J. Exp. Mar. Biol. Ecol., 154 (1991) 77-96 77 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-0981/91/$03.50 JEMBE 01685 Photosynthesis and growth of Nitzschia pungens f. multiseries Hasle, a neurotoxin producing diatom Youlian Pan ~, D u r v a s u l a V. Subba R a o 2 and Roderick E. W a m o c k 2 Ilnstitute of Oceanology, Academia Sinica, Qingdao, People's Republic of China; 2Biological Sciences Branch, Department of Fisheries at~d Oceans, Bedford Institute of Oceanography, Dartmouth, Nova Scotia, Canada (Received 23 April 1991; revision received 5 August 1991; accepted 10 August 1991) Abstract: The pennate diatom Nitzschia pungens f. multiseries Hasle implicated in the amnesic shellfish poisoning in bays of eastern Prince Edward Island, produces a neurotoxin, domoic acid. Batch cultures of this diatom were grown at photosynthet,_'¢ photon flux densities (PPFD) of 1100 (high light, HL) and 105 (low light, LL) #mol m -2 s-~ at 10 °C. The relationships between photosynthesis and PPFD were established on Days 1,4, 8, 15, 25 and 34 and were quantitatively described. Maximum specific growth rates (#max) for the HL and LL cultures were 0.74 and 0.56 d a y - ~ respectively. Cells grown ~t HL had higher assimilation number (Pmn: 2.2 #g C [#g Chl a] - ~ h - ~) but lower initial slopes (gn: 6.06 ng C [#g Chl a] I1- ! [#mol m - 2 s- t] --l)than those grown at LL (ProS: 1.7 #g C [#g Chl a] - i h - ~; 0ta: 8.69 ng C [#g Chl a] - i h - ~ [#tool m - 2 s - ~] - ~). Both PmB and 0tn increased from Day 1 to Day 4, and then decreased as the cells aged. Temporal variations in the rate of growth, cellular Chl a, cell concentration, carbon and nitrogen showed differences in their magnitude and lag phase. Key words: Age; Batch culture; Culture age; Growth; High light; Low light; Mass balance; Nitzschia pungens f. muitiseries; Photosynthetic photon flux density (PPFD) INTRODUCTION Formation ofmonospecific blooms of marine diatoms is rarely known, as for example, Asterionella japonica (Subba Rao, 1969), and production of any phycotoxin by such diatom blooms was unknewn until 1987. Blooms of the pennate diatom Nitzschia pungens f. mukiseries are unique because of the production of a neurotoxin, domoic acid, the first toxin reported for any diatom (Subba Rao et al., 1988). Also, since 1987, massive fall-winter blooms of this diatom have been associated with an annual outbreak of amnesic shellfish poisoning (ASP) in Mytilus edulis L. which is cultured in Cardigan Bay, eastern Prince Edward Island (PEI) (Subba Rao et al., 1988; Bates et al., 1989; Addison & Stewart, 1989). Although the occurrence of domoic acid was initially limited to eastern PEI, there is now evidence for its occurrence at low levels at coastal sites in Nova Scotia and New Brunswick as well as on Georges Bank (Gilgan et al., 1990). This Correspondence address: D.V. Subba Rao, Biological Sciences Branch, Department of Fisheries and Oceans, Bedford Institute of Oceanography, PO Box 1006, Dartmouth, Nova Scotia B2Y 4A2, Canada. 78 Y. PAN ET AL. is not surprising considering the ubiquitous distribution of the causative diatom N. pungens f. multiseries (Subba Rao & Wohlgeschaffen, 1990). The resulting serious commercial losses to the aquaculture industry and the loss of human lives due to the eastern PEi incident in i987 prompted us to carry out physiological ecology studies, similar to those on the toxic dinoflagellates Alexandrium tamarensis (Anderson, 1990), Dinophysis norvegica (Dahl & Yndestad, 1985) and D. accuminata (Sampayo et ai., 1990). In the Canadian Atlantic, especially in the Bay of Fundy, outbreaks of paralytic shellfish poisoning (PS P) have been an annual event restricted to summer (White, 1989) in contrast to the domoic acid problem which occurs in fall and winter. Photosynthesis and growth are the two basic aspects in the overall physiology of phytoplankton. A study ofthe relationship of photosynthesis and photosynthetic photon flux density (PPFD, I) is essential to understand the physiological ecology of algae. In both experimental and