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Journal of Applied Phycology 9: 55–63, 1997. c 1997 Kluwer Academic Publishers. Printed in Belgium.
Factors influencing growth and toxin production by cultures of the freshwater cyanobacterium Lyngbya wollei Farlow ex Gomont Qiqin Yin, Wayne W. Carmichael & William R. Evans Department of Biological Sciences, Wright State University, dayton, Ohio 45435, USA ( Author for correspondence; fax: +1-937-775-3320; e-mail:
[email protected]) Received 11 February 1997; revised 22 March 1997; accepted 24 March 1997
Key words: Lyngbya wollei, paralytic shellfish poisons, growth, toxin production
Abstract Collections of Lyngbya wollei were taken from Guntersville Reservoir, Alabama, over a period of three years. Healthy filaments were isolated and transferred to agar plates of Z-8 and LM6E media. Unialgal isolates were cultured for the study of growth and paralytic shellfish poison (PSP) production. Filaments were extracted and the toxins were detected using high performance liquid chromatography (HPLC) with post column oxidation followed by fluorescence detection. HPLC profiles show that laboratory cultures of L. wollei produced decarbamoyl gonyautoxin 2 and 3, plus several other PSP like toxins whose structures are under investigation. At 26 C and a light intensity of 11 or 22 mol m 2 s 1 optimum production of both biomass and toxins occurred. A decrease or increase in temperature or light flux caused a reduction in dry weight or toxicity. Compared to control levels, lower PO4 -P and NO3 -N and higher calcium levels gave rise to higher biomass and toxicity. Lower calcium, calcium- or PO4 -P deficient medium and high NO3 -N or PO4 -P caused a large decrease in dry weight and toxicity. Introduction The filamentous mat-forming cyanobacterium, Lyngbya wollei Farlow ex Gomont forms massive infestations in certain southeastern USA lakes and reservoirs. These mats restrict the recreational, agricultural and economic uses of the affected aquatic system (Speziale et al., 1991). Recent infestations of L. wollei in Guntersville Reservoir on the Tennessee River prompted this study on the possible production of biotoxins and (or) cytotoxins by L. wollei. The study was supported by a cooperative agreement between the Tennessee Valley Authority and Wright State University. Carmichael & Evans (1996) reported that L. wollei from Guntersville Reservoir, Alabama produced a potent neurotoxin against mice, Ceriodaphnia dubia and Pimephales promelas. Preliminary characterization indicated that the toxins were potent sodium channel blockers (Carmichael et al., accepted pending revision) which, after intraperitoneal injection into mice, caused symptoms including muscle fascicula-
*138093
tion, decreased movement, abdominal breathing, convulsion and death from respiratory paralysis. Further studies showed that L. wollei toxins could competitively inhibit the binding of saxitoxin to saxiphilin, a specific saxitoxin binding protein (Mahar et al., 1991; Carmichael et al., accepted pending revision), and that fluorescence of the toxins was oxidation-dependent. These findings suggest that the toxins produced by L. wollei belong to the saxitoxin group, also referred to as Paralytic Shellfish Poison (PSP) (Hall et al., 1990). PSP has been recognized as a major health risk for over 50 years. These toxins are produced primarily by certain marine dinoflagellates responsible for ‘red tide’ phenomenon. Two freshwater cyanobacteria, Aphanizomenon flos-aquae (Jackim & Gentile, 1968; Sasner et al., 1984; Mahmood & Carmichael, 1986) and Anabaena circinalis (Humpage et al., 1994), are also known to produce PSPs. The aim of this study was to isolate L. wollei into unialgal culture from field samples followed by studies on the influence of selected nutrient, irradiance and temperature regimes on growth and toxicity.
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56 Materials and methods Source of culture and sample collection Lyngbya wollei field samples were collected from Guntersville Reservoir, Alabama in November 1991; April, June, and November 1992; January, July and October 1993; and July 1994 from five locations of the reservoir: Boshart, Ossawintha, Siebold Cage, Siebold Marina and Waterfront. Several other sites were also sampled but on a less frequent basis. A complete list of sample sites and dates are given in Carmichael and Evans (1996). After collection, the samples were kept in an ice chest. Upon return to the laboratory, the samples were washed with distilled water and extraneous plant/animal material was removed. Approximately 20 g fresh material from each sample was removed for isolation and culture. The remainder was lyophilized and stored at 20 C or 80 C for subsequent use in toxicity tests and toxin isolation. Isolation and culture of L. wollei Before plating the filaments, they were washed with sterile media (Z-8 and LM6E) until the filaments were clean when viewed using a dissecting microscope. The Z-8 medium composition is after Kotai (1972) and Carmichael (1986) while the LM6E medium is from Speziale and Dyck (1992). Healthy, dark green, filaments were removed from the mat sample, cut into 0.5 cm long segments and plated at the center of Petri dishes containing Z-8 or LM6E media in 1% w/v agar. Plates were made in triplicate for each field sample. The sterile agar was autoclaved and washed 3 times over a 72-h period with distilled water before mixing with the filter-sterilized nutrient solution. The agar plates were stored at 4 C before use. Cycloheximide (50 mg L 1 ) and germanium dioxide (100 g mL 1 ) were used to eliminate contaminating eukaryotes. Imipenem (N-formimidoyl thienamycin monohydrate) and cycloserine were used to inhibit bacteria (final concentration, 100 g mL 1 ) (Vaara et al., 1979). All cultures were kept in an incubator at 26 C under 16/8 light and dark photoperiod (cool white fluorescence) with an irradiance of 22 mol m 2 s 1 photon flux (Speziale & Dyck, 1992). When new trichomes had grown out of the old filaments and away from the contaminated area, they were cut and transferred to another Petri dish using the same medium.
