Maximizing okadaic acid content from Prorocentrum hoffmannianum.pdf

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41ournal of Applied Phycology6: 41-44, 1994.( 1994 KluwerAcademicPublishers. Printed n Belgium.

Maximizing okadaic acid content from rorocentrum hoffmannianum Faust

Steve L.Mortonl' * & Jeffery W.Bomber2 ,3Southern Illinois University Dept. of Plant Biology Carbondale, IL 62901 USA;

2 Maritech, Inc, 150 E. Pleasant Hill Rd Carbondale, IL 62901 USA;3 Present address: WW.Knight Family Practice Center, 2051 Central Ave Toledo OH 43606, USA * Authorfor correspondence

Received 7 August 1993; revised 18 November 1993; accepted 21 November 1993.

Key words: Prorocentrum, semi-continuous culture, okadaic acid, growth, suspension culture

Abstract

Clonal cultures of Prorocentrum hoffmannianum Faust (clone 882a) were grown under optimal environmentalconditions for maximal okadaic acid production. The environmental conditions of 25 C and 86 mol photonm -2 s - were used to cultivate P. hoffmannianum in a semi-continuous 36-L culture vessel with continuous cellsuspension and pH control. Using these conditions, a 3-fold increase in harvestable biomass and okadaic acidcontent was observed when compared to batch culture techniques.

Introduction

Okadaic acid is a toxic polyether fatty acid first iso-lated from two sponges of the genus Halichondria, H.okadaii and H. melanodocia (Tachibana et al. 1981).Subsequent studies have shown that the progenitor ofokadaic acid is a number of dinoflagellates of the genusProrocentrum and Dinophysis which are accumulatedin the sponge by filter feeding (Kat, 1979; Murakamiet al., 1982; Lee et al., 1989; Dickey et al., 1990).Okadaic acid and its derivatives have been implicatedin the human diseases ciguatera and diarrhetic shellfishpoisoning.

The compound has become an important molec-ular probe for biological research (see review byCohen et al., 1990). The LD5 of okadaic acid is192 /g kg- (intraperitoneal (i.p.) injection) (Yasumo-to et al., 1984). Okadaic acid is a potent inhibitorof serine/thronine-specific protein phosphatases 1 and2A, also 2B at very high concentrations. However,the compound does not inhibit protein throsine phos-phatases or other phosphatases. Okadaic acid has alsobeen found to be a potent, non-phorbol ether tumorpromoter.

Okadaic acid has been synthesized from d-glucaltri-acetate and d-glucal tetra-acetate using 106 steps

(Isobe et al. 1987). The large number of steps in thechemical synthesis of okadaic acid led the authors to

conclude the unpracticality of the commercial synthe-sis of the compound. Thus, the precursor dinoflagel-lates are the only renewable source of okadaic acid.

Since, Dinophysis spp. have not been cultivatedin the laboratory successfully, only Prorocentrum pp.can be used as a renewable source of okadaic acid. P.hoffmannianum Faust clone 882a was chosen for thisexperiment due to the number of previous studies usingthis clone (Aikman et al. 1993; Morton et al., in press).The aim of the present study is to determine whether:(1) okadaic acid production can be manipulated by the

optimization of environmental parameters; (2) a semi-continuous, suspension culture can increase productionof the compound; (3) cell densities can be maintainedat maximal okadaic production.

Materials and methods

Stock cultures of Prorocentrum hoffmannianum, clone882a isolated from Little LameshurBay, St. John, U.S.Virgin Islands were maintained in a lighted, tempera-

ture controlled incubator on a 16:8 light:dark cycle at52 /tmol photon m- 2 s- . All stock cultures were main-


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ottl

Fig. 1. Diagram of the semi-continuous 36 L culturevessel.

tained at 28 C in K medium (Keller Guillard, 1985)which was modified by omitting Tris, copper and sil-ica (Morton Norris, 1990). Natural aged seawaterof 3 6 / 0 from Carolina Biological was used to pre-pare culture media which was sterilized by ultravioletlight illumination for 24 h. The sterile seawater wasenriched aseptically.

Optimal light and temperature condition formax-imal okadaic acid production were duplicated usinga lighted-temperature controlledincubator (Morton etal., in press). Semi-continuous mass culturesof P. hoff-mannianum was performed using twin 40 L culturevessels (Bellco Glass, Inc.) fitted with a model 7764Biotech Overhead Drive (Fig. 1).

Each Bellco jar had 36 L of sterile medium whichwas prepared aseptically. Each teflon paddle assemblywas set at a rotation of approximately 30 rpm. A Cole-Palmer pH/ORP controller model 5656-00attachedwith a submersible pH and a temperature probe was

used to measure pH of the culture medium. ThepH/ORP controller was connected to a CO 2 solenoidvalve, which maintained pHat 8.4. The air/CO 2 mix-ture (10 ) was sterile filtered using a 0.22 2m inlinefilter.