theoretical analyses of primary production of algae, several investigators (Steele, 1962; Platt et al., 1975, 1980; and others) utilized the curvilinear plots which relate the photosynthetic rates to the incident PPFD. Usually, Chl a and particulate carbon are used as indices of biomass for normalizing photosynthetic rates. Encouraged by our earlier studies (Platt & Subba Rao, 1970) we employed batch cultures of N. pungens f. multiseries as analogues of natural blooms to understand the physiological ecology of this toxin producing diatom. In this paper, we report variations in measured and derived P - I parameters of N. pungens f. multiseries grown under two levels of PPFD, at different phases of growth and the data are compared with other pennate diatoms, winter algal blooms and toxic red water blooms. MATERIALS AND METHODS Nonaxenic clonal batch cultures of N. pungens f. multiseries isolated from Cardigan Bay, PEI were grown in medium FE (Subba Rao et al., 1988), at 10-12 °C under continuous cool white fluorescent light of 105 and 1100 #mol m - z s- 1. Cultures were routinely grown at these two levels of PPFD and were subcultured every 5 days into fresh medium for 10 days. For stock cultures, 500-ml flasks each containing 200 ml FE medium were seeded with 25 ml of inoculum. At the beginning of the experiments, 3-1 Fernbach flasks containing 2 1of FE medium were seeded with 200 ml of exponentially growing (5 days old) N. pungens f. multiseries adapted to the experimental PPFD which was measured with a LICOR model Li-185B light meter. Cultures grown at 105 and 1100 #mol m - 2 s-~ were designated as low light (LL) and high light (HL) cultures respectively. Photosynthesis-PPFD (P-l) relationship was determined using the ~4C method (Steemann-Nielsen, 1952) for cells harvested after 1, 4, 8, 15, 25, and 34 days growth at 10°C. High specific activity ~4C-HCOf (111-222 C k Bq ml- ~ dependent on cell density) was added to ~, 65 ml culture and after mixing thoroughly, 1-ml aliquots of the culture were dispensed into 48 clean glass vials in a photosynthetron incubator and PHOTOSYNTHESIS AND GROWTH OF NITZSCHIA PUNGENS 79 incubated for 30 min at different PPFDs ranging from 11 to 5000 #mol m - 2 s - 1 (Lewis et al., 1985). Added activities were determined by adding 5 #1 of ~4C-labelled incubation medium into 5 ml Aquasol scintillation fluid containing 10 #1 6N NaOH solution. The incubation was terminated by adding 250 #l 6N HCI. The vials were shaken for > 1 h to remove the residual HCO3- (Li & Goldman, 1981). A Beckman two channel scintillation counter was used to determine the isotope activities. The relationship between photosynthesis (P: #g C [#g Chl a ] - ~ h - ~ or #g C [#g C ] - ~ h - ~) and PPFD (I" #mol m - 2 s - ! ) could be described by the photoinhibition model of Platt et al. (1980) with the addition of a single parameter: pB = [P~(1 - exp(-~BZ/Pp))exp(-flBl/p~)] + p~ (1) In this formulation, P~ is the maximum potential photosynthesis in the absence of photoinhibition; ~B (#g C [#g Chl a] - ~h - ~ [#mol m - 2 s - ~] - t or #g C [#g C] - ~h [#mol m - 2 s - ~ ] - a ) is the initial slope of P-I curve, that is photon efficiency, i.e., photosynthesis per unit PPFD, fib (same units as ~a) is the photoinhibition index and Pda (same units as P) is the intercept of the P-I curve on the y axis. The additional parameter Pda was found to be necessary to account for a background uptake of ~4C in all samples. P~, P dB, ~B, fib were calculated by fitting Eqn. 1 into experimental data points by non-linear regression using a commercial package employing the Marquardt algorithm (Marquardt, 1963). All the points were equally weighted. The derived parameters P~, Im, Ik, Is, were calculated from P~, ~B, fib following the relationship among these parameters suggested by Platt et al. (1980). Pma, the maximum biomass normalized photosynthetic rates: ( "B ) ( fib '~flll/~li ~B + ~B (2) lm, the PPFD corresponding to the maximum photosynthetic rate Pma: im PB=s ln(~B+flB) 0£B fiB (3) 4, = (4) Ik, photo-adaptive index: Is, the P P F D corresponding to the maximumpotential photosynthetic rate in the absence of photoinhibition: ts = es/ B B (5) where, Ira, Ik, Is have the same units as (#mol m - 2 s - i ) . Total carbon dioxide in the cultures was calculated from alkalinity determinations (Strickland & Parsons, 1972) based on salinity determined with Guildline Salinometer and pH with an Orion Research microprocessor ion-analyzer 901 at room temperature (20 °C). Inorganic nitrate, phosphate and silicate concentrations in the medium were measured with a Technicon Autoanalyzer II (Strickland & Parsons, 1972). 