This transfer process was repeated until an entire plate of unialgal cultures was observed under the dissecting microscope. When a unialgal culture plate was obtained, filaments were transferred to 125 mL Erlenmeyer flasks containing 100 mL Z-8 medium (Z-8 medium proved to be better than LM6E medium for growing L. wollei since the cultures in LM6E medium failed to grow after 1–3 months in culture). Only Z-8 was used for the liquid medium, while both Z-8 and LM6E were used in making agar plates for isolating field samples. Filaments were allowed to adapt to the liquid Z8 medium for 2 weeks and then transferred into 1-L Erlenmeyer flasks containing 800 mL Z-8 medium. These flasks were aerated at a rate 43–71 L h 1 . These production cultures supplied enough sample for toxicity and toxin isolation studies under various nutrient, irradiance and temperature conditions. Another isolation procedure for L. wollei was to place about 5 grams (fresh weight) of a field sample into a sterile nylon mesh bag which was suspended in 500 mL sterile Z-8 or LM6E medium in a 600 mL beaker (L. Dyck, pers. comm.). The medium was filtered through a 0.45 m membrane filter weekly to prevent the growth of epiphytes. When new filaments had grown out of the mesh bag, they were cut into 0.5 cm pieces and plated onto an agar plate. These steps were repeated until a unialgal culture was obtained. Extraction of toxins Mat samples from 1-L flasks were pooled and lyophilized. The lyophilized cells were extracted overnight in acidified (pH 3.5) 25% methanol, 50 mL g 1 at 4 C. The methanol extract was evaporated to dryness. The volume of this dried extract was brought to 50 ml g 1 , of original dry weight, with 50 mM acetic acid. Four different extraction methods were compared for toxicity and toxin profiles. These extraction procedures were: (1) 50 mM acetic acid plus treatment with a sonicator cell disrupter (Heat System Ultrasonics Inc., model W-200R, Plainview, NY) for 5 min using a thin probe setting number 9; (2) extraction with 0.1 M HCl plus sonication and boiling for 5 min. This is referred to as ‘Proctor Enhancement’ (Hall et al., 1980), a process which will hydrolyze the less toxic C toxins, if present, to the more toxic gonyautoxins; (3) 80% ethanol extraction overnight at 4 C and pH 3.5; (4) 25% methanol extraction overnight at 4 C and pH 3.5.
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57 Bioassay Toxicity tests were carried out using two related methods. The first was an estimation of the minimum lethal dose (LD100 ) by intraperitoneal (i.p.) injection of cell extract into ICR Swiss male mice (18–22 g). The mice were dosed at either 250, 500 or 1000 mg kg 1 using methanol extracts that had been dried and reconstituted to 50 mg mL 1 (dry weight cells) with 50 mM acetic acid. Toxicity signs, indicative of the PSP’s, were observed as muscle fasciculation, decreased movement, abdominal breathing, and convulsions followed by death from respiratory paralysis within 20 minutes. We compared this estimate of the LD100 with the AOAC method (Cunniff, 1995) for bioassay of PSP toxins in mice as developed by Sommer and Meyer (1937). In this procedure, the crude extracts were diluted until an i.p. injection of 1 mL caused at least two, 18–22 g, mice to die between 5 to 7 min. The toxicity was then expressed in Mouse Units (MU). The average survival time for the replicate injections was used to determine the number of MU from Sommer’s table (Cunniff, 1995). Toxin standards Standards of certain PSP’s were a generous gift from Dr Yasukatsu Oshima, Tohoku University, Japan. These standards include: saxitoxin (STX), neosaxitoxin (NeoSTX), decarbamoyl saxitoxin (dcSTX), gonyautoxin (GTX) 1–6, decarbamoyl gonyautoxin (dcGTX) 2 and 3 and C toxin 1–4. Standards of GTX 1–4, STX and NeoSTX were also provided by Dr Sherwood Hall, Office of Seafood Safety, Food and Drug Administration, Washington DC.
