Jun 1, 1992 - Kuenzler & Perras (1965) studied the phosphatase activity in whole cells of 27 strains belonging to six algal classes, and found both acid and ...
British Phycological Journal
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A comparative study of acid and alkaline phosphatase activities in several strains of Nannochloris (Chlorophyceae) and Nannochloropsis (Eustigmatophyceae) Luis M. Lubián , Julián Blasco & Rafael Establier To cite this article: Luis M. Lubián , Julián Blasco & Rafael Establier (1992) A comparative study of acid and alkaline phosphatase activities in several strains of Nannochloris (Chlorophyceae) and Nannochloropsis (Eustigmatophyceae), British Phycological Journal, 27:2, 119-130, DOI: 10.1080/00071619200650131 To link to this article: https://doi.org/10.1080/00071619200650131
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Br. phycol. J. 27:119-130 1 June 1992
A Comparative Study of Acid and Alkaline Phosphatase Activities in Several Strains of Nannochloris (Chlorophyceae) and Nannochloropsis (Eustigmatophyceae) By Luis M. LUBI,~N, JULIAN BLASCO and RAFAEL ESTABLIER Instituto de Ciencias Marinas de Andalucia, Poligono Rio San Pedro, Apartado Oficial, 11510 Puerto Real (Cadiz), Spain Acid and alkaline phosphatase activities in whole cells as well as in the particulate and soluble fractions, obtained from late exponential phase cultures of several marine strains of Nannochloris (Chlorophyceae) and Nannochloropsis (Eustigmatophyceae), have been studied. These enzymes exist in whole cells of all Nannochloris strains but only acid phosphatase was detected in particulate and soluble fractions. Nevertheless, at least in N. maculata, the existence of alkaline phosphatase in the soluble fraction has been revealed when the reaction mixture contains sea-water, but not in the presence of Mg 2+, Zn 2+, Co 2+, Mn 2+, Mo 6+, or Fe 3+. In addition to their different Km values, acid phosphatases from the particulate and soluble fractions are also differentiated by their optimum pH or their response to temperature. Of the three Nannoehloropsis species studied, N. salina and N. gaditana possessed only acid phosphatase, both in whole cells, and in particulate and soluble fractions, but no phosphatase activity was ever detected in N. oculata. Inorganic phosphate in the culture medium affected phosphatase synthesis in all cases, since the activity per cell was lower in cultures to which phosphate was added.
Many strains belonging to the genera Nannochloris and Nannochloropsis have been isolated from oceanic, coastal and brackish waters from all over the world (Hibberd, 1981; Sarokin & Carpenter, 1982; Lewin & Cheng, 1989). They have been found growing in great abundance in bays, brackish rock pools and salt-marshes (Ryther, 1954; Droop, 1955; Lubifin et al., 1985). Both genera belong to phylogenetically remote classes of algae, including tiny spheroid algae of similar appearance under the light microscope (Hibberd, 1981). As a consequence of their phylogenetic distance, previous studies on the ultrastructure and pigment composition in various species have revealed substantial differences between them (Antia et al., 1975a; Lubifin, 1982). However, with the exception of work by Antia et al. (1975b), Berland et al. (1979), Lubifin (1981) and Turner & Gowen (1984) on the utilization of different inorganic and organic nitrogen sources and vitamin requirements, there are 0007-1617/92/020119+ 12 $03.00/0 Published online 24 Feb 2007
