Microb Ecol (1992) 23:53-74

MICROBIAL ECOLOGY © Springer-VerlagNew York Inc, 1992

Extracellular Fibril Production by Freshwater Algae and Cyanobaeteria Tatiana Strycek, 1 J u d y Acreman, 2 Alison Kerry, 2 G a r y G. Leppard, 3 Milan V. Nermut, 4 and D o n n J. K u s h n e r ~,2 Department of Microbiology, University of Toronto, Toronto; Ontario M5S 1A8, Canada 2 Institute for Environmental Studies, University of Toronto, Toronto, Ontario M5S 1A4, Canada; 3 Lakes Research Branch, National Water Research Institute, Burlington, Ontario L7R 4A6, Canada; and 4National Institute for Medical Research, Mill Hill, London N.W. 7, UK

Received."March 12, 1991; Revised."September 11, 1991

Abstract. In order to study the ability o f freshwater algae and cyanobacteria to form extracellular fibrils, a screening test using r u t h e n i u m red (RR) staining was carried out on 28 species. Five o f these were e x a m i n e d for growth and p r o d u c t i o n o f fibrillar material in culture media o f different phosphate (P0 contents. RR-staining and uronic acid determinations at various stages o f algal growth were c o m p l e m e n t e d by electron microscopy o f the cells and o f fibrillar material released into the m e d i u m . The lower Pi concentrations enhanced growth ofMicrasterias radiata, Eremosphaera sp., and Microcystis aeruginosa, and had little or no effect on growth o f a Xanthidium sp. and Scenedesmus quadricauda. Extracellular uronic acid production, which was higher in low Pi m e d i u m in M. radiata, M. aeruginosa, and Xanthidium sp., could reach levels o f 50 mg/liter or more. Algae with high proportions o f RR-positive cells (M. radiata, Eremosphaera sp., Xanthidium sp., and M. aeruginosa) p r o d u c e d high levels o f slime-like material and distinct fibrils that were often seen attached to the cell surface and only slowly released into the m e d i u m . N o such material was found in cultures (or supernatants) o f Sc. quadricauda, which also p r o d u c e d relatively low a m o u n t s o f polyuronic acids. Specific types o f filaments, often forming "fascicles" with rectangular arrays o f globular particles were observed by negative staining electron microscopy o f some algal cultures. RR-positive material was also observed in the cytoplasm and on the cell walls and surfaces o f M. radiata and M. aeruginosa.

Introduction M a n y aquatic organisms produce extracellular mucilage c o m p o s e d o f fibrils. Fibrils o f bacterial, algal, and plant origin make up a very substantial part, on

Offprint requeststo: D. J. Kushner.

54

T. Strycek et al.

an episodic basis, of the dissolved fraction of organic carbon (DOC) in Canadian lakes; concentrations as high as 33% of total DOC have been recorded [2]. These fibrils are likely to interact with some contaminants in the water column, so that, eventually, these contaminants may be bound to them and transported to sediments by fibril coagulation [7]. There is evidence that nutrient levels, especially levels of available phosphate, strongly influence fibril production by some algal species [9]. This suggests that there may be a tie-in between nutrition and control of pollutant dispersion. Earlier work on the attachment of microbes in both lakes [12] and marine waters [4] suggested that fibril formation might be very widespread indeed, but quantification of this phenomenon has usually been left for further investigation. Extensive comparisons between the ability of different species to produce fibrils have not been made. We have now investigated fibril production by a number of different algae and cyanobacteria in the University of Toronto Culture Collection, Toronto, Canada, which contains about 180 pure cultures of algae and cyanobacteria, 55 of them axenic. Mucilagenous fibril production was measured, in a correlative manner, by microscopic and chemical methods that indicate such structures, including binding of ruthenium red (RR), analyses of uronic acids, and by different methods of electron microscopy.

Materials and Methods

Organisms Unicellular algae and cyanobacteria, recently isolated from fresh waters in Ontario and cultivated in the University of Toronto Culture Collection (UTCC) in the Department of Botany of the University of Toronto, used for this study are listed in Table 1. Some algal cultures also contained very small proportions of unidentified bacteria.

Culture Media The medium used for most of the experiments reported here was Bold's Basic Medium (BBM) [15], as modified by J. Acreman. It had the following composition (in mg/liter): KH2PO4, 17.5; K2HPO4, 7.5; CaCI2'2H20, 2.5; NaNO3, 25; NaC1, 2.5; Na2EDTA, 10; KOH (combined with EDTA), 6.2; FeSO,.7H20, 4.98 (combined with 1 ~1 H2SO4); H3BO3, 2.86; MnC12.4H20, 1.81; Na2MoO4"2H20, 0.39; CuSO4.5H20, 0.08; Co(NO3)2"6H20, 0.05; ZnSO4.7H20, 0.22; NaSiO3. 9H20, 28.4; cyanocobalamin, 0.01; biotin, 0.001; thiamine HC1, 0.002; final pH 6.8. The phosphate (Pi) content of this medium was designated as 100%; other Pi concentrations are based on this value. In the first screening, the BG-11 medium of Rippka et al. [13] and the M 169 medium of Wehr and Brown [16] were also used, but neither gave as good growth as the modified BBM medium.

Conditions of Cultivation All species were grown at 18-20°C under four 15 W Sylvania Cool-White fluorescent lamps (ca. 50 uEinsteins m 2 sec-~) in an environmental chamber. The 13 species selected for further study

Extracellular Fibril Production

55

Table 1. Preliminary survey of algae and cyanobacteria in the University of Toronto Culture collection for ruthenium red staining UTCC no.