80 Y. PAN ET AL. Biomass samples of the cultures were taken on 1, 4, 8, 15, 25 and 34 days. Cells were enumerated on an l-ml aliquot settled in plankton chambers and counted using an inverted microscope. Chl a determinations were based on duplicate samples employing the fluorometric method (Strickland & Parsons, 1972), whereby cells retained on 25-mm G F / F filters from 10-ml samples of cultures were transferred into vials containing 10 ml of 90% acetone and refrigerated at 0 °C for 24-36 h before analysis. Particulate carbon and nitrogen were analyzed using Perkin-Elmer 240B C H N Elemental Analyzer. Cells from 40-ml samples of culture retained on 25-mm G F / F filters were dried at 60 °C overnight and then rolled in silver filters for combustion (Strickland & Parsons, 1972). The growth ofN. pungens f. multiseries in batch culture was found to be well described by the growth model of Gompertz (Zwietering et al., 1990). When growth is defined in terms of the logarithm of cell concentrations (or other biomass index) as a function of time, changes in growth result in a sigmoidal curve such that there is a lag phase at beginning (t - 0), tbllowed by an exponential phase ofgrowth and finally by a stationary phase. The Gompertz equation may be expressed as: y = a e x p ( - exp(b - ct)) (6) where y = In (N/No), N is the cell concentration at different time of gro~eth, No is the initial cell concentration, t is the time and the parameters a, b and c describe the shape of the curve. Parameter a describes the asymptote (ln(N~/No), the maximum value of y reached. The maximum growth rate (#m,,x) is given by ac/e, where e is the base of Naperian logarithms (~2.718). The duration of the lag phase is given by ( b - l)/c (Zwietering et ai., 1990). Growth curves as a function of time, normalized to cell concentration and other biomass indices, were fitted by nonlinear regression. Growth rates throughout the culture experiments were computed from the derivative of Eqn. 6 with respect to time: dy/dt = ac e x p [ - exp(b - ct)] exp(b - ct) (7) using the parameter estimates obtained from nonlinear regression of Eqn. 6. RESULTS BIOMASS INDICES Cell concentrations (in both low light (LL) and high light (HL) cultures) increased exponentially for 8 days attaining a maximum concentration of 1.41-1.56 x 10s cells 1-~ by the 25th day (Fig. 1A). Subsequently, there was no marked change in cell concentration in the stationary phase. PHOTOSYNTHESIS AND GROWTH OF NITZSCHIA PUNGENS ,,#/,"'"° 10 8 I- .T 10 81 ~~ concentration A: Cell cJ 1 08 I I I I I I [ zx ,........-z~.............................................. ~ ......................-~, T - !00 ¢1 B: Chlorophyll a 10 I T I X I 1 X I000 - o E / """ "l 1 O0 ~ Particulat: carbon ""1 . I ......................... " '~ ~ I ....................... " .2 100 - 7 i--i z .~~ o E 10 0 D: Particulate nitrogen I 1 I I I I 5 I0 I5 20 25 30 35 Culture age (days) Fig. I. Growth curves based on different indices of biomass. Solid circles are experimental data for HL culture, open triangles are experimental data for LL culture, solid lines are modelled values for HL cultu.,'e, broken lines are modelled values, for LL culture (see text). 