1.1370 x 10 -6 9.000 X 1 0 - 6
0.899 0.970 0.890 0.938 0.979
detected (see Fig. 1), different modified limitationby-substrate models were tested. For the interval of 0-372 #M, the modified model of partial inhibition by the substrate was tested. The one which best reproduced the experimental #/So variation was the modified Monod model, eqn (2): /~=
#miS+/gm2Ks
Ks+S
(2)
The modification of the kinetic models consisted of adding a term that accounted for the specific finite growth rate with P absent from the culture medium. This could be explained by proposing growth mechanisms which take into account the utilization of the P reserves of the biomass (Martinez et al., 1997). Table 1 presents the kinetic parameters #ml, maximum value for p; Pro2, constant value for p at high concentrations of P; ~tm3, constant value for ~t in the absence of P in the culture medium, So = 0; and the coordinates of the maxima, #maxand S=ax for the model of partial inhibition by the substrate at the 4 temperatures, as well as the parameters #ml, maximum value for p and #m2, constant value for # in the absence of external P of the modified Monod model for 30°C. The parameters were obtained by nonlinear regression and used to trace the continuous lines of Fig. 1. At 30°C the line corresponds to eqn (2). The growth rate parameters #max, #=1, #=2 rose with temperature to 30°C and fell at 35°C. This dependence is evident in Fig. 2(a), where for example the variation in /t=~/T clearly corresponds to a mesophilic microorganism, with a steeper slope above the optimal temperature. For Pro3, this variation was not observed, but, as/2m3 represents the growth rate in the absence of external P, it should have been influenced less by temperature than were the other parameters. Figure 2(b) shows the variations of Ks, saturation constant, and K~, inhibition constant, with temperature. The variation of these two parameters was to be expected. As Ks is an inverse function of the microalga-phosphorus affinity, this affinity increased on raising the temperature until it reached the optimal temperature, and therefore Ks decreased. The opposite happened when the temperature was raised to above-
optimal levels. The KI increased on raising the temperature until the optimal temperature was reached, and therefore the inhibitory effect of P diminished. Beyond this temperature, KI declined, 35°C being the temperature at which the inhibitory effect of P was most pronounced. These experimental results agree with the work of Soeder et al. (1985). These authors showed that Scenedesmus obliquus SAG.276-3a was a mesophilic microalga with an optimal temperature of 31-32°C and a maximum growth temperature of 34-36°C. The higher the temperature, the less was the tolerance time; this microalga tolerated 37°C for 6 days, 38°C for 3 days and 39°C for 2days, after which the cells died. Studying Scenedesmus crassus, Guerri et al. (1981) found that the specific growth rate increased within the interval of 15-33°C from 0.033 to 0.069 h-I. These optimal growth temperatures are dependent on the intensity of the illumination. Thus, for Scenedesmus bijugates, Castillo et al. (1980) reported that the optimum temperature for growth is 37°(2 under light intensities of between 100 and 50 #E m -2 s -1, and 32°C under illumination of less than 50/~Em-2s -1. The positive and negative effects of temperature, before and after the optimum, increase under conditions of light saturation. 3.2. Biomass/substrate yield The biomass/phosphorus yield, expressed in mg dry biomass/#mol P used, was calculated using the equation: C-Co= Yp (So-S)
(3)
Figure 3 presents, by way of example, biomass formation (C-Co) mg 1-1, plotted against P consumption, (S0-S) pM, at 20°C. Experimentally this relationship, [eqn (3)], was linear and therefore Yp = constant at residual P concentration values of S > 3 pM in the culture medium. Fig. 3(a) indicates this linear relationship for the experiment at So = 344 #M. For the experiments where So > 65 pM, this relationship held during the exponential and linear growth phases, since the residual P concentration at the end of the experiments was consistently greater
237
S.M.E. Maninez et aL/Bioresource Technology 67 (1999) 233-240
than 3/~M. For the experiments with 4 #M
curve i n ' t h e s e cases indicated that Yp increased on decreasing the P concentration in the culture medium. This observation was confirmed on plotting Yp mg dry biomass//~mol of P used, at 20°C, against the initial P concentration So (Fig. 4). Similar curves were obtained at the other temperatures. Increased
"t
125
O.O6
/
100
~75
T
o/ /J
V
::It
O.M
1
o
50
o.~ 25 0.01 16
i 28
t
i 28
i M
~0
40
C¢
0
T ('C)
/
'
I
I
I
40
80
120
160
.)
200
(so - s) . .
lO00r
1J
8oo
I.¢
.) 12"5 1 0
lo I i
V4oo
~,
2801-
o
16
-
/I
~
20
-- l V
. o.s ~
-IOS
o
28
30
38
40
T ('C)
b) Fig. 2. (a) Variation of the kinetic parameters with temperature: ~, /Zm~,G t~=.,/~ and o / ~ . (b) Variation of the suburation constant Ks and inh~ition constant K] with temperature.
0
0.08
!
!
