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INFLUENCE OF HUMIC SUBSTANCES ON THE GROWTH OF MARINE PHYTOPLANKTON: DINOFLAGELLATES A. Prakash and M. A. Rashid Fisheries Research Board Bedford of Canada and Atlantic Oceanographic Institute, Dartmouth, Nova Scotia Laboratory, ABSTRACT Humic substances, in small amounts, exert a stimulatory effect on marine dinoflagellates that is reflected in increased yield, growth rate, and ‘“C uptake. Humic acid was found to be more active than fulvic acid; in both cases the growth responses were dependent on concentration. Of the various fractions isolated, the low molecular weight fractions of humic acid produced the greatest growth response in unialgal cultures of dinoflagellates. The positive effect of humic substances on phytoplankton growth is, for the most part, independent of nutrient concentration and cannot be attributed entirely to chelation processes. It appears that growth enhancement in the presence of humic substances is linked with stimulation of algal cell metabolism. Because of their high concentrations in coastal waters, humic substances maythus be regarded as an ecologically significant entity influencing phytoplanktonic prod&ion. INTRODUCTION tivity of soil extract has been attributed primarily to the chelating action of the humic component, resulting in a reduction of the toxicity or an increase in the availability of trace metals. Soil extract may also be a source of vitamins, auxins, and other micronutrients. Sweeney ( 1954) was able to replace soil extract with vitamin B12 and cthylenediaminetetraacetate (EDTA) which was added to aged seawater. In several of the completely synthetic media developed at the Haskins Laboratories, New York, and at Millport, Scotland, soil extract has been entirely replaced by organic additives (Provasoli et al. 1957). We have used one such medium, ASPP ( Provasoli 1963a), for growing several species of armored dinoflagellates of the genus Gonyaulax, but seawater medium ( Erd-Schreibcr) enriched with soil cxtract has always supported better growth ( Prakash 1967). In a study of the nutritional characteristics of Gonyaulax tamarensis of the Bay of Fundy, natural seawater with high humic content was able to support reasonable growth of this dinoflagellate without further enrichment. Similarly, yellow humusladen river water added to artificial seawater gave a positive growth response for this and several other species of dinoflagellates. Wilson and Collier ( 19%) Instiobserved an improvement in growth of 598 Our knowledge of the growth requirements and ecological interactions of marine dinoflagellates, which show significant seasonal pulses in coastal waters, is fairly restricted. Dinoflagellate maxima in temperate waters usually occur towards the end of the spring diatom bloom when major nutrients (N, P, and Si) are in low concentrations. However, the prodigious growths of dinoflagellates leading to red-water conditions have not been convincingly explained. Relatively few species of marine dinoflagellates have been brought into unialgal and bacteria-free cultures; many of the successful media have soil extract as their common ingredient. While a few dinoflagellates can apparently be grown in inorganic medium, most marine dinoflagellates seem to require some organic growth factors apparently supplied by soil extract. The history of the development of completely artificial seawater media is essentially the history of the elimination of soil extract and its replacement with vitamins, chelators, trace metals, and other organic additives. This aspect has been adequately reviewed by Provasoli, McLaughlin, and Droop ( 1957). The growth-promoting ac1 Contribution tu tc. No. 102 from the Bedford EFFECT OF HUMIC SUBSTANCES Gymnodinium brevis in a seawater medium to which water from a Florida river was added, but a much better growth occurred when both river water and extract of peat soil were added. Since substantial amounts of humic matter enter the inshore waters of the Bay of Fundy from land drainage just before the onset of the dinoflagellate maximum, it appeared that runoff was providing natural soil extract which stimulated the growth of the dinoflagellates. WC have examined the nature of these substances and their influence on the growth of marine dinoflagellates. THE ORIGIN AND SUBSTANCES NATURE IN OF HUMIC ON 599 DINOFLAGELLATES uble in water; however, recent evidence shows that certain low molecular weight fractions of it may be ( Flaig 1960). Fulvic acid is less complex in structure and more soluble in water than humic acid and shows fluorescence in UV light. Humus present in the dissolved state in natural waters and aqueous soil extracts is largely composed of fulvo-hu.mic fractions that are biologacid is an ically active. Hymatomelonic alcohol-soluble fraction of humic acid and is a simpl.er form ( Kukharcnko 1948). Some workers regard fulvic, hymatomelonic, and humic acids as related compounds and not as distinct chemical entities (Thiele and