Immunological quantitation of a polysaccharide formed by Emiliania huxleyi

JoumaO of Marine Systems 9

I19969 67-74

Ccohiochemistry Department. Gorkurus Luhorutc~rirs. Leidm University. P.O. Box 9502, 2300 R.4 Leiden. The Nethrrlunds

Received26 May

1995; revised IO November 1995; accepted IO November 1995

Abstract The principle of ELBA was adapted to measure quantitatively the concentration of an extracellular polysaccharide of the unicellular alga EnJihJiu huxleyi.This so called “coccolith polysacchavide” could be detected at concentrations as low as 0.02 mg I- ‘. The concentration of coccolith polysaccharide was determined in the supematant of actively growing calcifymg and naked batch cultures of E. huxleyi and amounted to 0.56 and 0.87 pg cell- ‘, respectively. In these cultures, maximum concentrations of 0.26 and IO.I mg I- ’ coccolith polysaccharide, respectively, were reached in the late stationary phase of growth. These results suggest that in a bloom of E. huxleyi, the dissolved coccolith polysaccharide makes a modest contribution to the pool of dissolved organic carbon (DOC), and forms a small part of the E. hrcxkyi primary production.

The unicellular algal species E0&&i huxleyi is an important constituent of the oceanic phytoplankton (Braarud, 1962; McIntyre and BB, 1967; Balch et al., 1991; Kleijne, 1993). This cosmopolitan species regularly forms vast blooms at mid-latitudes, covering thousands of square kilometres (Holligan, 1986; Ackleson et al., 1988; Brown and Yoder, 1994). The “Global Emiliuniu Modelling Initiative” (GEM), aims to model climate forcing by oceanic ecosystems (Westbroek et al., 1993) with E. huxleyi as the model organism. An understanding of the carbon metabolism of this species is, therefore, important. An essential organic compound formed by E. huxleyi is coccolith polysaccharide. However, the amount of this compound formed by E. huxleyi is

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Corresponding author.

09247963/%/$15.00 Copyright0 1996Elsevier Science PII SO924-7963(96)00017-6

not known. In addition, no data are available concerning the release of this polysaccharide to tbe ocean where it may contribute to the level of dissolved organic carbon (DOC). Coccolith polysaccharide is thought to be involved in the morphogenesis of coccolitbs by inhibition of the growth of certain crystal faces (Van Emburg et al., 1986). Coccoliths are minute CaCO, scales which are produced in an intracellular compartment, the Golgi-derived coccolith vesicle. As soon as their formation is completed they are transported to the outer cell surface where they form an extrncellular cover or coccosphere (De Vrind-de Jong et al., 1994). With immunological techniques the presence of coccolith polysaccharide has been demonstrated at the surface of both cells and coccoliths (Van Emburg et al., 1986). Thus, the polysaccharide may also function as an organic matrix to establish a coherent coccosphere (De Vrind-de Jong

B.V. All rights reserved.

et al., 1994). The coccolith polysaccharide of E. hrc_t-Ieyi has been isolated and its structure partly elucidated. It is a complex carboxylated compound, weakly acidic, sulphated and water soluble, containing at least 13 different monosaccharides(De Jong et al., 1976; Fichtinger-Schepmanet al., 1979, 1981). The molecular weight is 88,600 f 3200 g mol-’ (Bormanet al., 1986).Coccolith polysaccharide may also be involved in the agglutination of cells. This subject, however, is beyond the scope of this paper and will be included in another report. As coccolith polysaccharide is water soluble it is likely that a substantial portion will dissolve upon excretion. Here we study the amount of coccolith polysaccharidewhich dissolves into the medium during growth of E. huxleyi and the importance of this compound for the oceanic DOC level. To quantify the concentration of excreted dissolved coccolitb polysaccharide, an immunological assay was developed and applied in preliminary growth experiments.

2. Materials and methods 2.1. Organism and cultivation

condtions

The growth experiments were conducted with axenic cultures af E. huxleyi strain, BQF92, which was kindly provided by Prof. E. Paasche (Oslo University). This calcifying strain was maintained on Eppley medium (Eppley et al., 1967) and in F/25 medium. F/25 medium is identical to Eppley medium except for the added amounts of nitrate and phosphate which are both reduced by a factor of 25. The culture maintained in F/25 remained calcifying. The culture maintained on Eppley medium, however, became naked. This phenomenon that cells do not form coccoliths when cultivated continuously on relatively nutrient-rich media has been reported before (Paasche, 1964; Guillard, 1975). The production of polysaccharideswas assayed in batch cultures. The naked culture was grown on Eppley medium and the calcifying culture on F/25. Both cultures were grown under a light regime in which a 15 hour light period alternated with a 9 hour dark period. The ambient intensity of white light was 4Sf 15 kErns2 s-l and the temperature was 18°C. The batch cultures were incubated on a gyratory

shaker (120 rpmj in conical flasks (5 1) wi initial volume of 1.5 I. Samples for analysis were always taken half-way through the light period. immnosorbent 2.2. Enqwe-linked for coccolith polysaccharide

assay

fELtSA)