82 Y. PAN ET AL. Chl a levels (Fig. 1B) were initially low (2.4 #g 1- ~in HL culture), despite the similar cell concentrations in both HL and LL cultures (Fig. 1A). The HL cells contained less Chl a than LL cells throughout the experiments (Fig. IB). As with cell concentration, Chl a concentrations increased in a sigmoidal pattern with increasing age of the culture. Maximum Chl a concentrations attained were 40.2 #g 1 - ' in the HL cukure and 103.3 #g 1- ' in the LL culture. A sigmoidal increase in both particulate carbon and nitrogen over time also occurred at both levels of PPFD (Fig. 1C,D). The principal features of interest are: (i)the maximum concentrations of carbon in both HL and LL cultures were very close (Fig. 1C); so were the maximum concentrations of nitrogen (Fig. ID); (ii) accumulation of carbon, however, seemed to be more rapid than that of nitrogen (Fig. 1C,D); and (iii) the lag phase for the accumulation of nitrogen seemed to be longer in HL cells (Fig. 1D). Of the three macronutrients, P O 4 and N O 2 + NO3 were abundant over the entire 34 days of growth (Table I). However, the initial levels of SiOa (84-86 #M) were reduced to 11 and 8% in the HL and LL cultures, respectively, by Day 8 due to utilization by diatoms for frustule formation. This feature corresponds with the onset of the stationary phase. The decrease in silicate concentrations (Table I) between Days 1 and 15 was 72/aM (HL) and 73 #M (LL) and the corresponding increase in cell concentrations was 1.0 x 10s cells 1 - ' and 1.14 x 10s cells 1-' (Fig. 1). Utilizing a calculated cellular silicon of 20.2 pg cell- ' (HL) and 15.5 pg cell- t (LL), it is concluded that the residual silicate (2.1-3.2/~M) available by Day 15 would be insufficient for further division of cells. Using Eqn. 7, growth rates (#) were calculated based on all indices of biomass. However, because of the scarcity of data on the exponential phase, caution should be TABLE I Nutrient concentrations (#M) in culture media throughout experiment. PPFD (#mol in - 2 s - ' ) Growth time (day) SiO3 PO4 NO2 +NO3 1100 1 4 8 15 25 34 75.30 49.30 9.26 3.19 3.72 5.64 102.0 53.3 63.4 47.3 59.1 80.4 3028 3001 2961 2813 2892 2770 105 1 4 8 15 25 34 75.60 55.80 6.06 2.05 9.25 6.42 101.0 77.3 62.9 70.4 89.7 84.9 3085 2941 3032 2987 2937 2969 PHOTOSYNTHESIS AND GROWTH OF NITZSCHIA PUNGENS 83 applied when interpreting these values. The maximum growth rates (#max) based on cell concentrations calculated from the derivative of Eqn. 6 for the HL culture was 0.56 day- ~with a lag phase of 1.07 days. The corresponding values for LL culture were 0.74 day- ~ and 1.85 days (Fig. 2A). C ,8 -°,°°°°-..% 0.6 ,, % ., -- 0.40.T2 ",,, A: Cell concentration "", 0.0 1.2 B: Chl r o p h y l l 0.9 a 0.6I 0.3- "-'/' 0.0 l ............. • ', I-, "=_j 0.4 -h .: O ""',, C: C a r b o n ,j 0.2 0.4/ 0.3+ 0.2± 0.0 , ', I i • • 0.1t- "" .°- 0.0 0 3 6 Culture 9 12 5 age (days) Fig. 2. Modelled growth rates (#, day- i)of different indices ofbiomass for first 15 days of culture. Solid lines are of HL cultures, broken lines are of LL cultures. 84 Y. P A N E T AL. Similar time-dependent variations in the accumulation rate of Chl a, particulate carbon and nitrogen were observed. In the case of Chl a, the ,//max was 0.95 (LL) and 1.34 (HL) day- ' (Fig. 2B). The maximum growth rates based on Chl a was much higher than those based on nitrogen. It occurred much earlier (Fig. 2B) compared to those calculated for other biomass indices (Fig. 2A,C,D). The ~max for nitrogen was 0.2-0.3 (Fig. 2D), which was the lowest and the lag phase was the longest compared to the others (Figs. 1D, 2D). During the early stages of growth, Chl a:Cell, Chl a:C and 500 400 A: C a r b o n cJ 300 200 100 0 ~,00 w i i i • I l Ix I I T L 80i. " B: N"t t r o g e n ~ 60- o Z ~020. . . . . . . . . . -. . . . . . . . . . i . