lunula Schimper. Limnol. Oceanogr. 14:448-449. 13. Scott, W. E., and F. M. Chutter. 1981. Introduction: infections and predators. Univ. Orange Free State Publ.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1989, p. 2990-2994 0099-2240/89/112990-05$02.00/0 Copyright © 1989, American Society for Microbiology
Vol. 55, No. 11
Relationship between the Unicellular Red Alga Porphyridium and Its Predator, the Dinoflagellate Gymnodinium sp.
sp.
MICHAL UCKO,"2 EPHRAIM COHEN,3 HILLEL GORDIN,2 AND SHOSHANA (MALIS) ARAD3*
Department of Biology' and The Institutes for Applied Research, P.O. Box 1025,3 Ben-Gurion University Beer-Sheva, and Israel Oceanographic and Limnologic Research Ltd., Elat,2 Israel
of the Negev,
Received 17 April 1989/Accepted 1 August 1989
Contamination of algae cultivated outdoors by various microorganisms, such as bacteria, fungi, algae, and can affect growth and product quality, sometimes causing fast collapse of the cultures. The main contaminant of Porphyridium cultures grown outdoors in Israel is a Gymnodinium sp., a dinoflagellate that feeds on the alga. Comparison of the effects of various environmental conditions, i.e., pH, salinity, and temperature, on Gymnodinium and Porphyridium species revealed that the Gymnodinium sp. has sharp optimum curves, whereas the Porphyridium sp. has a wider range of optimum conditions and is also more resistant to extreme environmental variables. The mode of preying on the alga was observed, and the specificity of the Gymnodinium sp. for the Porphyridium sp. was shown. In addition, Gymnodinium extract was shown to contain enzymatic degrading activity specific to the Porphyridium sp. cell wall polysaccharide. protozoa,
nodiniumfungiforme was found to ingest 13 Dunaliella cells day (14). Higher ingestion rates were reported for the dinoflagellate Oxyrrhis marina, the rates being inversely dependent on the cell volume of the prey. Specifically, one dinoflagellate cell ingests about 350 algal cells -40 ,um3 in volume per day and about 50 cells -200 ,um3 in volume per day (7). An in-depth understanding of the relationship between the alga (the Porphyridium sp.) and its predator, the Gymnodinium sp., may help us find ways of controlling contamination of the outdoor cultures. (This work was done by M. Ucko in partial fulfillment of the requirements for the M.Sc. degree from Ben-Gurion University of the Negev, Beer-Sheva, Israel, 1989.)
Large-scale algal cultivation is usually performed outdoors. The cultures are thus exposed to contamination by bacteria, fungi, protozoa, and undesirable algae. The contaminant microorganisms can hinder growth and production of the desirable alga, impair product quality, and cause collapse of the culture. For example, it has been shown that protozoa can cause a decrease in the concentration of a population of colonial algae by more than 99% within 1 or 2 weeks (4). In a culture of the blue-green alga (cyanobacterium) Anacystis nidulans that was contaminated by the ciliate Colpoda ateinii, the predator doubled every 3 to 4 h, resulting in the collapse of the algal culture (13). Grobbelaar (8) described an event in which a culture of a Chlorella species was first contaminated by a Stylonichia species and 5 days later taken over by a Scenedesmus species. Thus, as a result of selective preying, the alga dominating the culture was replaced. One way to prevent contamination of outdoor algal ponds is to grow the algae under extreme environmental conditions. For example, a Dunaliella species, an alga currently being cultivated on a large scale, is grown in a highly saline medium, a condition favorable to the alga but prohibitive to most other microorganisms (3). Experiments with Phaeodactylum tricornutum growing in seawater have shown that various concentrations of NaCl in the medium have different effects on its predators, Actinomonas mirabilis, Paraphysomonas vestita, and Ciliophrys marina. Cultivation of the alga without NaCl or with 32 g liter of NaCI-1 inhibited the growth of the predators (L. W. Haas, unpublished results). A Porphyridium sp. is grown in outdoor ponds in southern Israel for the purpose of production of valuable biochemicals. One important product is a very viscous cell wall sulfated polysaccharide, the external part of which dissolves in the growth medium. This polysaccharide can be used as a substitute for carrageenans in various applications (2). The main contaminant in these ponds was found to be a Gymnodinium sp., a dinoflagellate that ingests the algal cells and can cause collapse of the cultures. Dinoflagellates are known to feed on bacteria and phytoplankton (10) as well as on other smaller dinoflagellates (12). The dinoflagellate Gym*
per
