NITROGEN FIXATION IN CLEAR LAKE, CALIFORNIA. II. SYNOPTIC STUDIES ON THE AUTUMN ANABAENA BLOOM’ Clear Lake Algal Alexander J. Home”, J. E. Dillard, Research Institute Unit, of Ecology, University of California, Davis D. I<. Fujita, and C. R. Goldman Division of Environmental Studies, University of California, Davis ABSTRACT Nitrogen fixation at three stages of an autumnal bloom of Anabaena circinulis was measured after almost simultaneous collection at up to 32 stations in Clear Lake and algal hetcrocysts, phytoplankton cell numbers, NO,-N, NIL-N, dissolved organic-N, POh-P, Fe, primary production, particulate carbon, and chlorophyll a were also measured. Nitrogen fixation was significantly and positively correlated to Anabaena heterocyst numbers (P > correlated to dissolved 0.001 ), negatively correlated to NO,-N (P > 0.01 ), and positively A negative correlation with NH4 is proborganic-N (P > 0.01) and PO,-P (I’ > 0.05). able; no significant relationship was found with the other variables mcasurcd. An cxplanation of the apparent restriction of substantial cyanophyccan Nz fixation to nonoligotrophic waters is proposed. The results arc consistent with the theory that heterocysts are responsible for Nz fixation in situ under oxic conditions. INTRODUCTION Combined inorganic nitrogen is the major limiting nutrient in many aquatic ecosystems (Thomas 1969; Ryther and Dunstan 1971; Home and Vincr 1971). Nitrogen fixation is the only method by which plants can provide new combined nitrogen an d overcome this dcficicncy. In oligotrophic lakes Ng fixation can usually be dismissed as negligible, but it frequently plays a major role in the nitrogen mctabolism of blue-green algae (Horne and Fogg 1970) and that of whole lakes and marshy areas (Horne and Vincr 1971; Hornc 1971; Hornc and Goldman 1972; IIorne 1972). The contribution of biological Nz fixation to lake nitrogen budgets has been measured in several lakes including Windermcrc (Hornc and Fogg 1970), Lake Gcorgc ( Horne and Viner 1971)) and 1 This study was supported by the County of Lake, the California Department of Water Resources, the Institute of Ecology (University of California, Davis ), and the Soap & Detergent Association. 2 Present address: Division of Sanitary Engincering, University of California, Bcrkclcy 94720. LIMNOLOGY AND OCEANOGRAPIIY Clear Lake (Horne and Goldman 1972). However, the synoptic technique, measuring many stations virtually simultaneously, has not previously been successful for Ng fixation. The high seasonally and spatially variable rates of N2 fixation in Clear Lake (IIornc 1971; Horne and Goldman 1972) provide an ideal opportunity to study NZ fixation by the synoptic technique. The object of these studies is to understand Nz fixation in relation to blue-green algal blooms, nitrogen compounds in the lake, and the role of the hctcrocyst. Some of thcsc can best bc shown by seasonal analysis (Horne and Goldman 1972) but because many measurements of blooms will be irregular and made only during the bloom period some method of interpreting incomplete data is nccdcd. The synoptic tcchniquc cnablcs instantaneous relationships to bc intcrpretcd in the light of seasonally gathered information. WC wish to thank E. de Amezaga for the computer analysis, C. Carmiggclt and B. Jones for drawing the figures, and the many others who assisted in the collection and processing of the samples. 693 SEPTEMBER 1972, V. 17(5) 694 HORNE, TABLE 1. Significant DILLAW, relationships between d f = 6; Het = heterocysts; FUJITA, algae’ key: >Om Vol. GOLDMAN and N2 fixation on 26 August synoptic >0.9?