The effect of naturally occurring organic substances on the growth of a red tide organism

!.l,ar, r Re~¢urch Vol. '~, pp. 601 to 606. Per zarnon Press 19"4. Printed in Great Britain.

THE EFFECT OF NATURALLY OCCURRING ORGANIC SUBSTANCES ON THE GROWTH OF A RED TIDE ORGANISM MARION T. DOIG, III and DeAN F. MaRViN Department of Chemistry, University of South Florida, Tampa, FL 33620, U.S.A. (Recewed

18 M a r c h

1974)

Abstract -The effect of soil extracts on the growth of the Florida red tide organism. Gym,odinium brt,rc. was investigated to determine the potential effect of organic matter in rivers on red tide outbreaks. Soil from a peat bog was extracted with water, filtered through a membrane filter (0.22/~m), then fractionated into four molecular-weight fractions (0--500; 500-1000; 1000-10.000; and 10.000) by means of Amicon Diaflo ultramembranes. Fractions were analyzed for organic carbon, iron and manganese. The effect of different amounts of the last three fractions on the rate of growth and maximum cell population of G. brece cultures was determined. No substantial change in the growth constant (K,,) with change of concentration of added fraction was noted, nor was there a marked variation from one fraction to another. The maximum cell population increased with an increase in the amount of added organic extract; and using the second and third fractions, the increase was related to the amount of iron present (linear correlation coefficient, r = 0.994, P = 0.0011; the last molecular-weight fraction showed a similar relationship. though the effectiveness in increasing Nr.~,was less (on the basis of amount or iron added). The results were compared with the effect of adding gibberellic acid. which is a naturally occurring hormone that reportedly reduces the time for maximum cell populations to be attained.

INTRODUCTION Many investigations have indicated that humic The factors affecting the growth of the unarmored materials stimulate plant growth (Burk et al., 193l; dinoflageUate, Gymnodinium breve, have been of con- Flaig and Otto, 1951; Dekock, 1955). and the growth siderable interest since G. breve was identified (Davis, rates of cultures of dinoflagellates have been enhanced 1948) and associated with mass mortalitites of marine by the addition of soil extracts (Wilson and Collier. organisms in the Gulf of Mexico (Davis, 1948; Gait1955). Prakash and Rashid (1968) have studied the soft. 1948, 1949; Gunter et al., 1948; Wilson and Ray, effect of selected soil fractions on the growth of several 1956). Blooms of G. breve have been termed "red tides", species of armored dinoflagellates of the genus and outbreaks usually occur in late spring or early fall Gonyaulax. The stimulatory effect of the humic acid along the west coast of Florida, most often in the fraction (alkali-soluble, acid- and alcohol-insoluble) area from Cape Sable to Tarpon Springs (Finucane, was greater than that of the fulvic acid (acid-soluble) 19641. fraction. The humic acid was further fractionated by Red tide outbreaks are often preceded by periods of gel-filtration on Sephadex G-10, G-15, G-25 and G heavy rainfall (Slobokin, 1953; Collier, 1954; Aldrich 50 gels. The low molecular weight fractions produced and Wilson, 1960). Presumably, river discharge has the greatest growth enhancement. Three reservoirs of naturally occurring organic subsome effect other than reducing the salinity of coastal waters because lowered salinity is not biologically stances are available, directly or indirectly, to red tide essential for G. breve growth (Aldrich and Wilson, organisms: terrestrial, sediment, and in situ (cf. Martin 1960). Also, the incidence of G. breve blooms does not and Martin, 1973). Of these, the first has been emphaappear to be related to phosphorus or nitrogen con- sized because of convenience, though available evicentrations in Florida's west coast rivers (Bein, 1957; dence suggests the second may be very significant Dragovich, 1960; Rounsefell and Dragovich, 1966). In- because several Florida red tide outbreaks appear to gle (1965) suggested that flooded rivers, following per- have initiated in the benthos (cf. Steidinger and Ingle, iods of heavy rainfall, could leach humie substances 1972). Also, Prakash and Rashid (1968)actually studfrom inland bogs and transport these natural chelators ied the effect of humic substances isolated from marine and associated metals to potential outbreak areas. sediments. Consideration of the third reservoir has 601