Cell densities were measured every three to fourdays by withdrawing a 10 ml sample which was count-ed using a Palmer-Moloney counting chamber. Wheneach Bellco reached late-log phase, 4 L were with-drawn every third day. Four litres of fresh mediumreplaced the 4-L harvest, leaving a total 36 L at all

times. Cells were concentrated by cold (5 C) cen-trifugation. To remove salts, distilled deionized water

(DDIW) was added to the resulting cell pellet that istransferred to a preweighed vial and centrifuged. TheDDIW is decanted and the cell pellet is immediatelyfrozen at -20 C and lyophilized for48 h until dry.

Okadaic acid content was determined using a mod-ified High Pressure Liquid Chromatographic methodof Lee et al. (1987). Briefly, cells were sonicat-ed in 80 (v:v) methanol for five minutes andcentrifuged. The resulting supernatent was extract-ed with petroleum ether (2x5 ml) and chloroform(2x4 ml). The chloroform phase was dried undera stream of nitrogen and redissolved in 0.5 mlmethanol. The extracts were fluorescence-labelledwith 1 pyrenyldiazomethane (Molecular Probes,Inc.).The solution was sonicated for 10 min, heated at 75C for 30 min, and dried under a stream of nitro-gen. The dried sample was redissolved in 1 ml hex-ane:chloroform (1:1). The reaction mixturewas passedthrough a silica gel solid-phase extraction columnasdescribed by Lee et al. (1987). Ethers of okadaic acidwas extracted from the column using 5 ml chloro-form:methanol (95:5 v:v) and dried under a streamof nitrogen, redissolved in 250 tl acetonitrile.

Okadaic acid content was determined using a SSIModel 300 HPLC equipped with a Turner Model 10AU Fluorometer with a 1-mm flow cell and a Macin-tosh Classic II computer. Analyses of the toxin deriva-tives was carried out at room temperature on a What-man ODS column (4.6x 125 mm) using 75 acetoni-trile as the mobile phase. The flow rate was maintainedat 2 ml min - '. The excitation and emission wave-lengths were set at 390 nm and 400 nm respectively.Peak heights of the standards (95 pure okadaic acid,L.C. Services, Inc.) wascorrelated to the concentrationof the standard using the Pearson Test (Sokal Rohlf,1985).

Results

From previous experiments (Mortonet al., in press),the environmental conditions of 25 C and 86 Amolphoton m- 2 s - were used to determine the maximumokadaic acid content using the twin Bellcojars. Totalcell biomass (cell ml - l) during the growth cycle isshown in Fig. 2; the highest value was 48.000 cellml-l.

Cell harvest started once the cultures reached late-log growth phase where okadaic acid production has

been found to maximize (Aikmanetal., 1993). Growthrate ranged from 0.5 division d -1 at early log growth


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0000

9 j LUU

0

DAY

Fig. 2. Growth cycle of Prorocentrumhoffmannianumin asemi-continuous culturevessel.

_- 0.20_

- 0.15Co

3 0o.1o

W 0.05

I 0

IBII4 6HARVEST NUMBER s

1.00o

0.80

0.60

0.401 0

0 20 3cp

Fig. 3. Harvest weight and okadaic acid content of Prom-centrum hoffinannianumgrown in a semi-continuousculturevessel.

phase to 0.38 division d - at late-log growth phase.Once biomass reached late-logphase,6 L was removedfrom each Bellco every fifth day. The semi-continuousculture was replenished with 6 L of fresh mediumafter the cells had been removed. The harvestable cellweight is shown in Fig. 3 for eight continuous har-vest days (20 d total after late-log growth phase wasreached). The weight of the resultant cell pellet rangedfrom 130 to 114 mg L- . The resultant okadaic acidcontent of the cells ranged from 0.8 to 0.5 mg L ofmedium (Fig. 3).

Discussion

of the favorable combination of high total okadaicacidcontent coupled with high growth rate (Mortonet al.,in press). The total okadaic acid and total biomassdid not correlate with optimal okadaic acid contentper cell or maximum growth rate (Morton et al. inpress). Maximal biomass, total harvestable okadaicacid, growth rate, and okadaic acid content per cellare all independent parameters. Each maximized orminimized with different environmental conditions.

Highest cell densities was 48.000 cells ml- 1; thishigh cell density is approximately three times thatfound in 20 L carboy batch cultures at late-log phase(S.L. Morton, unpublished). Harvestable cell weightwas, like cell density, almost three times that of batchcultures. Three factors may account for this increasein cell density: (1) optimization of environmental fac-tors, (2) suspension of the cells from the teflon paddlethroughout the growth cycle, and (3) control of pH.