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . - ~ o I I I I I t /\ T C" C h l o r o p h y l l a O 6' L) ", 21 0 0 I i I I [ I 5 10 15 20 25 30 .35 Culture age (days) Fig. 3. Variations with time ofcarbon (A), nitrogen (B), and Chl a (C) per cell. Solid circles are experimental data for HL culture, open triangles are experimental data for LL culture, solid lines are modelled values for HL culture, broken lines are modelled values for LL culture. PHOTOSYNTHESIS AND GROWTH OF NITZSCHIAPUNGENS 85 Chl a:N were all increasing. Ratios observed in the biomass indices seem to change systematically with the age of the culture. Cellular carbon and nitrogen were at their maximum on Day 1 and drastically decreased by Day 4 but exhibited no marked differences between IlL and LL cells (Fig. 3A,B). Carbon:Chl a followed the same pattern of decrease but was consistently lower in LL than in HL cells (Fig. 4A). However, cellular Chl a in general, increased and reached a maximum after ~owth of 3.2-3.5 days and then decreased (Fig. 3C). The levels were always higher in LL cells than in IlL cells. The C: N ratio in the cells ~bllowed a similar pattern, values peaked by Day 4 in IlL and Day 8 in LL cells, followed by a gradual decrease (Fig. 4B). A • ,,.-i o 700 600 °,,., A ..i.a f,,, 500 ,..,. ,,... 400 K. - o o .C o o 300 - 200 - Q I~,. • 1 0 0 - /. • 9 A --,~. . . . . . . . . z~ . . . . . . . . . . . . . . . . 7< . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 0 t - I I I 14 . ,,,,,d E o ---I o . ,...J 12 B 10 J o L ..,,=I o =o ,,o ~ " "°'"-4 :/ ~ ............... " ......... i,,- ...................... ® 4. L u 2 0 I I l l l i 5 I0 15 20 25 50 55 Culture age (days) Fig. 4. Changes with time in ratios of(A) carbon to Chl a by weight and (B) carbon to nitrogen by atom. Solid circles are experimental data for HL culture, open triangles are experimental data for LL culture, solid lines are modelled values for HL culture, broken lines are modelled values for LL culture. 86 Y. PAN ET AL. PHOTOSYNTHETIC PARAMETERS Photosynthesis-PPFD (P-I) curves exhibited reductions in photosynthetic rates at high PPFDs ( > 1500 #tool m - 2 s - ~, Fig. 5) and so required an expression that included a photoinhibition description. The empirical formulae of Platt et al. (1980) provided with an additional parameter (a constant) describe our data quantitatively. Photosynthetic rates in our experiments were corrected for Pde because our data show that P~ varied significantly with the age of the culture: in exponentially growing cultures it was < 10% of the maximum corrected photosynthesis (Pmn), in postexponential cultures, on .5 , : " ' T ! T iii..ii ~.l. ",'.5 T ~ I/. | ::l. .=.i, o o ~, "",, 7 ,.o) ~ " • '":: " "'-. o,s ~I!, . ......~-" . . . . . . . . . . . ~ ~ . . . . . . ~ .... ~---- ~m ~i eO 0 .0 A A A ............. l[ ~ "I ] l -- I 'I " 0.06 0.05 i I A°'"° ...A'-,~ H I -= O.Oa w 0.03 4 ",,% A A I _.__1 w 0.02 e.4] ::L 0.0! • r~ 17,/," ~ .....'......... - . ' . ~ .,. o.0o ,# ,' I 0 1000 2000 ,-~ 30C0 I 4.000 Photosynthetic photon flux density (~mol m 5000 -2 s -t) Fig. 5. Relationships between photosynthesis and PPFD in cultures on 4 days growth. Solid circles are experimental data for HL culture, open triangles are experimental data for LL culture, solid lines are modelled values for HL culture, broken lines are modelled values for LL culture. (A) Normalized to Chl a (#g C [#g Chla] - ~ h - ~); (B) Normalized to carbon (#g C [#g C]-n b-n). PHOTOSYNTHESIS AND GROWTH OF NITZSCHIA PUNGENS 87 the other hand, it was 3 - 4 times higher than the corrected photosynthesis, similar to the findings of Laws & Caperon ( 1 9 7 6 ) o n culture of a flagellate Monochrysis lutheri. The photosynthetic response to P P F D varied systematically with the age of the culture in both H L and LL cells. With increasing culture age, 0ca increased to attain a m a x i m u m on Day 4 (6.06 ng C [#g Chl a] - ~ h - ~ [#mol m - 2 S - l ] - I at HL, 8.69 ng C [#g Chl a] - ~h - ~ [#mol m - 2 S - l ] - i at LL; Fig. 6A), after which there was a drastic decrease. The variations of Pma over time were similar to those of ~a, i.e., they attained a m a x i m u m on Day 4 (2.2 Fg C [#g Chl a] - ~ h - ~ in H L 1.7 #g C [#g Chl a ] - i h in LL) and subsequently decreased (Fig. 7A). T "IT "~ I 10 -o 8-.- { - '= 6 , } ,,, i , A Z1 ii;"... ~ ". ",.- i i ": em .~. 