602

M. T, DOIG.III and D. F. ~,I.¢RrI\

been neglected, though Nissenbaum and Kaplan I 19721re~ iewed evidence lbr the in situ origin of marine humic substances and proposed a five-step pathway tbr marine formation and transformation in sediment. Regardless of the source of humic substances, an)' stud~ of their effect on G. hret'e cultures must first overcome a serious problem: the procedures emplo3ed in the extraction of humic and fulvic acids from soil humus are rigorous (Felbeck. 1965)and may result m the alteration or complete destruction of compounds of ecological significance. For example, the growth of G. l,rerc ma? be influenced by the availability of vitamins. auxins or hormones. Mild extraction conditions, however. followed by molecular size fractionation, should provide material for a realistic appraisal of the growthpromoting potential of soil-organic matter for the Florida red tide organism. The use of a single bog sample seemed reasonable in view of previous observations IPierce, 1970) on the similarities of humic substances isolated from different soils along west Florida rivers. MATERIALS

Or qanism,s

Samples of Gymnodinium breve were originally obtained as bacteria-free unialgal cultures from S. M. Ray and W. B. Wilson (Texas A & M Marine Station, Galveston) and were cultured in an enriched sea water medium tB-5: cf. Brydon et al., 1971). Stock cultures were maintained in the enriched sea water medium in 8 1. carboys and aliquots were aseptically removed for bioassays. Sea water

Samples used in the bioassays and in the preparation of B--5 medium were collected at sampling stations from three to I0 miles due west of St. Petersburg. Florida, in the Gulf of Mexico. Samples were filtered through sand and stored in the dark in 5-gal glass carboys for approx. 3 months. The "aged" sea water was stirred with activated charcoal (ca. 4 g 5- t gal) for 30 min and filtered through 0.45 #m and 0.22 t~m membrane filters (Millipore Corp.). The filtrate was then autoclaved at 15 psi for 15 rain and stored for at least 3 days prior to use. The nutrient composition of the sea water after filtration and sterilization is summarized in Table 1. Aliquots (200 ml) of this water were used in the algal assays, and all enrichments were added aseptically through Swinnex (MiUipore Corp.) filter units. Soil extracts

Soil from a peat bog located in the Tampa, Florida, area was picked free of macroscopic organisms and

Table 1. Nutrient composition of u~cd as assa~ media Parameter

,;ea ~ a t , : r

Concentration

Salinit,, pH Total POa-P NH.~ N NO 3 -:- N O : - \ SiO:

34.5 ppt ~.2t) units

0.125 ppm IV)OI ppm 0.0075 ppm 0.4I ppm

large debris and air-dried at room temp. !25 -- 2 C) tbr 24 h. The soil sample I200 g) was extracted with 3 1. of distilled water tpH 6.5J for 36 h with constant mixing. The mixture was filtered through 0.22 #m membrane filters (Millipore Corp.) and the characteristics of the filtrate, hereafter referred to as the crude soil extract. were determined (Table 2). The crude Soil extract was separated into four molecular-weight fractions by means of Amicon Diaflo ultramembranes (UM 05, UM 2, UM 10) using an Amicon filtration cell under a nitrogen pressure of 50 psi. The fraction retained by each membrane was washed and concentrated as described by Gjessing (1970). Table 2. Characteristics of the crude soil extract Characteristic Final pH Total volume Total nonvolatile material Total organic carbon Humic acid Iron

Value 4.5 units 2500 ml 275 ppm 6l ppm 0.77 ppm 0.096 ppm

METHODS

Chernical analyses

Chemical analyses were performed by the standard procedures described in the EPA methods manual (Environmental Protection Agency, 1971). with several exceptions. Humic acid concentrations were determined spectrophotometricatly after extraction (Martin and Pierce, 1971)and trace metal concentrations were determined by atomic absorption spectroscopy using a Perkin-Elmer Model 403 Atomic Absorption Spectrophotometer equipped with a graphite furnace. Salinities were determined with a hand-held reffactometer. Cell em~meration