I
O. 16
0.24
0,32
0.4
(so- s) j
b) Fig. 3. Relationship of biomass formed (C-Co) and phosphorus consumed (So-S) at 20°C: (a) So = 344/~M, (b) So = 1.16tzM.
S.M.E. Martinez et aL/Bioresource Technology 67 (1999) 233-240
238
m
II
0
E =L E
I0
0
m
.Q t~ v
E
|
~L
>-
¢, Z
4/
0 0
I
4
1
I
;/
II
t~l
o
J
~
I
tN
..,
¢,
I
I
IMO
tOO
400
so Fig. 4. Variation in yield of biomass/phosphorus used, Yp, with So at 20°C.
efficiency in P utilization by the microalga when growth is limited by this nutrient has been mentioned by several authors (Healey and Hendzel, 1975; Fitzgerald and Nelson, 1966) and appears to be related to a rise in phosphatase production. The variation of Yp with S was adjusted to the equation: rp =
KYp_+S K+
1
(4)
S
YPmin
which gives a constant value for Yp equal to YPm,, at high S values and a YP_x in the absence of external phosphorus. The values for the parameters of eqn (4) at the temperatures assayed were obtained by nonlinear regression (Table 2). At 20°C these values were used to plot the continuous line in Fig. 4. Without external P, the efficiency of utilization of the cell reserves of P, Yp.... was independent of the temperature of the culture medium. The specific growth rate,/~m3, was also independent of temperature under the same conditions. However, YPm,. increased
with temperature, reaching a maximum value at nearly 25°C and then decreased. This variation showed that temperature affects not only growth kinetics, but also the efficiency of converting external P into biomass. The difference in the optimal conversion temperature (25°C) and optimal temperature for growth (30°C) has been discussed by Moser (1985), and this disparity implies that in the designing of the cultures, temperature must be set according to the original aim - - to obtain biomass or eliminate P.
3.3. Specific rate of P consumption The specific rates of phosphorus consumption, qs, were calculated as the quotient between the specific growth rate, #, and the apparent biomass/substrate yield, Yp, in the exponential growth phase. # qs = - Yp
(5)
since Table 2 Parameters of eqn (4) T, °C
20 25 30 35
1 dC
Yp...... mg biom//tmol P
lip...... mg biom/#mol P
K, #tool 21-~ mg
P-r 2
18.2 18.0 18.4 18.0
0.65 1.00 0.71 0.39
1.05 0.42 0.33 0.57
0.956 0.998 0.974 0.999
C dt 1 dS qs . . . . . . dC C dt
(6)
dS In Fig. 5, the values of qs are represented against So. In the range of initial P concentrations considered
239
S.M.E. Maninezet aL/BioresourceTechnology67 (1999) 233-240
the elimination of P by the microalga Scenedesmus crassus in the interval of 15-35°C.
(0
3.4. Biomass productivity during the linear growth phase
Figure 6 presents the values for the volumetric productivity of biomass, PB = dC/dt, obtained by fitting data of C/t during the linear growth phase, against So (#M), at 25°C. The existence of this linear-growth phase again confirms that the alga is capable of growing at the expense of its internal phosphorus reserves, given that when this phase began, except in the experiments carried out at very high P concentration (So> 65 pM), P was not detected in the culture medium. Generally, the appearance of this linear growth is explained by limited carbon dioxide or light, or both. This may be true at P concentrations of 3 pM < So< 10 #M. At lower concentrations, however, the effect of the initial P concentration on biomass productivity or growth rate during this phase must be exerted by influencing the intracellular P content. Although all the experiments began with cells of the same concentration of internal P (0.0072 mg P/mg dry biomass), the Ps/So variation appears to indicate that in the range 0-3 #M the cells generated during the exponential phase were deficient in phosphorus. The internal concentration varied with changes in the external concentration, So, and behaved differently on reaching the linear growth. In the experiments with 3#M
E.
,~: 200
% lOO
, I 0
2.5
5
7.5
10
12.5
So (.M) Fig. 5. Variation of the specific rates of phosphorus consumption with the initial phosphorus concentrations at temperatures: o 20°C,
[] 25°C,o 30°Cand ~ ; 35°C.
e- 2 .d 13
E m
D.
/
G
1
O
n
0 0
I 2
i 4
i IS
I 8
/
/
i / /( 10 / / 5 0
So
i 120
i 150
i 250
i 330
(.M)
Fig. 6. Variationof the volumetricbiomassproductivity,PB,with the initial phosphorusconcentrations,Sdat 25°C.
400
240
S.M.E. Martinez et al./Bioresource Technology 67 (1999) 233-240
concentrations, So> 65/zM, the inhibition observed during the exponential growth phase persisted during the linear growth phase. The highest biomass productivity, 1.84 mg 1-1 h -1, was reached at 30°C. At 20 and 25°C, the maximum productivities were similar at approximately 1.5mgl -I h -1, and at 35°C productivity was lower (1.1 mg 1-1 h-l).