MATERIALS AND METHODS Organisms and growth conditions. Porphyridium sp. strain UTEX 637 was grown in the artificial seawater (ASW) of Jones et al. (9), as previously described (1). The Gymnodinium sp., isolated from outdoor ponds of the Porphyridium sp. with a micropipette, was grown in ASW containing Porphyridium cells. The Gymnodinium sp. was cultured at 20 ± 1C in 250-ml Erlenmeyer flasks containing 50 ml of medium placed on a shaker (70 rpm) under continuous illumination supplied from above at an intensity of 32 microeinsteins m-2 S-1. In the search for a suitable medium for the Gymnodinium sp., the following media were tried: ASW (9), Sweeney medium (15), the medium of Tuttle and Loeblich (16), and ASP 8A (M. D. Ahles, Ph.D. thesis, Fordham University, New York, N.Y., 1967). A manure extract and a soil extract were also added at a concentration of 4% to ASW or ASP 8A. For the manure extract, 0.5 liter of cow manure was incubated in 5 liters of deionized water for 24 h, filtered, and autoclaved. For the soil extract, 0.5 liter of soil was incubated in 1.5 liters of deionized water for 24 h, filtered, and autoclaved. In addition, ASW enriched with one of the following supplements was examined: the Porphyridium sp. polysaccharide (1 mg ml-1), glucose, galactose, sodium citrate (10 mg liter-1 each), yeast extract, commercial yeast extract (Difco), living bakers' yeast (Saccharomyces cerevisiae) extract, broken Porphyridium cells (broken in a Sonifier Cell Disruptor B-30, output control 6, 60% duty cycle,
Corresponding author. 2990
VOL. 55, 1989
two sessions of 20-s min-1 pulses at 60-s intervals; Branson Sonic Power Co., Danbury, Conn.), lyophilized Porphyridium cells, glutaraldehyde-fixed Porphyridium cells (2% for 1.5 h), and living or boiled Porphyridium cells. Growth. Growth was followed by periodic monitoring of cell numbers with a light microscope by means of a hemacytometer. The movement of Gymnodinium cells was arrested with 4% Formalin. Preparation of algal cell wall polysaccharides and measurement of their viscosity. Cell wall polysaccharides of the Porphyridium sp., Porphyridium aerugineum, and Rhodella reticulata were prepared by separating the cells from the growth medium containing the soluble fraction of the cell wall polysaccharide and dialyzing the supematant against doubly distilled water (until the conductivity of the water reached 25 to 30 mS). The supernatant was then frozen and lyophilized. The powder produced was dissolved (0.2%) in phosphate buffer (0.1 M, pH 6.7), and sodium azide (0. 125%) was added to the solution. The viscosity of the polysaccharide was measured with a Brookfield digital viscometer. Preparation of Gymnodinium extract and test of its polysaccharide-degrading activity. Five liters of Gymnodinium culture was centrifuged for 20 min at 27,500 x g. The resulting pellet was suspended in phosphate buffer (0.1 M, pH 6.7). The cells were broken in a sonicator, as described above for Porphyridium cells, and the suspension was designated the crude extract. The supernatant was prepared by centrifuging the crude extract (20 min at 47,000 x g). The crude extract or the supernatant (1.6 ml) was added to the polysaccharide solution (6.4 ml), and the mixture was incubated at 30°C in a shaker bath. Porphyridium extract was prepared from stationary-phase cells that were broken in a sonicator and centrifuged (47,000 x g for 20 min). The supernatant was used as the enzyme preparation. RESULTS Growth medium. The difficulty of growing heterotrophic dinoflagellates in a synthetic medium lies in the fact that they usually grow on algae, bacteria, or fungi. We attempted to find a suitable synthetic medium, since we deemed it important to grow the Gymnodinium sp. and the Porphyridium sp. independently. Various synthetic media were examined (detailed in Materials and Methods), none of which supported growth of Gymnodinium cells, not even those enriched with yeast or with broken or dried algae. Gymnodinium cells grew only in ASW (or seawater) to which living Porphyridium cells had been added. Growth curve of the Gymnodinium sp. The growth curve of the Gymnodinium sp. inoculated into a Porphyridium culture at the logarithmic phase of growth is shown in Fig. 1. A typical growth curve consisting of a logarithmic phase until day 2, a peak at days 3 and 4, and a decline thereafter was observed. Porphyridium cell concentration started decreasing at the end of the Gymnodinium logarithmic phase. However, the main decline paralleled that of the Gymnodinium sp. The experiment was performed in the light and in the dark without significant differences. The decline of Gymnodinium culture is apparently not caused by the removal of Porphyridium cells due to preying, since the decline was not prevented by the addition of Porphyridium cells to Gymnodinium culture after the growth peak (not shown). The Gymnodinium growth curve was not affected by the phase of growth of Porphyridium cells present in the culture.