To determine the concentration of dissolved coccolith polysaccharide, culture samples were centrifuged (10 min at 13,000 g) to remove cells and coccoliths. Salts were removed from the supernatant using Sephadex columns, type PD-10 (Pharmacia), equilibrated and eluted with 10 mM NH,HCOJ. The eluent was freeze-dried and the remaining compounds were dissolved in TBS buffer (see below). For analysis of intracellular coccolith polysaccharide and coccolith polysaccharide at the surface of cells and coccolitbs, the pellet from centrifuged culture samples (see above) was resuspended in 100 mM NH,HCO, and centrifuged (10 min at 13,000 g). After removal of the supematant the pellet was freeze-dried and resuspended in acetate buffer (0.1 M, pH 5). At this pH coccoliths dissolve although sometimes additional HCI must be added due to calcite buffering. Subsequently, the cells were disrupted by sonication, the suspension was neutralized with NaOH and centrifuged (30 min at 13,000 g) to obtain a cell-free extract. The cell-free extract was desalted over a Sephadex column (see above). The eluent of the Sephadex column was freeze-dried and finally resuspended in TBS buffer (see below). In the ELISA the following solutions were used: poly-L-lysine (0.1% w/v) from Sigma diagnostics (P8920); TBS buffer (NaCI (9.0 g I-’ ) and tris(hydroxymethyl)aminomethane(1.21 g l- ’i in demineralized water and adjusted with concentrated HCI to a pH of 7.5); TBS/Tween (TBS buffer containing 0.05% v/v Tween 20). Gelatin (2% w/v) was dissolved in demineralized water by boiling for 10 minutes and cooled to 37°C before use. The first antibody (K4772) was obtained from a rabbit in which it was raised against a coccolith polysaccharide-albumin conjugate (Borman et al., 1987). In addition to this antiserum, a preimmune serum of this rabbit was available for control experiments. When preserum was used instead of the antiserum, coccolitb polysaccharide was not detected. The second antibody, goat-anti-rabbit labelled with alkaline

Fig. I. Schematic represer,tationof the ELlSA for coccolitb polysaccharide:(a) polystyrene,ib) polylysine, (c) coccolith polysaccharide, (d) blocking by gelatin, (e) antibody for coccolith polysaccharme raised in a rabbit, (f) goat-anti-rabbit antibody labelled with, (g) alkaline phosphatase.(h) phosphause staining reaction.

phosphatase, was purchased (Sigma Immunochemicals, A8025). The antiserum with the first antibody and the solution with the second antibody were diluted 1000 and 30,000 times, respectively, in freshly prepared TBS/Tween with 0.2% w/v gelatin. The substrate solution contained di-sodiump-nitrofenylphosphate (0.05% w/v) in substrate buffer. The substrate buffer was diethanolamine (97 ml I-’ ) in demineralized water brought to pH 9.8 with concentrated HCl and supplied with NaN, (0.2 g l- i 1. The dilutions of the first and second antibody and the substrate solution were always prepared just before use. The ELISA procedure, which is schematically shown in Fig. I, included the following steps: (1) The wells of a polystyrene microtitre plate (NUNC immunoplate, type Maxisorp I F96) were filled with 100 pL poiy-L-lysine and incubated during I hour. During this incubation period the polystyrene is coated with poly-L-lysine (see Fig. IB). (2) The microtitre plate was emptied and rinsed 3 times with 200 p.1 TBS/Tween. (3) From sample solutions in TBS buffer, 100 p.1 was added per well ‘and incubated for 1.5 hours. At pH 7.5 poly-L-lysine is