Celt counts were determined electronciatly with a Model B Coulter Counter after sterile removal of aliquots from test cultures. The aliquots were obtained

The ~owth of a red tide organism

603

Table 3. Effect of naturally-occurring organic substances on the growth of Gymnodinium brere Lag time (days)

Culture* Control FI (0.5 mll FI (l.0 ml) F2 (0.5 ml} F2 11.0 mli F3 ~0.5 ml) F3 (l.0 ml} GA (10 -s M} GA ( I 0 - : M} GA (10 -~' st)

2.3 2.5 2.8 2.0 1.8 2.3 2.5 t.8 1.7 0.9

Max. cell count (cells ml- ')

K (days- t)

1020 + 50 1375 ___80 t860 4- 85 1490 -+- 90 2125 -- 105 1200 + 60 1600 _ 75 II00 + 60 1090 _+ 55 1030 -4- 75

0.230 0.223 0.231 0.224 0.241 0.202 0.179 0.201 0.194 0.173

t,, (days)

-- 0.006 ~ 0.010 ___0.003 -.7_0.003 - 0.011 ___0.014 -4- 0.016 ..4-0.025 -2_ 0.009 -,- 0.002

3.0 3.l 3.0 3.1 2.9 3.4 3.9 3.4 3.6 4.0

* F I, F2 and F3 refer to molecular-weight fractions of 500-1000. 1000-10.000, and > 10,000. respectively. GA. Gibberellic acid. from triplicate cultures which were counted every two to three days. Aliquots from each culture were counted five to seven times, and all results were used to calculate the mean cell count and the standard deviation of the mean. Cell viability was determined visually at 100 x with a binocular microscope.

Bioassays A modification of the Flask Test of the Provisional Algal Assay Procedure of the Joint Industry/Government Task Force on Eutrophication (1969)was used to determine the response of G. breve to enrichment with the soil extract fractions, lnoculum was aseptically transferred to 200 ml aliquots of test medium in 250 ml Erlenmeyer flasks when the parent (stock) culture had reached the stationary phase ofgrowth. The inoculated flasks (initial count ca. 150cells m l - ,) were maintained at 25 + 2~C under constant illumination of approximately 600 ft-c., provided by dual banks of 40 W coolwhite fluorescent lamps. The response of G. breve to the enrichments was evaluated in terms of four growth parameters that were obtained from semi-logarithmic plots of mean cell count (N) as a function of time (t}. Typically, the plots consisted of four phases. The lag phase was the period after inoculation during which little or no growth occurred, and often the cell count decreased during

this period. Following the lag phase, the cell count increased exponentially (log phase) until the supply of some nutrient was exhausted and a plateau period was then observed. The plateau phase, which was often of long duration, was followed by a decline in the cell population (death phase). Lag times and maximum cell counts were taken directly from the semi-logarithmic plots. The growth constant (K,.) was calculated from the data corresponding to the linear portion of the semi-logarithmic plot using the expression: 1

N

K,, = (t - to)In N--o where N and No are mean cell counts at times t and to. The mean generation time (t.~) was determined from the relationship: ty = In 2,,K,.