References Castillo, S.J., Merino, M.F., Heussler, P., 1980. Production and ecological implications of algae mass culture under Peruvian conditions. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass, Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 123-134. Dauta, A., Brunel, L., Guerri, M., 1982. Drtermination experimentale des param~tres lirs h l'assimilation de l'azote et du phosphore par Scenedesmus crassus. Annls. Limnol. 18, 33-40. De la Noiie, J., Pruix, D., 1988. Biological tertiary treatment of urban wastewater with chitosan-immobilized Phormidium. Appl. Microbiol. Biotechnol. 29, 292-297. Droop, M.R., 1974. The nutrient status of algal cells in continuous culture. J. Mar. Biol. Ass. U.K. 54, 825-855. Fitzgerald, G.P., Nelson, T.C., 1966. Extractive and enzymatic analyses for limiting or surplus phosphorus in algae. J. Phycol. 2, 32-37. Guerri, M., Brunel, L., Dauta, A., 1981. Interaction de la lumi~re et de la temperature sur le taux de croissance de Scenedesmus crassus. Annls. Limnol. 17, 97-104. Healey, F.P., 1978. Phosphate uptake. In: Heilebust, J.A., Craigie, J.S. (Eds.), Handbook of Phycological Methods, Physiological and Biochemical Methods, Cambridge University Press, Cambridge, pp. 411-417. Healey, F.P., Hendzel, L.L., 1975. Effect of phosphorus deficiency on two algae growing in chemostats. J. Phycol. 11, 303-309.
Kaplan, D., Richmond, A.E., Dubinsky, Z., Aaronson, S., 1986. Algal nutrition. In: Richmond, A. (Ed.)~ CRC Handbook of Microalgai Mass Culture. CRC Press, Boca Raton, FL, pp. 147-198. Koopman, B., Benemann, J.R., Oswald, W.J., 1980. Pond isolation and phase isolation for control of suspended solids concentration in sewage oxidation pond effluents. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 135-161. Lavoie, A., De la Noiie, J., 1985. Hyperconcentrated cultures of Scenedesmus obliquus: a new approach for wastewater biological tertiary treatment. Water Res. 19, 1437-1442. Martinez, S.M.E., Jimrnez, C.J.M., Espinola, L.J.B., El Yousfi, F., 1993. Consumo de f6sforo y crecimiento del alga Scenedesmus obliquus. Alinidad 50, 33-39. Martinez, S.U.E., Jimdnez, C.J.U., El Yousfi, F., 1997. Influence of phosphorous concentration on the growth kinetics and stoichiometry of the microalgae Scenedesmus obliquus. Process Biochemistry 32, 657-664. Moser, A., 1985. Rate equations for enzyme kinetics. In: Brauer, H. (Ed.), Biochemical Engineering. VCH, Weinheim, pp. 199-226. Nyholm, N., 1977. Kinetics of phosphate limited algal growth. Biotechnol. Bioeng. 19, 467-492. Okada, M., Sudo, R., Alba, S., 1982. Phosphorus uptake and growth of blue-green alga Microcystis aeruginosa. Biotechnol. Bioeng. 24, 143-152. Oswald, W.J., Benemann, J.R., 1980. Algal bacterial systems. In: San Pietro, A. (Ed.), Biochemical and Photosynthetic Aspects of Energy Production. Academic Press, New York, pp. 59-80. Rhee, G.Y., 1973. A continuous culture study of phosphate uptake, growth rate and polyphosphate in Scenedesmus sp. J. Phycol. 9, 495-506. Rhode, W., 1948. Environmental requirements of freshwater plankton alga. Symbol. Bot. Ups. 10, 1-10. Soeder, C.J., Hegewald, E., 1988. Scenedesmus. In: Borowitzka, M.A., Borowitzka, L.J. (Eds.), Micro-algal Biotechnology. Cambridge University Press, Cambridge, pp. 59-84. Soeder, C.J., Hegewald, E., Fiolitakis, E., Grobbelaar, J.U., 1985. Temperature dependence of population growth in a green microalga: thermodynamic characteristics of growth intensity and the influence of cell concentration. Zeitsch. Natur. 40, 227-233.