few comparative studies of Nannochloris and Nannochloropsis nutrition. These studies revealed that, unlike Nannochloris, none of the Nannochloropsis species require exogenous vitamins for growth, and that they cannot use arginine as a nitrogen source, at least up to a concentration of 1 mM. The two genera also differ with respect to the enzyme involved in the catabolism of urea, which in Nannochloris is ATP: urea-amidolyase, and in Nannochloropsis is urease (Leftley & Syrett, 1973). It is known that some species from each genus are able to grow with glycerophosphate as the only source of phosphorus (Lubifin, 1981), which implies the presence of phosphatases in these algae. The presence of phosphatases in algae has been widely studied both in fresh water (Talpasayi, 1962, Blum, 1965; Knutsen, 1968; Guerrini, Cremona & Preddie, 1971; Lien & Knutsen, 1973; Patni et aL, 1974; Nagy et aL, 1981) and marine (Kuenzler & Perras, 1965; Antia © 1992 British PhycologicalSociety
120
L.M. Lubifin, J. Blasco and R. Establier
& Watt, 1965; Moher, Miklestad & Haug, 1975; Swanson & Floyd, 1979; Rivkin & Swift, 1980; Sakshaug et al., 1984; Carpen~ & Wynne, 1986) species, but in no case has any Nannochloris or Nannochloropsis species been studied. The presence of acid and/or alkaline phosphatases has been revealed in species from different groups of marine microalgae. Kuenzler & Perras (1965) studied the phosphatase activity in whole cells of 27 strains belonging to six algal classes, and found both acid and alkaline phosphatases in 13 of them, alkaline phosphatase alone in 11, and acid phosphatase alone in three. However, when Antia & Watt (1965) analysed the phosphatase activity from celt extracts of six species belonging to three classes, of which five coincided with those studied by Kuenzler and Perras, they detected only acid phosphatase. This inability to detect alkaline phosphatase might have been due to various factors, such as an insufficient extraction treatment to solubilize the membrane-bound enzyme (Kuenzler & Perras, 1965), the presence of inhibitors (Antia & Watt, 1965; Patni et al., 1974) or the absence of Mg 2+ (Patni et al., 1974; Rivkin & Swift, 1980). In this paper we present comparative results of acid and alkaline phosphatase activities in several strains from both Nannochloris and Nannochloropsis, cultured under the same conditions.
MATERIALS AND METHODS
Algae used and culture conditions The strains used were: Nannochloris macutata Butcher (CCAP, strain No 251/3); Nannochloris atomus Butcher (CCAP, strain No 251/6); Nannochloris sp. (ICMA, Palau strain); Nannochloropsis oculata (Droop) Hibberd (Millport, strain No 66); Nannochloropsis salina Hibberd (ICMA, Monasal strain); Nannochloropsis gaditana Lubifin (ICMA, B-3 strain). Nannochloris sp. (Palau strain) was supplied by R. A. Lewin of Scripps Institution of Oceanography, La Jolla, California. N, salina (Monasal strain) was sent by S. Y. Maestrini of Station Marine d'Endoume, Marseille. Algae were cultured in f/2 medium-enriched (Guillard & Ryther, 1962) natural sea-water (36%0 salinity) containing a double amount of nitrate
and phosphate. Cultures were maintained aseptically in 500 ml flasks with continuous agitation by means of CO2-enriched air, keeping the pH between 7'7 and 8'0 (Mourente, Lubi/m & Odriozola, 1990), at constant temperature of 20°C and continuous lighting (100 g E m -2 s -1) with day-light fluorescent lamps. Cell densities were estimated in a Neubauer haemocytometer. Observations under phasecontrast microscopy never revealed bacterial contamination of cultures. Analyses of phosphatase activity were performed in samples from late exponential phase cultures once phosphate was severely limiting in the medium.