10 15 16 17 20 23 26 30

61 62 63 65 98 105

121 124 127 128

131 135 150

151 153 157 158

163 166 173

Taxaa/species

Scenedesmus acutus Selenastrum sp. Coelastrum sp. Navicula sp. Scenedesmus sp. Chlorellasp. Tabellaria sp. Cosmarium botrytis Diatoma vulgaris Diatoma elongatum Ankistrodesmus falcatus Tabellaria flocculosa var. flocculosa Aphanocapsa sp. Anabaena variabilis Chlamydornonas acidophila Microcystis aeruginosa Eremosphaera sp. Staurastrum johnsonii Micrasterias radiata Xanthidium sp. Chrysochromulina breviturrita Chrysochromulina breviturrita Scenedesmus denticulata Scenedesmus obliquus Scenedesrnus quadricauda Scenedesmus quadricauda Tabellaria sp. Coelastrum sp.

RR stain

Remarks

+

+

Variable to extreme; some very light, some dark

++ ++ + ++

Promising for experiments Many cells stained Stain only positive in clumps

+

Weak Weakly positive; some pink cells

+ + ++

+ ++ ++

? ? + + + + +

Stained pieces come off the cells; spikes stained pink around cells Very few cells, test indefinite Very few cells, test indefinite Weak, on walls or fine strands Clumped cells stained pink

Weak, more intense staining at colony edge

All of the organisms were members of the Chlorophyceae, except for Navicula, Tabellaria, and Diatoma (Chrysophyceae), Chrysochromulina (Haptophyceae) and Aphanocapsa, Anabaena, and Anacystis (Cyanophyceae or cyanobacteria) = No stain; + = cells stained; + + = cells and extracellular material stained; ? = too few cells to be certain of staining; with Xanthidium and Eremosphaera species, color fades with time -

after the first screening were first grown in quadruplicate in standing tubes, 18 m m diameter, shaken once every day. Six of these species were also grown on a reciprocal shaker at 100 oscillations/ min in quadruplicate, in 50 ml of culture in 125 ml Erlenmeyer flasks fitted with 18 m m tubes as side arms. The growth medium used for these cultures contained different amounts of phosphate, as indicated. Growth was measured as turbidity (O.D. at 450 nm).

R u t h e n i u m R e d S t a i n i n g o f Cell S u s p e n s i o n s Ruthenium red (RR) powder (Sigma Chemicals Co.) was dissolved at a concentration of 5 mg/mt by heating to 60°C, stored in the dark at 4°C, and filtered, when necessary, to remove any undissolved clumps [3, 10]. A drop of RR was added to a drop of culture on a glass slide, a coverslip placed

56

T. Strycek et al.

over the liquid, and cells observed in a light microscope (Carl Zeiss, Germany) using phase contrast at 625 x and, if required, 1,000 x magnification, at 15, 30, and 45 rain intervals. Location of the dye on and around the cells was determined using bright-field illumination, and the percentage of red-stained cells was calculated after observing a m i n i m u m of 10 fields.

Ruthenium Red Uptake RR (0.05 mg/ml) was incubated with cell suspensions for 1 hour at room temperature, with frequent agitation. The suspension was centrifuged at 1,300 × g for 15 rain, and absorbance ofsupernatants was measured at 535 nm. Experiments were carried out in duplicate. The adsorption was calculated as the difference between the control dye and that remaining following interaction with algae.

Assay of Uronic Acid-Containing Polysaccharides These were assayed by the method of Bitter and Muir [1]. In some samples, a green color appeared instead of the pink/red (adsorbance maximum 535 nm) of pure uronic acids. This green color, which may be due to peroxides [1], could usually be avoided by including 2% L-cysteine hydrochloride (Sigma Chemical Co.) in the material to be analyzed.

Electron Microscopy: Negative-Staining Techniques Carbon-coated, formvar-covered grids were ionized for 30 min in ultraviolet light before being used. A drop of supernatant or pellet mixed with 3 volumes of 0.1 M a m m o n i u m acetate was placed on the grids, and allowed to stand for 20 min. The grids were washed three times with 0.1 M a m m o n i u m acetate and stained with 1% uranyl acetate pH 4.4 for 20, 30, and 60 sec each.

Electron Microscopy: Rotary Shadowing Technique Drops of the suspension, either from the whole culture, cell pellet, or supernatant, were adsorbed to grids, as described for negative staining. After washing in a m m o n i u m acetate, grids were floated on 2% glutaraldehyde in 0.07 M cacodylate buffer, pH 7.3 for 2 min, washed twice with the same buffer, once with distilled water, then dried from 96% ethyl alcohol. Rotary shadowing was carried out in a Leybold P-100 vacuum coating unit equipped with an electron gun. Pure tungsten was evaporated from an 8* angle.

Electron Microscopy: Preparation of Whole Mounts for Scanning Electron Microscopy (1) A pellet of the culture, obtained by centrifugation, was fixed with 2% glutaraldehyde in 0.07 M cacodylate buffer, pH 7.4, washed, and then post-fixed in 1% OsO4 in 0.067 M phosphate buffer, pH 7.2, for 1 hour at room temperature. It was then washed with distilled water, placed on W h a t m a n fiber-glass filters, dried from alcohol, and sputter-coated with gold (6 nm). (2) Algae cultivated on cover glasses coated with 0.1% poly-L-lysine were fixed 24 hours in 2% glutaraldehyde and 0.1% R R in cacodylate buffer as above. Fixed samples were dehydrated in an ethyl alcohol series of 50, 70, 95, and 100% and kept in the last concentration until critical-point drying was carded out. After critical-point drying, samples were stored in a vacuum desiccator over silica gel and then sputter-coated with gold.