RESULTS

Typically, soil extracts are excellent sources of chelated metals (e.g. iron), and the addition of soil extracts to cultures of dinoflagellates is a common practice (Wilson, 1966). The effect of several molecular-weight fractions of a soil extract on the growth of G. breve was determined and compared to the effects of various other naturally-occvrring organic substances on the growth parameters (cf. Table 31. In order to evaluate

Table 4. Characteristics of the soil extract fractions Molecular weight range 0-500 500-1000 1000-10.000 > 10.000 w.R. ~ 9--n

Volume (ml) 3000 25 25 25

Organic carbon (ppm) (mg) 8 660 2400 1200

24 16.5 60 30

of total organic carbon

(ppm)

(/ag}

?'o of total iron

18.4 12.6 46.0 23.0

0.005 2.040 2.460 2.380

15.0 51.0 61.6 59.6

8.0 27.3 32.9 31.8

Iron

ug Fe rag- ' C 0.62 3.09 1.03 1.93

604

M. T. DOlG, lll and D. F..M.~,arIx Table 5. Effect of selected organic substances on the grov, th of G. brece in enriched med:a" Culture + Control GA (10 -s ~,~) GA(10- M) GA 10-" M) TA (I ppm)

Lag time (da~s)

Max.. cell count tcells mi- t)

K (da~s ~)

:~ Ida,,~l

3.4 3.3 3.S 3.1 5.0

2595 : 125 2500 '_--2160 2465 - [15 _4__~ - 120 2455 _+ 145

0.156 -,- 0.003 0.15S : 0.009 0.158 = 0.006 0.156 __7_0.01)3 0.168 2 0.002

,t.4 -.4 4.4 -t J, 4. I

* Gulf of Mexico water with B-5 enrichments. + GA, Gibberellic acid: TA, tannic acid.

better the results of the study, an examination of the characteristics of the soil extract fractions (cf. Table 4) is necessary. A large percentage (46 per cent) of the organic substances present in the crude extract were in the 100010,000 molecular-weight range; however, the substances in the 500-1000 tool. wt range are associated with more iron (on a t~g Fe mg- t C basis). The percentages given (cf. Table 4) are valid for the recovered material, but may not be valid for the initial crude extract. Although 85.5 per cent of the total organic carbon (and 78.0 per cent of the total iron) in the crude extract was present in the combined fractions, the recovery of different components was probably variable. Losses or organic material are due mainly to adsorption onto the filters, and the recovery of high molecular-weight material should be less efficient than that of low molecular-weight substances because the high molecular-weight fractions come into contact with more filters. Each of the soil extract fractions was effective in increasing the maximum cell population (cf. Table 3); however, the substances in the 500--1000 tool. wt (iron rich) fraction were the most effective on a mg C-added basis. The growth constant was not affected by enrichment with low molecular-weight compounds but was significantly lowered by the addition of the high molecular-weight substances. Responses of G. breve to gibberellic acid (a plant growth hormone) enrichment were similar to those observed by Paster and Abbott (1970) with respect to the lag time (which decreased); however, the maximum cell population was not increased, and the growth constant decreased. In enriched media (cf. Table 5), the addition of gibberellic or tannic acid (1 ppm) did not significantly affect the growth parameters. Higher concentrations of tannic acid (5 and 10 ppm) caused the cells to die within a day. The final pH of the media was unaffected by the addition of tannic acid (up to 10 ppm), and, therefore, the demise of the G. breve cells must be attributable to some other phenomenon such

as precipitation of the tannic acid at the salinity of the media (35 ppt}. CONCLUSIONS Previous studies (Doig and Martin, 1974) have indicated that ongoing red tide outbreaks in Florida coastal waters are probably limited by the available sources of nitrogen: however, the factor or factors which "trigger" red tide outbreaks are as yet undetermined. Certainly the presence of a suitable environment is necessary before an outbreak can occur, but fluxes of certain micronutrients (e.g. trace metals, vitamins, etc.) may well cause the onset of logarithmic growth. Ingle and Martin (1971) have suggested that the amount of iron entering a potential outbreak area may provide a means of predicting the likelihood of a red tide occurring. The present study appears to confirm the stimulatory effect of iron that is associated with soil organic materials. A linear relationship exists between the maximum cell population attained and the a m o u n t of iron added to the assay system (cf. Fig. It. The correlation between these parameters is significant (linear correlation coefficient, r = 0.994) at a probability level of 0.001 using control FI and F2; data (Table 3). The data for F3 show a stimulatory effect of iron, but the dependence of growth response, N . . . . on iron concentration is different as is evident from Fig. I. It appears that the iron associated with high molecular weight fraction ( > 10,000) F3 is less effective in causing an increase in Nm,, than the lower molecular weight fractions Fl (500-1000) and F2 (101X)--10,000). Prakash and Rashid (1968) also noted the high molecular weight (5000-10,000) produced a lower growth response than low molecular weight fractions (<700, 700-1500) for several marine dinoflagellates. These workers did not report the trace-metal compositions of their humic acid fractions, though they noted the ash content was reduced to less than 3 per cent by means of exchange resin columns. Thus they suggested