2991
PORPHYRIDIUM-GYMNODINIUM RELATIONSHIP
00
0
1
2
3
TIME
4
5
6
[days]
FIG. 1. Cell numbers of the Gymnodinium sp. (L, *) and the Porphyridium sp. (A, A) cultured together under light (open symbols) and dark (closed symbols). Results are averages of three replicates from two different experiments.
Effect of environmental conditions. The effects of environmental conditions (salinity, temperature, and pH) on the growth of the Gymnodinium sp. and the Porphyridium sp. (grown separately) are shown in Fig. 2. Growth kinetics was determined under all conditions, but the values in the figure represent only the state at the stationary phase, at days 7 and 8 for the Porphyridium sp. and at days 3 and 4 for the Gymnodinium sp. Salinity. ASW was prepared with increasing concentrations of NaCl as follows: 0.23, 0.46, 0.92, 1.38, and 1.84 M (seawater and ASW contain 0.46 M NaCl). For Gymnodinium cells (Fig. 2A), optimum growth was obtained at 0.46 M NaCl. At a concentration of 0.23 M, growth was slightly inhibited (P > 0.05); without NaCl or at 0.92 M NaCl, growth was inhibited significantly (P < 0.05). At concentrations of 1.38 and 1.84 M NaCl, i.e., three or four times the concentration in seawater, the cells died immediately, as judged by the arrest of cell movement followed by their disintegration. For Porphyridium cells (Fig. 2A), there were no significant differences in cell densities at salt concentrations ranging between 0 and 0.92 M NaCl (P > 0.05). Inhibition of growth was observed at 1.38 M NaCl. At 1.84 M, the algal cell density did not change (P < 0.05). Temperature. The experiments were performed in aerated Erlenmeyer flasks at 10, 15, 20, 25, 30, and 35 + MC. Optimum growth of Gymnodinium cells (Fig. 2B) was observed at 20°C; the growth decreased somewhat at 25°C (P > 0.05). At 15 and 10°C, there was a lag after inoculation that was followed by slow growth, but the maximum number of cells obtained was similar to that obtained at 20°C, albeit at a later time (on day 8). Both lag period and time to maximum cell number were inversely proportional to the temperature (data not shown). At 30°C growth was severely inhibited (P < 0.05), and at 35°C the cells died. The highest number of Porphyridium cells (Fig. 2B) was observed at 20 to 30°C. At 15 and 35°C the cell number was lower (P < 0.05), and at 10°C no growth was observed. pH. Growth was measured in citrate buffer (0.1 M) at pH 3.5 and 5.0 and in Tris buffer (0.1 M) at pH 6.0, 7.5, and 8.5.
APPL. ENVIRON. MICROBIOL.
UCKO ET AL.
2992
19
,
0
3.0
I00
w-
-.r
A 30
0P-
a: w m
Z -i -i
cv,
E
A
10
w
I
C)
-J
2
-1-1 w
0
z
E E
5 z
I
C)
0 C'
00 00
0
1
2
3
4
5
6
7
8
9 10 11 12 13
TIME [days]
Z) _
D _
FIG. 3. Change in the volume of Gymnodinium cells and number of Porphyridium cells they contain during growth. Symbols: O, cell number; _, cell volume. The numbers above the bars represent the average numbers of Porphyridium cells inside the Gymnodinium cells. Cell volume was calculated from the cell diameter measured microscopically with an ocular micrometer.