BioresourceTechnology67 (1999) 233-240
Influence of phosphorus concentration and temperature on growth and phosphorus uptake by the microalga Scenedesmus obliquus M.E. Martinez a*, J.M. Jim6nez a, F. E1 Yousfi b ~lnstituto de Biotecnolog[a, Departamento de Ingenieria Quimica, Universidadde Granada, Granada, Spain hDepartamento de Ingenier[aQu[mica, Universidadde Granada, Granada, Spain Received 18 December 1997; revised 20 June 1998; accepted 1 July 1998
Abstract
The growth of the freshwater microalga Scenedesmus obliquus has been studied in a mineral medium with P concentrations of between 0 and 372/~M and temperatures of 20, 25, 30 and 35°C. At all the temperatures, growth was inhibited by the P. The inhibition by P was highly dependent on temperature, detected at concentrations on the order of 1/aM at 35°C, 2/zM at 20 and 25°C, and 350/zM at 300C. The greatest specific growth rate, 0.047 h -t, was registered at 30°C. The specific growth rates were adjusted to a semistructured model of inhibition by the substrate. The inhibition constants, KI (/~M), increased with rising temperatures from 20 to 25 and 30°C, and decreased at higher temperatures. At 35°C, the lowest value was recorded for Kj 1.20 #M, and thus the inhibitory effect was the most pronounced. At 30°C, within the interval of P concentrations of 0-300/~M, it was possible for the variation in l~/Soto be fitted to a semistructured Monod kinetic model of limitation by the substrate. Yield of biomass varied with temperature. The highest yields were reached at 20°C. The specific rates of P consumption increased with So, tending towards a constant value. The highest values, obtained at 30°C and 35°C, were practically the same, and the lowest were obtained at 20°C. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Microalgae; Scenedesmus obliquus; Phosphorus removal; Batch growth
YPm,,
Nomenclature
A560 C
C0 K
KI Ks P-F PB qs S So Smax
SSQ t
T
Y~
absorbance of the cell suspension at 560 nm biomass concentration, mg 1-1 initial biomass concentration, mg 1-t parameter of eqn (4),/zmol 2 1-1 mg-1 inhibition constant,/~M saturation constant,/~M pseudo-regression coefficient biomass productivity, mg 1-~ h-t specific rate of phosphorus consumption, /~mol mg- 1 h - 1 phosphorus concentration,/~M initial phosphorus concentration,/~M phosphorus concentration corresponding to the maximum,/~M residual sum of squares culture time, h temperature, °C biomass/phosphorus yield, mg/lmol-t
*Corresponding author.
YPm,o
maximum mg/~mol- t minimum mg/~mol -t
biomass/phosphorus
yield,
biomass/phosphorus
yield,
Greek symbols #
]'/rn3
specific specific (1) and specific (1) and specific
/[/max
maximum specific growth rate, h-t
~/mt Jim2
growth rate, h-t growth rate, h-t, parameters of eqn eqn (2) growth rate, h-t, parameters of eqn eqn (2) growth rate, h -t, parameter of eqn
(1)
1. I n t r o d u c t i o n
Cultured microalgae, in secondarily treated sewage, can help eliminate inorganic substances such as ammonium and phosphorus, and at the same time can
0960-8524/99/$ - - see front matter © 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 8 ) 0 0 1 2 0 - 5
234
S.M.E. Martinezet al./Bioresource Technology67 (1999) 233-240
provide a source of maketable biomass, pigments and chemical products (Oswald and Benemann, 1980; Lavoie and De la Noiie, 1985; De la Noiie and Pruix, 1988). Nitrogen and phosphorus are considered the principal nutrients to be eliminated in order to control eutrophy, although other elements, such as iron, molybdenum, zinc and manganese, can constitute important factors in phytoplankton growth in waters exposed to secondarily treated sewage (Koopman et al., 1980). For reducing inorganic P, the most widely used procedure up to now has been physico-chemical dephosphatization. For the reduction of inorganic N, the use of biological processes of nitrification and denitrification are common. The primary disadvantage of these two types of elimination processes concerns the high operational costs of adding chemical products in the case of P, and strong aeration in the case of N. Algae can use a wide variety of nitrogenous compounds, both inorganic and organic, as a nitrogen source to synthesize amino acids. Phosphorus, used in the cellular processes related to energy transfer and nucleic-acid synthesis, is taken preferentially from inorganic phosphates in the forms of H2PO4 and HPO 2-. The consumption rate of P depends on the concentration of P in the medium, the intracellular P content, pH, the concentrations of the ions Na ÷, K + and Mg2+, and temperature (Kaplan et al., 1986). Ambient temperatures outside the cells substantially affects the growth of all microorganisms, not only directly acting on the chemistry of cell growth and death but also indirectly influencing the solubility of CO2 and 02 in the nutrient medium. Most microalgae, including Scenedesmus obliquus, are mesophilic and can grow at temperatures between 15 and 40°C. There are few studies available which focus on the influence of temperature on the microalgal growth and substrate consumption. Nevertheless, the optimal temperatures for growth, substrate conversion and product synthesis differ from each other, and thus the optimal temperature for the culture depends on the aim to be achieved (Soeder and Hegewald, 1988). Phosphorus in cells is used for the formation both of organic and inorganic compounds. Algae use three different processes to transform P into high energy organic compounds: phosphorylation at the substrate level, oxidative phosphorylation, and photophosphorylation. The general reaction can be represented by: A_n Energy l.Jr+ri • ATP n
In the first two processes, the energy comes either from the oxidation of the respiratory substrates or from the electron transport system of the mitochondria. In the
third process, light energy is transformed and incorporated into ATP. In the present work, we examined the growth and P consumption of the miroalga Scenedesmus obliquus under conditions of excess mineral nutrients and with P and temperature within the ranges 0 to 372 ktM and 20 to 35°C.