The growth of a red tide organism

605

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Fig. l. Growth response of Gymnodinium breve to different fractions of soil extract, expressed as maximum cell yield, N,,~(cells ml- ~k as a function of volume of fraction and amount of iron added. Arrows indicate the exact amount of iron added for purposes of comparison. Vertical bars indicate one standard deviation. that growth-promoting activity of humic fractions used could not be attributed to iron and other tracemetal concentrations. Though the iron concentrations of fractions FI, F2 and F3 are known, the data must be interpreted with caution because additional, unknown factors may be involved. For example, the iron-rich fraction (F1) is also manganese-rich (ttg Mn m g - t C for fractions FI, F2 and F3 are 0.36, 0.15 and 0.16, respectively). Nevertheless, this study has indicated that naturally occurring organic substances (and associated trace-metals) have a biostimulatory effect on G. breve and the study has indicated it is plausible to suggest these substances may be involved in the "triggering mechanism". The nature of the involvement of organic substances and associated trace metal species in the triggering mechanism is uncertain. Prakash and Rashid (1968) suggested two roles: as specific sensitizing agents to enhance the uptake of nutrients; as respiratory catalysts. Whether these agents are involved in stimulation of a microbloom, whether they stimulate the emergence of G. breve from an encistment stage to a motile form, or whether they have some related role in growth stimulation of G. breve remains to be ascertained. Acknowledgements--We gratefully acknowledge the financial support of the Procter & Gamble Company and a PHS Research Career Award (to D.F.M. I K04 GM 42569-04}

from the National Institute of General Medical Sciences. The technical assistance of Mrs. Carol B. Stackhouse is gratefully acknowledged. REFERENCES Aldrich D. V. and Wilson W. (1960) The effect of salinity on the growth of Gymnodinium breve Davis. Biol. Bull. Mar. biol. Lab., Woods Hole 119, 57-64. Bein S. J. (1957) The relationship of total phosphorus concentration in sea water to red tide blooms. Bull. Mar. Sci. GulfCarib. 7, 316--329. Brydon G. A., Martin D. F. and Olander W. K. (1971) Laboratory culturing of the Florida red tide organism Gyranodinium breve. Environ. Lett. 1,235-244. Burk D., Lineweaver H., Homer C. K. and Allison F. E. (1931)The relation between iron, humic acid, and organic matter in the nutrition and stimulation of plant growth. Science, N.Y. 74, 522-524. Collier A. W. (1954) Gulf Fishery lnvestigatio. In Annual Report for Fiscal Year 1964, Branch of Fishery Biology, U.S. Fish and IJ/ildl. Serv, 23-25. Davis C. C. (1948) Gymnodinium brevis sp. nov.. a cause of discolored water and animal mortality in the Gulf of Mexico. Botan. Ga-. 109, 358-360. Dekock P. C. (1955) Influence of humic acids on plant growth. Science, N.Y. 121, ,173-474. Doig M. T. III and Martin D. F. (L974) The response of Gyranodinium breve to Municipal Waste Materials. Mar. Biol. 24, 223-228. Dragovich A. (1960) Hydrology of Tampa Bay and Adjacent water, In Galveston Biological Laboratory Fishery Research for the Year Ending June 30, 1960. U.$. Fish an~ Wildl. Serv.. Circ. 92, 48-51.

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M T l~)lo. I11 and D. F. NI~RT~\

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