Enzyme assay Phosphatase activity was estimated by the liberation of p-nitrophenol from p-nitrophenyl orthophosphate (disodium salt, Merck) at saturating concentration. Buffers were 0"1 M citric acid/ sodium citrate for the acid range and 0-1 M Tris/HC1 for the neutral and alkaline range. When analysing whole cells, buffers were made up with sea-water. For whole cell phosphatase activity, three series of tubes were prepared with a reaction mixture containing: (a) 0-5 ml buffer and 25 gl substrate; (b) 0.5 ml buffer, 0"5 ml culture filtrate (0.45 gm) and 25 ~tl substrate; and (c) 0"5 ml buffer, 0"5 ml 1:5 diluted microalgae culture and 25 gl substrate. 0.5 ml of 1:5 diluted microalgae culture were added to series (a) tubes once the reaction was stopped. Final substrate concentration was 8 mM. When studying the effect of inorganic phosphate (Pi), 25 gl of NaH2PO42H20 at a final concentration of 0-100 gM was added to the reaction mixture. Assay temperature was 20°C and incubation time 120 rain. For cell fractioning, the culture was centrifuged at 1600 g for 15 rain., the supernatant discharged and the pellet washed twice in 0.6 M NaC1, 3 ml of 100 mM Tris-HC1 at pH 7 were finally added and sonicated for 20 min. in two steps. The extract was again centrifuged for 15 rain. at 1600g and the supernatant employed as an enzyme source for the determination of phosphatase activities in the soluble fraction. A third sonication never produced activity in the supernatant. The pellet resulting from the third centrifugation was resuspended in water (Milli-Q system) and sonicated for 5 min. to resuspend it. The suspension was used for measuring phosphatase activities in the particulate fraction. Light microscope observations of the particulate fraction revealed that, despite ultrasonic treatment, no visible cell lysis occurred in any of the algae. The reaction mixture contained 1-0 ml buffer, 0.1 ml substrate and 0"1 ml of extract or 0-1 ml of
Phosphatase in Nannochloris and Nannochloropsis
was determined at optimum pH within a range between 20 and 60°C, with the exception of the particulate fraction of N. atomus (upper limit of
suspension, depending on whether the soluble or the particulate fraction was analysed. Final substrate concentration in both cases was 25 mM. To study the effect of Pi, 0-1 ml of NaH2PO4.2H20 at a final concentration of 0-100 gM was added to the reaction mixture. T h e assay temperature was 40°C and incubation time 60 min. The reaction was stopped by adding 5 ml of 0-1 M NaOH to the reaction mixture in all cases. Phosphatase activities were calculated using a molar extinction coefficient for p-nitrophenol of 18"5 cm2.gmol -~ at 405 nm (Walter & Schiitt, 1974). Results were expressed as /amol p-nitrophenol.min-L(10m cell)-L The temperature response of phosphatase activity in the particulate and soluble fractions Whole cell
121
50°C).
To determine kinetic parameters, substrate concentration ranged from 0 50 mM p-nitrophenylphosphate for the soluble and particulate fractions, at each optimum pH and at temperature 40°C. Km and gmax w e r e calculated using a non-linear regression analysis. To determine any possible regulating effect of phosphate in the culture medium on the enzymatic activity, 400 ml of culture of each alga at the end of the exponential phase of growth were subcultured in half of the initial volume. One subculture was added to 200 ml of f/2 enriched fresh medium without phosphate ( - P ) , while the
Soluble fraction
Particulate fraction
50
40
40
40
30
30
20
20
30 2O I0
o
IO
,
,
,
,
,
,
,
0
I0
'
'
'
'
'
'
'
0
Alannochlor/s sp.
7
2 o 5
I
I
pH
FIG. 1. pH profiles of phosphatase activities (U = gmol p-nitrophenol.min L) in whole cells, soluble fractions and particulate fractions of cultured Nannochloris species.
122
L, M. Lubifin, J. Blasco and R. Establier
other had a concentration of 64 gM NaH4PO2.2H20 (+P). Cell density, phosphorus concentration in the medium and whole cell phosphatase activity were measured before starting the trial, and after 24 and 48 h. Phosphate analyses were performed in a Technicon TRAACS 800 autoanalyser, according to Strickland & Parsons (1968).