2. M e t h o d s
2.1. Microalga and culture media
The freshwater microalga Scenedesmus obliquus 276-3a was used (from the algal collection of the University of G6ttingen, Germany). The culture medium composition in gl -~ was: 1.011 KNO3, 0.247 MgSO47H20, 0.015 CaCI22H/O, 0.007 FeSO4 7HzO (complexed with EDTA). The micronutrients added in mg 1-1 were: 0.06 Mn, 0.07 Zn, 0.06 Cu, 0.01 B, 0.001 Mo. A total of 73 different initial concentrations of P (So), determined by the analysis of the culture media after sterilization by filtration, were used. The phosphorus was added in the form of NaH2PO42H20, and the pH of the culture medium was adjusted to an initial value of 6.5 2.2. Experimental conditions
All experiments were carried out in a batch-culture system consisting of four photobioreactors, each of 1 1 working capacity, jacketed for the circulation of water, equipped with a thermostat, stirred magnetically and having a continuous supply of air. The working temperatures were 20, 25, 30 and 35°C. All cultures were supplied with air, sterilized by filtration, at a specific rate of 0.16v/vmin -~. Continuous illumination was provided by OSRAM L40W/10 daylight fluorescent lamps. The intensity of the illumination, measured in the interior of the bioreactors by a Gossen-Mavolux luxometer, was 11.334 Klux. The inoculum was a culture in mineral medium, 5.06 mM in P, solidified with agar and incubated for 4 days under continuous illumination at room temperature. The cells were transferred to the culture medium by a platinum loop to obtain an initial biomass concentration (Co) of approximately 8 mg 1-1. 2.3. Analytical methods
Cell concentrations (C), mgl -~, were indirectly measured by the absorbance of the cell suspension at 2 = 5 6 0 n m . A straight-line calibration of A560-dry weight, mg 1-I, was obtained. The P concentration in the culture medium was determined by a colorimetric method of reduction with
S.M.E. Martinez et aL/Bioresource Technology 67 (1999) 233-240
ascorbic acid in samples from which the cells had been previously separated by centrifugation (Healey, 1978). The intracellular P concentration was determined by the same method after digesting the cells with persulphate (Okada et al., 1982).
microalgae, can grow at the expense of its internal P reserves. At each temperature, /~ rose with an increase in So, reached a maximum value and then decreased. This decrease indicated a certain inhibitory effect of the P. This effect, known for some time, enabled Rhode (1948) to differentiate 3 groups of freshwater microalgae according to their capacity to tolerate concentrations of P: So < 0.65/~M; So = 0.65 #M and So > 0.65/~M. The inhibition by P was highly dependent on temperature, occurring at concentrations of roughly 1/~M at 35°C, 2~M at 20 and 25°C and 350 #M at 30°C. In addition, # rose at temperatures of 20 to 25 and 30°C. At P concentrations lower than 0.5/~M, there were no significant differences between the values of/~ at 25, 30 and 35°C, these being greater than at 20°C. Nevertheless, inhibition by the substrate at 35°C appeared at a very low P concentration, compared with the other temperatures; thus, at So > 2.55 .uM, the specific rates at 35°C were lower than those at 20°C. Different modified models of inhibition by substrate and toxicity were tested at 20, 25, and 35°C. The one which best reproduced the experimental tz/So variations observed was the one of partial inhibition by the substrate, eqn (1):
3.Resuland tsdiscussion In the present work, the influence of temperature was analysed in relation to changes in the specific growth rate, biomass/substrate yield, specific rate of P consumption and biomass productivity, in experiments with different initial P concentrations. For all temperatures and initial P concentrations, growth curves without lag phases were obtained, the first phase being exponential growth followed by a phase of linear increase in biomass with time. The development of the cultures was tracked for a minimum time of 27 h and a maximum of 177 h.
3.1. Specific growth rate The specific growth rates, /~ = dlnC/dt, during the exponential-growth phases, were plotted against the initial phosphorus concentration (So) in /zM for the 4 temperatures tested (Fig. 1). In all cases, when the P concentration in the culture medium approached zero, the specific growth rate approached a finite value other than zero. This observation, confirmed experimentally by cultures with phosphorus-free nutrient medium and discussed by different authors (Rhee, 1973; Droop, 1974), showed that Scenedesrnus obliquus, like other
0.051
235
[dinIKIS+ldmzS2+tZm3KsKl lt =
(1)
KsKr+K1S+S z
At 30°C, in the interval of P concentrations of 0 to 300 pM, where no inhibition by the substrate was
0
/ // O
0
0
/
0
0 0
0
00
0
~J
0"02 0
~ tt"
2
4
6
8
10
12
50
~
!
120
190
~ !
i 0
260
330
400
So Fig. 1. Variation of the specific growth rate, p, with the initial phosphorus concentration, S~, at temperatures: 35°C.