_49 = P?O.OOl; ‘,0.8343 =: P>O.Ol; = P:,O.O5; .,0.6215 ---- Anabaena N2 fixed AND No. Het % Het (IO stations). = P:aO.lO Aphani- Micro- Oscil- Melo- Crypto- zomenon cystis latoria s ira monads Vol. oss!5 Het 0.8754 LZ.4 0.9621 II-. 5-E % Het 0.4308 0.3916 0.6121 Aphanizomenon -0.0192 0.1463 0.0326 -0.3548 Microcystis -0.2461 -0.2459 -0.3756 -0.7293 .- 0.4734 Oscillatoria 0.7446 0.8652 ~ 0.8951 -1z- 0.4356 -0.0125 -0.1909 Melosira 0.6352 0.3862 0.3294 0.0671 0.0092 0.3012 0.1731 Cryptomonads 0.7310 0.5714 0.5539 0.1770 -0.1399 0.1344 &69&O --0.6251 Ciliates 0_h 6482 0.5901 0.4626 -0.0813 -0.3176 -0.2127 0.4,502 0.2906 0.4385 0.9321 -- 0 9406 +,- 0.I.- 8930 0.3657 0.0634 -0.1377 0.7855 Lv- !I,6384 0.7256 -- Anabaena Total No. biomass Ciliates 0.5705 -- tNo significant flagellates, relationship Scenedesmus, was found between N2 fixation and the following algae: Chroococcus, Oocystis, Cyclotella, Pandorina. METHODS Collections were made at up to 32 stations from two or three boats so that all samples were collected within 2 hr. This synoptic technique has been described by Goldman and Carter (1965). Samples were collected on 8 and 21 Scptcmber using O-3-m tube samplers with a 4-cm diameter. On 26 August samples were taken at 0 to 0.6 m with a Van Dorn bottle. The method of assay for acctylcne rcduction, based on that of Stewart et al. ( 1967)) has been described previously (Hornc and Goldman 1972). The only difference for the synoptic work is that all samples were incubated at the standard depth of 0.5 m. The methods used for the mcasurcmcnt of the biological, chemical, and physical parameters wcrc given by IIornc and Goldman ( 1972). For each of the synoptics the simple correlation cocfficicnt “8’ has been computcd for all variables measured taken by pairs. Computation of r was done using the maximum available data for each pair of variables. This gave r 6 dE for the synoptic of 26 August, 14 df for 8 September, and 28 df for 21 September, with the cxccption of 27 dff for all r computed for carbon fixed on 21 September, since station 32 was not assayed for carbon fixation on that day. Studies using pair correlations with large amounts of data collected without refcrcncc to the final analysis occasionally show biologically meaningless but statistically significant results. We cxcludcd most parameters known from culture studies not to affect Nz fixation in any direct biological sense, or do not show them in de tail. This in troduccs some improvcmcnt to this rclativcly simple analysis, along with a certain clement of subjectivity. Bccause it was impossible to measure all the variables that may affect Na fixation (c.g. MO) we feel this approach justified. The major rcscrvation with regard to the USCof a simple pair analysis is that a high degree of direct correlation bctwccn two parameters (e.g. Nz fixation and hctcro- NITROGEN TABLE 2. Significant Het = heterocysts; CLEAR LAKE 695 2. relationships between algae+ and N2 fixation for 8 September synoptic (17 stations). ;O 7420 = P>O.OOl; bO.6226 = P>O.Ol; >0.4973 = P>0.05; d f = 14; key: *-__L_ >0.4259 Chl N2 fixed Chl IN FIXATION = P>O.iO Aphanizomenon Anabaena a Vol. No. Het No. Het Vol. % Het % Het Micro- Oscilla- cystis toria 0.9337 a Vol. 0.9319 ----- __ 0.9606 :m Het 0.9499 _-z 0.9681 -_zYzzzzz % Het -0.2828 -0.3882 -0.3913 -0.3270 Vol. 0.0741 -0.0508 -0.0059 -0.0252 -0.3648 Het -0.0540 -0.1699 -0.0652 -0.0895 -0.3782 0.6116 % Het -0.1189 -0.1695 0.0894 -0.1002 -0.1825 0.3918 O.J893 Le.