Arch. Milcrobiol. 84, 234--242 (1972) �9 by Springer-Verlag 1972

Antimicrobial Effects of Cellvibrio on Blue-Green Algae

ULF GRANItALL and BJORN BERG

Department of Microbiology, Agricultural College, and Institute of Biochemistry, University of Uppsala, Uppsala, Sweden

Received January 11, 1972

Summary. The culture fluids from two Cellvibrio strains, in the stationary phase of growth, are shown to contain heat-resistant, low molecular weight substances with antibiotic-like effects on blue-green algae. Morphological changes and lysis of cells were observed in various species of blue-green algae; ultrastructural changes were noted in the cell wails of growing vegetative ceils of Anabaena inaegualis. The viability of resting cells, including heterocysts and akinetes was not affected.

Many blue-green algae are ecologically important microorganisms in view of their nitrogen-fixing ability. They can, however, constitute a severe nuisance when mass development occurs. Their mucilaginous sheaths provide an excellent carbon source for bacteria. Whereas syner- gism is usually exerted between the algae and the bacteria, the lat ter have also been reported to kill or lyse cells of various species of blue-green algae (Shilo, 1967, 1970; Stewart and Brown, 1969; Wu et al., 1968). I teterocysts and akinetes have been found to be less affected than vege- tat ive cells. The process of lysis has generally been found to be enzymatic. Blue-green algae are also known to be sensitive to antibiotics such as streptomycin and penicillin (Lamont, 1969 ; Srivastava, 1970).

In the present study, however, two Cellvibrio strains are reported to affect the growth of blue-green algae by producing low molecular sub- stances. Cellvibrio spp., which are common in polluted waters and in soils (Michoustine, 1968), have not been reported earlier to be antagonistic to microorganisms other than the fungus Stachybotrys atra and this anta- gonism was interpreted merely as competition for substrate (Henis et al., 1961).

Cellvibrio spp. occur in the polluted water of the river Fyris, running through Uppsala. The river is characterized by an annual waterbloom of blue-green algae, including Anabaena spp. Cellvibrio may contribute to the degradation of these algae.

Material and Methods Microorganisms and Cultivation Techniques. Cellvibrio /ulvus (NCIB 8634) and

a Cellvibrio strain isolated from the river/~yris (Berg et al., 1968) were investigated for possible inhibiting effects on the growth of the following microorganisms. ]31ue-

Antimicrobial Effects of Cellvibrio 235

green algae: Anabaena inaequalis, Chlorogloea/ritschii (pure cultures from the Algal Collection, Department of Botany, West field College, London), Spirulina platensis (Department of Biochemical Technology, Royal Insti tute of Technology, Stock- holm), Nodularia spumigena, Nostoc puncti/orme and 2~ T. muscorum isolated in axenic cultures fl'om Swedish soils (Granhall and Henriksson, 1969). Bacteria: Escherichia coli K-12, Serratia marcescens, Bacillus subtilis, B. megaterium (culture collection, Insti tute of Biochemistry) and Staphylococcus aureus (Wallenberg Laboratory, Uppsala). The cellulolytic Cellvibrio strains were cultivated at 28~ in shaken 5-1 Fernbach flasks, each provided with 2 1 mineral salts medium (Berg et al., 1972) of pH 7.3, and containing l~ Munktell 400 cellulose powder or glucose. Centrifuged culture fluids were generally drawn off after 7--8 days. The blue-green algae were cultivated at 24~ in stationary 250 ml Erlenmeyer flasks each containing 100 ml nitrogen-free salts medium (Henriksson, 1951) of p i t 7.3, under continuous illumi- nation from white fluorescent tubes. Spirulina, which does not fix nitrogen, was cultivated in a medium containing combined nitrogen (Zarrouk, 1966).

The bacteria used for sensitivity tests, were cultivated at 30~ in Nutrient Broth (Difco).

Examination o/Inhibiting E]/ects o/ Cellvibrio Filtrates on Blue-Green Algae and Bacteria. The supernatant liquids, centrifuged at 8000 g, from the 7--8 days old Cellvibrio cultures were ultrafiltered through a Diafio PM-10 membrane to remove enzymes and sterile filtered. The antagonistic effects of the ultrafiltrates were deter- mined by adding 2 ml of the test solutions to tubes with 2 ml suspensions of algae in the logarithmic growth phase. The tubes were incubated at 24 ~ C in a light chamber. After four days, samples were withdrawn and the algal cells were examined under the light microscope. Effects recorded were: trichome breakage, swelling, sphero- plast formation, impaired cell division and lysis.