ing 30 minutes. Gelatin was used to block binding sites of poly-L-lysine which were not occupied by coccolith po~ysaccb~i~e. In addition, gelatin cov open spaces on the polystyrene (see I$. e gelatin was remove and I (6) t.~l of the diluted solution of the first antibody (tie antibody against coccolith olysaccharide) was add followed by an incubation per Fig. IE). (7) The solution with removed and the wells were rinsed 4 times with TBS/Tween. (8) 100 p,l of the second antibody (goat-anti-rabbit labelled with alkaline ~~os~~atase) was added to each well and incubated for 1.5 hours (see Fig. 1F and 6). (9) The solution with the second antibody was removed and the wells were pinsed 5 times with TBS/Tween. (IO) 100 pI substrate solution was added to ea well and the microtitre plate was incubated (Fig. ). (1 I) The absorbance was measured every IO minutes. (12) When the absorbance of the wells with the highest amount of coccolith polysaccharide had reached values of OS0.7, the enzymatic reaction of alkaline phosphatase NaQ was stoppeci by adding 100 pl of a 1.0 solution. Thus, the amount of bound alkaline phosphatase was determined by means of a calorimetric assay. This amount is related to the concentration of coccolith polysaccharide in the sample. All incubation steps were performed at 37°C. During incubation the microtitre plates were sealed with an adhesive cover. On each microtitre plate a triplicate calibration curve with purified coccolith polysaccharide was included. This coccolith polysaccharide was previously isolated from E. huxleyi strain 92D (Barman et al., 1987) according to the method described by De Jong et al. (1976). Absorbances were measured on a Titertek Multiscan Plus spectrophotometer from Flow Laboratories. An example of an ELISA calibration curve is shown in Fig. 2. In unconcentrated samples a concentration of coccolith polysaccharide as IOW as 0.02

I

0.3

0.2 cps

I

0.4

Imgslw')

Fig. 2. Calibration curve of the quantitative ELBA for coccolith polysaccharide. The absorbance ( A) at 405 nm is plotted against the concentration of coccolith polysaccharide (cps).

were disrupted by sonication after which the cell debris was removed by centrifugation (20 min at 13,000 g). The cell-free extracts were desalted using a Sephadex column (see ELISA) and finally freezedried. In the freeze-dried samples from supematants and cell-free extracts, the total polysaccharide content was determined (see below). Furthermore, the sugar composition was determined by liquid-gas chromatography, after methanolysis in 2 M methanolic HCI for 24 hours at 85°C and subsequent derivatization into trimethylsilylated methyl glycosides, as described by Kamerling and Vliegenthart (i 982). The following sugars were assayed: arabinose, galactose, galacturonic acid, glucose, mannose, rhamnose and xylose. 2.4. Miscellaneousmethods

mg 1-i could be measured, Generally, the association rate between the antigen (coccolith polysaccharide) and an antibody is influenced by pH, temperature and ionic strength. In the assay the pH and temperature were easy to control. Initially, differences in ionic strength were a source of error. This problem was solved when the samples were desalted by means of a Sephadex column. In control experiments the loss of coccolith polysaccharide from a standard solution in sea water was assayed after desalination in a Sephadex column and freeze-drying of the eluent. The loss of coccolith polysaccharide d to be less than 5%. The specificity of the ainst coccolith polysaccharide (K4772) has been shown in previous research (e.g. Bormnn et al., 1987; Van Bleijswijk et al., 1991). 2.3. Analysisof sugar composition To analyse the sugar composition of polysaccharides of E. huxleyi, cell suspensions were centrifuged (30 min at 1600 g). The supernatant containing the dissolved polysaccharides was then dialysed in Spectraporemembrane tubing (MW cut-off 3500) against demineralized water at 4°C. The dialysed liquid was subsequently freeze-dried. The pellets Icells plus coccoliths) were resuspended in 100 mM NaH,PO~. In the case of calcified cells HCI was added until all coccoliths were dissolved, The cells

Total saccharide concentrations were determined as glucose equivalents by the anthrone method (Herbert et al., 19’71). Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard. Cell numbers were determined in a Blirker haemocytometer with a depth of 0.100 mm. To determine the presence of coccoliths, at the cell surface a light-microscope with cross~polarized illumination was used. Cells with I or more attached coccoliths were counted as calcified. 2.5. Biodegradationof coccolithpolysacchwide A test was set up to investigate the biodcgradability of the E. huxleyi coccolith polysaccharide. Marine bacteria from North Sea samples were enriched in two different aerobic Eppley media. One of these media was supplied with cell-free extract of E. huxleyi cells and the other with several monosaccharides. From both enrichments several bacterial strains were isolated. For the test, one strain fire n the enrichment with cell-free extract and two strains from the monosaccharide enrichment were selected. Supematant obtained from the naked culture on day 18 (Fig. 4) was devided into 4 aliquots. Each aliquot was supplied with 10% (v/v) sterile, freshly prepared Eppley medium to provide sufficient nutrients for bacterial growth. Three aliquots were inoculated