-- Microcystis o.9200 0.9753 ---- 0.9765 E- -0.9697 -0.3913 -0.0975 -0.1934 -0.1700 Oscillatoria 0.4914 0.3558 0.1967 0.2504 -0.0300 0.0343 -0.2019 -0.1782 0.2929 Cryptomonads 0.9137 ~- 0.8535 -__ __- 0.8552 -_ -__ 0.8488 z=z -0.3428 0.0303 -0.1645 -0.1425 0.8958 Anabaena No. Aphanizomenon +No significant Pandorina, No. relationship Cyclotella, was flagellates, found between Peridinitrm, 0.9943 -- N2 fixation Navicula, and the following Planktococcus, cysts) dots not, in itself, mean that a dircct relationship actually exists. But when such a relationship is already known to exist from laboratory culture studies, then the level of probability of the correlation is useful to define the relationship more precisely than could be done using the raw data. The validity of our choice of revclant paramctcrs is partially dcmonstratcd in Tables l-3 and their respcctivc footnotes, which show that most of the significant relationships involve paramcters already known to be rclatcd to blucgreen algal N2 fixation, In view of the occasional and sometimes valid claims for Nz fixation involving nonhctcrocys tous Cyanophyta or cpiphytic bacteria, we felt that some negative correlations dcscrvcd brief mention in the table footnotes. The level of significance of the correlation coefficients was dctcrmincd with the table of Fisher and Yates ( 1953). Results and level of significance arc shown in Tables l-3. Nitrogen fixation (acctylenc reduction) algae Scenedesmus, tested: Oocystis, ciliates, Melosira, 0.5466 Coelastrum, Chroococcus. is expressed as nmolcs cthylcnc liter-l hr-I. produced RESULTS Small synoptic: 26 August 1970 (10 stations) Nitrogen fixation in Oaks Arm was very high and almost zero clsewherc (Fig. IA). Values in Oaks Arm ranged from 102-750 ( mean = 400) t-moles liter-l hr-I. Average values in Lower Arm and Upper Arm wcrc 40 and 5. Nitrogen fixation was dircctly related to the amount of Anabaena prcscnt ( P > 0.01) and also to the number of Anuhaenu hctcrocysts (P > 0.01) (Tablc 1). IIcterocysts varied from 1,012 4,148 (mean = 2,800) ml-l (Fig. 1B) and pcrccnt hcterocysts from 3.85-6.7, but percent hcterocysts wcrc not significantly rclatcd to Na fixation. Aphanixomenon flosaquae, the other main potential N,-fixing alga, showed no significant relationship with Nz fixation (Table l), presumably because thcrc were no Aphanizomenon hctcrocysts at this time. 0.1083 0.0066 No. Het % Het ZOllle”OIl -0.3881 ?r\/\-\/ -0.3727 7,,c\- P Total a Achnanthes. for carbon biomass -Values Total Chl Dlatomata, -0.2786 0.1330 -0.3186 Mollomonas, -0.0207 0.0337 -0.0500 -0.2255 -0.1328 0.3620 \,\,\ L 0.0669 0.2472 -0.3223 -0.1875 -0.1777 -0.3488 -0.0680 -0.0694 0.2456 0.3862 ,‘,‘\1\1 -0.2099 -0.0040 -0.0316 0.0888 -0.2803 -0.2660 Vol. 0.8947 - -0.1994 -0.0125 -0.0651 -0.0246 -0.0658 0.1473 0.1346 -0.1603 -0.2113 0.5316 -__ -0.1933 -0.1079 -0.1842 Cryptomonas for C fixed. 0.3288 -0.0441 -0.2616 O.i438 -0.0018 -0.3056 -0.2952 0.0233 0.2129 -0.2034 ~0 3363 -0.1374 0.2900 No. Het Aphaniromenon -b.lOSS -0.1137 given between % Het Key: algae*. ‘No marssonii, -0.5541 -0.2266 0.2975 -0.2665 -0.0112 relationship -0.1809 0.0728 -0.0006 -0.2314 -0.0743 0.2721 C. reflexa, 0.5281 -0.4593 -0.3230 0.4305 -ml\-\, -0.4751 -0.6360 - -0.64820.2587 0.2243 0.3917 T-i.‘,-2 -0.3824 z--i\. 0.3039 -0.2198 C. curvata, was found 0.2075 0.0586 0.2028 o.:e3! -- 0.2317 -0 L- 4641 -0.4955 - 0.0637 0.3740 ,* “J -0.2717 Schr. -0.2028 0.6159 E -0.6113 -- -0.0093 -0.5407 ___ Coelastrum. between -0.4512 - -0.0880 0.2806 -E parameters SOI. 0.0752 -0.2430 -0.2384 Oocystis, -0.2129 0.5650 - Pediastrum, algae: -0.3521 -0.3316 h,. , / * -0.2064 m 0.5478 -0.7276 __ Scenedesmus, 0.1027 0.1784 -0.2980 - N2 fixationand -0.4241 y,,-, I -0.2520 -0.3657 -1 -0.3762 ?