<>
20°C, [] 25°C,
o
30°C, and []
S.M.E. Martinez et al./Bioresource Technology 67 (1999) 233-240
236
Table 1 Influence of temperature on kinetic parameters T, *C
Model
/zm~, h -l
Sin=, #M
#ml, h -I
#m2, h - l
,td,m3, h -I
Ks, #M
KI, #M
SSQ
P-r 2
20 25 30 35 30
Partial inh. Partial inh. Partial inh. Partial inh. Monod modif.
0.0355 0.0405 0.0466 0.0384
3.20 3.31 20.08 0.56
0.0438 0.0454 0.0471 0.0446 0.0466
0.0219 0.0287 0.0350 0.0317 0.0256
0.0255 0.0320 0.0272 0.0356
1.33 0.96 0.25 0.62 0.20
10.50 15.80 955.72 1.20
3.7701x 10-5 8.4140X 1 0 - 6 2.6025x 10 -5 1.1370 x 10 -6 9.000 X 1 0 - 6
0.899 0.970 0.890 0.938 0.979
detected (see Fig. 1), different modified limitationby-substrate models were tested. For the interval of 0-372 #M, the modified model of partial inhibition by the substrate was tested. The one which best reproduced the experimental #/So variation was the modified Monod model, eqn (2): /~=
#miS+/gm2Ks
Ks+S
(2)
The modification of the kinetic models consisted of adding a term that accounted for the specific finite growth rate with P absent from the culture medium. This could be explained by proposing growth mechanisms which take into account the utilization of the P reserves of the biomass (Martinez et al., 1997). Table 1 presents the kinetic parameters #ml, maximum value for p; Pro2, constant value for p at high concentrations of P; ~tm3, constant value for ~t in the absence of P in the culture medium, So = 0; and the coordinates of the maxima, #maxand S=ax for the model of partial inhibition by the substrate at the 4 temperatures, as well as the parameters #ml, maximum value for p and #m2, constant value for # in the absence of external P of the modified Monod model for 30°C. The parameters were obtained by nonlinear regression and used to trace the continuous lines of Fig. 1. At 30°C the line corresponds to eqn (2). The growth rate parameters #max, #=1, #=2 rose with temperature to 30°C and fell at 35°C. This dependence is evident in Fig. 2(a), where for example the variation in /t=~/T clearly corresponds to a mesophilic microorganism, with a steeper slope above the optimal temperature. For Pro3, this variation was not observed, but, as/2m3 represents the growth rate in the absence of external P, it should have been influenced less by temperature than were the other parameters. Figure 2(b) shows the variations of Ks, saturation constant, and K~, inhibition constant, with temperature. The variation of these two parameters was to be expected. As Ks is an inverse function of the microalga-phosphorus affinity, this affinity increased on raising the temperature until it reached the optimal temperature, and therefore Ks decreased. The opposite happened when the temperature was raised to above-
optimal levels. The KI increased on raising the temperature until the optimal temperature was reached, and therefore the inhibitory effect of P diminished. Beyond this temperature, KI declined, 35°C being the temperature at which the inhibitory effect of P was most pronounced. These experimental results agree with the work of Soeder et al. (1985). These authors showed that Scenedesmus obliquus SAG.276-3a was a mesophilic microalga with an optimal temperature of 31-32°C and a maximum growth temperature of 34-36°C. The higher the temperature, the less was the tolerance time; this microalga tolerated 37°C for 6 days, 38°C for 3 days and 39°C for 2days, after which the cells died. Studying Scenedesmus crassus, Guerri et al. (1981) found that the specific growth rate increased within the interval of 15-33°C from 0.033 to 0.069 h-I. These optimal growth temperatures are dependent on the intensity of the illumination. Thus, for Scenedesmus bijugates, Castillo et al. (1980) reported that the optimum temperature for growth is 37°(2 under light intensities of between 100 and 50 #E m -2 s -1, and 32°C under illumination of less than 50/~Em-2s -1. The positive and negative effects of temperature, before and after the optimum, increase under conditions of light saturation. 3.2. Biomass/substrate yield The biomass/phosphorus yield, expressed in mg dry biomass/#mol P used, was calculated using the equation: C-Co= Yp (So-S)
(3)
Figure 3 presents, by way of example, biomass formation (C-Co) mg 1-1, plotted against P consumption, (S0-S) pM, at 20°C. Experimentally this relationship, [eqn (3)], was linear and therefore Yp = constant at residual P concentration values of S > 3 pM in the culture medium. Fig. 3(a) indicates this linear relationship for the experiment at So = 344 #M. For the experiments where So > 65 pM, this relationship held during the exponential and linear growth phases, since the residual P concentration at the end of the experiments was consistently greater
237
S.M.E. Maninez et aL/Bioresource Technology 67 (1999) 233-240
than 3/~M. For the experiments with 4 #M
curve i n ' t h e s e cases indicated that Yp increased on decreasing the P concentration in the culture medium. This observation was confirmed on plotting Yp mg dry biomass//~mol of P used, at 20°C, against the initial P concentration So (Fig. 4). Similar curves were obtained at the other temperatures. Increased
"t
125
O.O6
/
100
~75
T
o/ /J
V
::It
O.M
1
o
50
o.~ 25 0.01 16
i 28
t
i 28
i M
~0
40
C¢
0
T ('C)
/
'
I
I
I
40
80
120
160
.)