The growth of both calcifying and naked cultures of E. huxleyi was followed in atcb cultures. In the I ~~rnber incrtased calcifying culture (Fi from 1.8 X IO5 cells cells ml-’ decrease by cell lysis was probably caused by depletion of a nutrient. Almost simultaneously the concentration of total saccharides in the biomass fraction decreased from 5.0 mg I- ’ on day 11 to 3.0 mg I - ’ on day 18. Such a decrease was not found for the concentration of coccolith polysaccharide dissolved in the medium. This latter concentration increased steadily from 35.0 to 260.0 pg I-‘. The results from the naked culture are depicted in Fig. 4. As this culture was grown in Eppley medium, which is rich in nitrate and phosphate, much higher cell numbers and polysaccharide concentrations were reached in comparison with the calcifying culture. In addition to the results presented in Figs. 3 and 4 several other data were obtained. The concentration of coccolith polysaccharide in cell-free extracts

Fig.

4. Growth

Epplcv

polysacchmide bioma.is

of Ihe

me4hum:

Cl = (elk.

expressed

naked cell

cps); as glucose

cukurc ~~~~r; =

time

(dl

of

Emi/io,ricl

0 =

dissolved

total saccharide

huskyi

in

coccolilh

content

CA the

equivalenls.

rifuged culture samples was alpg 1-I for botb cultures. Ihis low value may be due to incomplete extraction of poiysaccharide from the pellets ing the rocedure. To obtain quanritative ta concerning coccolith polysaccharide the cellular fraction more research is required, e protein content was always less than I .O mg 1-l in the calcifying culture, but in the naked culture the protein content gradually increased from 1.0 to 16.6 mg I- ’ on day 3 and 18, respectively. Between day 3 and I I, for both cultures, the percentage of naked cells aud the appearance of cell-aggregates were monitored microscopically. The calcifying culture always contained less than 2% naked cells and aggregates were not observed. In the naked culture the percentage of naked cells varied between $6 and 94% and on day 11 small aggregates consisting of 5-10 cells were present. Although in the naked culture approximately 10% of the cells carried coccoliths, it must be noted that the calcified cells usually possessed 1 or 2 attached coccoliths only. 3.2. Monosaccharidecompositionof polysaccharides

0

2

Fig. 3. Growd~ af F/25 medium: Cl saccharide ( diw. biomass expressed

r,

6

8

10

12

14 time Id1

16

lb

the calcifying culture of EmiliwCu huxleyi in = cell number; 0 = dissolved coccolilh ply= lotal saccharide content of the cps); as glucose equivalenls.

In previous research the monosaccharide composition of purified coccolith polysaccharide has been determined (Fichtinger-Schepman et al., 1979). The principal monosaccharides are listed in Table 1. As a control for the ELBA, similar analyses were performed with samples obtained on day 18 from the

Table I Composition of purified coccolilh polysaccharide. analysrd using methanolysis and expressed as the molar ratio to mannose (Fichtinger-Schcpman c: a!.. 1979)

Table 3 Composition of polysaccharides extracted from disrupted cells of u calcifying and a naked CUltWe of ~nJi/hJnJu hJ&yi, expressed as the molar ratio lo m‘annose

Monosaccharide

Molar mtio to mannose

Monosaccharide

Calcifying

arabinose rhamtiose + ribose xylosc mannose galactose galacluronic acid glucose di-methyl-rhamnose methyl-xylose methyl-mannose

0.10 0.73 0.57 1.00 0.13 0.83 0.03 0.27 0.23 0.27

arabinose rhamnose xylose mannose galactose galacturonic acid glucose

co.15 0.63 0.27 1.00 0.29 0.47 IO.16

culture

Naked culture 0.79 0.63 0.60 I .oo I.57 0.38 7.40

lith polysaccharide was indeed present. In Table 3 the glucose content is very high for both cultures. In growth experiments shown in Figs. 3 and 4. For 7 of

fact, the proportion of glucose is greater than the

the 11 monosaccharides mentioned in Table 1, the relative molar quantities were determined. In order to allow comparison with Table 1 all quantities were related to mannose, which was arbitrarily fixed at 1.0 mol. In the supernatant of the calcifying culture (Table 2) the rhamnose and galacturonic acid content were relatively low whereas the glucose content was relatively high. In this sample, however, errors cannot be excluded because the concentrations were very low and near the detection limits of the equipment. The monosaccharide composition in the supernatant of the naked culture (Table 2) agreed fairly well with the data shown in Table I, in extracts from disrupted

sum of t’le other monosacchCarides,which are assumed r~ be a constituent of coccolith polysaccharide. A possible explanation is the formation of an intracellular glucose-containing storage compound. Indeed, E. huxleyi can form an intracellular P-Dglucan, which consists of more than 99% glucose (V&rumet al., 1986).