230 nm and collected in 4-mL fractions. Fractions were collected up to 200% of the column bed volume. Two to three fractions were pooled and lyophilized. Lethal potency of these fractions was determined by mouse bioassay. Toxic fractions were further characterized by HPLC. Toxin analysis by HPLC Crude extracts were filtered through a 0.22 m filter. The filtrates were passed through either a 5000 MW filter or a SepPak C18 cartridge column prior to HPLC. HPLC using ion pair chromatography with post column oxidation was done using a 5 m, 250 4.6 mm Inertsil C-8 column (Alltech) at a flow rate of 0.8 mL min 1 (Oshima et al., 1993). The HPLC system used a Water’s 600E system controller combined with a Water’s model 470 fluorescence detector and post column oxidation system. Three mobile phases modified from Oshima et al. (1993) were used to separate the three different toxin groups: (A) 2 mM 1-heptanesulfonic acid in 10 mM ammonium phosphate buffer (pH 7.1) for the gonyautoxin group; (B) 2 mM 1-heptanesulfonic acid in 10 mM ammonium phosphate buffer (pH 7.1): acetonitrile (92:8) for the saxitoxin group; and (C) 1 mM tetrabutyl-ammonium phosphate solution adjusted to pH 6.0 with acetic acid for the C1-C4 toxins. Eluent from the column was continuously mixed with 7 mM periodic acid in 50 mM sodium phosphate buffer (pH 9.0) at 0.4 mL min 1 , heated at 65 C, and then mixed with 0.5 N acetic acid at 0.4 mL min 1 just before entering the monitor. The fluorescence detector used excitation wavelength of 330 nm and an emission wavelength of 390 nm (Oshima et al., 1993).
Toxin separation and purification Bio Gel P-2, fine grade (200–400 mesh, Bio-Rad laboratory), was swollen and deaerated in 0.1 M acetic acid overnight. The gel was packed to a 300 mL bed volume in an Amicon column (2.2 90 cm). Dried crude toxin extract, from 1–2 g dry weight of cell material, was redissolved in 3.0 mL of 0.1 M acetic acid and filtered through a 5000 MW filter (Diaflo YM-5 ultrafiltration membrane, Amicon Corp). The filtrate was passed through a Sep-Pak C18 Cartridge to remove photosynthetic pigments. The toxin containing eluant was applied to the Bio Gel P-2 column and eluted with two bed volumes of 0.1 M acetic acid at a flow rate of about 0.67 mL min 1 . The effluent was monitored at
Testing the influence of several environmental factors on dry weight and toxicity of L. wollei The five environmental factors varied in this study were nitrogen (NO3 -N), phosphorus (PO4 -P), calcium, photosynthetically active radiation (PAR) and temperature. In these experiments, one variable was changed at a time. The three major plant nutrients varied included sodium nitrate, potassium phosphate dibasic and calcium nitrate. PO4 -P and calcium were applied at 0%, 10%, 100% and 500% of the normal concentration in Z-8 medium. NO3 -N was tested at 8%, 17%, 100% and 500% of the Z-8 medium normal levels. These levels were chosen to improve growth and toxin production
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58 in L. wollei, not to duplicate environmental conditions from the source waters for L. wollei. In the phosphate-deprived experiments, samples were grown in PO4 -P-deprived Z-8 medium for 5 days and then transferred into new phosphate-deficient medium. This was done to minimize transfer of these nutrients on the Lyngbya filaments. Initial toxicity, chlorophyll a and phycobilins were measured before each test. Three to eight grams fresh weight of the production culture was removed from the flask and blotted dry using a sterile paper towel. This sample was divided into 4 equal parts by weight and then transferred into one liter flasks for culturing under different experimental conditions. Cultures were harvested after three weeks. The toxicity, fresh weight, dry weight, chlorophyll a and phycobilins were determined. The toxin profiles were also determined when there was significant toxicity change in the samples. For the irradiance experiment, plant pigment composition including carotenoids as well as chlorophyll a and phycobilins were determined (Parsons et al., 1984; Talling & Driver, 1963; Tandeau & Houmard, 1988). Statistical analysis Data were analyzed by a one-way analysis of variance (Software Package: Super-Anova). When a significant P value was obtained, Fisher’s protected LSD was used to analyze dry weight and toxicity data under each experimental condition with the means being compared for significant difference. Significance was based on a P = 0.05.