2 0.30 T ! I T 0.25 - I I 0 13 0.20 / • o.!5 T T I 0.1o : T/ :' 0.00 ± .............. , 0 S !0 j j 15 20 , 25 30 35 C u l t u r e age (days) Fig. 6. Variations of ~a in cultures of various phases of growth. Solid lines and circles are values of HL culture, open triangles and broken lines are values of LL culture. (A) Normalized to Chla (Fg C [#g Chla] -! h -~ [#molm-2s-~]-I); (B) Normalized to carbon (Fg C [#g C] -~ h -~ [Fmoi m- 2 s- i] - i). 88 Y. PAN ET AL. 2.5 T - 2.0- I i i---i 1.5- m. i . o - 0.5 | 0.0 --I I 0.06 T 0.05 z : : o e4) B , 1 _T'~ 0.04 ~ ', ; 0.03- ~L , o 0.02~L 0.01 0.00 - 0 I I 5 _ I , 10 I , 15 -7 ...... L . . . . A , ~ 20 25 .. j 30 _ ~5 Culture age (days) Fig. 7. Variation of Pm n in cultures of various phases of growth. Solid lines and circles are values of HL culture, open triangles and broken lines are values of LL culture. (A) Normalized to Chl a (#g C [#g Chl a] - i h - i ) ; (B) Normalized to carbon (#g C [#g C]- i h - l ) . Interpretation of the P - I responses in HL and LL depends upon the biomass index used (Fig. 5A,B). For example, if exponentially growing cells are normalized to Chl a, HL cells achieve a greater maximum photosynthetic rate (Pm n" assimilation number) than the LL cells (2.2 compared to 1.7 #g C [#g Chl a ] - ~ h - ~; Figs. 5A, 7A). In contrast, when normalized to cellular carbon, HL cells attain a Pma that is only 40~o of that of LL cells (0.02 compared to 0.05 #g C [#g C ] - ~ h-~; Figs. 5B, 7B). This is a consequence of differences in the C'Chl a ratio in HL and LL cells. Irrespective of biomass index chosen, HL cells attain P~ at a higher PPFD than LL cells (Im(HL)= 1272, Im(LL) = 489 #tool m - 2 s - I, Table II). A similar result is seen with the photosynthetic parameter describing the initial slope of P - I response (~B) which was of the same order PHOTOSYNI'HESIS AND GROWTH OF N I T Z S C H I A P U N G E N S 89 TABLE II Photoadaptive parameters of N. pungenr f. multiseries over time in cultures grown at two levels of PPFD (/~molm - 2 s - ~). PPFD (#mol m - 2 Age (days) Im [k Is II00 1 4 8 15 25 34 1006 1272 1716 1049 1086 593 172 415 399 217 244 156 212 569 427 239 346 154 105 ! 4 8 15 25 34 599 489 379 197 426 276 86 140 102 35 114 46 311 339 134 31 106 42 S - I) of magnitude both in HL and LL cells when normalized to Chl a (Fig. 6A). However, in terms of cellular carbon, although the patterns were similar, the ~B in HL cells was usually lower (Fig. 6B) because C : Chl a ratio was 3 times higher in HL cells (Fig. 4A). The parameters Im, Ik and Is are independent of the biomass index chosen (Table II). Consistent differences existed between the HL and LL cells. The Im of HL cells are always higher (by a factor of 1.7-5.3) than those of LL cells; for Ik the factor is 2-6.2. Ik seems to show similar changes to ~B, pmB and growth (Table II, Figs. 6, 7, 2). DISCUSSION The maximum photosynthetic rates attained on Day 4 of growth (Fig. 7) corresponds to the end of the exponential phase based on Chl a and mid exponential phase based on cell numbers, carbon and nitrogen. The Pn~ decreased as the cultures aged, which is consistent with the findings of Humphrey & Subba Rao (1967) and Glover (1980). The relative magnitudes of the maximum photosynthetic rate occurring during thc exponential phase compared to that in the early stationary phase (P~ (Day 4): PmB (Day 8)) were 4.7 and 3.2 for HL and LL cells, respectively, which is similar to published values. For example, for cultures of Thalassiosira pseudonana 3H, grown at 18 °C and 200 #mol m - 2 s - ~ (Glover, 1980), the corresponding ratio was 6. Our HL cultures yielded higher assimilation numbers than those grown at PPFD an order of magnitude less, consistent with the concept of light and shade populations (Falkowski, 1981). The actual P - I values ofN. pungens f. multiseries differed from those transformed and tabulated for other algae (Table III). The initial slopes ~B are low when normalized to 90 Y. P A N ET AL. TABLE III Summary of photosynthetic parameters of various algae. Culture conditions Taxa Growth phase °C P' L:D 10-12 1100 105 24L ]m lk ls (/~moi m - 2 S - I ) Pennate diatoms Nitzschia pungens f. multiseries Exp Exp Amphiprora paradoxa 10 30 10 140 12:!2 18 200 24L 569 339 Exp Sta 27-57 