-w-Y-/z-u -n - 7i73 w-w Tota I P 0.6311 Partic. C C fixed Fragilaria, -0.1884 -0.1139 Total Fe Rhiocosphaerium. Melosira, 0.1718 0.3152 -0.2977 Closterium, Cyc/ofe/fa, 0.0631 0.8571 - -0.0718 = P-O.10 -0.3646 Yryu-.z 0-z SOI. P04-P the following Org-N 20.2960 September - P. 0.05; for 21 “.3!85 0.1444 -0.1629 =zztzzZ -0 6528 -0.6753 -- 0.3291 N03-N -= P.%O.Ol; ‘0.34~4 and chemical :0.4487 - cost. physical - P>O.OOl; Oxill. N2 fixation, Micro. significant -0.2915 0.0121 -0.1291 -0.0031 -0.0070 0.0307 0.0239 -0.1677 -0.1813 0 3401 L -0.2025 -0.1131 -0.1936 Het = heterocysts; relationships =28: Rhodomonas, to those Gymnodrnium, are identical -0.0695 -0.1670 efficiency -0.0717 0.1415 -0.3712 IV\A/X?* -0.2457 O.s01_7 - 0.0812 -0.0071 Total -0.0890 ~14;089 -0.2308 Fe 1 0.4896-0.2771 --0.4969 0.4344 0.5097-i-^ 0.3843 .- / I_, __ “” C fixed- Particulate P04-P Sol. Sol. organic-N NO34 0.2553 0.3698 -vu Schroederia $364 -0.3256 Coscinodiscus 0.5257 0.2739 0.5540 -0.1777 0.1536 -o.T?5J oscr//atorra 0.0196 -0.1388 % Wet d f Significant No. Het Anabaena -0;49 OUvS83 0.1584 0.3226 __ -0.1077 -0.1223 0,3682 Vol. MKrocystis fixation -0.1930 Vol. Aphani. C 0.8990 -0.1067 % Het Anabaena No. Het N2 fixed 3. (32 stations). TABLE synoptic a 0.1024 Chl - ---_. ----- NI’I‘IIUGJAN l!lXA’I‘lUN IN CLEAR LAKE,. 7 697 2. 4. Nutrient concentrations (yg liter-‘) for 26 August and 8 September at representative stations in Cleaf Lalce. (u = <lO pg liter-‘) TABLE upper 26 N114-N surface bottom Aug Arm Lower Q Scp 26 Aug Oaks mm Q sep 26 Aug Arm Q sep u u Ll " 40 Go I.7 14 - 4:" 2:o 20 N03-N (O-3 m) Soluble orqanic-N N2 FIXATION n moles (O-3 AS C2H4 liter-’ 519 m) Soluble P04-P hr-’ (O-3 Total 17 533 - - - 340 360 - 160 17 - 410 895 m) Pe to-3 160 m) 1,430 1,485 B u HETEROCYSTS ml” C PHYTOPLANKTON [black VOLUME area is 740 pm3 x lo6 ml-’ Anabaena) FIG. 1. Nitrogen fixation (A), hetcrocysts (B), and phytoplankton (C) on the 26 August synoptic. Diameters of circles indicate quantities measured. Oaks Arm is enlarged to clarify results (synoptic station letters are given in B). Thcrc was a statistically significant rclationship of Nz fixation with Oscillatoria (P > 0.01) and also cryptomonads (P > 0.05) but not with any other algae counted (Table 1). This is the result of an indircct relationship because the abundance of OsciL!atoria and Anabaena are significantly related to each other (P > O,Ol) and explains the apparent relationship of OScillatoria and Na fixation. Cryptomonads arc rclatcd to OsciZZatoria (P > 0.1) and the relationship between them and NB fixation is again indirect. Nitrogen fixation was significantly rclated to total biomass ( P > 0.001) mainly because Anabaena was the codominant spccics (with Microcystis) at this time, but Anabaena’s larger size gives it much in biomass measurcmore importance ments. Measured as a pcrccnt of total phytoplankton biomass, Anabaena accounted .for 24-51 (mean = 40%) in Oaks Arm. Mcasurcmcnts at one central station from Upper and Lower Arms gave 4 and 30% Anabaena (Fig. 1C). Bccausc Nz fixation was known from routinc measurements to be appreciably high only in Oaks Arm at this time, only the 8 stations in Oaks Arm wcrc trcatcd separately and avcragc valucs for the other two basins wcrc obtained using samples from 5 offshore stations in each. Table 4 shows the major nutrients: At this time the concentration of Non-N was low, there was little intcrbasin variation, and N&-N was undetcctablc (<lo pg liter-’ ) . Phosphate-P was high, especially in Upper Arm, Nitrogen fixation rates in the 26 August 698 IIORNE, Nq FIXATION FUJITA, A n moles liter HETEROCYSTS PHYTOPLANKTON (black DILLARD, -1 hr” ml-’ VOLUME area pm3 x lo6 nli"i is % Anabaena] FIG. 2. Nitrogen