200
(so - s) . .
lO00r
1J
8oo
I.¢
.) 12"5 1 0
lo I i
V4oo
~,
2801-
o
16
-
/I
~
20
-- l V
. o.s ~
-IOS
o
28
30
38
40
T ('C)
b) Fig. 2. (a) Variation of the kinetic parameters with temperature: ~, /Zm~,G t~=.,/~ and o / ~ . (b) Variation of the suburation constant Ks and inh~ition constant K] with temperature.
0
0.08
!
!
I
O. 16
0.24
0,32
0.4
(so- s) j
b) Fig. 3. Relationship of biomass formed (C-Co) and phosphorus consumed (So-S) at 20°C: (a) So = 344/~M, (b) So = 1.16tzM.
S.M.E. Martinez et aL/Bioresource Technology 67 (1999) 233-240
238
m
II
0
E =L E
I0
0
m
.Q t~ v
E
|
~L
>-
¢, Z
4/
0 0
I
4
1
I
;/
II
t~l
o
J
~
I
tN
..,
¢,
I
I
IMO
tOO
400
so Fig. 4. Variation in yield of biomass/phosphorus used, Yp, with So at 20°C.
efficiency in P utilization by the microalga when growth is limited by this nutrient has been mentioned by several authors (Healey and Hendzel, 1975; Fitzgerald and Nelson, 1966) and appears to be related to a rise in phosphatase production. The variation of Yp with S was adjusted to the equation: rp =
KYp_+S K+
1
(4)
S
YPmin
which gives a constant value for Yp equal to YPm,, at high S values and a YP_x in the absence of external phosphorus. The values for the parameters of eqn (4) at the temperatures assayed were obtained by nonlinear regression (Table 2). At 20°C these values were used to plot the continuous line in Fig. 4. Without external P, the efficiency of utilization of the cell reserves of P, Yp.... was independent of the temperature of the culture medium. The specific growth rate,/~m3, was also independent of temperature under the same conditions. However, YPm,. increased
with temperature, reaching a maximum value at nearly 25°C and then decreased. This variation showed that temperature affects not only growth kinetics, but also the efficiency of converting external P into biomass. The difference in the optimal conversion temperature (25°C) and optimal temperature for growth (30°C) has been discussed by Moser (1985), and this disparity implies that in the designing of the cultures, temperature must be set according to the original aim - - to obtain biomass or eliminate P.
3.3. Specific rate of P consumption The specific rates of phosphorus consumption, qs, were calculated as the quotient between the specific growth rate, #, and the apparent biomass/substrate yield, Yp, in the exponential growth phase. # qs = - Yp
(5)
since Table 2 Parameters of eqn (4) T, °C
20 25 30 35
1 dC
Yp...... mg biom//tmol P
lip...... mg biom/#mol P
K, #tool 21-~ mg
P-r 2
18.2 18.0 18.4 18.0
0.65 1.00 0.71 0.39
1.05 0.42 0.33 0.57
0.956 0.998 0.974 0.999
C dt 1 dS qs . . . . . . dC C dt
(6)
dS In Fig. 5, the values of qs are represented against So. In the range of initial P concentrations considered
239
S.M.E. Maninezet aL/BioresourceTechnology67 (1999) 233-240
the elimination of P by the microalga Scenedesmus crassus in the interval of 15-35°C.
(0
3.4. Biomass productivity during the linear growth phase
Figure 6 presents the values for the volumetric productivity of biomass, PB = dC/dt, obtained by fitting data of C/t during the linear growth phase, against So (#M), at 25°C. The existence of this linear-growth phase again confirms that the alga is capable of growing at the expense of its internal phosphorus reserves, given that when this phase began, except in the experiments carried out at very high P concentration (So> 65 pM), P was not detected in the culture medium. Generally, the appearance of this linear growth is explained by limited carbon dioxide or light, or both. This may be true at P concentrations of 3 pM < So< 10 #M. At lower concentrations, however, the effect of the initial P concentration on biomass productivity or growth rate during this phase must be exerted by influencing the intracellular P content. Although all the experiments began with cells of the same concentration of internal P (0.0072 mg P/mg dry biomass), the Ps/So variation appears to indicate that in the range 0-3 #M the cells generated during the exponential phase were deficient in phosphorus. The internal concentration varied with changes in the external concentration, So, and behaved differently on reaching the linear growth. In the experiments with 3#M
E.
,~: 200
% lOO
, I 0
2.5
5
7.5
10
12.5
So (.M) Fig. 5. Variation of the specific rates of phosphorus consumption with the initial phosphorus concentrations at temperatures: o 20°C,
[] 25°C,o 30°Cand ~ ; 35°C.
e- 2 .d 13
E m
D.