Kettncr 1953). SEAWATER The formation of humic substances rcsults from the biochemical transformation of plant and animal tissues incorporated in soils or sediments. Lignins, proteins, and carbohydrates are apparently first broken down by microorganisms into simpler substances which are then resynthesized into the more complex molecules of humic matter. The humic material found in coastal waters is primarily of terrestrial origin and is present in both dissolved and colloidal states, imparting a yellowish-brown color to the seawater. Planktonic decomposition products referred to as “gelbstoff” by Kalle (1966) and as “water humus” by Skopintsev ( 1959) are also a source of yellowish color in seawater and appear to have characteristics similar to those of humic compounds. Based on their solubility characteristics and also on their molecular configurations, humic substances derived from soil organic matter have been classified into four main groups : humins and ulmins, humic, fulvic, and hymatomelonic acids (Kononova 1966). Because of the hetcrogenous and dynamic nature of these compounds, their properties have not been defined precisely, Since humins are insoluble, only humic, fulvic, and hymatomclonic acids can be rcgardcd as biologically utilizable by phytoplankton. It is also unlikely that humic substances present in the colloidal state in seawater are efficiently utilized by algal cells. It is generally held that humic acid is not sol- MATERIAL Culture AND METHODS conditions Bacterized and bacteria-free unialgal cultures of G. tamarensis (Bay of Fundy and Plymouth strains), G. catenekza, G. acatenella, and G. monilata were grown in 125-ml capacity screw-cap erlenmcyer flasks containing 100 ml of medium. All cultures except G. monilata were maintained in a constant temperature room at 10 * 1C under constant overhead illumination of 3,000-4,000 lux from banks of 40-w “cool white” fluorescent lamps. G. moniZata was grown in an illuminated incubator at 25C. Test cultures were maintained in a marine synthetic medium ESWA (Table 1) as well as in artificial seawater ( ASW) that was enriched with only humic additives. The culture medium was sterilized by filtration through 0.22-p Millipore filters, and all1 glassware was autoclaved at 1.02 atm for 15 min. All humic additives were filter-sterilized before introduction into tither ASW or ESWA. For growth experiments, an inoculum was usually taken when the parent culture had reached the stationary phase of growth. Growth responses to added humic compounds were measured as changes in ccl1 numbers at frequent intcrvals throughout the growth phase. Cell counts were made using a sedimentation method, as well as with a Coultcr counter (model B ) . Growth was expressed in 600 A. TABLE 1. I. Artificial Parsons Preparation PRAKASII of marine synthetic ESWA seawater ( AS W )-Strickland ( 1960) Salinity 31%0 NaCl 2.2 g MgCI,~6HzO 9.7 g NazSOa 3.7 g CaCL 1.0 g KC1 0.65 g NaHC03 0.17 g 0.02 g HaI Distilled water to 1 liter 2. ES enrichment-Provasoli Hz0 NaNOs Naz* glycero PO, Fe EDTA P II metals* * . Vitamin BU Thiamine Biotin Tris buffer pH 7.8 AND medium * One (as Cl-), A. RASHID TABLE 2. Growth response of Gonyaulax arensis in different media with and without acid enrichment tamhumic and (unpublished) 100 ml 350 mg 50 mg 2.5 mg 25 ml 10 /-e 0.5 mg 5.0 I-G 500 mg 3. Final concentration: 20 ml of ES enrichment to each liter of charcoal treated ASW 0.2 mg; M. ml of P II metal mixture contains: B (as H,BO,), Fe (as Cl-), 0.01 mg; Mn (as Cl-), 0.04 mg; Zn 5 pg; Co (as Cl-), 1 pg; Na,-EDTA, 1 mg. terms of either relative yield or relative growth constant (K) and mean generation time ( tg ) represented by the expressions: 1 N2 Ic = ( t2 - tl> log2 xq and t, = l/K, where N1 and N2 are cell numbers at times tl and t2. The response of dinoflagellates to humic additives was also determined by 14C incorporation after various periods of cxposure to the isotope. One ml of 14C labeled Na2EIC03 solution containing 1 &i of activity was added to each test culture and incubated from l-24 hr under constant temperature and continuous light. The activity was measured with a thin window gas-flow counter (Baird Atomic, model FC-1) . Corrections for dark assimilation of 14C were made in all experiments. Medium Final yield (cells /ml X 103) ASW no growth ASW (autoclaved) 2.1 ASW + humic acid (5 a/ml) ESWA 4:; ESWA + humic acid (5 b&ml) 14.0 Growth constant &&days)-1 Mean gcncration time tu(hr) 0.062 117 0.087 0.108 82 67 0.154 46 Isolation and purification of humic substances The soil used for extraction of humic substances was obtained from wooded areas of New Brunswick and Nova Scotia, and samples of marine sediment were collected from Cow Pen Basin on the Scotian Shelf at a depth of 198 m. The procedure used for the extraction and purification of fulvic and humic acid fractions is described by King ( 1967). The hymatomelonic acid was extracted according to the method suggested by Kononova ( 1966). The humic substances dissolved in