fixation (A), heterocysts (B), and phytoplankton (C) on the 8 September synoptic. Diameters of circles indicate quantities measured. synoptic (0-0.6-m samples) are higher than the later synoptics (O-3-m samples ) but are not directly comparable bccausc of the sampling diEfcrenccs. Surface samples gcncrally fix the most NB ( Horne and Goldman 1972). Minor synoptic: 8 September 1970 (16 stations) Nitrogen fixation occurred towards the castcrn end of every basin (Fig. 2A) but high rates were confined to Oaks and Lower Arms, especially the latter whcrc values of O-209 (mean = 105) nmoles liter-l hr-1 were recorded. This compares with rates of O-82 (mean = 25) for Oaks and 17-60 ( mean = 30) for Upper Arm. AND GOLDMAN That this pattern of fixation was due to Anabaena and its hctcrocysts is clearly shown in Fig. 218 and Table 2. Fixation was highly significantly directly related to Anabaena hcterocyst numbers (P > 0.001). The concentration of hcterocysts per milliliter ranged from 400-850 (mean = 600) in Upper Arm, 40-1,780 (mean = 600) in Oaks Arm, and 180-5,600 (mean = 2,190) in Lower Arm. The other phytoplanktonic algae showed no significant relationship with fixation, with the exception of Microcystis (P > 0.001) and cryptomonads (P > 0.001). This is a similar situation to that found For the 26 August synoptic and is presumably again due to indirect relationships of these algae with the main Nzfixing species. Anabaena was related to Microcystis ( P > 0.001) and to cryptomonads ( P > 0.001). There was a significant correlation with N2 fixation and chlorophyll a, owing to the dominance of Anabaena biomass in the phytoplankton. Measured as a percentage of total phytoplankton volume ( Fig. 2C), Anabaena accounted for 7.9-15.6010 (mean = 10.4%) in Upper Arm, 0516.3% (mean = 7.7%) in Oaks Arm, and 9.2-81% (mean = 43%) in Lower Arm. In this synoptic, few chemical mcasuremcnts were made but rcprcscntative samples indicate the general trend of each basin (Table 4),, In the surface water of the central area of Lower Arm, NOS-N was low but still considerably higher than that in cquivalcnt areas of the other two arms. Not unexpectedly ammonia was gcncrally very low (5-17 pg liter’) and showed a pattern opposite to that found for NOR-N. Total-P and Pod-P were very low in Oaks Arm where Na fjixation was low, but much higher in the two basins where high NZ fixation was occurring, suggesting the possibility of a lasting N and P limitation in Oaks Arm following the peak of the bloom at the end oE August. Dissolved organicN and total iron showed no significant Howcvcr, bccausc intcrbasin variation. thcsc values rcflcct only the gcncral trend of each basin more reliance should bc NITROGEN 2 FIXATION n moles PHYTOPLANKTON (black VOLUME area pm3 FIXATION AS liter” x lo6 IN CLEAR LAKE. 2. 699 C2 H4 hr” r-l ml” is % Anabaena) FIG, 3. Nitrogen fixation tember synoptic. Diameters (A), hcterocysts (B), phytoplankton (C), and Non-N of circles indicate quantities mcasurcd. placed in the final synoptic whcrc cnts wcrc measured at all stations. nutri- Major synoptic: 21 September 1970 (32 stations) Nitrogen fixation was confined to Upper Arm and its connection with the other basins at the Narrows (see Fig. 3A). Rates of N2 fixation in Upper Arm varied from 32425 ( mean = 92) nmoles liter-l hr-l and were highest at the nearshore sites 9, 10, and 11, reflecting the abundance of the potential Na-fixing algae and heterocysts at these places (Fig. 3B). There was a highly significant positive correlation between Ns fixation and Anahnena heterocyst numbers (P > 0.001) and a significant relationship with Anabaena volume (P > 0.05) (Table 3). There was no significant relationship between NB fixation and Aphanixomenon hctcrocysts. Nitrogen fixation was significantly and negatively correlated to the concentration of NOs-N (P > 0.05), directly corrclatcd to dissolved organic-N (P > 0.01 ), total-l? (D) for the 21 Sep- (P > O.OS), and P04-P (P > 0.05) (Table 3, Figs. 3 and 4). There was no significant relationship bctwecn Na fixation and chlorophyll a, particulate-C, carbon fixed, total iron, total biomass, or efficiency of C fixation (Table 3). As opposed to the earlier synoptics there was a significant negative (rather than positive) correlation bctwcen Nz fixation and Microcystis (P > 0.05). Thcrc was a significant positive relationship between Nz fixation and Oscillatoria (P > 0.01) and Schroederia ( P > 0.05). Once again this was probably due to an indirect relationship bccausc these two species were both highly correlated to total-l? and PO,+-P ( P > O.OOl), which is itself significantly corrclatcd to N2 fixation (P > 0.05). No other of the 23 algal species counted were significantly rclatcd to N2 fixation. Measured as a pcrccnt of total phytoplankton volume Anabaena accounted for 1.3-32% (mean = 16%) in Upper Arm, 1.4-11% (mean = 4%) in Oaks Arm, and O-69% (mean = 12%) in Lower Arm. An interesting effect shown by this syn- 700 pg IIORNE, DILLARD, N liter-’ (O-3m) FUJITA, AND GOLDMAN (light area is % POT-P) D IMARY mg PRODUCTIVITY C mw3 day’] mg mm2 (O-3 m) FIG 4. Dissolved organic-N (A), total-P and PO,-P (B), p rimary productivity (C), and chlorophyll a (D) for the 21 September synoptic. Diameters of circles indicate quantities measured. Synoptic station numlxrs are given in D. optic is the biologically scparatc behavior in IJppcr Arm and higher concentrations of the basins as demonstrated by the con- of 0.1-0.93 mg literl in Oaks and Lower Arms rcspcctively. centration of NOS-N and P04-I?. In Upper Arm, where Na fixation was high, NO:%-N The data for C fixed show what appears concentrations were very low, ranging to be an cxamplc of limitation of photosynthesis by lack of nitrogen in Upper from lo-83 (mean = 21) PI; N liter-l; but Arm relative to the other two arms (Figs. in Lower and Oaks Arms whcrc fixation 3 and 4). In Lower and Oaks Arms where was insignificant, N03-N was much higher, ranging from 129-192 (mean = 162) in nitrogen was more concentrated than in Upper Arm, C fixation ranged from 279Oaks Arm and from 15-130 (mean = 79) in Lower Arm. Phosphate-P showed the 677 (mean = 370) mg C m-3 day-” in opposite picture, being low where NO.?-N Lower Arm and from 142-163 (mean = was high and Nz fixation low. In Upper 210) in Oaks Arm; in Upper Arm values Arm, Pod-P ranged from 260404 (mean of only 49-191 ( mean = 140) wcrc found = 352) pg liter-l, and in Oaks and Lower (Fig. 4C). This suggests that Nz fixation Arms values ranged from 160-190 (mean alone was not able fully to supply the = 165) and .from 50-100 (mean = 70) algal demand for nitrogen. rcspcctively. Chlorophyll a and particulate-C showed Ammonia concentrations wcrc not mcaa relationship to N2 fixation in Upper Arm sured at all synoptic stations, but mcasurcbut mainly bccausc Anabaena dominated mcnts taken at the time of the synoptics the phytoplankton there. High standing indicate that there was almost no ammonia crops of Chl a and particulate-C were in the surface water layers of any basin produced in the other arms without high On 21 on 26 August or 8 Scptembcr. N2 fixation or C fixation at this stage September, however, there was a surface although active Nz fixation did occur in ammonia concentration of 0.06 mg liter-’ NITROGL;.N