P. hoffmannianum is the first benthic dinoflagel-late to be grown successfully in a semi-continuous,suspension culture vessel. The benthic dinoflagellate,Gambierdiscus oxicus has been cultivated in smallersuspension culture vessels of 8 L total volume (Babin-chak et al. 1986). However, these vessels are com-monly used as batch cultures and not semi-continuouscultures (J.A. Babinchak, pers. comm.). Planktonicdinoflagellates are generally considered to be sensitiveto stirring in culture, displaying reduction in growthrates and cellular damage. Amphidinium carteri (Gol-man et al. 1976), Cryptotecodinium cohnii (TuttleLoeblich, 1975), Gonyaulax excavata (White, 1967)and Alexandrium fundyense (Anderson et al., 1990)have all shown cellular damage and/or reduced growthrates to suspension cultureconditions. These attemptsat suspension culture may have been unsuccessful,because of the sensitivity of the planktonic organismto constant turbulance. Benthic species such as P. hoff-mannianum and G. toxicus generally have thicker the-

cal plates than planktonic species, which may makethem less sensitive to turbulance.

Dinoflagellates such as P. hoffmannianum are theonly renewable source for the economically importanttoxin okadaic acid. This study shows that toxin yieldcan be manipulated using optimal environmental con-ditions of light intensity and temperature. P. hoffman-nianum can be cultured in a semi-continuous culturevessel such as a Bellco jar with marked increase in celldensities and okadaic acid production.

The environmental conditions were chosen for masssemi-continuous culture using the Bellco units because

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Acknowledgements

This research was supported by the National Cancer

Institute and MariTech, Inc. The authors thank Paul

Driedger of L.C. Services Laboratory for the generousgift of the okadaic acid standard. Valuable assistancewas given by Julie Morton and Dana Madden through-

out this study.

References

Aikman KE, Tindall DR, Morton SL(1993) Physiology andpotency of the dinoflagellate Prorocentrum hoffmanni-anum during one complete growth cycle. In Smayda T,Shimizu Y (eds), Toxic Phytoplankton Bloomsin the Sea.Elsevier, Amsterdam: 463-468.

Babinchak JA, Jollow DJ, Voegtline MS, Higerd TB (1986)Toxin production by Gambierdiscus oxicus isolated fromthe Florida Keys. Marine Fisheries Review 48: 53-56.

Cohen P, Holmes CFB, Tsukitani Y (1990) Okadaic acid: anew probe for the study of cellular regulation. TIBS 15:98-102.

Dickey RW, Bobzin SC, Faulkner DJ, Bencsath FA, Andrz-ijewski P (1990) Identification of okadaic acid from aCaribbean dinoflagellate, Prorocentrum oncavum. Toxi-con 28: 371-377.

Galleron C (1976) Synchronizationof the marine dinoflagel-late Amphidinium carteri n dense cultures. J. Phycol. 12:69-73.

Kat M (1979) The occurrence of Prorocentrum species andcoincidental gastrointestinal illnessof mussel consumers.In Taylor DL, Seliger HH (eds), Toxic Dinoflagellates.Elsevier, Amsterdam: 215-222.

Keller MD, Guillard RRL (1985) Factors significant tomarine dinoflagellate culture.In Anderson DM, White

AW, Baden DG (eds), Toxic Dinoflagellates. Elsevier,Amsterdam: 113-116.

Lee JS, Igarashi T, Fraga S, Dahl E, Hovgaard P, YasumotoT (1989) Determination of diarrhetic shellfish toxins invarious dinoflagellate species. J. appl. Phycol. 1: 147-

152.Morton SL,Bomber JW, Tindall PA (in press) Environmentaleffects on the production of okadaic acid fromProrocen-trum hoffmannianum: temperature, light and salinity. J.exp. mar. Biol. Ecol.

Morton SL, Norris DR (1990) The role of temperature, salin-ity, and light on the seasonality of Prorocentrum ima. InGraneli E, Sundstrom B, Edler L, Anderson DM (eds),Toxic Marine Phytoplankton. Elsevier, Amsterdam:201-205.

Morton SL, Norris DR, Bomber JW (1992) Effect of temper-ature, salinity, and light intensity on the growth and sea-sonality of toxic dinoflagellates associated with ciguatera.J. exp. mar. Biol. Ecol. 157: 79-90.

Murakami Y, Oshima Y, Yasumoto T (1982) Identification ofokadaic acid as a toxic component of a marine dinoflag-ellate Prorocentrum lima. Bull. Jap. Soc. Sci. Fish. 48:69-72.

Tachibana K, Scheuer PJ, Tsukitani T, Kikuchi H, Engen D,Clardy J, Gopichand T, Schmitz FJ (1981) Okadaic acid, acytotoxic polyether fromtwo marine spongesof the genusHalichondria. . Am. Chem. Soc. 103: 2469-1471.

Tuttle RC, Loeblich AR (1975) An optimal growth mediumfor the dinoflagellate Cryptothecodinium cohnii. Phycolo-gia 14: 1-8.

White AW (1967) Growth inhibition caused by turbulencein the toxic marine dinoflagellate Gonyaulax excavata. J.

Fish Res. Bd Can. 33: 2598-2602.Yasumoto T, Murata M, Oshima Y, Matsumoto GK, Clardy J(1984) Identification of Dinophysisfortii as the causativeorganism of diarrhetic shellfish poisoning.Nippon SuisanGakkaishi 46: 1405-1411.

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Maximizing okadaic acid content from Prorocentrum hoffmannianum.pdf