Extracellular Fibril Production

57

Electron Microscopy: Preparation of Samples for Ultrathin Sectioning A pellet of the culture washed three times with 0.07 M cacodylate buffer pH 7.3 was fixed with 2% glutaraldehyde and 0.1% ruthenium red in cacodylate buffer, pH 7.3 for 2 hours at ambient temperature (22°C). After fixation, the samples were washed twice with cacodylate buffer and once with distilled water, placed into 1% uranyl acetate for 4 hours in the dark, washed once with distilled water and embedded in water-miscible Nanoplast FB 101 resin [10 g MME 7002 melamine resin and 0.20 g B 52 acid catalyst (J.B. EM Services Inc., Dorval, Quebec)] [8]. The BEEM capsules, half-filled with Nanoplast-embedded cells, were dried in an oven for 2 days at 40°C and then 2 days at 60°C. After hardening, the capsules were filled with Spurr's resin [14] or Epon and allowed to polymerize for two days at 60-70°C.

Electron Microscopic Examination Samples were examined in a Philips EM 300 transmission electron microscope (TEM) and in a JEM 1200 EX electron microscope. Scanning electron microscopy was performed on both JEM 1200 EX (fitted with a scanningattachment) and JSM-35 CF scanningelectron microscopes(SEM).

Results

Preliminary Studies of RR Staining At first, 28 species were screened for their reaction to R R staining, using cultures grown for one to three m o n t h s in BM, BG-11, or M169 m e d i u m . Eighteen cultures gave positive tests and 6 p r o d u c e d large a m o u n t s o f extracellular polyanions, as measured b y R R staining (Table 1). Thirteen U T C C species (nos. 17, 26, 61, 62, 63, 65, 121, 124, 127, 131, 135, 157, and 158; Table 1) were then studied in m o r e detail. In the first experiments, cultures were grown in standing tubes, shaken once a day. G r o w t h was m e a s u r e d as optical density (O.D.) at 450 n m and fibril p r o d u c t i o n by R R staining. R R uptake was also measured. Results are summarized in Table 2. U n d e r these culture conditions, growth was limited, being greatest in Navicula sp., Scenedesmus obliquus, Scenedesmus quadricauda, and Ankistrodesmus sp. T w o o f the cultures (Navicula sp. and Scenedesmus obliquus) stained moderately with R R early in growth and no better late in the growth cycle. One species (Micrasterias radiata) stained very well at the beginning and at the end o f growth. This m a y have been due to staining o f old cells in the three-week-old inoculum. The greatest degree o f R R staining was observed in Eremosphaera sp., Micrasterias radiata, a n d Microcystis aeruginosa, and it usually developed with increasing culture age. There was no clear c o n n e c t i o n between growth or staining and R R uptake. Low degrees o f growth a n d / o r R R staining could be correlated with high a m o u n t s o f R R uptake. Cultures that stained well t o o k up smaller a m o u n t s o f the dye than cultures that stained poorly, except for Eremosphaera sp. Although R R

58 Table 2.

T. Strycek et al. Growth, ruthenium red (RR) staining, and RR uptake by static cultures

Property measured

Time (days)

1

2

Species~ 3

4

5

Growth (O.D.450.m)

2 32

0.009 0.030

0.027 0.070

0.010 0.050

0.011 0.024

0.004 0.010

59 4 35 60

0.070 + 0 +

0.100 + 0 0

0.082 0 0 +

0.040 0 0 0

0.020 0 ++ +++

RR stainingb

RR uptake (ug)

32 65

13.5 3.5

13.7 17.3

15.7 20.3

11.4 7.4

9.1 12.4

a Species: 1. Navicula; 2. Scenedesmus obliquus; 3. Scenedesmus quadricauda; 4.

Chlamydomonas; 5. Eremosphaera; 6. Ankistrodesrnus; 7. Micrasterias; 8. Tabellaria; 9. Diatoma elongatum; 10. Diatorna vulgaris; 11. Tabellaria flocculosa; 12. Microcystis aeruginosa; 13. Xanthidium. See text for UTCC numbers b RR staining indicated as in Table 1. ND = Not determined The O.D. readings are the mean values of 4 tubes up to 32 days. At 35 days, one tube was tested for RR uptake; the following O.D. values are the means of 3 tubes S.D. was _+ 5-10%

uptake has been used to measure mucilagenous fibrillar content of Zoogloea flocs in activated sludge [3], our results suggest that such uptake is by no means a good quantitative measurement of staining, and possibly of polyanion production in algal cultures. The difficulty of separating cells of some species from mucilage adds to the uncertainty and difficulty of interpreting uptake measurements. In subsequent experiments carried out with five of these cultures under more favorable growth conditions, polyuronic acid was measured directly. Experiments (not shown) were also started with Diatoma elongatum, but the cultures became badly contaminated with bacteria. Despite relatively low algal growth, Diatoma cultures did produce substantial amounts (up to 20 mg/liter) of polyuronic acid.