FTXATION these basins earlier in autumn (Figs. 1 and 2). Micronutricnts are suspected as a possiblc limiting factor for the growth of blucgreen algal blooms in Clear Lake (Horne and Goldman 1972). The only “micronutricnt” measured, total iron, showed a trend towards lower values of 1.28-2.43 (mean = 1.60) pg liter-” and 1.58-3.08 ( mean = 2.05) in Lower and Oaks Arms respectively, rclativc to the higher values of 1.54-3.72 (mean = 2.29) found for Upper Arm. Our total nutrient results show that a scvcrc N limitation in Upper Arm occurred during a time when Pod-P was in cxccss and that Na fixation was unable to meet the need for added nitrogen. At this time there seemed to bc a phosphorus or iron ( or other trace element) limitation or possibly an inhibitor in the other basins. However, this limitation or inhibition occurrcd a&r a large peak of Na fixation in thcsc b‘asins. DISCUSSION In Clear Lake the autumnal bloom of Anabaena circinulis fixes N2 where there arc large numbers of hcterocysts, relatively low concentrations of nitrate and ammonia, high concentrations of phosphate, and moderately high concentrations of dissolved organic nitrogen. Only when thcsc five conditions wcrc satisfied clid very much N2 fixation occur. Perhaps the most important of our findings is that of the role of the hcterocyst bccausc there has been sonic controversy over its function (Stewart ct al. 1969; Smith and Evans 1970). This study supports previous claims that in oxic lake cnvironmcnts hcterocysts give a good mcasure of in situ rates of N2 fixation by Anuhaena (Hornc and Fogg 1970; IIornc and Goldman 1972). Our data rcprcscnt a step forward in the understanding of in situ N2 fixation by planktonic blue-green algae bccausc until now there has been no clear relationship shown between Na fixation and o thcr chemical parameters. Early studies have shown various types of rcsponsc to inor- IN CLEAR LAKE. 2. 701 ganic-N added tither from natural sources or added artificially (Dugdale and Dugdale 1962; Gocring and Necss 1964). Our study of an Anabamu bloom, showing a clear invcrsc relationship between Nz fixation and the instantaneous conccntrations of N03-N, now clarifies this uncertainty, at least for N03-N for which turnover rates arc slow. Blue-green algae appear to need a certain amount of combincd nitrogen as a “starter” if they are to fix appreciable quantities of Na. Seasonal studies (Horne and Fogg 1970; IIornc and Goldman 1972) show various slightly positivc, or uncertain relationships between N03-N and Nz fixation. Because high N2 fixation by cyanophyceans occurs in situ only when other inorganic nitrogen sources arc used (Dugdalc and Dugdale 1965; Billaud 1967, 1968)) whole-season studies can bc cxpectcd to show occasional indircct positive relationships between Nz fixation and N03-N, NHJ-N, or both, at least in cutrophic lakes. When measurements are made at the height of Nz fixation of the bloom most inorganic-N will already have been dcplcted to product the high N2 fixation. This situation would not ncccssarily hold for NHd-N, where regeneration rates may be sufficiently rapid to supply much of the nitrogen used in growth but insufficiently fast to build up a significant standing concentration. The relationship between the growth oE blue-green algae in lake waters and organic-N has been much discussed (Pearsall 1932; Hutchinson 1957, 1967; Fogg 1965; Lund 1965). According to Fogg (1965) thcrc is probably some general rclationship bctwecn the two but the basis for this is still obscure. Home and Fogg (1970) showed a noncausativc relationship bctwccn N2 fixation by blue-green algae and dissolved