I I
1
O >Z x
OL
TEMPERATURE
4 lUU
50
rcj
C
- -
--
___
20A-
10 1
-
-~~~~~~~~1
0.5 . ,
0.2
0.1
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
pH
FIG. 2. Cell numbers of the Gymnodinium sp. (O) and the Porphyridium sp. (A) at the stationary phase as a function of NaCl concentration (A), temperature (B), and pH (C). The results are averages of five replicates from three different experiments. Cell numbers upon inoculation were 1 x 104 to 2 x 104 ml-' and ca. 1 x 106 ml-' for the Gymnodinium sp. and the Porphyridium sp., respectively.
The pH was adjusted daily. The highest number of Gymnodinium cells (Fig. 2C) was obtained at pH 7.5. At pH 6.0, there was a lag of 4 days followed by a slow increase in cell number (data not shown). At pH 5.0 and 8.5, no growth was observed (P < 0.05). At pH 3.5, the cells died immediately (data not shown). For Porphyridium cells (Fig. 2C), the optimum pH ranged between 6.0 and 8.5 (P < 0.05). Significant inhibition (P < 0.05) was evident at pH 5.0. At pH 3.5, the cells did not grow.
Gymnodinium cell volume and number of Porphyridium cells ingested. The number of alga-containing Gymnodinium cells increased with the time of contact between the two organisms up to day 1 or 2 of growth, reaching ca. 99% of the cell population, and decreased thereafter (data not shown). The volume of Gymnodinium cells and the number of algal cells consumed during growth seem to be correlated (Fig. 3). The average diameter of Gymnodinium cells on day 0 was 7.87 ,um. On the second day of contact with Porphyridium cells, the average diameter doubled, and the volume was 10 times larger. On day 13, the average cell diameter returned to its
original size, i.e., 7.8 ,uam. The change in size correlated with the number of algal cells consumed, i.e., an average of 0.21 and 6.89 algal cells on days 0 and 2, respectively. The correlation between cell size and number of algal cells ingested is demonstrated in Table 1. The largest Gymnodinium cell, which had a diameter of 24 ,um, contained 15 Porphyridium cells. Specificity. The specificity of the Gymnodinium sp. for the Porphyridium sp. was studied by feeding Gymnodinium cells with various unicellular algae. The Gymnodinium sp. did not consume a Nanochloropsis sp., a marine chrysophyte. It did randomly (up to 50% of the Gymnodinium population) ingest the freshwater red alga P. aerugineum, the brackish-water red alga R. reticulata, the marine chrysophyte Isochrysis sp., and the green algae Chlorella and Dunaliella spp., but only one to two cells of these algae (versus up to 15 Porphyridium cells) were found in Gymnodinium cells. However, none of the algae other than the Porphyridium sp. seemed to be digested, as no further growth, either in cell volume or cell number, occurred in Gymnodinium cultures fed on them. TABLE 1. Relationship between the size of a Gymnodinium cell and the number of algal cells ingesteda Avg
No. of
Gyllmodineituerm Gymnodinium ces (pLm) 4 8 12 16
20 24
70 375 318 71 91 2
% of Gymnodinium cells containing the
following no. of algal cells: 0-1 2-3 4-5 6-7 8-9 10-11 12-13 14-15
100 98 2 78 14 5 2.5 0.5 20 15 20 17 17 11 3 4 11 10 26 20 50
16
10 50
a Results are taken from the growth curve presented in Fig. 3. Measurements are results of two counts of nine squares each that were measured with the ocular scale of the microscope. The number of algal cells in a Gymnodinium cell was counted with an epifluorescence microscope. The results are the averages of all values determined during the 13 days of the experiment.
VOL. 55, 1989
PORPHYRIDIUM-GYMNODINIUM RELATIONSHIP
2993
TABLE 2. Effect of Gymnodinium cells and extract on the viscosity of polysaccharides of various unicellular red algaea Source of digesting
Source of
activity
polysaccharide
Gymnodinium whole cells Gymnodinium crude extract Gymnodinium supernatant Gymnodinium boiled supernatant Gymnodinium supernatant Gymnodinium supernatant Porphyridium sp. supematant
Porphyridium sp. Porphyridium sp. Porphyridium sp. Porphyridium sp. P. aerugineum R. reticulata Porphyridium sp.