Amphiprora kufferathii 18 400 24L Exp Sta Fragilaria sp. 5-8 47 12:12 Exp Cylindrotheca closterium 20 23-27 800 86 237 24L 24L 24L Navicula pelliculosa 415 140 106 95 Nitzschia delicatissima (dominant sp. in natural sampling) Nitzschia atnericana 1272 489 Centric diatoms Porosira pseudodenticulata 26-60 Thalassiosira scotia 28-50 Leptocylindrus danicus I0 !0 12:12 9:i5 Dinoflagellates Gonyaulax polyedra 330 12:!2 Exp early Sta late Sta Ceratium lineatum HL LL 190 45 Ceratium fusus HL LL 325 190 Ceratium tripos HL LL 325 160 ~ #mol m- 2 s - ' cell concentration; but are comparable to those of Nitzschia delicatissima (Erga, 1989) and Nitzschia americana (Miller & Kamykowski, 1986b) when normalized with respect to Chl a. The photosynthetic rates are also comparable when normalized to Chl a. When normalized to carbon they are of the same magnitude as other pennate diatoms but are PHOTOSYNTHESIS AND GROWTH an( • h - J (#mol m - -" s - i )- i ) cell - i ).017 ).039 ng C pg Chi a - t 6.06 8.69 ng C pg C - i 0.06 0.26 pg C cell - ~ 5.43 7.32 2.20 1.66 /,lg C/,/g C 0.021 0.049 !.2 !.7 0. !-0.5 0.28-1.30 39 21 !!-0.91 0.003-0.015 .2-9.2 50 5.8 8.7 Rivkin & Putt (1988) 0.01 0.006 4.9 2.0 0.083 2.76 3.8 1.3 2.32 2.92 Glover(1980) 2.3 Taguchi(1976) Humphrey (1979) Humphrey& Subba Rao (1967) Rivkin & Putt (1988) Rivkin & Putt (1988) 70-172 29.6 21.9 Present study Present study Glover (1980) 74-276 .5-6.3 I M i l l e r & Kamykowski (1986a) M i l l e r & Kamykowski (1986b) 9.9-19 0.60 - Erga (1989) 0.54-2.16 ~2-2.22 75 pg C pg Chl a - I 91 Reference P,~( • h - i ) 11.28 17.84 ~9-0.043 O F ¥1TZSCHIA PUNGENS Verity (1981) 4.3 2.9 20.4 8.8 5.3 8.95 4.42 4.76 3.7 2.4 i.8 Prezelin & Matlick (1983) 0.40 1.20 18.2 26.7 78 55 3.55 1.22 Rivkin & Voytek (1985) 0.40 0.48 11.4 9.6 130 90 3.71 1.80 Rivkin & Voytek (1985) 1.1 !.1 14.7 12.2 390 190 5.2 2.11 Rivkin & Voytek (1985) low compared to Amphiprora paradoxa (Glover, 1980). Photosynthetic rates based on particulate carbon are two orders of magnitude lower than other algae tabulated. The carbon assimilation numbers for N. pungens f. multiseries are comparable with those for some of the dinoflagellates summarized by Subba Rao (1988). 92 Y. PAN ET AL. T T ,.,.,,, ,--.,,, 2.5 i I 4 2.0 ~ 1 1.5 ~L) ::l. B ,0 --- ¢D O 0.5- 15 .m 0.0 - O o -0.5 0 : I I ! I 2 3 4 Cellular chlorophyll 5 a (pg cell -I) Fig. 8. Relationship between photosynthetic rate ( p a ) and cellular Chl a. (A) Solid circles are values of i l L culture: y = 0.98x - 0.27, r = 0.96; (B) open triangles are value of LL culture: y = 0.46x - 0.71, r = 0.87. Number at each point denotes culture age (days). The maximum carbon assimilation rates (#g C [#g Chl a ] - ' h - i ) are positively correlated with cellular Chl a (Fig. 8, p < 0.01). The highest assimilation rates associated with the highest cellular Chl a were in the Day 4 cells and their lowest values were in the advanced stationary cultures (34 days). Assimilation numbers increased between Day 1 and Day 4 corresponding with a decrease in C : Chl a ratio. Following this, the assimilation numbers decreased rapidly while the C: Chl a slightly increased and then remained relatively constant in stationary phase (Figs. 7A, 4A). The pattern of changes in assimilation numbers resembled that of C : N but with no strict correspondence (Fig. 4B). This relationship of assimilation number (pB, #g C [#g Chl a ] - ' h - 1) to carbon: Chl a or to cellular carbon : nitrogen for N. pungens f. multiseries also differed from those for Leptocylindrus danicus (Verity, 1981) and could be attributed to the effects of physiological stage of the cells. In N. pungens t. multiseries culture of 4 days growth, assimilation number, C : Chl a ratio, N :Chl a increased with an increase in PPFD; which is consistent with the observation on L. danicus (Verity, 1981). Similar to the relationship between assimilation rate and cellular Chl a, the relationship between specific growth rate and cellular Chl a was positive (Fig. 9) and significant (p < 0.01). The values were highest in exponentially growing cells and lowest in advanced stationary phase cells (Fig. 9). Growth rate is also positively correlated to assimilation rate (Fig. 10, p < 0.01). 