Results Preliminary culture and toxicity testing of L. wollei Fifty single filament isolates from both toxic (n = 37) and nontoxic (n = 13) field samples, representing eight collection sites on Guntersville Reservoir, Alabama, were obtained. The majority of the isolates (20 out of 37) decreased or lost their toxicity during culture. However, there were five isolates in which toxicity increased (data not shown). Two of these were toxic even when the original field sample was non-toxic in the mouse assay.
Minimum lethal dose and mouse units We compared the AOAC mouse unit (MU) bioassay method with the LD100 method. We were unable to establish a correlation between the LD100 and MU since two different samples with the same LD100 could have different MU values. MU were used as the accepted method for toxicity although the LD100 was useful in estimating toxicity when a more defined toxicity level such as MU was not needed. Toxicity and toxin composition with different extraction procedures Among the four different extraction procedures, no consistent differences in toxicity were obtained. Toxin profiles of acid and methanol extractions were similar. Both acetic acid and hydrochloric acid extraction with boiling for 5 min caused a higher proportion of dcGTX compared to an unknown toxin eluting before GTXs. No conversion to GTXs (‘Proctor Enhancement’) was observed. The efficiency of toxin extraction with acetic acid or hydrochloric acid was dependent on the length of sonication. Increasing sonication time produced more photosynthetic pigments. The 25% methanol extraction caused the least amount of photosynthetic pigments, so this was the method of choice used for Lyngbya toxin extraction. Comparison of toxin profile of toxic and nontoxic strains with and without lyophilization Some of the nontoxic samples had similar retention times for HPLC peaks compared with toxic samples except that the peak heights were much less in the nontoxic samples. In some cases, nontoxic strains had a totally different HPLC profile to the toxic ones. There were no significant differences in toxicity and toxin profiles between fresh and lyophilized cells. Bio Gel P-2 gel filtration and high performance liquid chromatography Bio Gel P-2 has been successfully used in PSP separation (Buckley et al., 1975; Hall et al., 1980), especially at the initial purification steps. The Lyngbya toxins we isolated were well resolved from most of the nontoxic constituents and the toxic fractions were pure enough to be applied to HPLC. Our results from Bio Gel P-2 show that the toxins were eluted between 69%–81% of
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59 column bed volume with isocratic acid elution. Even though two major peaks were observed between 90%– 120% of bed volume, at a point where saxitoxin and its derivatives should elute, no toxicity was detected. When these nontoxic fractions were boiled in 0.1 M HCl at 100 C for 5 min, no ‘Proctor Enhancement’ occurred. Not all of the toxicity in the crude extract applied to the Bio-Gel P-2 was recovered following acid elution. In one sample from ‘Siebold Park’, only 23% of the toxicity was recovered, with 12% being contributed by decarbamoyl gonyautoxins and 11% by the unknown toxins. This reduction in toxicity from Bio-Gel P-2 fractions compared with the initial crude extract might be due to degradation of the toxins during storage and handling, tailing during column elution or retention to the column. The first toxic fraction from Bio-Gel P-2 (69–73% of column bed volume) contained the unknown toxins (data not shown) and the second toxic fraction (73–81% of column bed volume) had a similar retention time to dcGTX2 and dcGrX3 (Figure 1a, b). We compared the toxin profile of toxic fractions from Bio Gel P-2 with that of the crude extract. Crude extracts also showed the presence of dcGTX but in a lesser amount relative to the major unknown peaks. Some crude extract showed no dcGTX. This may be because dcGTX2 and 3 did not exist or existed in trace amounts and were not detected on our system. Samples sent to Dr Yasukatsu Oshima, Tohoku University, Japan, confirmed the presence of dcGTX2 and 3 in L. wollei. No STX, NeoSTX, GTX or C toxins were found in L. wollei cultures that we examined. Figure 1d shows a typical HPLC toxin profile of toxic L. wollei strains. As mentioned, there were some unknown toxic peaks in the profile which did not match the profiles of the standards tested (Figure 1c, d). These samples are being examined by mass spectrometry and nuclear magnetic resonance. Their structures will be reported later. Dry weight, toxicity and toxin profile variation with different experimental conditions There was no correlation between pigment content in the cell, the dry weight and fresh weight or toxin levels. There was however a high correlation between fresh weight and dry weight. We therefore opted to represent biomass production, for the different parameters tested, by dry weight only.
Figure 1. HPLC toxin profile of L. wollei cultures. (1) Mobile Phase (A) for GTX: (a) dcGTX2 and 3 standard; (b) Toxic fraction of Siebold-7 from Bio Gel P-2, 75–81% of column bed volume. (2) Mobile Phase (B) for GTX and STX group: (C) Standard mixture of gonyautoxin and saxitoxin group; (d) 20 l Oss-D4, 50 mg ml 1 , toxicity 500 mg kg 1 .
There was a significant increase in dry weight over initial inoculum at PO4 -P concentrations of 0.55 (p
