Appl Microbiol Biotechnol (1990)34:103-107

Short contribution

Applied Microbiology

Biotechnology © Springer-Verlag 1990

The influence of flow rate and the composition of supplied COz/air mixtures on discontinuous growth of Tetraselmis sp.

Emilio Mol ina 1, M a Eugenia Martinez 2, Sebastihn S~nchez 3, Francisco Garcia 1, and Antonio Contreras 1

~ Departamento de Ingenieria Quimica, Colegio Universitario de Almeria, 04071 Almerla, Spain 2 Departamento de Ingenieria Quimica, Facultad de Ciencias, 18071 Granada, Spain 3 Departamento de lngenieria Quimica, Colegio Universitario de Ja6n, 23071 Ja&n, Spain

Received 3 January 1990/Accepted 17 May 1990

Summary. This paper studies the influence of the flow rate of gaseous mixtures on the kinetics of growth and the fatty acid composi t ion of Tetraselmis sp. at CO2/air ratios of 3 x 10 - 4 and 2 x 10 -5. The specific growth rate rises with increased flow rate up to values of ap- proximately 0.086 h -1 and 0.063 h -1 at CO2/air ratios of 3 x 10-4 and 2 × 10-5 respectively, when the flow rate is approximate ly 3 v / v per minute. At higher flow rates, the specific growth rate decreases. The polyunsa- turated fatty acid content decreases slightly as the ga- seous mixture flow rate increases, whereby the ratio o93/006 remains between 2 and 3, indicating good nutri- tional values.

Introduction

Interest in microalgal cultures has grown in recent years due to various socio-economic factors: the need for proteins, the quality of the environment, sources of energy and commercial chemical products (Camacho et al. 1988). At the present time, this interest centres on the use of microalgae in aquaculture as food for mol- luscs, herbivorous fishes, crustacean larvae and zoo- plankton (Coll 1983). The marine microalga Tetraselmis is widely used for this purpose and may be adapted to a varied range of culture conditions (Persoone and Claus 1980). Modifications in pH, temperature and nutrient concentration, among other factors, however, do not only influence the growth rate of the alga but also the quality of the biomass which is obtained.

Different authors have studied the influence of nu- trient concentrat ion and salinity of the medium on the growth and protein content of Tetraselrnis (Ffibregas et al. 1984, 1985). Less information, however, is available on the factors which determine the quality of biomass for use in aquaculture and, more particularly, on the polyunsaturated fatty acid content in the biomass (De

Offprint requests to: E. Molina

Paw et al. 1984). Moreover, given that the majority of green algae may only use free CO2 for photosynthesis (Goldman et al. 1981), CO2 supply to the medium is, therefore, one of the principal operat ional costs in- volved in large-scale production.

From a practical point of view, it is economical to supply CO2 by bubbling air (0.03% CO2) into the cul- ture medium. Under such conditions and with refer- ence to 02, the supply of 02 as well as its generation by photosynthesis should be taken into account. Maintain- ing appropr ia te concentrations of CO2 and 02 in order to avoid CO; restriction and O2 inhibition is closely linked to the degree of mixing in the culture and is one of the prior requirements to optimize algal product ion for use in aquaculture (Pruder 1981 ; M~irkl and Mather 1985).

The aim of this paper is to analyse the influence of different aeration flow rates at two CO2/air ratios, as- sociated with different degrees of mixing in the culture, on the parameters for growth and polyunsaturated fatty acid content in Tetraselm& sp.

Materials and methods

The microalga used was Tetraselmis sp. provided by the Torre de la Sal Aquaculture Institute (Castell6n, Spain).

The experiments for this paper were perfo-rmed in a batch culture installation. One-litre flasks were used as growth vessels, into which 500 ml enriched sea water was poured, according to a modified version of the method as described by Ukeles (Ffibregas et al. 1984).

The sea-water was sterilized in an autoclave at 120°C for 30 rain, and the complete culture medium was sterilized by filtra- tion through 0.2 ~tm pore membranes. In all cases the initial pH was 8.0.

Air was pumped into the growth vessels by means of a com- pressor. The operative flow rate was selected and the air sterilized by a 0.2-~tm pore filter. The selected flow rates ranged from 0.5-10 v/v per minute and the bubbling itself provided mixing for the suspension.

Two ratios of CO2/air were used: 3 x 10 -4 and 2 x 10 -5. The former corresponded to air and the second was obtained by mix- ing two streams: air and CO2-free air obtained by CO2 absorption in a concentrated solution of sodium hydroxide.

104

The cultures were continuously exposed to light from two Philips (Mfilaga, Spain) TLD 36W/54 fluorescent lamps, which had an incident light intensity (I0) of 50 W.m -2 measured at the surface of the culture. The temperature was 24+ 0.5 ° C.