the river water and in aqueous extracts of soil were recovered by evaporation under reduced pressure. Concentration of humic substances was estimated by evaporating to dryness and weighing known volumes of purified extracts. Molecular weight measurements and fractionation of humic acids The molecular weight measurements of humic acids extracted from soil were made by the Sephadex gel-filtration tcchniquc described by Mehta, Dubach, and Deuel ( 1963) and modified by Rashid and King ( 1968). Fractionation of humic acid based on molecular weight measurements was accomplished by the same technique using Sephadex gel G-10, G-15, G-25, and G-50. The humic acid excluded from the lowest grade gel was concentrated and introduced to the next higher grade gel and so on. The fraction of humic acid absorbed on EFFECT OF Gonyaulax HUMIC SUBSTANCES ON 601 DINOFLAGELLATES tamarensis 32 pg/ml 13 pg/ml 6pg/ml Gonyaulax catenella 0,105 pg/ml HUMIC ACID FIG. 1. Response of Gonyaulux tamarensis (13 days growth) and G. cuter&a (6 days growth) to various concentrations of humic acid. Cultures grown under identical conditions of light and temperature. Medium was ASW with different concentrations of filter-sterilized humic acid extractcd from marine scdimcnt. each gel was elutcd with a Tris buffer solution of 0.5 M concentration. Molecular weight measurements of humic substances dissolved in aqueous soil extract and river water were made in the same manner. RESULTS Growth responses to humic acid enrichment Test cultures of G. tamarensis were grown in ASW and ESWA with and without the addition of the humic acid fraction of soil humus. The growth responses are shown in Table 2. In media devoid of humic acid, the yield as well as growth rates of dinoflagellates tended to be approximately 30-50% lower than in the enhanceenriched media. A significant ment in growth was also seen when ASW, which by itself is incapable of supporting any growth, was autoclaved at 1.02 atm for 15 min and used as a culture medium. The reason for this rcsponsc is not clearly understood, but it has been observed by others (Pramcr, Carlucci, and Scarpino 1963; Jones 1967). Therefore, we have used only filter-sterilized ASW for subsequent enrichment cxperimcnts. Growth was also enhanced when yellow humic water from some Nova Scotia and New Brunswick rivers (avg humic acid ‘\ 5 IO DAYS \ I I5 AFTER 20 I 25 I 30 INOCULATION FIG. 2. [Growth of Gonyacdax tamarensis ASW enriched with different concentrations humic acid extracted from marine sediment. in of concn, 22 pg/ml) was added to ASW. Dinoflagellates grew reasonably well in ASW medium diluted up to 50% with yellow river water, until the change in salinity became too great to permit growth. Controls with charcoal-treated river water failed to show any growth at all. Dosage effect Responses of G. tamarensis and G. catenella to various concentrations of humic acid are shown as dosage-yield curves (Fig. 1). Similar curves have been obtaincd for other species. At concentrations less than 2 pg/ml of humic acid in the medium, the growth was erratic and the cells tended to be sluggish, enlarged, and with a high incidcncc of aberrant forms. On the other hand, at concentrations over 35 pg/ml, a definite decrease in growth rate as well as yield was noticed. Another aspect of the response of dinoflagellates to concentrations of humic acid is seen in Fig. 2. The cultures of G. tamarensis grown with 6-32 lug/ml of humic acid in the media showed practically no change in rate during the exponential phase of growth, as is evident from identical values for K and ts. However, they differed considerably in final yields, which were approximately proportional to the initial concentrations of humic acid in the 602 A. PRAKASH AND M. A. RASIIID TABLE 3. Rc~sponse of Gonyaulax tamarensis to various concentrutions of humic acid as measured by ‘“C assimilution PHOSPHATE Humic acid concn b.WmU &z-z~=-- Incubation 4 hr period 6 8 hr (counts/min) hr r-a / NITRATE t I 10-S 10-4 I 10-3 t 10-Z I 10-I CONCENTRATION I I A- Without 0- With HUMIC HUMIC ACID 7 14 21 28 430 500 1,220 550’ 570 630 1,340 770 870 1,530 680 920 ACID t IO - mg /ml FIG. 3. Cell yields at different levels of N and P, with and without 7 PgIml of humic acid. culture media. Both K and final yields tended to be significantly reduced in cultures grown in humic acid concentrations over 32 pg/ml and under 6 pg/ml. These observations suggest that the difference in yield under identical K values are related to differences in duration of the exponential as well as of the lag phase of growth and are brought about by the presence in the media of differing amounts of humic matter. Uptake of IJC A definite increase in the rate of 14C assimilation by G. tamarensis was noticcable when soil humic acid was added to ESWA. Dinoflagellates were exposed to several concentrations of humic acid and the radiocarbon uptake measured over 2-8 hr incubation (Table 3). As indicated by counts per minute, the rate of 14C assimilation