Nitrogen fixation was measured by the acetylene reduction technique (Stewart et al., 1967).

Electron Microscopy. Anabaena inaequalis cells, before and after treatment were prefixed in 2.5~ glutaraldehyde in a veronal-acetate buffer, pH 7.2 (Palade, 1952), fixed and stained in buffered 2~ KMnO4-solution , mounted in agar, dehydrated in ethanol (increasing concentrations) and propylene oxide, and poststained in 2o/o uranyl acetate. Embedding was done in Epon (v. Hofsten and Holm, 1968), sec- tioning with a L.K.B. "ultrotome", and examinations in an Akashi TRS-50 or an AEI 80 electron microscope operated at 50 and 80 ku respectively.

Methods /or the Examination o] Some Properties o/Algicides Produced by Cell- vibrio. The substances were concentrated either by freeze-drying, rotary evapora- tion or by ultrafiltrations through a PSAC 1000 membrane (allowing substances under 1000 in molecular weights to pass). A rough estimation of molecular size was obtained by the use of these membranes and by gel filtrations on a Sephadex G-10 column. The pH stability was tested by incubating the filtrates (10 ml) at different pH's for 1 h at 24~ and titrating back to p i t 7.3 with dilute HC1 or NaOtt.

Thermal stability was tested by incubating 10 ml samples at various tempera- tures for 4 h up to the boiling point.

The presence of peptide bonds was tested for by incubations with the proteolytic enzymes papain (activated with cysteine and HC1), Arthrobacter protease and pepsin.

Resu l t and Discuss ion

T h e s ter i le sa l t s m e d i u m for g rowing Cellvibrio was f o u n d to be wi th -

o u t a d v e r s e effect on a lgal g r o w t h a n d d id n o t cause changes in t h e mor -

p h o l o g y of a lgae w h e n a d d e d to t h e cu l tu re of g rowing algae. U l t r a f i l t e r e d

236 U. Granhall and B. Berg:

Fig. 1. A. inaequalis treated with PM-10 filtered culture medium from C./ulvus. The figure shows cells of normal appearance (A) and the extensive increase in cell volume of other ceils (B). Heterocysts (H) are not affected. Metabolic changes are indicated

by the formation of great inclusions (I). Light microscopic picture

culture fluids from fully outgrown Cellvibrio cultures (pH 8.0--8.5), how- ever, caused trichome breakage of blue-green algae, increased cell volume of vegetative cells--resulting in the formation of osmotically sensitive spheroplasts and occurrence of lysis. All these effects were detected by light microscopy (Fig. 1). The rise in p i t of the medium during the culti- vation of Cellvibrio was not responsible for any of these effects.

To ascertain whether only growing algal cells are affected, dark incu- bated algal cells were treated with ultrafiltered fluids from Cellvibrio. No lysis or any other typical effects were noted.

Metabolic changes of growing cells, such as decreased nitrogen fixation were recorded in the presence of Cellvibrio culture filtrates. Cell division of all blue-green algae continued, but was found to be substantially im- paired in the presence of such filtrates. Lower concentrations of the active substances led to the appearance of rows of unseparated vegetative cells, suggesting tha t septum formation is especially affected. Treated cells of A. inaequalis clearly demonstrated the formation of enlarged septa and great inclusions acting as mechanical barriers for the growing septa (Fig.2). The inclusions were stained red with iodine, indicating the

Antimierobial Effects of Cellvibrio 237

Fig.2. A. inaequalis treated with culture filtrates from C./ulvus. The thylakoid- bound inclusions (I) act as mechanical barriers for the growing enlarged septa (S), and cause an impaired cell division. :Note the accumulation of material in the septa

(AEI 80i)

presence of glycogen or amylopeetin. Fully developed akinetes and heterocysts were not affected, but defects could be seen in the cell walls of proheteroeysts.

The effects of the Cellvibrio culture filtrates are very similar to the antibiotic action of penicillin on blue-green algae and bacteria (Lamont, 1969; Fitz-James and Hancock, 1965). The longitudinal walls of blue- green algae bulge outward in growing cells in ~ similar way when treated with diaflo-filtered Cellvibrio culture fluids or low concentrations of peni- cillin (10 IE/ml) and the inner edges of the septa become expanded into a fibrous mass (Fig. 2; ef. Lamont, Fig. 6). Spheroplasts are formed like the osmotically sensitive spheroplasts formed in the presence of peni- cillin.