Cell concentration (c) was determined by measuring the opti- cal density (OD) at 530 nm. The relationship found experimen- tally was c(g-1-])=0.302 x ODs~o.

The nature and content of fatty acids in the biomass were de- termined from the lipid fraction obtained by extraction with chlo- roform-methanol in the ratio 2:1 (Kochert 1978). Methylation of the fatty acids was performed with boron trifluoride in methanol (14%) and the analysis of methyl esters was carried out by gas chromatography using a 30-m capillary column of fused silica (SP2330, Supelco, Bellefonte, Pa, USA), with an internal diameter of 0.25 mm and a flame-ionisation detector.

Results and discussion

As an example, a representation in semilogarithmic co- ordinates f rom the adimensional biomass concentration (C/Co) versus time (t) in Fig. 1 is given for the experi- ments per formed with a CO2/air ratio of 3 x 10 - 4 and flows of 0.7, 3.6 and 5.5 v / v per minute.

In all experiments the presence of an initial adapta- tion phase was observed. Exponential growth zones were characterized by calculation of the specific growth rate #=d(ln(c/co))/dt, determined at the beginning of growth. Figure 2 shows the values for the specific growth rate versus the specific gaseous mixture supply rate (Q) for the two ratios of CO2/air considered.

At both CO2/air ratios, an increase in # was ob- served when the gaseous mixture flow rate increased, up to a value for the latter of about 3 v / v per minute.

102

A A ° 0

~ 101 o

]3

o

Q ,

100 ~ , , , 0

~ . . . . i , , _ , , i ,

50 100 150 Time (h)

Fig. 1. Variation in the adimensional biomass concentration with time at a CO2/air ratio of 3 x 10 4 and specific gaseous mixture supply rates: 0.7 (O), 3,6 (A) and 5.5 (©) v/v per minute

0.10

v

d

L

2 0.05 c ~

.~_

.~

~ _

0 5 10 Speci f ic g a s e o u s - m i x t u r e supply rate,

Q(v/v.min)

Fig. 2. Relationship between the maximum specific growth rate and the specific gaseous mixture supply rate, Q: CO2/air 3 x 10 - 4

(E)), and 2 x 10 -5 (0)

At higher flow rates # decreased gradually, which points to the presence of an inhibitory effect on the cul- ture. Maximal values for # were 0.086 h -1 for a CO2/ air ratio of 3 x 1 0 -4 and 0.063h -1 for the 2 x 1 0 -5 CO2/air ratio.

Since the inhibiting effect of excessive oxygen on photosynthesis and algal growth is well known, it is likely that this may also be responsible for the decrease in specific rates. Thus, Ogawa et al. (1980) consider that the effect of O2 on specific growth rates and CO2 con- sumption may be represented by a competit ive inhibi- tion of the r ibulose-l ,5-bisphosphate carboxylase /oxy- genase enzyme by 02. Since the same active centre is involved in CO2 and O2 reactions, each substrate is a competit ive inhibitor of the other (Merret and Armitage 1982).

It is clear that in the experiments per formed at a given ratio of CO2/air, partial pressure of 02, and, therefore, the solubility of O2 in the culture medium is identical. The degree of mixing, however, provided by the bubbling at the different aeration flow rates modif- ies the t ransport capacity of the system. Oxygen inhibi- tion increases when the relative O2/CO2 proport ion is increased, which may cause lower specific growth rates as seen in the experiments carried out at the 2 x 10 -5 CO2/air ratio (Fig. 2).

The experimental results have been related to the gaseous mixture flow, which is indicative of the degree of mixing in the culture and of the concentrations of CO2 and O2 available to the cells, through an inhibi- t ion-type equation, Eq. 1, represented in Fig. 2 by con- tinuous lines that acceptably reproduce the experimen- • tal variation observed.

a.Q # - f l+ Q + Q3/~/ ( l )

105

Table 1. Variation of AXr, dc/dt, AX, and %(CO~)~ according to the specific gaseous-mixture supply rate

CO2/air ratio=3 x 1 0 4

Q AXe-. 103 dc/dt. 103 r 2 AX. 103 %(CO2) c

0.5 6.10 1.60 0.999 0.80 13.0 0.7 8.20 1.59 0.991 0.79 9.7 1.1 13.00 1.39 0.980 0.70 5.4 1.3 16.00 3.70 0.999 1.85 11.6 2.5 30.00 5.36 0.991 2.62 8.9 2.7 32.50 4.27 0.994 2.14 6.6 3.6 42.80 6.32 0.999 3.16 7.4 3.9 46.90 4.72 0.998 2.36 5.0 4.4 53.40 5.53 0.986 2.78 5.2 5.5 66.30 6.22 0.998 3.11 4.5 6.7 80.30 8.42 0.999 4.11 5.2