Polysaccharide viscosity (cP) At
zero
21.7 36.1 29.4 27.1 39.5 46.5 20.6
time'
+ 0.6 ± 0.5 ± 0.5 ± 0.4 ± 2.2 ± 0.1 ± 0.6
After 21 h
22.0 14.2 6.4 22.3 37.5 48.6 20.4
± ± ± ± ± ± ±
0.2 0.6 1.7 0.3 0.9 0.5 0.4
a Results are averages of triplicates in each of two different experiments. b Differences in zero-time viscosities of the Porphyridium sp. polysaccharide are due to the different extracts added.
Degradation of polysaccharide. We assumed that Gymnodinium cells utilize the polysaccharide of Porphyridium cells by means of enzymatic cleavage. To verify this assumption, we added a crude extract of broken Gymnodinium cells and its supernatant to purified polysaccharide of the Porphyridium sp. (Table 2). It can be seen that the viscosity of the polysaccharide was significantly reduced by the crude extract and to an even greater extent by the supernatant, indicating that the molecule had indeed been split. After the crude extract was boiled, almost no change in viscosity was observed, probably because of denaturation of the digesting enzymes. Whole Gymnodinium cells did not exhibit any enzymatic activity towards the polysaccharide, showing that the activity is located inside the cells. The supernatant had no effect on the polysaccharides of R. reticulata or of P. aerugineum, which is in agreement with the inability of the dinoflagellate to digest these two algae. When extract supernatant of the Porphyridium sp. cells was tested, no change in the viscosity of the isolated polysaccharide was observed. DISCUSSION Until now, we have been unsuccessful in finding synthetic media to support the growth of the Gymnodinium sp., as has been achieved for other dinoflagellates, such as 0. marina (5), Noctiluca scintillans (11), Amphidinium holleri (6), and Crypthecodinium cohnii (16). The failure of synthetic media to support growth of the Gymnodinium sp., even when enriched with broken or dried cells of the Porphyridium sp. or with its polysaccharide (which was found to be cleaved by Gymnodinium extract), might indicate that the cells require live algal cells, rather than a specific constituent of the cells, for growth. The Gymnodinium sp. used in our study may be defined as a phagotroph, as is another species of Gymnodinium, G. fungiforme (14), since it was found to contain intact Gymnodinium cells. However, we have not been able to observe the mode of preying. The comparison of the effects of various environmental conditions on the growth of Porphyridium and Gymnodinium species showed that the Porphyridium sp. is the more resistant of the two organisms; it can tolerate and grow under a wider range and more extreme conditions of pH, temperature, and salinity. It is possible that the polysaccharidic envelope surrounding the algal cells in the form of gel serves as a barrier between the cell and its environment, enabling the cells to survive under extreme environmental conditions. This tolerance can be used to confer an advantage on the alga over its predator in an open outdoor system. Control of contamination is one of the most important objectives in large-scale algal production in open ponds. Grobbelaar (8) summarized several approaches to the con-
trol of exposed cultures: (i) optimization of growth conditions; (ii) application of chemicals, such as herbicides, which act only against the predator; and (iii) exploiting physical parameters that affect the contaminant selectively. We have started applying the second concept and have already produced a Porphyridium mutant, SMR1, resistant to a herbicide of the sulfometuron methyl group (17). The third approach could also be applied on the basis of the greater tolerance of the Porphyridium sp. over that of its predator to extreme environmental conditions, e.g., by treating the culture with pH shocks, high salinity, or high temperature that will affect only the Gymnodinium sp. A most interesting finding of this study is the specificity of the Gymnodinium sp. for its prey, which was expressed both by its limited preying on algae other than the Porphyridium sp. and by its inability to digest these algae. Although it ingested red unicells other than the Porphyridium sp. that are also encapsulated with sulfated polysaccharides, e.g., P. aerugineum and R. reticulata, as well as two species of green algae and one species of chrysophyte, it could not grow on them, nor could its extract digest the cell wall polysaccharide of other red microalgae. The chemical composition and structure of the polysaccharides of the red unicells Porphyridium sp., P. aerugineum, and R. reticulata are under detailed study in our laboratory (2). These organisms are similar in several characteristics: all show similar general rheological behavior, i.e., they are resistant to a wide range of pH, temperatures, and salinities; all are negatively charged, although to a different degree, because of glucuronic acid and sulfate esters in the polysaccharide; and the main sugars of each are xylose, glucose, and galactose. However, the exact chemical compositions and the ratios between the various sugars and their minor sugar components differ from one species to another. Thus, it is especially interesting to find out which characteristic(s) of the polysaccharide controls its digestibility by Gymnodinium extract. The importance of studying the enzymatic activity of the Gymnodinium sp. that cleaves the Porphyridium sp. polysaccharide resides in its potential application for determining the composition and structure of the polysaccharide and for obtaining algal protoplasts, which may be used in genetic engineering studies. This study is now under way in our laboratory. ACKNOWLEDGMENTS We thank B. Kimor for identifying the dinoflagellate isolated outdoors as a Gymnodinium sp. and Inez Mureinik and Dorot Imber for editing the manuscript.