238 U. Granhall and B. Berg:

Fig. 3. Treated cells, after transfer to fresh medium showing the formation of healthy cells (identical with untreated controls). Polyhedral bodies (P) are again formed. Note the absence of inclusions, and the sharp growing edges of the septa (S). A

heterocyst is also shown (H) (Akashi TRS-50)

As is the case with penicillin treated algal cells, cells treated with Cellvibrio culture filtrates resume growth and division on subculture in drug-free medium. In the case of A. inaequalis, growth, metabolism and ultrastrueture became identical with that of control cells within some days (Fig.3). Polyhedral bodies and cyanophycin granules were again formed (those granules were absent at growth in the presence of Cellvibrio filtrates), the glycogen-like inclusions vanished, and cell division and septa formation occurred undisturbed. The effect of the Cellvibrio filtrates was thus not persistent. A small number of resistant cells was present as shown by the formation of single colonies on agar plates containing sterile filtered Cellvibrio culture filtrates. In view of the rapid renewed growth other cells were probably not totally deprived of cell wall primers for reinitiating a new cell wall.

The sensitivity of other species of blue-green algae is shown in Table I. All algal species investigated were sensitive. The algicides thus have a broad spectrum. None of the bacteria investigated were affected.

Cellvibrio spp. form cellulases, which are capable of dissolving the outer cellulOse walls of heteroeysts (Granhal], unpublished), but these enzymes were totally excluded by the Diaflo PM-10 membrane.

Antimicrobial Effects of Cellvibrio

Table 1 Effects o/ultra/iltered culture medium ]rom C. fulvus on some

239

blue.green algae

Trichome Swelling Spheroplast Impaired Lysis breakage formation cell

division

Anabaena inaequalis + + + + + Chlorogloea /ritschii -~- + + -]- ~- Nodularia spumigena + + + + -~-

Nostoc puncti]orme + + + + ~- iYostoc muscorum -- ~- + -+- + Spiruliua platensis -~- -~ -- -~- --

Fig. 4. A. inaequalis t reated with culture filtrates from C. ]ulvus. The figure shows loss of the outer layers of the cell walls except a t local areas (CW), and par t ly loss of the total cell wall. The cell next to the top cell has completely collapsed. In this cell, the inner layer of the cell wall (LI) and the plasma membrane (PM) are broken a t different places (arrows). Most of the nuclear region (N) is dissolved (Akashi

TRS-50)

16 Arch. ]~ikrobiol., Bd. 84

240 U. Granhall and B. Berg:

Fig. 5. Treated cells. The loss of cell wall rigidity is demonstrated by a "loop" on the outer membrane (OM) at a weak point (AEI 801)

The action of the algicides seems to be restricted to an inhibition of the cell wall synthesis in blue-green algae. No direct a t t ack on the wall occurs, as growth is necessary for effect, and the viabil i ty of resting vegetat ive cells (dark incubated), akinetes or heterocysts is not affected. As several cell wall layers are part ial ly or total ly lacking (Figs. 4 and 5), the act ion of the Cellvibrio culture filtrates m a y be explained as an interference with murein formation, leading to loss of cell-wail rigidity, spherop]ast forma- t ion and osmotic lysis of cells. This hypothesis is fur ther suppor ted by the accumula t ion of material in the enlarged septa, possibly cell wall precur- sors.

The conditions for the format ion of algicides were invest igated by subsampling culture fluids at intervals during growth of Cellvibrio. The algicides did no t appear in the culture medium of Cellvibrio/ulvus until the s ta t ionary phase of growth was reached (7--8 days). Part ia l lysis of bacterial cells seems to be involved in the release of the algicides. The carbon source did not affect the format ion of active substances. No significant differences could be recorded in act ion spect rum by filtrates f rom the two Cellvibrio strains. The a]gieides passed th rough a Diaflo PM-10 membrane, bu t were retained by a PSAC 1000 membrane. The molecular weights are thus between 10000 and 1000.

Concentrates were also obtained by freeze-drying or rotary evaporation. Gel filtrations of fivefold concentrated culture filtrates on Sephadex G-10 (fraetionation range for peptides 0--700) gave two distinct peaks well behind the void volume material. The eluted components had an identical action (as already described) on Anabaena. The retardation could possibly be due to the presence of absorbing groups.

Antimicrobial Effects of CeIlvibrio 241

The algicides were not destroyed by boiling for 15 rain, and were stable within the pH-interval 2 to 13. Papain caused a two-third reduction in activity, whereas pepsin and Arthrobacter protease had no effect. The algicides might thus contain peptide bonds.