08] PHOTOSYNTHESIS AND GROWTH OF NITZSCHIA PUNGENS 93 4 A 0.6]- 4, o o.'° I .°" ooO° B"" i ...-" o.4 ,,i ID • Q °° °°° .° .-"" 0.2 "'" o 2~ 0.0- A n ? • 0 2 Is a z~ , , , i i i i 1 2 ~ 4. Cellular chlorophyll , a (pg cell 5 -I ) Fig. 9. Relationship between specific growth rate and cellular Chl a. (A) Solid line and circles are values of HL culture: 3' = 0.26x - 0.06, r = 0.94; (B)Broken line and open triangles are value of LL culture: y = 0.2Ix - 0.32, r = 0.92. Number at each point denotes culture age (days). 0.8 A .6 -- O I i 0.4-0.2-- O L., r,..3 iO 0.0--0.2 i -0.5 0.0 - I i i 0.5 1.0 1.5 ! 2.0 2.5 Photosynthetic rate (#~gC [#~g Chl a] -I h -i) Fig. 10. Relationship between growth rate (#) and photosynthetic rate (pn). y = 0.23x + 0.06, r = 0.70, solid circles are values of HL culture, open triangles are values of LL culture. 94 Y. PAN ET AL. Comparison of growth rates based on the 4 biomass indices, i.e., Chl a, cell number, carbon, and nitrogen showed that cell division was not internally consistent (Table IV). In HL, Chl a-based growth rate increased and reached its maximum after 1.8 days (Table IV). Cells were actively dividing as the photosynthetic capacity increased resulting from the rapid increase of Chl a per unit volume (Figs. 1B, 2B) and reached the maximum division rate after 3.5 days, i.e., 1.7 days later than Chl a based rate TABLE IV Maximum growth rates (#m.~x) and time it was reached (Tmax) based on cell concentration, Chl a, carbon and nitrogen (based on growth model, Fig. 2), where HL and LL denote high and low PPFD cultures, respectively. Biomass parameters Cell concentration Chl a Carbon Nitrogen HL LL ~max (day- l ) Tmax (day) ~/max (day- t ) ~max (day) 0.56 !.34 0.87 0.30 3.5 1.8 4.4 5.2 0.74 0.95 0.50 0.19 3.5 2.7 3.8 3.9 (Table IV). Particulate carbon reached its maximum growth by 4.4 days, followed by particulate nitrogen that reached the maximum 5.2 days after incubation (Table IV). The magnitudes of #max attained, based on the 4 biomass indices differed as well. In LL culture, the maximum growth based on Chl a was attained after 2.7 days as compared to 1.8 days at HL (Table IV). This could be due to variation in chemical composition of algae as a function of growth rate. Shuter (1979) divided the cellular carbon into four compartments and emphasized that differences in the flows of material between compartments and between cells would result in unbalanced growth. Caperon & Meyer (1972a,b) demonstrated that # based on ~4C, carbon or Chla cannot be equated to the growth prediction based on nutrient uptake. Although similarities exist between N. pungens f. multiseries and other diatoms (in the decrease of photosynthetic rates as the cells aged, higher assimilation rate (Pro B) and lower photosynthesis per unit photon (~8) in HL culture than LL culture), there are obvious physiological differences (such as the low Pm n, 0~a). Apparently unique to N. pungens f. multiseries is the production and intracellular accumulation of domoic acid, particularly in the postexponential phase in F and FE medium (Subba Rao et al., 1990) and later exponential phase in NH4 enriched medium (Bates et al., 1990, presentation on the 2nd Canadian Workshop on Harmful Algae). Usually, this occurs when the cells are metabolically inactive or when the cells are physiologically stressed, i.e., photosynthetic rate, cellular materials and growth rate are all decreasing. The unbalanced variation in cellular materials (Figs. 1, 2) and low PHOTOSYNTHESIS AND GROWTH OF NITZSCHIA PUNGENS 95 photosynthetic rate (Fig. 7) may also be related to the production of the neurotoxin. Reasons for such a timing of domoic acid production still need to be determined. ACKNOWLEDGEMENTS We are grateful to our colleagues K.H. Mann, J. Stewart, D. Gordon and W. K. W. Li for criticism of the manuscript. We are thankful to T. 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