2994
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LITERATURE CITED 1. Adda, M., J. G. Merchuk, and S. (Malis) Arad. 1986. Effect of nitrate on growth and production of cell wall polysaccharide by the unicellular red alga Porphyridium. Biomass 10:131-140. 2. Arad, S. (Malis). 1988. Production of sulfated polysaccharides from unicellular red algae, p. 65-87. In T. Stadler, J. Mollion, M.-C. Verdas, Y. Karamanos, H. Morvan, and D. Christiaen (ed.), Algal biotechnology. Elsevier Science Publishing, Inc., New York. 3. Ben-Amotz, A. 1988. The production of p-carotene by the alga Dunaliella: a new venture in biotechnology, p. 4-7. In J. Ramus and M. C. Jones (ed.), Polysaccharides from microalgae: a new agroindustry. Duke University Marine Laboratory, Beaufort, N.C. 4. Canter, H. M., and J. W. G. Lund. 1968. The importance of protozoa in controlling the abundance of planktonic algae in lakes. Proc. Linn. Soc. London 179:203-219. 5. Droop, M. R. 1970. Nutritional investigation of phagotrophic protozoa under axenic conditions. Helgolander Wiss. Meeresunters. 20:272-277. 6. Elbrachter, M. 1972. Begrengte Heterotrophie bei Amphidinium (Dinoflagellata). Kiel. Meeresforsch. 28:84-91. 7. Goldman, J. C., M. R. Dennett, and H. Gordin. 1989. Dynamics of herbivorous grazing by the heterotrophic dinoflagellate Oxyrrhis marina. J. Plankton Res. 11:391-407. 8. Grobbelaar, J. U. 1981. Infections: experiences in miniponds.
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Univ. Orange Free State Publ. Ser. C 3:116-123. 9. Jones, R. F., H. L. Speer, and W. Kury. 1963. Studies on the growth of the red alga Porphyridium cruentum. Physiol. Plant. 16:636-643. 10. Lessard, E. J., and E. Swift. 1985. Species-specific grazing rate of heterotrophic dinoflagellates in oceanic waters, measured with a dual-label radioisotope technique. Mar. Biol. (Berlin) 87:289-296. 11. McGinn, M. P., and K. Gold. 1969. Axenic cultivation of Noctiluca scintillans. J. Protozool. 16(Suppl.):13. 12. Norris, D. R. 1969. Possible phagotrophic feeding in Ceratium lunula Schimper. Limnol. Oceanogr. 14:448-449. 13. Scott, W. E., and F. M. Chutter. 1981. Introduction: infections and predators. Univ. Orange Free State Publ. Ser. C 3:103-109. 14. Spero, H. J., and M. D. Moree. 1981. Phagotrophic feeding and its importance to the life cycle of the holozoic dinoflagellate, Gymnodinium fungiforme. J. Phycol. 17:43-51. 15. Sweeney, B. M. 1951. Culture of the dinoflagellate Gymnodinium with soil extract. Am. J. Bot. 38:669-677. 16. Tuttle, R. C., and A. R. Loeblich. 1975. An optimal growth medium for the dinoflagellate Crypthecodinium cohnii. Phycologia 14:1-8. 17. van Moppes, D., Z. Barak, D. M. Chipman, N. Gollop, and S. (Malis) Arad. 1989. An herbicide (sulfometuron methyl) resistant mutant in Porphyridium (Rhodophyta). J. Phycol. 25: 108-112.