/
G
1
O
n
0 0
I 2
i 4
i IS
I 8
/
/
i / /( 10 / / 5 0
So
i 120
i 150
i 250
i 330
(.M)
Fig. 6. Variationof the volumetricbiomassproductivity,PB,with the initial phosphorusconcentrations,Sdat 25°C.
400
240
S.M.E. Martinez et al./Bioresource Technology 67 (1999) 233-240
concentrations, So> 65/zM, the inhibition observed during the exponential growth phase persisted during the linear growth phase. The highest biomass productivity, 1.84 mg 1-1 h -1, was reached at 30°C. At 20 and 25°C, the maximum productivities were similar at approximately 1.5mgl -I h -1, and at 35°C productivity was lower (1.1 mg 1-1 h-l).
References Castillo, S.J., Merino, M.F., Heussler, P., 1980. Production and ecological implications of algae mass culture under Peruvian conditions. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass, Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 123-134. Dauta, A., Brunel, L., Guerri, M., 1982. Drtermination experimentale des param~tres lirs h l'assimilation de l'azote et du phosphore par Scenedesmus crassus. Annls. Limnol. 18, 33-40. De la Noiie, J., Pruix, D., 1988. Biological tertiary treatment of urban wastewater with chitosan-immobilized Phormidium. Appl. Microbiol. Biotechnol. 29, 292-297. Droop, M.R., 1974. The nutrient status of algal cells in continuous culture. J. Mar. Biol. Ass. U.K. 54, 825-855. Fitzgerald, G.P., Nelson, T.C., 1966. Extractive and enzymatic analyses for limiting or surplus phosphorus in algae. J. Phycol. 2, 32-37. Guerri, M., Brunel, L., Dauta, A., 1981. Interaction de la lumi~re et de la temperature sur le taux de croissance de Scenedesmus crassus. Annls. Limnol. 17, 97-104. Healey, F.P., 1978. Phosphate uptake. In: Heilebust, J.A., Craigie, J.S. (Eds.), Handbook of Phycological Methods, Physiological and Biochemical Methods, Cambridge University Press, Cambridge, pp. 411-417. Healey, F.P., Hendzel, L.L., 1975. Effect of phosphorus deficiency on two algae growing in chemostats. J. Phycol. 11, 303-309.
Kaplan, D., Richmond, A.E., Dubinsky, Z., Aaronson, S., 1986. Algal nutrition. In: Richmond, A. (Ed.)~ CRC Handbook of Microalgai Mass Culture. CRC Press, Boca Raton, FL, pp. 147-198. Koopman, B., Benemann, J.R., Oswald, W.J., 1980. Pond isolation and phase isolation for control of suspended solids concentration in sewage oxidation pond effluents. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 135-161. Lavoie, A., De la Noiie, J., 1985. Hyperconcentrated cultures of Scenedesmus obliquus: a new approach for wastewater biological tertiary treatment. Water Res. 19, 1437-1442. Martinez, S.M.E., Jimrnez, C.J.M., Espinola, L.J.B., El Yousfi, F., 1993. Consumo de f6sforo y crecimiento del alga Scenedesmus obliquus. Alinidad 50, 33-39. Martinez, S.U.E., Jimdnez, C.J.U., El Yousfi, F., 1997. Influence of phosphorous concentration on the growth kinetics and stoichiometry of the microalgae Scenedesmus obliquus. Process Biochemistry 32, 657-664. Moser, A., 1985. Rate equations for enzyme kinetics. In: Brauer, H. (Ed.), Biochemical Engineering. VCH, Weinheim, pp. 199-226. Nyholm, N., 1977. Kinetics of phosphate limited algal growth. Biotechnol. Bioeng. 19, 467-492. Okada, M., Sudo, R., Alba, S., 1982. Phosphorus uptake and growth of blue-green alga Microcystis aeruginosa. Biotechnol. Bioeng. 24, 143-152. Oswald, W.J., Benemann, J.R., 1980. Algal bacterial systems. In: San Pietro, A. (Ed.), Biochemical and Photosynthetic Aspects of Energy Production. Academic Press, New York, pp. 59-80. Rhee, G.Y., 1973. A continuous culture study of phosphate uptake, growth rate and polyphosphate in Scenedesmus sp. J. Phycol. 9, 495-506. Rhode, W., 1948. Environmental requirements of freshwater plankton alga. Symbol. Bot. Ups. 10, 1-10. Soeder, C.J., Hegewald, E., 1988. Scenedesmus. In: Borowitzka, M.A., Borowitzka, L.J. (Eds.), Micro-algal Biotechnology. Cambridge University Press, Cambridge, pp. 59-84. Soeder, C.J., Hegewald, E., Fiolitakis, E., Grobbelaar, J.U., 1985. Temperature dependence of population growth in a green microalga: thermodynamic characteristics of growth intensity and the influence of cell concentration. Zeitsch. Natur. 40, 227-233.