Biochemical and Microscopic Studies of Shaken Cultures Growth, staining with RR, and polyuronic acid production were studied during the growth o f shaken cultures with the normal amount of Pi (100% P0 and with 2% Pi- The percentage figures indicate the amount in the bulk media, but it must be emphasized that some Pi was transferred with the inoculum, usually 5 ml o f a culture on 100% Pi to 50 ml of the medium indicated. Higher levels of growth were found in shaken than in static cultures. Cultures o f Scenedesmus quadricauda grew best of the algae tested and reached an O.D. o f 0.2 in 20 days (Fig. 1). The level of Pi (100% or 2) had little effect on growth. Polyuronic acid, of which very little appeared in the supernatant, was higher in the earlier stages of growth and not affected by the level of Pi in the medium (Table 3). Because staining with R R occurred only between the cells of these

Extracellular Fibril Production Table

59

2. Continued Speciesa

6 0.024 0.100 0.120 0 0 0 8.0 16.6

7 0.010 0.013 0.020 +++ ++ +++ 9.7 8.3

8

9

10

11

0.003 0.012 0.017 ND 0 + 13.7 10.8

0.010 0.017 0.027 ND 0 + 14.8 12.1

0.010 0.016 0.030 ND 0 + 14.1 9.8

0.008 0.018 0.026 ND 0 ++ 10.3 8.3

12 0.017 0.100 ND ND ++ +++ ND ND

13 0.010 0.033 0.010 ND ++ +++ 5.7 1.6

"E 0.400 c 0

0.350

v~" 0.300 ;,I-

z

o. 2 5 0

o zoo 0.150

..J 0 . 1 0 0 o -

0.050

I.D.. O O.OOO

0

I0

20

50 DAYS

40

50

60

70

Fig. 1. Growth of Scenedesmus quadricauda ((3, 0) and Micrasterias radiata (A, A) at different Pi concentrations. Open symbols = 100% Pi medium. Closed symbols = 2% Pi medium. Figures are means of quadruplicate determinations; SD was 1--4% for Sc. quadricauda and 5-10% for M. radiata.

algae, i t w a s difficult t o q u a n t i f y t h i s p r o c e s s ; h o w e v e r , it d i d n o t s e e m t o b e affected b y Pi ( n o t s h o w n ) . T h i s c o r r e l a t e s w e l l w i t h t h e e l e c t r o n m i c r o s c o p i c i m a g e o f Sc. quadricauda s h o w i n g n o c o n s p i c u o u s f i b r i l l a r m a t e r i a l a s s o c i a t e d w i t h t h e cells (Fig. 2), b u t s o m e t r a n s p a r e n t l a y e r a t t h e cell e d g e s a n d s h o r t c y l i n d e r s ( a r r o w h e a d s ) o n t h e m o s a i c - l i k e cell s u r f a c e w e r e seen. Micrasterias radiata c u l t u r e s r e a c h e d a s t e a d y O . D . o f ca. 0.13 in 30 d a y s (Fig. 1). L o w e r Pi h a d n o effect o n g r o w t h b u t i n c r e a s e d t h e d e g r e e o f R R s t a i n i n g (Fig. 3) a n d s l i g h t l y i n c r e a s e d u r o n i c a c i d c o n t e n t o f w h o l e c u l t u r e s ( T a b l e 3). S c a n n i n g e l e c t r o n m i c r o g r a p h s s h o w e d a cell s u r f a c e c o v e r e d w i t h r a t h e r u n i f o r m s m a l l g l o b u l a r p r o t r u s i o n s w h i c h w e r e o b s c u r e d in p l a c e s b y a l a y e r of "slimy" material. Similar material was seen bridging the gap between spines (Fig. 4, a r r o w s ) . U l t r a t h i n s e c t i o n s o f Micrasterias radiata (Fig. 5), a n d l i g h t microscopy revealed RR-positive material in the cytoplasm; some RR-positive m a t e r i a l w a s c o n c e n t r a t e d m a i n l y in " p o c k e t s " b e t w e e n a n i n d e n t e d " p l a s m a m e m b r a n e " a n d t h e cell wall. C o r r e l a t i v e T E M s h o w e d t h a t e l e c t r o n - d e n s e t u b e s s e e m e d t o c o n n e c t t h e " p o c k e t s " w i t h t h e o u t e r s u r f a c e o f t h e cell w a l l w h e r e d i s t i n c t b u n d l e s o f s h o r t f i l a m e n t s s u r r o u n d e d b y a large, b r i g h t h a l o w e r e u s u a l l y o b s e r v e d . I t is t e m p t i n g t o s p e c u l a t e t h a t R R - s t a i n e d m a t e r i a l is

60

T. Strycek et al.

Fig, 2. Cell surface of Scenedesmus quadricauda. Two-week-old culture in 100% Pi BBM. Rotary shadowed with tungsten. Dark areas may be short cylinders. Arrowheads show some of them viewed from an angle. Note transparent layer at upper edge of cell. Bar = 1 ~zm.

Table 3. Effect of phosphate concentration on polyuronic acid content of cultures and culture supematants

Polyuronic acid (mg/liter) Whole culture Alga Sc. quadricauda

M. radiata

Eremosphaera sp. Xanthidium sp,

31. aeruginosa

Culture supematant

Time (days)

100% Pi

2% Pia

100% Pi

2% Pi~

21 28 50 21 28 50 26 54 33 43 54 9 29 51

42 21 19 15 26 38 41 57 10 27 23 0 0 0

37 13 16 35 33 51 22 17 3 18 26 5 19 16

0 2 1 3 5 11 2 3 4 26 4 3 0 6

0 3 4 1 1 14 4 4 4 7 7 3 22 13

Microcystis aeruginosa cultures in 0 P~, instead of 2% P~

transported to the cell surface by a trans-wall tube, which there releases distinct fibrils (Fig. 5 a - c ) . I n a l i g h t m i c r o s c o p e w e r e s e e n i r r e g u l a r p a t c h e s s t a i n e d w i t h R R , s o m e t i m e s c o n n e c t e d t o t h e cell. T h e d i a m e t e r o f t h e " b u n c h o f f i b r i l s " (ca. 7 0 0 n m ) o n t h e c e l l s u r f a c e , t o g e t h e r w i t h t h e b r i g h t h a l o w a s a b o u t 2 # m ,

Extracellular Fibril Production

61

120 a z o') ct~

._i

iO0-

8060

._I la_l ~u -

40-

o

N

ZO o • 0

i I0

20

i

i

70

40

DAYS

I

50

60

70

Fig. 3. R u t h e n i u m red staining o f Micrasterias radiata growing at different Pi concentrations. Cells from the cultures shown in Fig. 1 were examined. Hatched bars = 100% P~. Solid bars = 2% Pi.