cells all 7 monosaccharides were unambiguously detectable, with exception of arabinose in the calcifying culture (Table 3). In general, these results indicate that in the samples used for the ELBA, coccoTable 2 Composilion of polysaccharides in the supematant of a calcifying and naked culture of Emiliwriu huvleyi, expressed as le molar mtio to mannose Monosaccharide

Calcifying culture

naked culture

arabinose rbamnosc XYlOSe mannose

0.04 < 0.03 0.42 1.00 0.43 0.05 I .26

O*SO 0.61 0.54 1.00 0.5 I 0.77 0.65

&tllWlOS~

galacturonic acid glucose

3.3. Degradation of coccolithpoiysaccharide The initial concentration of coccolith polysaccharide in the experimental cultures was 9.1 + 2 mg I- ’. After 2.5 weeks no significant decrease in the concn>ntrationof coccolith polysaccharide was found in either of the cultures. This is an indication that coccolith polysaccharide may be rather resistant to bacterial degradation, which is in agreement with earlier experiments at our laboratory (E.W. De Vrind-de Jong, pers. commun., 1995).

4. Discussion The ELBA-technique can be used for a quantitative, non-destructive analysis of coccolith polysaccharide. The lowest detectable concentration was 0.02 mg I-‘. In batch cultures both calcifying and naked cells appeared to be a source of dissolved coccolith polysaccharide. In an actively growing batch culture of mainly naked cells (5 1.5 x 10’cells ml- ’) the concentration of dissolved coccolith poly-

pellet fraction of cells was very Pow. nary result indicates that in cells o coccolith polysaccharide is functi results of Borconcentrations. This agrees with saccharide inhibited CaCO, precipitation in vitro at relatively low concentrations of 3-8 mg I-‘. It is unlikely that in the naked culture the dissolved coccolith polysaccharide originated from the calcifying cells only for two reasons. The first involves the amount of coccolith polysaccharide produced per cell. For example, in the naked culture at day 6 the concentration of dissolved polysaccharide amounted to I.01 mg 1-l and the total cell number was 231 .O X 10’ cells ml- I. When it is assumed that 10% of the cells was caIcified (23.1 X IO4 cells ml-’ ) the amount of coccolith polysaccharide produced per calcified cell would be 4.4 pg cell - ’_ In the calcifying culture at day 6 the number of calcified CP!ISwas comparable (26.0 X IO4 cells ml- ‘), but the irmolmt of coccolith polysaccharide produced was only 0.56 pg cell - ’. The second reason is that naked cells are covered by a layer of coccolith

polysaccharide. This has been demonstrated by cytochemical staining (Van der Wal et al., 1983) and by fluorescence ELISA (results not shown). As coccolith polysaccharide is water soluble, it is likely that a part of this extracellular polysaccharide will dissolve. This is in agreement with the results of De Jong et al. (1979) who reported the excretion of coccolitb polysaccharide into the medium by naked cells. The results of this paper cannot be translated

directly to oceanic blooms as in the batch cultures several important conditions were not comparable with those in oceans. Nevertheless, the results allow to speculate about concentration ranges that may be found in natural blooms. As extracellular dissolved coccolith polysaccharide seems to be difficult to degrade, it may accumulate during a bloom of E. huxleyi. In natural blooms

OC ranges generally from I .O zw et al., 1993; ~e~kay et al., ccharide may be a very -pool in a bloom of E. sity of IO” cells ml ~-’ and a cellular organic carbon content of IO-20 pg C cell-’ (Van Bl particulate organic carbon 0.1-0.2 mg C 1-l. This i entioEed above is 0.4-4.0% of the small part of the primary

We wish to thank I. Janse (Croningen University, The Netherlands) for sugar composition analysis, E.W. de Vrind-de Jong and J.P.M. de Vrind (Leiden University) for useful suggestions and W. Zeven-

boom (North Sea-Directorate, Kijswijk, The Netherlands) for making available marine facilities for sampling of the North Sea. This study was funded by Dutch and European research programmcs (NOP and MAST II; MAST II Contract no. MAS2-~T~2-003~)~ This paper is EHUX contribution number 54 and NSG publication no. 950507.

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