IO
a)
RESULTS p H effect The effect of p H on phosphatase activity in whole cell, particulate and soluble fractions of the studied Nannochloris strains is shown in Fig. 1. Whole cells of N. maculata, N. atomus and Nannochloris sp. had maxim u m activities at p H 5.5, 4.5-5-0 and 3-0 respectively, and at p H 9.0 in all cases. A maximum of activity at p H 5.0-6.0 existed in the particulate fraction from N. maculata, while in Nannochloris sp. m a x i m u m activity was obtained at p H 3.0, from which point activity diminished as p H increased. In N. atomus the activity of the particulate fraction was low and a clearly defined m a x i m u m was not found, although the highest value at low p H was obtained at p H 5"0. Likewise, the soluble fraction from N. maculata, N. atomus and Nannochloris sp. showed a single m a x i m u m at p H 5.0, 5.0 and 6"0 respectively. A m a x i m u m of activity only detected in the soluble fraction (Fig. 2) was found for N. maculata, when sea-water was ~clded to the reaction. Addition to the f/2 medium of 1 0 m M Mg 2+ (MgC12) , or 38.8 m M N a + (NaCI), as well as trace metals, Zn 2+ ( Z n S O 4 ) , C o 2+ (CoCl2) ' M n 2+ (MnC12), M o 6+ (Na2MoO4) and Fe 3+ (FeC13), did not alter m a x i m u m activity in either particulate or soluble fractions. Enzymatic activity in whole cells of Nannochloropsis salina and N. gaditana (Fig. 3) showed only one m a x i m u m at p H 5-5. Both particulate and soluble fractions showed an identical response. N o phosphatase activity was ever detected for N. oculata. Temperature effect and substrate affinity
Acid phosphatase activity in the particulate and soluble fractions of all algae
g
0
*6
I0
o-
8
6
4
2
Oi 2
I 3
I 4
I 5
I 6
I 7
I 8
I 9
I0
pH
FIG. 2. Effect of t0 mM Mg2+ (MgCI2) (empty circles), 38.8 mM Na + (NaC1) (filled squares) and natural sea-water (filled circles) on phosphatase activity in particulate (A) and soluble fractions (B). studied increased with temperature within the range assayed (Figs 4 and 5), with the exception of Nannochloris maculata (maxima at 50°C and 30°C for the particulate and soluble fractions respectively) and N. atomus (maximum at 30°C for the soluble fraction). Activation energy values (Arrhenius plot) in the particulate and soluble fractions of Nannochloris sp. were different, while Nannochloropsis salina and N. gaditana both showed similar values (Table I). K m and Vm~x values obtained for acid phosphatase from the particulate and soluble fractions are shown in Table II. Phosphatase
Phosphatase in NannocMoris and Nannochloropsis
123
TABLEI. Activation energy values (Kcal.mol 1) for acid phosphatase for Nannochloris and Nannochloropsis species
250
AZsalma 200
Algae species 150
Nannochloris maculata Nannochloris atomus Nannochloris sp. Nannochloropsis salina Nannochloropsis gaditana
100
Particulate fraction
Soluble fraction
4.49 1.77 7.93 9-10 9.53
n.d. n.d. 6"00 9.30 9-21
n.d. = undetermined. 50 uble fraction, whereas in N. maculata K m was noticeably higher in the particulate fraction. Conversely, substrate affinity in Nannochloropsis salina a n d N. gaditana was higher in the soluble fraction. A low Vmax was f o u n d in the particulate fraction o f N. atomus unlike the other two Nannochloris species, while in Nannochloris sp. a lower activity was f o u n d in the soluble fraction. N. salina a n d N. gaditana showed generally high Vmax values, m a i n l y in the particulate fraction.