Fig. 4. Scanning electron micrograph (SEM) ofMicrasterias radiata; 15 week culture in 100% Pi BBM showing spines with distinct globular (cylindrical) structures (about 2 ~tm across; arrow) and spines covered with " s l i m y " layers (arrowheads). × 1,300; bar = 10 ~m.

62

T. Strycek et al.

Fig. 5. a Thin section through a spine of Micrasterias racliata showing RR-positive tubular structures penetrating the cell wall (CW). Small RR-positive "pockets" are seen at the "roots" of tubules in the cytoplasm (arrows). Clusters of thin fibrils are on the outer cell wall surface (arrowheads) and white "cups" (about 2 t~m; small arrows) surround clusters of fibrils. No post-staining. × 8,500; bar = 1 ~m. Inset shows a RR positive tube and "pocket" at × 30,000. b Oblique section through RR-stained tube (in cell wall) and "'pocket." Post-stained with uranyl acetate and lead citrate. Bar = 0.5 ~m. c This picture shows a "pocket," an oblique section of a "tube" and a bunch of RR-positive fibrils on the cell wall outer surface (white space below the fibrils is artificial). No post-staining. Bar = 0.5/~m. d Slightly oblique section through a spine ofM. radiata reveals periodic structure associated with plasma membrane (arrows). Post-stained with uranyl acetate and lead citrate, x 50,000; bar = 0.1 urn.

Extracellular Fibril P r o d u c t i o n

63

Fig. 6. a S E M o f X a n t h i d i u m sp., cultured one week o n a cover glass in 100% Pi BBM, s h o w i n g a n apparently dividing cell with distinct spines (arrows) a n d a surface network. Bar = 10/zm. b S E M o f Xanthidium sp. f r o m the s a m e culture as in a, s h o w i n g a surface envelope "peeling off" the structural layer. Bar = 10 era. c Higher magnification o f the structural layer o f t h e Xanthidiurn sp. s h o w n in b. T h i n a n d thick filaments connect "globular projections." Fenestrated wall is seen below fibers. Bar = 1 /~m.

64

T. Strycek et al.

"= E0 . 4 0 0 0

0.350

t~ v

>I--

Z

0.500 O. 2 5 0

o zoo

~r~ 0 . 1 5 0 -J

O. I 0 0

0 -i--

0.050

(~

0.000 I0

20

30

40

50

60

70

DAYS

Fig. 7. Growth of Eremosphaerasp. at different Pi concentrations. Open circles = 100% Pi. Closed circles = 2% Pi. Determinations and SD as for Sc. quadricauda (Fig. 1).

120t~

ta l O 0 Z

I.o") O3

.J

BO60-

.J hi

o

40

LL

0

N

20

I0

20

30

40

DAYS

50

60

70

Fig. 8. R u t h e n i u m red staining o f Eremosphaera sp. growing at different Pj concentrations. Cells from the cultures shown in Fig. 7 were examined. Hatched bars = 100% Pi. Solid bars = 2% Pi.

which is close to the size o f the globular projections seen in scanning micrographs (Fig. 4). T h e plasma membrane-cell wall interface showed some periodic structure, possibly a protein layer or crystalline cellulose synthetic complex external to the m e m b r a n e (Fig. 5d). The cylindrical shape o f the tube in the cell wall was apparent in the longitudinal section o f the spine and is shown at higher magnification in Fig. 5a, inset. With the X a n t h i d i u m sp., P i levels had little effect on growth, which had reached an O.D. o f only 0.06 in 60 days, or on R R staining, which occurred throughout growth on all ceils at b o t h concentrations tested and showed a distinct halo a r o u n d each cell. In the m e d i u m with 100% Pi, m o r e uronic acid was produced than in the 2% Pi m e d i u m . In the f o r m e r m e d i u m , but not in the latter, almost all the uronic acid was extracellular at 43 hours growth (Table 3). The high level o f RR-stained cells is well illustrated by the scanning electron micrograph images o f this species. Figure 6a--c shows that the surface o f the cells is covered with a regular network o f thick fibers, 300--400 n m in diameter, which in some cells is obscured by a layer o f slimy material (the capsule, seen open in Fig. 6b). The release o f such a capsule could influence the level o f uronic acid in the supernatants. The network o f fibers was found mainly on

Extracellular Fibril Production

65

Fig. 9. a Erernosphaera sp. cultured in 100% P~ BBM for 1 week on a cover glass. SEM shows cells covered with both fibrillar and slimy material. Bar = 100 #m. b Detail ofEremosphaera sp. surface (preparation as in a) revealing high density of globular structures. Bar = 1 #m. d i v i d i n g cells (Fig. 6a, c), a n d c o n i c a l p r o j e c t i o n s were seen at the c r o s s - p o i n t s o f fibers (Fig. 6b), w h i c h is best seen in stereo m i c r o g r a p h s , n o t s h o w n here. Eremosphaera sp. grew b e t t e r at the low Pi c o n c e n t r a t i o n (Fig. 7), a n d m a n y m o r e o f the cells were s t a i n e d w i t h R R d u r i n g the g r o w t h cycle (Fig. 8). Curiously, h o w e v e r , l o w e r i n g the Pi c o n c e n t r a t i o n l o w e r e d the u r o n i c acid c o n t e n t o f the cultures (Table 3). R e l a t i v e l y little u r o n i c a c i d was f o u n d in the supern a t a n t s o f cells g r o w n at either Pi c o n c e n t r a t i o n (Table 3). S c a n n i n g electron m i c r o g r a p h s o f Eremosphaera sp. g r o w n o n coverslips