increased with increasing concentration of humic acid, but at concentrations over 21 pg/ml, it tended to be considerably reduced. This inhibition may bc due to selective absorption of light by the yellow of the humic acid, resulting in rcduccd photosynthesis. In our growth experiments discussed earlier, inhibition was noticed only after the concentration of humic acid in the medium reached over 35 pg/ml, whereas in 14C experiments, the inhibition was detected at the 21 pg/ml level. This inconsistency can be attributed partly to the difference between photosynthesis and growth and partly to the different properties of humic acids derived from marine sediments and from forest soil. Relative responses to fulvic, humic, and hymatomelonic acids Growth responses to differential enrichment of ASW with humic, fulvic, and hymatomelonic acids, as measured by cell yields, growth constants, and generation times are shown in Table 4. Growth responses to fulvic acid enrichment were also dependent on concentration, but for the same concentrations, humic acid gave approximately twice as much growth as TABLE 4. melonic Effect of humic, fulvic, and hymutoacids enrichment on the growth of Gonyaulax tamarensis Concn bglml) Humic Fulvic acid acid IIymatomelonic 14.1 ;::g (2!,s 1,179 7.0 1,086 3.5 837 7.5 602 3m.7 451 0.9 283 10.5 5.3 K&lay+-1 0.10 0.10 0.08 0,.07 0.05 0.02 no appreciable no appreciable tub9 72.4 72.61 90.3’ 103.2 144.5 361.2 growth growth EFFECT OF HUMIC SUBSTANCES FIG. 4. Growth response of dinoflagellates media at 4 &ml concentration. N Earlier studies on the growth of G. tamarensis indicated specific requirements for nitrate, vitamin B12, and thiamine (Prakash 1967). H owever, the possibility that the growth stimulating effect of humic compounds on dinoflagellates is due to the presence of vitamin B12 and thiamine in humic fractions can bc ruled out because the extraction and purification procedures used to obtain these fractions would result in destruction of these vitamins. In order to examinc the possibility that the growth enhancement was due to increased nitrogen and phosphorus contribution from humic material, test cultures of G. tamarensis were grown in ESWA with various concentrations of sodium nitrate ,and sodium glycerophosphate for 32 and 11 days respectively. The yield increased with the increase in N and P levels, until 603 DINOFLAGELLATES to the various fulvic acid. In media enriched with hymatomclonic acid, dinoflagellates remained viable only for two days and no growth response could be detcctcd. Effect on yield at different and P levels ON fractions of humic acid added to the at high concentrations an inhibitory effect became apparent. In cultures with humic acid cnrichmcnt, although the response curves followed the same trend as the controls, the yields for a given concentration of N and P were considerably higher ( Fig, 3). These experiments suggest that the growth response of dinoflagellates in the presence of humic acid is to a large extent independent of the N and I? levels in the medium. Humic compounds do not appear to be contributing significant amounts of N and P to the organisms as our fulvic and humic acid fractions never contained more than 3.4% of nitrogen and 1.2% of phosphorus. Growth responses to different molecular weight fractions Although humic fractions of soil humus arc generally regarded as compounds of high molecular weight, there is wide variation in the molecular weight distribution and this variation is related in some measure to the soil type. In our samples, humic acid extracted from marinc sediment was predominantly represented by fractions 604 A. PRAKASH AND TABLE 5. Moleculur weight distribution of humic acids extracted from various sources --~Soil Mol wt range <700 700-1,500 1,500-5,000 5,000-10,000 >10,000 extract (%) River water (%) Forest soil (%I Marine sediment (o/o) 43.8 56.2 - 27.4 7.3 65.3 - 33.2 7.7 11.9 47.2 - 6.5 5.0 5.6, 9.9 73.0 ( aqueous ) having molecular weights over 10,000, whereas humic acid cxtractcd from forest soil was devoid of any fraction over 10,000. Water soluble humic acids rccovcred from soil extract and river water were represented almost exclusively by fractions lower than 5,000 (Table 5). To determine the relative effectiveness in growth stimulation of each of these fractions, cultures of three species of Gonyaulax were grown separately in ESWA and ASW under identical enrichment conditions with different molecular weight fractions added (Fig. 4). Although the three spccics responded somewhat differcntly, in general the greatest response was found in the lower molecular weight fractions. DISCUSSION While the role of humic matter in the development and growth of terrestrial plants has been the subject of fairly extensive study, comparable studies on marine algae are almost nonexistent. That humic substances may have some stimulating effect on the phytoplankton production in the sea has been suggested by a number of authors (Strickland 1965; Provasoli 1963b), but there