10.0 121.00 8.76 0.999 4.38 3.6

CO2/air ratio = 2 x 10 -5

Q AXv. 103 dc/dt. 103 r e AX- 103 %(CO2)o

1.2 0 . 9 6 . . . . 3.0 2.41 1.79 0.993 0.89 36.9 5.0 4.02 1.79 0.990 1.22 30.0 6.0 4.83 2.56 0.994 1.28 27.0 7.0 5.64 2.42 0.982 1.20 21.0

Q = specific gaseous-mixture supply rate, v/v- rain; AXr = theore- tical increase in biomass, g.h-~; dc/dt=linear growth phase slope, g. 1 ~ h- ~ ; r e = linear regression coefficient; AX= actual in- crease in biomass, g.h-~; %(CO2)~=percentage of CO2 con- sumed

Through non-l inear regression, the following values for the parameters have been obtained: a = 0.096, f l=0.258 and 7/=168.372 with an S S Q = 1 . 1 7 9 × 10 -4 (residual sum of squares values).

At the end of the exponential growth phase (Fig. 1), a linear growth phase appeared in the cultures. The val- ues for dc/dt expressed in g . l - l . h -1 for all experi- ments per formed are shown in Table 1 together with the corresponding regression coefficients. The fact that the growth rate remained constant and independent of the biomass concentrat ion indicates that this rate is de- termined by the supply of CO2 or light, or both.

For all the experiments performed, the CO2 supply rates to the culture medium have been calculated and expressed in mol . h - l ; the theoretical increases in bio- mass, AX:~, are expressed in g .h -~. These theoretical increases have been calculated on the assumption that all the CO2 supplied will be fixed in the cells in the carbohydrate state of oxidation (CH20) (Beale and Appleman 1971). These values, together with the in- creases in biomass observed, AX, in g. h-1 , determined from dc/dt, are shown in Table 1.

It is clear, by comparing the values for AXv and AX, that this calculation reveals, al though only approxi- mately, that the CO~ supplied may cause a higher in- crease in biomass than indeed takes place. The CO2 supply may, however, be higher than consumption, but its transfer through the liquid phase may control the rate of the process.

15

~ • • •

S 10 • •

~s

0 10 20 30 LO

Supplied CO 2, OCO 2 . lO-4(mol.h -1)

F i g . 3. Relationship between consumed CO2 (N) and supplied CO2, Qco2, at CO2/air ratios of 3 × 10-4 (0 ) and 2 × 10 _5 ((3). The straight line represents 100% assimilation efficiency

The biochemical consumption of C O 2 , N , expressed in t oo l . h -1 may be calculated f rom the CO2 supplied to the culture medium and the quotient AX/AXT. These values, at both ratios for CO2/air used, are shown in Fig. 3 between the CO2 supplied and consumed versus Qco2 in tool. h-1 . In approximate terms, a linear rela- t ionship is observed for low values of Qco2, whereas a saturation effect is observed at high values of Q at the CO2/air ratio of 3 × 10-4

The influence of the degree of mixing on the trans- port capacity of the system is shown in Fig. 3. At the CO2/air ratio of 2 × 10 -5, almost a 37% assimilation ef- ficiency rate was achieved, while at the CO2/air ratio of 3 × 10 -4, maximum assimilation efficiency is slightly over 13% (Table 1).

In order to compare the results based on the degree of mixing in the culture, values of N versus the specific supply rates of the gaseous mixture are shown in Fig. 4. This consumpt ion rises as Q, v / v per minute, increases tending towards a value for N o f 15 × 10 -5 m o l . h -1 for the CO2/air ratio of 3 × 10 -4 (line I) and a value of 4 .5x 10 -5 m o l . h -1 for the CO2/air ratio of 2 x 10 -5 (line II).

The restriction on CO2 consumption in the first zone in both curves may be due to one or both of th~ following reasons. (a) If, in the linear zone, the restrict- ing factor for growth is the light received by the cells, light will increase as turbulence increases, and, there- fore the amount of CO2 consumed will also rise (Terry and Kenneth 1986). (b) I f CO2 is the restricting factor, as the total flow rate of the gaseous mixture increases, then the degree of mixing in the culture increases, thereby facilitating the tansport of CO2 and its con- sumption. .