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o°.°°°1 "~ 0.050 1 v)- 0.040 F-

--

0.030 Z tl.I

0.020 _1 tO 0.010 13_ 0 0.000

i

,0

2'0

i

30 DAYS

.o

5'0

60

Fig. 10. Growth of Microcystis aeruginosa at different Pi concentrations. Open circles = 100% PiClosed circles = 0% Pi. Determinations and SD as for M. radiata (Fig. 1).

showed a large a m o u n t o f fibrillar material associated with m a n y cells (Fig. 9a). This could account for the low levels o f uronic acids in the medium. On the s m o o t h cell surface a dense population o f small uniform projections o f about 180 n m diameter, was discovered at higher magnifications (Fig. 9b). Experiments with Mierocystis aeruginosa were undertaken with the idea o f inducing m o r e severe Pi starvation. Precultures were grown in 0% Pi to reduce the m a x i m u m Pi concentration in the test culture to 1%. These cyanobacteria grew better in the absence o f added Pi, though growth at both Pi concentrations was marginal (Fig. 10). Cells in the higher P~ m e d i u m initially stained better with R R than those in lower Pi (Fig. 11). However, the cells in the " O Pi" m e d i u m m a d e m u c h m o r e uronic acid than the control cells; m u c h or all o f this uronic acid was extracellular (Table 3). The light microscopic image was different from that o f the previous species: no halo appeared a r o u n d the cells, but the cells were evenly stained. This is in agreement with R R staining o f ultrathin sections in correlative TEM-light microscope analYSiS , where some cells showed a thin but distinct stained layer on the surface, and others had RR-positive linear structures in the cytoplasm (Fig. 12a, b). The length and thickness o f the fibrillar structures varied. An overall view o f uronic acid production in these experiments showed that it could reach high levels, i.e., up to 50 mg/liter. In some cases, as in Micrasterias radiata, this material was mainly cell-associated, but in others, notably Microcystis aeruginosa and in some stages o f growth o f Xanthidium sp., it was extracellular.

Morphology of Fibrillar Material Released by the Organisms The presence o f fibrillar material released into the m e d i u m was studied by both negative staining and rotary shadowing o f the supernatants obtained by pelleting the cells at 1,300 × g for 15 min. These preparations showed that the morphology o f such material was by no means uniform. Well-defined filaments were observed in supernatants from Scenedesmus quadricauda, Xanthidium sp. and Eremosphaera sp., while different kinds o f slime-like material were seen in abundance in Micrasterias radiata, Microcystis aeruginosa, and to some extent in Eremosphaera sp.

Extracellular Fibril Production

c',, b.I

120

Z

I00

Ico

8O

o") ._1 _1 W o 0

67

6O 40' 2O 0 ' 10

' 20

-

' 30

DAYS

40'

5'0

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70

Fig. 11. Ruthenium red staining of Microcystis aeruginosa growing at different P~ concentrations. Cells from the cultures shown in Fig. 10 were examined. Hatched bars = 100% Pi- Solid bars = 0% Pi.

Fig. 12. a Cross section of Microcystis aeruginosa stained with RR, post-stained with uranyl acetate and lead citrate. Note distinctive staining of the cell wall (arrow). Bar = 0.5 gin. b Thin section ofM. aeruginosa stained with RR, post-stained with uranyl acetate and lead citrate. Note linear structures (arrowheads) and also round RR-positive "bodies" in cytoplasm. Bar = 0.5 gm.

Scenedesmus quadricauda. I n r o t a r y s h a d o w e d p r e p a r a t i o n s t w o t y p e s o f fibrils w e r e o b s e r v e d (Fig. 13a). O n e t y p e a p p e a r e d as s t r a i g h t , s e g m e n t e d fibrils t h a t a g g r e g a t e d i n t o fascicles. A n o t h e r t y p e a p p e a r e d as t h i n , c o n t i n u o u s filam e n t s , w h i c h w e r e w a v y , b e n t , o r b r a n c h e d (arrow). F a s c i c l e s w e r e o f t e n s e e n close to the spines, but not really attached to them. The same types of structures w e r e s e e n i n n e g a t i v e l y - s t a i n e d p r e p a r a t i o n s (Fig. 13b), b u t n e g a t i v e s t a i n i n g r e v e a l e d a r e c t a n g u l a r p a t t e r n m a i n l y i n t h e c e n t r a l p a r t o f t h e fascicles. M e a surements obtained from optical diffraction showed subunits 5-6 nm in dia m e t e r (Fig. 13b, inset). Micrasterias radiata. A d e n s e n e t w o r k o f b u n d l e d f i l a m e n t s , c o n s i s t e n t in g e n e r a l a s p e c t s w i t h m u l t i - m e t h o d v i s u a l i z a t i o n [9] o f algal a n d c y a n o b a c t e r i a l