T
0
400
IV. god/tono
320
24O
160
Effect of added phosphorus on phosphatases activities
8O
0 2
5
4
5
6
7
8
9
I0
pH
FIG. 3. pH profiles of phosphatase activities (U = pmol p-nitrophenol.min-t) in whole ceils of cultured Nannochloropsis species. Particulate and soluble fractions have the same pH profiles in all cases. activity in the particulate fraction o f N. atomus a n d Nannochloris sp. showed a higher substrate affinity t h a n that in the sol-
W h e n p h o s p h a t e was added to the i n c u b a tion m e d i u m up to a c o n c e n t r a t i o n of 100 laM, n o i n h i b i t i o n in the acid or alkaline p h o s p h a t a s e activity was detected either in the particulate or soluble fraction, or in whole cells of a n y o f the studied algae, Figures 6 a n d 7 show the d e v e l o p m e n t t h r o u g h time o f cell densities a n d p h o s p h a tase activities in subcultures with a n d w i t h o u t phosphate. Nannochloris atomus showed such a low level of activity as to
TABLEII. Km (mM) and Vmax [tam01.rain t (1012 cell) -t] values (means _+standard deviation of three replicates) of acid phosphatase for Nannochloris and Nannochloropsis species Soluble fraction
Particulate fraction Algae species Nannochloris maculata Nannochloris atomus Nannochloris sp. Nannochloropsis salina Nannochloropsis gaditana
K,~ 5-51 +0-14 0.98 _+0"38 0.28_+0-17 1.99_ 0.16 2.59 -+0.14
Vm~ 55.54_+0.23 2"72_-_0"11 93'14__+2-46 741.44 ± 9.19 688-86+ 9.06
K,~ 1-91_+0"18 1"52-+_0"17 1.70-+0.26 0.85 _+0.11 1.15_+0"15
Vm~x 91"13+ 1"58 26.75 _+0'47 15.01_+0-58 94.53 -+ 1-50 116-25-+_3"07
124
L . M . Lubifin, J. Blasco and R. Establier
Soluble fraction
Particulate fraction 16C F
40
,4/. maculata
N moculoto
30
120
20
80
I
I0
I
I
I
I
I
40
I
I
300
200 Nonnoch/oms sp.
Nonnochloris sp.
J
/
25O 150 T
2OO I00 0
~50
/
50
I00
I
I
1
I
I
50
I
2.75
I
I
I
I
30
40
50
60
5O ,/V. otomus
/V. atomu$ 25
2.50 2O 2.25
15 I0
2.00 5 1.75
I0
I
I
I
I
I
20
30
40
50
60
0
70
I0
20
70
Temperature (%)
FIG. 4. Temperature profiles of acid phosphatase activities (U = pmol p-nitrophenol.min. ~) in particulate and soluble fractions of cultured Nannochloris species.
Phosphatase in Nannochloris
and Nannochloropsis
125
Soluble fraction
Particulate fraction 60 2400
N. salma
/o
2000
40 1600 50 1200 20 8O0 I0 i
400 ~I
O
I
I
I
1600
I
I
0
I
I
I
OOC
A/. gadifano
N 9a~tana
1400
/0
1200
800
~000
600
800! 400
600 400
200 200 0 I0
I
I
I
I
I
20
30
40
50
60
0
70
l0
I
I
I
I
I
20
30
40
50
60
70
Temperature (°C) FIG. 5. Temperature profiles of acid phosphatase activities (U = pmol p-nitrophenol.min -L) in particulate and soluble fractions of cultured Nannochloropsis species.
preclude reliable results using present methodology. Before subculturing, P concentration in the medium of all algae ranged from 0.16 gg-atom.1 -] for Nannochloris sp. to 0-61 gg-atom.1 ~ for Nannoehloropsis gaditana, with the exception of Nannochloris maculata for which the value was 3.1 pg-atom.l ~. Twenty-four hours after subculturing, Nannochloropsis salina and N. gaditana had similar P levels in the medium, around 0'3 pg-atom.1 -~, both in cultures with ( + P) and without ( - P ) added phosphate. No phosphate was detected after 48 h. There was no phosphorus in the culture medium of N. maculata and Nannochloris sp. after 24 h.
Once subcultured, + P and - P cultures of all algae continued to grow, although growth in the latter was not clearly revealed after 48 h, with a lower cell density than + P cultures. Acid phosphatase activity in the studied species developed in a similar way all through the assay. In general, 24 h after addition of new phosphate-enriched medium, the activity per cell was lower or had not varied, and in all instances was lower than in cultures without phosphate addition. In these cultures, phosphatase activity noticeably increased. Once phosphate was completely consumed after 48 h, cellular phosphatase activity always
126
L . M . Lubi~n, J. Blasco and R. Establier -p
+P
250
200
250 ~
100
IV. macu/ata
IILrnocu/ato 200
~, I \, \
,oo
,,
o
150
200 -
,oo