is little direct evidence available. In his study on a marine diatom Skeletonema costatum, Droop ( 1962) found that its growth was enhanced when humic acid was added to the medium and that humic fractions were more active than fulvic fractions. Our results arc in general agreement with his obscrva tions except that while he found the maximum response with the high molecular weight fraction of humic acid, the maximum activity in our cultures was observed with low M. A. RASHID molecular weight fractions. Although the evidence for diatoms is limited, there arc grounds for believing that humic substances stimulate growth oE both diatoms and dinoflagellates and are thus of potential ecological significance in phytoplankton productivity. The growth-promoting activity of humic substances in our experiments cannot be accounted for in terms of vitamin B12 or thiamine, as the tcchniqucs used to extract humic and fulvic acids from soil humus result in complete destruction of these vitamins. Nor can the growth enhancement in our cultures be attributed entirely to Fe and other trace metal concentrations in the humic additives, as most metallic cations originally present in the humic fractions were removed in exchange resin columns; the ash content was rcduccd to less than 3%. The N and P contribution from humic compounds also has limited effect, since increasing N and P beyond a certain concentration in the medium did not proportionately increase the growth of the dinoflagellates. On the contrary, while at high concentrations of N and P, both the growth rate and the total yield decreased, our results suggest that humic acid is capable of removing the negative effect of high dosage of N and P. The identical phenomena have been reported by Chaminade (1958) who grew rye grass in various concentrations of nitrogen with and without sodium humate. Since the positive growth response of marine phytoplankton in the presence of humic substances cannot be adequately explained on the basis of N, P, trace metals, and vitamin requirements alone, it appears that other processes are involved. The most plausible suggestion is that humic and fulvic components of soil humus, in small amounts, act as stimulants for marine algal cells and may be involved in cellular metabolic processes in the same manner that has been suggested for terrestrial plants [ Prozorovskaya ( cited in Kononova 1966) ; Khristeva 1951; Chaminade 19561. However, the mechanism underlying the physiological stimulation by humic sub- EFFECT OF IIUMIC SUBSTANCES ON DINOFLAGELLATES stances and the pathways leading to growth enhancement are little undcrs tood. A limited amount of experimental work suggests that humic substances act as specific sensitizing agents enhancing the permeability of the plant cell membrane and thereby increasing the uptake of nutrients from the surrounding medium. IIumic compounds may also act as respiratory catalysts since oxygen is absorbed more intensively by plant tissue in the presence of humic acid (Biber and Magaziner 1951; Khristeva 1953; Smidova 1960). Prjt and Pospisil ( 1959) and P&t, Smidova, and Cincerova ( 1961) using 14C-labeled humic acid showed that both humic and fulvic acids are capable of penetrating the plant cell, but the latter is more efficient bccausc of its smaller molecular size. Since coastal waters rcceivc significant quantities of humic substances and other nutrients as a result of river discharge <and runoff, it is reasonable to assume that these substances may influence the production and succession of phytoplankton species. The ecological significance of humic substances in coastal waters might not be rcstrictcd to stimulation of processes within the algal cell. Chelation could also be an important factor; De Kock (1955) showed that iron became readily available to the plant cell in the presence of humic acid and stimulated growth, and Gran (1933) linked the seasonal abundance of phytoplankton in the Gulf of Maine with iron concentration, nutrients, and humic compounds washed from the shore. Apparently, algal excretion and decomposition products are biologically similar to terrestrial humic substances, Nordli ( 1957) found that aqueous extracts of benthic algae stimulated growth of a number of marinc dinoflagellates. In one expcrimcnt, we found that decomposition products of Nitzschia stimulated growth of Gonyaulax, and the growth response was conccntration dependent ( Prakash, unpublished ) , Yentsch and Reichert (1962) observed an increase in oxygen uptake by bacteria when yellow filtrate from algal cultures was added to raw seawater. 605 The stimulatory cffcct of freshwater runoff on dinoflagellate production in coastal waters could be due to two indcpendcnt mechanisms. In addition to bringing in ecologically significant amounts of humic substances, the freshwater runoff creates low salinity conditions which favor faster division rates among marine dinoflagellates (Braarud 1951; Prakash 1967). 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