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Fig. 13. a Scenedesmus quadricauda. Fascicle of fibrils in supernatant (2 weeks culture in 100% P~ BBM). Thin filaments (arrow) are also present here. Rotary shadowed with tungsten × 40,000. Bar = 1 um. h S. quadricauda. Fascicles as in a, negatively stained with uranyl acetate. Bar = 0.1 urn. Higher magnification ( x 150,000) (inset) reveals a rectangular periodic pattern. slime-like materials, was o b s e r v e d in r o t a r y s h a d o w e d s p e c i m e n s o f M . r a d i a t a (Fig. 14). H o w e v e r , distinct i n d i v i d u a l filaments were also p r e s e n t (arrow). X a n t h i d i u m sp. T w o types o f fibrils were o b s e r v e d in r o t a r y s h a d o w e d p r e p arations: long, well-defined fibrils a n d ill-defined networks, p o s s i b l y f o r m e d b y slimy m a t e r i a l (Fig. 15a). I n negatively stained p r e p a r a t i o n s m o s t filaments were 7 - 8 n m thick, t h o u g h a few filaments ca. 3 - 4 n m in d i a m e t e r were occasionally o b s e r v e d . T h e slime-like m a t e r i a l in the b a c k g r o u n d was n o t clearly visualized b y negative staining (Fig. 15b).

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Fig. 14. Micrasteriasradiata. Material in supernatant of 8 week culture in 100% Pi BBM. Single (arrow) or bundled fibrils and "slimy" materials are seen. Rotary shadowing with tungsten. Bar = 0.1 gm.

Eremosphaera sp. In rotary shadowed preparations, a slimy material, forming irregular Curved branching filamentous structures o f differing thickness, was seen when cells were cultured in 100% Pi BBM (Fig. 16a). Similar material was found in supernatants o f 2% Pi BBM (Fig. 16b), but negatively stained preparations also showed a dense network o f short, curved, and twisted fibrils about 7 n m in diameter; some o f the fibrils showed signs o f branching or fusion (Fig. 16c, arrow). Microcystis aeruginosa. In rotary shadowed preparations a network o f long, flexible fibrils fusing into thick fascicles was observed in supernatants o f these algae growing b o t h in 100% Pi BBM (not shown) and 0% P~ BBM (Fig. 17).

Discussion

Eventually, we hope that the i n f o r m a t i o n obtained in the present survey m a y p e r m i t us or others to harvest large a m o u n t s o f reproducibly obtainable and

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Fig. 15. a Supernatant from Xanthidium sp. (8 weeks culture in 100% Pi BBM) showing long twisted fibrils on a less-defined "slimy" material. Rotary shadowing with tungsten. Bar = 0.1 #m. b Supernatant from 3fanthidium sp. (3 weeks culture in 2% Pi BBM), Negative staining with uranyl acetate. Most fibrils are slightly curved, thickened, or twisted in places (arrows); only a few are straight and thin (arrowhead). Bar = 0.1 #m. h o m o g e n e o u s fibrils, a n d to s t u d y t h e r o l e o f s u c h m a t e r i a l in n a t u r a l w a t e r s . A s s t a t e d i n t h e I n t r o d u c t i o n , algal fibrils a l m o s t c e r t a i n l y m a k e u p a s u b s t a n t i a l part of the dissolved organic carbon (DOC) fraction in thesewaters; but which a l g a e a r e r e s p o n s i b l e a n d w h a t c o n t r o l s t h e i r fibril p r o d u c t i o n r e m a i n u n known. W e w e r e e s p e c i a l l y i n t e r e s t e d i n t h e effect o f n u t r i e n t d e p l e t i o n o n fibril p r o d u c t i o n . W i t h Micrasterias radiata, Xanthidium sp., a n d Microcystis aeruginosa, as w i t h o t h e r s [9], l o w e r i n g t h e p h o s p h a t e c o n t e n t in t h e b u l k m e d i u m

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Fig. 16. a Eremosphaera sp. Supernatant from 8 weeks culture in 100% Pi BBM, showing slimelike mucilagenous material. Rotary shadowed with tungsten. Bar = 0.1 /zm. b Eremosphaera sp. Supernatant from 3 weeks culture in 2% Pi BBM. Rotary shadowed with tungsten. Bar = 0.1 um.

c Material as in b. Negatively stained with uranyl acetate, showing "branching" fibrils (arrow). Bar = 0.1 #m.

did stimulate growth and fibril production, as measured by extracellular and cell-associated polyuronic acids. However, it is unlikely that the organisms were really " s t a r v e d " o f phosphate in most m e d i a tested, with the possible exception o f Microcystis aeruginosa in 0% Pi m e d i u m . M a n y algae can accumulate large a m o u n t s o f phosphate, as polyphosphate granules, when growing in phosphate-rich m e d i a [6], as were the media used here, c o m p a r e d with freshwater. M a n y m o r e generations o f growth than were practical here, in low phosphate or none at all, might have been needed to free cells o f such storage

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Fig. 17. Microcyst&aeruginosa. Supernatant from 3 weeks culture in 0% Pi BBM. A dense "meshwork" of bundles of fibrils is seen. Rotary shadowed with tungsten. Bar = 0.1 urn. products. In general, the physiological determinants of fibril formation in the species examined here certainly deserve much deeper study. We chose the most convenient method of measuring growth: by turbidity (O.D.) readings. This method seems suitable for a first survey, though we are well aware of its shortcomings. The size of the cells of different species and the prevalence of pigmentation (chlorophyll and carotenoid pigments), reflected, for example, in the yellow-brown color of the Xanthidiurn sp. in contrast to the green color of most other cultures, could have affected the O.D. of a given cell mass. The organisms differ in size, with Microcystis aeruginosa being 1532 ~tm in diameter, Scenedesmus quadricauda 35 tzm x 12 ~m, Micrasterias sp. having a diameter of 145/~m with spines and 109 ~m without spines, and Erernosphaera sp. a diameter of 170-200 gm. Such differences must be considered in estimating cell mass, but they probably do not greatly influence determination of the relative phases of algal growth and their relation to fibril production. We were able to show that the algae studied produce fibrils, and we were able to characterize many of these fibrils structurally in some detail. One of our major findings was that the relatively simple techniques of negative staining and rotary shadowing, suitable for rapid survey of a number of samples, gave a good deal of structural information. Electron microscopy showed a basic difference between the fibrillar material found in cultures of Scenedesmus quadricauda and that observed in the four other species, which all produced large amounts of slime-like material with varying proportions of well-defined fibrils (e.g., Xanthidium sp.). Whereas the