organic-N (which was rclatcd to phytoplankton biomass) in Windcrmcrc and Esthwaite Water. The results of our study confirm this result correlating Nz fixation and dissolved organic-N ( P > 0.01). In particular on 21 Scptcmbcr N2 fixation. was highest at site 9 (421 nmolcs liter-1 hr-I) and was corrclatcd to the high- 702 HORNE, DILLARD, FUJITA, est dissolved organic-N value (5,600 ,ug N liter-l). The mean for Upper Arm was only 910 lug liter l. Our studies show that Nn-fixing Cyanophyta also arc associated with quite high levels of dissolved organicN (0.35 mg litcr’l ) , Our data could provide a rcasonablc answer as to why high NP fixation is only found at certain times and only in nonoligotrophic lakes. In oligotrophic lakes thcsc conditions of some combined inorganic-N plus sufficient dissolved organic-N may never occur. In stratified nonoligotrophic lakes these conditions are normally only provided toward the end of a spring bloom and during autumn overturn, In nonstratificd, nonoligotrophic lakes these conditions may occur at irregular intervals throughout the year, depending on nitrogen turnover rates. It is thus obvious that the rate of supply and uptake, as well as just the standing concentration, of nitrogen should bc considered, at least in the metabolism of Nzfixing cyanophycean blooms. We disagree with IIutchinson (1967) and believe it is to be expected, not paradoxical, that standing concentrations of the limiting nutricnt (s ) will be low during the development of a healthy blue-green algal bloom, bccause uptake rates may exceed supply rates. The probable controlling factor in these fast, large blooms is the rate of turnover of limiting nutrient ( s ) , and the standing concentration is a secondary factor. The methods of mathematical analysis used in these studies will not show any significance for a variable that fluctuates above the critical level for participation in For example photosynthesis Nz fixation. in Clear Lake has no significant rclationship with NP fixation bccausc it is probably ,always adcquatc to power Na fixation, despite the fact that the large amounts of energy needed to break the NsN bond may be biologically disadvantageous in seasonal competition with other algae that can use existing low levels of NIL-N. Thus in Clear Lake the alga Oocystis which does not fix Nz dominates the summer AND GOLDMAN phytoplankton after the collapse of the spring Aphanixomenon bloom which does fix Nz. The seasonal variations of P04-P in Clear Lake arc now well defined (Hornc 1971) but because of the huge relcasc of this compound in summer there is ncvcr any real prolonged shortage. The results of our study are thus not readily applicable to other lakes whcrc P04-P concentration and rate of turnover may bc low in summer-autumn. We do not know how these conclusions will apply to other lakes whcrc Nz fixation plays a significant role. Certainly Nz fixation occurs usually when N03-N and NHd-N levels are much less than maximum in most lakes (Dugdalc and Dugdalc 1962; Goering and Neess 1964; Horne and Fogg 1970 ) , although this is not always true (Horne and Fogg, 1970). The data obtained for Clear Lake suggest that when the demand for inorganic-N exceeds the supply (ic. nitrogen stress) then Nz fixation will occur regardless of the ambient concentration of inorganic-N-unless this is toxic. Most algal species not known to fix N:! showed no significant relationship with Na fixation, hetcrocysts, or Ns-fixing algae. The few which did show a significant relationship are all obviously related to some third factor ( Tables l-3). 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