Now, tu rn ing to ecological considerations Myxobacter (Shilo, 1967, 1970; W u et al., 1968), Cytophaga (Stewart and Brown, 1969) and cyano- phages (Safferman and Morris, 1964 ; Granhall , 1972) are though t to contri- bute to the often observed sudden disappearance of blue-green algal blooms in aquat ic habitats . Perhaps Cellvibrio should also be t aken in to considerat ion as it occurs in polluted waters characterized by algal blooms.

The antibiotic-l ike substances formed by Cellvibrio may result in lysis of blue-green algal vegetat ive cells; the cellulases formed by Cellvibrio m a y dissolve heterocysts (Granhall, unpublished). Whether Cellvibrio could be used to control algal growth in ponds and lakes is no t known, bu t biological means for such control are certainly needed.

Aelcnowledgements. We should like to thank Dr. Bengt v. Hofsten, Ass. Prof. Hans Ljunggren and Mr. l~ajesh Kumar for much help and advice. The most skilful technical assistance of Miss Ingvor Andersson is gratefully acknowledged. Grants have been received from the Swedish Natural I~esearch Council and from the Novo Industri A/S, Copenhagen, Denmark.

References

Berg, B., v. Hofsten, B., Pettersson, G. : Some properties of cellnlolytic Cellvibrio strain from polluted water. Appl. Microbiol. 16, 1424 (1968).

-- -- -- Growth and cellulase formation in CeUvibrio /ulvus. J. appl. Bact. 85 (1972).

Fitz-James, P., Hancock, R. : The initial structural lesion of penicillin action in Bacillus megaterium. J. Cell Biol. 26, 657 (1965).

Granhall, U. : Aphanizomenon ]los aquae: infection by cyanophages. Physiol. Plant. (in press, 1972).

- - Henriksson, E. : Nitrogen-fixing blue-green algae in Swedish soils. Oikos 20, 175 (1969).

Henis, Y., Keller, P., Keynan, A. : Inhibition of fungal growth by bacteria during cellulose-decomposition. Canad. J. Microbiol. 7, 857 (1961).

Henriksson, E. : Nitrogen fixation by a bacteria-free symbiotic Nostoc strain isolated from Collema. Physiol. Plant. 4, 542 (1951).

v. Hofsten, A., Holm, L. : Studies on the fine structure of ascospores. L Grana Paly- nologica 8, 231 (1968).

Lamont, It. C. : Sacrificial cell death and tricbome breakage in Oscillatoriacean blue- green algae: the role of murein. Arch. l~ikrobiol. 69, 237 (1969).

Michoustine, E. : Microorganismes cellulolytiques des sols de I'U.R.S.S. Ann. Inst. Pasteur 115, 596 (1968).

Palade, G. E. : A study of fixation for electron microscopy. J. exp. Mad. 95, 285 (1952).

Safferman, R. S., Morris, M. E.: Control of algae with viruses. J. Amer. Water. Works Ass. 56, 1217 (1964).

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942 U. Granhall and B. Berg: Antimierobial Effects of Cellvibrio

Shilo, M. : Formation and mode of action of algal toxins. Bact. Rev. 31, 180 (1967). - - Lysis of blue-green algae by Myxobacter. J. Bact. 104, 453 (1970). Srivastava, B. S. �9 Sensitivity and resistance of a blue-green alga Phormidium muci-

cola to Streptomycin and penicillin. Arch. Mikrobiol. 72, 182 (1970). Stewart, J. R., Brown, R. M., Jr. : Cytophaga that kills or lyses algae. Science 27,

1523 (1969). Stewart, W. D. P., Fitzgerald, G. E., Burris, R. H. : In situ studies on 1~ 2 fixation

using the acetylene reduction technique. Proc. nat. Acad. Sci. (Wash.) 58, 2071 (1967).

Wu, B., Handy, M. K., Branch Howe, H., Jr. : Antimicrobial activity o fa myxobac- terium against blue-green algae. Bact. Proc. 91, 48 (1968).

Zarrouk, C. : Contribution ~ l'6tude d'une cyanophyc6e. Influence de divers facteurs physics e~ chemiques sur la croissance et la photosynth~se de Spirulina maxima (Sctch et Gardner) Geitler. Th~se, Paris 1966.

Dr. Ulf Granhall Department of Microbiology Agricultural College Uppsala, Sweden