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fibrillar structures o f Scenedesmus and, in part, o f Xanthidium and Eremosphaera showed distinct morphological features, the images o f slimy material associated with cultures o f Micrasterias, Erernosphaera, Microcystis, and, in part, o f Xanthidium were obviously influenced by the preparative conditions, such as air-drying from ethanol, although 0.1 M a m m o n i u m acetate should have preserved samples from condensation effects due to dehydration. This survey o f fibril-producers has yielded the knowledge base for initiating morphological investigations o f hydrated fibrils in their native state. We hope to continue the research on the physical parameters o f minimally perturbed fibrils using (1) a correlative electron microscope approach which includes selective staining and freeze-etching, and (2) cultures grown in sufficient quantity to p e r m i t detailed analysis o f fibril c o m p o n e n t s by conventional wet chemistry. T h e fibrillar material is probably o f polyanionic nature since it stained well with RR. However, our studies do not allow any closer d e t e r m i n a t i o n o f the chemical nature o f the observed fibrillar structures, other than a correlation with uronic acid-rich molecules. Ultrathin sections o f RR-stained cells o f Micrasterias radiata showed the presence o f polyanionic material in the cytoplasm (in "granular" form) and on the cell surface (in fibrillar form). On the other hand, in Microcystis aeruginosa, RR-positive filaments were observed in the cytoplasm while the cell surface was stained diffusely. We were also able to show, by chemical analysis for uronic acids, that some o f the species p r o d u c e d substantial a m o u n t s (50 mg/liter or higher) o f extracellular and cell-associated polyuronic acid. Cultures probably contain additional polysaccharides [5]. Our ultrastructural studies showed not only a good deal o f variation between the fibrils produced by different algae, but also production o f m o r e than one kind o f fibril by individual algal species. Our findings point to the need for m u c h m o r e detailed correlation o f morphological, chemical, and physiological studies o f algal fibril formation. The morphological observations relate well to past observations o f Canadian freshwater algae in which natural populations were prepared for electron microscopy directly u p o n sampling at the lake site [11].

Acknowledgments. We wish to thank Professor F. Doane of the Department of Microbiology, University of Toronto, for professional advice regarding electron microscopy and for allowing us to use the electron microscope facilities, as well as Mr. S. Doyle, and Dr. A. G. Clark for providing help and facilities. Dr. A. Khan gave valuable help with some analyses. This work was supported by a contract from the Department of Supply and Services, Canada and by a grant from the Natural Sciences and Engineering Research Council of Canada to DJK.

References 1. Bitter T, Muir HM (1962) A modified uronic acid carbazole reaction. Anal Biochem 4:330-334 2. Burnison BK, Leppard GG (1983) Isolation of colloidal fibrils from lake water by physical separation techniques. Can J Fish Aquat Sci 40:373-381 3. Figueroa LA, Silverstein JA (1989) Ruthenium red adsorption method for measurement of extracellular polysaccharides in sludge flocs. Biotechnol Bioengineering 33:941-947

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4. Fletcher M, Floodgate GD (1973) An electron-microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J Gen Microbiol 74:325-334 5. Hellebust, JA (1974) Extracellular products. In: Stewart WDP (ed) Algal physiology and biochemistry. University of California Press, Berkeley and Los Angeles, pp 838-863 6. Kuhl A (1974) Phosphorus. In: Stewart WDP (ed) Algal physiology and biochemistry. University of California Press, Berkeley and Los Angeles, pp 636-654 7. Leppard GG (1984) Relationships between fibrils, colloids, chemical speciation, and the bioavailability of trace heavy metals in surface waters--A review. National Water Research Institute Contribution No. 84-45. Burlington, Ontario, Canada, pp 1-53 8. Leppard GG, Burnison BK, Buflte J (1990) Transmission electron microscopy of the natural organic matter of surface waters. Anal Claim Acta 232:107-121 9. Leppard GG, Massalski A, Lean DRS (1977) Electron-opaque microscopic fibrils in lakes: Their demonstration, their biological derivation and their potential significance in the redistribution of cations. Protoplasma 92:289-309 10. Luft JH (1971) Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy, and mechanism of action. Anat Rec 171:347-368 11. Massalski A, Leppard GG (1979) Morphological examination of fibrillar colloids associated with algae and bacteria in lakes. J Fish Res Board Can 36:922-938 12. Paerl HW (1973) Detritus in Lake Tahoe: Structural modification by attached microflora. Science 180:496-498 13. Rippka R, Deruelles J, Waterbury JB, Herdmann M, Stanier RY (1979) Generic arrangement, strain histories, and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1-61 14. Spurt AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31-43 15. Stein JR (1973) Handbook of phycological methods, culture methods, and growth measurements. Cambridge University Press, New York 16. Wehr JD, Brown LM (1985) Selenium requirement of a bloom-forming planktonic alga from soft water and acidified lakes. Can J Fish Aquat Sci 42:1783-1788.