In an at tempt to discern which of these two reasons may cause the restriction observed and in order to ex- plain the presence of the plateau of both curves, a fur- ther two experiments were performed at the CO2/air

106

2s I

~ 15

E w o

:; 10 O ( A

~ 3

E ~ 5 m ~-

o ~.3

~ / ~ j / / ~

0 ' i ; Specific gaseous-mixture supply rate, O(v/v.m[n)

Fig. 4. Relationship of consumed CO2 (N) and specific gaseous mixture supply rate, Q, at CO2/air ratios of 3 x 10 -4 (©) and 2x 10 _5 (~), incident light intensity (Io)=50 W/m-2: (A) ex- periments performed at COz/air= 3 x 10 -4 and lo = 150 W-m 2

ratio of 3 x 10-4 and at values for Q of 1.5 and 10 v / v per minute. In each experiment, the intensity of light was increased to 150 W - m -2. At 10 v / v per minute there is a considerable increase in biomass productivity and CO2 consumption, reaching a value of N = 2 5 x 10 _5 m o l . h -1, while at 1.5 v / v per minute N remains unchanged from its original value obtained at 50 W. m-2 . These results seem to indicate that the con- sumption of CO2 in the first zones of the curves is lim- ited by CO2 transfer. The CO2 available to the cells rises as the degree of mixing increases.

At the higher CO2/air ratio the constant consump- tion of CO2 at high values for Q is determined by the value of the incident light intensity, 50 W - m -2. In the experiments performed at the CO2/air ratio of 2 x 10 .5 and Io = 50 W. m-2 , however, the constant values for N must be determined by the CO2 available to the cells. This conclusion is confirmed by the increase in carbon dioxide consumpt ion as the partial pressure of carbon dioxide is increased (line I). Moreover, it is interesting to point out that in the zones of linear growth of the cultures, no harmful effect of O2 was observed at the higher specific supply rates for the gaseous mixture used, possibly due to the considerable increase in cell concentration, compared with a concentration of dis- solved oxygen similar to that present at the beginning of the experiment.

The analysis of fatty acid content in the lipid frac- tion of the biomass is indicated in Fig. 5. With regard to the group of unsaturated fatty acids e)7, (99, and o11, an increase in content appears with a rise in the flow rate up to a value of 6 v /v per minute.

The fraction of polyunsaturated fatty acids tends t o decrease as Q increases, which is also revealed with the fractions of these acids in the co3 and co6 groups. This behaviour has been explained by Barber and Berheim (1967) by considering that an increase in O2 in the cul-

-u /.0

~030 D [~

~ 2o

~2 I0

~ 0

~ 80 ~ 70

g ~ 6 0 ~2 O"

2 50

= LO

~ 30 c . _

g~ 0 x~ ~ 2

O0 0 0 0 • 0 0 • 0 ~

0

•

• 0 0 •0• 0 O0 O0 0

• •

0 ° 0 o 2

O © o

•

•

0

0 2 /. 6 8 10 Specific gaseous-mixture supply rate, Q(v/v. rain)

(a)

=40 o; ~3o o~ 10

40

3 ~ 30

20

20

Y o~ 10

0%

o o o 0 o o

• •

•

o •

• @

oOO o 0 • o •

o 0 o o o

• •

• •

o 0 o o o o o o o

0 o

$ I I I ~

2 4 6 8 10 Specific gaseous-mixture supply rate, Q(v/v .min)

(b)

Fig. 5. Variation in fatty acid content versus the specific gaseous mixture supply rate: (a) percentage of the fractions of saturated (C14; C15; C16; C18), unsaturated (C16:10)6,(o9; C16:20)6; C16:30)3; C18: lo)7, 0)9; C18:2~o6; C18:30)3; C18:40)3 ; C20:10)9; C20:406; C22:1; C20:50)3) and polyunsaturated fatty acids (C16:30)3; C18:3o)3; C18:40)3; C20:40)6; C20:5~03); (b) percent- age of the 0)7/9/11, co3 and co6 groups. CO2/air=3 x 10 -4 (©); CO2/air = 2 x 10-5 (O)

il o ~ O 0 o 0 0 O "~r~ C, 0 • •

• •

0 . ~ I ~ I ~ I I

0 2 4 6 8 10 Specific gaseous-mixture supply rate, Q(v/v.min)

Fig. 6. Relationship between acids of the 0)3 and 0)6 groups versus the gaseous-mixture flow rate: CO2/air=3×10 -4 (O); CO~/ air=2 × 10 -5 (O)

107

ture m e d i u m m a y d imin i sh the p o l y u n s a t u r a t e d fa t ty ac id con ten t o f the cell by a r eac t ion o f O2 wi th d o u b l e C-C l inks, wh ich m a y l ead to the fast p e r o x i d a t i o n o f the l ip ids t h r o u g h a cha in r eac t ion o f free rad ica ls .

Last ly , the nu t r i t i ona l va lue o f the cu l tu red mic roa l - gae has b e e n a n a l y s e d acco rd ing to the c l a s s i f i ca t ion e s t a b l i s h e d by W e b b a n d C h u (1982), b a s e d on the ra t io o f fa t ty ac ids in the 0 6 and 0)3 g roups (Fig. 6). In mos t cases, the c03/0)6 ra t io fel l wi th in the range 1.34-4.15, va lues w h i c h are c o n s i d e r e d to ind ica te a g o o d nutr i - t iona l va lue by W e b b a n d C h u (1982).

References

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