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Title NITROGEN FIXATION IN CLEAR LAKE, CALIFORNIA. 4. DIEL STUDIES ON APHANIZOMENON AND ANABAENA BLOOMS.

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Author Home, A.J.

Publication Date 1976-12-01

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NITROGEN FIXATION IN CLEAR LAKE, CALIFORNIA. 4. DIEL STUDIES ON APHANIZOMENON AND ANABAENA BLOOMS

RECE1VD A. J. Home LAWRE E BERKLY LA...AT0RV LV 2o YI9 December 1976 L1RARY AND OCUMgNTS sECTION

Prepared for the U. S. Department of Energy under Contract W-7405-ENG-48

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This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor the Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or the Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or the Regents of the University of California. LlmnoI. Oceanogr., 24(2), 1979, 329-341 © 1979, by the Amencan Society of Limnology and Oceanography, Inc. Nitrogen fixation in Clear Lake, California. 4. Diel studies on Aphanizomenon and Anabaena blooms 1

A.J.Horne Department of Sanitary Engineering, and Sanitary Engineering Research Laboratory, University of California, Berkeley 94720

Abstract Day and night measurements of N. fixation (as acetylene reduction) were made during spring blooms of Aphanizomenonflos-aquae and two autumn blooms of Anabaena spp. From 9 to 23% of the 24-h fixation occurred between 1100 and 1300 hours. Nitrogen fixation in spring showed complex, physically shallow but optically deep and mobile subsurface peaks of nitrogenase activity, which were totally unrelated to Aphanizomenon biomass but may have been due to diel changes in light penetrating the relatively clear water. Nocturnal fixation was uniformly distributed with depth and accounted for 1/ to Y3 of daylight fixation. In more turbid autumn waters, the pattern of N, fixation for Anabaena blooms was simpler, with a surface (or near-surface) peak decreasing with depth. Nocturnal cixation was more uniformly distributed with depth. The difference in fixation patterns between the two species is attributable to the interactions of oxygen with the nItrogenase enzyme system. The diel changes in nitrogenase activity suggest a need to establish whether the precursors of nitrogenase accumulate in an oxygen-stable form.

Nitrogen fixation requires more energy photorespiration may not be inevitable than any other biological process. Thus since the algae can regulate their position it should be highly light-dependent in in the waterco1umn (Waisby 1972; Reyn- 'lakes, since only photosynthetic organ- olds 1972, 1973). Thus there may be less isms are quantitatively significant in lake need for nocturnal N 2 fixation as after- N2 fixation (Home 1975a; Fogg et al. noon activity should be less depressed- 1973). However, phytoplanktonic N 2 fix- at least in lakes with several meters of ation may occur at low light levels and photic zone. The work reported here was even in total darkness (Home and Fogg designed to test this hypothesis and to 1970; Duong 1972; Vanderhoef et al. provide a quantitative method of calcu- 1975; Burns and Peterson 1978). lating daily and hence annual N 2 fixation Quantities fixed are generally but not al- amounts using incubation normally for ways low; there are disadvantages to high only a couple of hours at midday. photosynthesis, particularly the onset of A further reason for carrying out five photorespiration which successfully diel studies in both blooms spread over competes with N 2 fixation for available 3 years was to improve the meaningful- energy (Home 1975b). Photorespiration ness of the data. Generalizations based is enhanced by the afternoon low CO 2 on any one of the diel studies reported and high 02 levels, which are possible in here would not be significant without planktonic algal colonies and inevitable guidance from the other four. inside gelatinous Nostoc communities in I thank C. J. W. Carmiggelt and P. streams, despite the constant 0 2 and CO2 Kellar for technical assistance. environment provided by the well aer- ated stream water (Home and Carmiggelt 1975). Methods In the more open "flake" association of Samples were usually collected at .1 Aphanizomenon or coils of Anabaena, depths of 0, 0.5, 2.0, 3.0, and 4.0 m with an opaque Van Dorn bottle. The photic 'This study was supported by the Clear Lake zone is generally between 1 and 4 m in Algal Research Unit, Lake County, California, and Clear Lake, shallowest in autumn and the California Department of Water Resources. winter. Since the main purpose of the ex- 329 330 Home

Table 1. Diel variation in nutrients (in g !iter 1 ) spring and early summer clouds are rare during an Aphanizomenon bloom, 21-22 June and some near-surface thermal stratifica- 1972. tion re-forms each day. Afternoon winds

Period Time P0P NH-N NO,-N usually destroyed thermal stratification for at least part of each day on which 3 0930 42 11 14 studies were carried out and the regular 5 1500 45 45 1 7 2200 43 1 1 stirring mixed such nutrients as were 9 0600 10 15 1 available from the sediments into the photic zone. Nevertheless, biologically important nutrients were scarce, having periments was to document the diel been depleted by the first nitrate-fueled changes in N2 fixation, relatively few oth- stages of the massive Aphanizomenon er measurements were made. Collections bloom. For example, in June 1972 nitrate were taken at intervals of a few hours ranged from 1-14 gliter', ammonium either from dawn to dusk (3 days) or over from 1-45 jLgliter 1 , and phosphate from a 36-h period (2 days) during spring 1970, 10-45 gliter 1 (Table 1). The imbal- 1971, 1972 (Aphanizomenon bloom), and ance between P and N produced a severe autumn 1970, 1971 (Anabaena bloom). nitrogen stress and was the reason for the Details of methods were given by Home N2 fixation at this time (Home and Gold- and Goldman (1972). Measurements man 1972). were made of N 2 fixation as acetylene re- An overall view of the diurnal, changes duction, carbon fixation, chlorophyll a, in N2 fixation and chlorophyll a in the general chemistry, algal species, biomass water column for the three seasons and heterocyst counts, water transparen- (1970-1972) is given in Fig. 1. In general cy as Secchi depths, solar energy with chlorophyll a, which was contained time using a Belfort pyrheliometer, and mainly in Aphanizomenon, showed no estimated wind speed and direction. Al- regular pattern but fluctuated consider- gae were counted in spring 1972 at each ably due to patchiness (Wrigley and depth sampled for all nine periods over Home 1975) and vertical migration 36 h, to distinguish the effects of Apha- (Reynolds and Walsby 1975). Nitrogen nizomenon from those of other less abun- fixation showed a broadly constant pat- dant algae that may have contributed tern in all 3 years but considerable vari- disproportionately to photosynthesis and ation within that general trend. Early nutrient uptake (Watt 1971; Dozier 1976; sunlight around 0700-0800 hours pro- Stull et al. 1973). Such influence is un- duced only a small increase in N2 fixation likely in eutrophic lakes where the most over nocturnal rates, but by midmorning common algae physically can dominate rates were almost as high as at any other the struggle to gain the most favorable time of day. Early morning activity was light climate. definitely dependent on sunlight, since a cloudy start to the day depressed fixa- Results tion below normal nocturnal levels. Pre- All studies were carried out at the sumably nocturnal fixation after an ab- height of N 2 fixation activities in Upper normal (totally cloudy) day would be Arm, which comprises 70% of the area of negligible (Home 1975b). During the the lake. There were many similarities major part of the day activity was high and differences between the three spring, and quite constant (200-600 ttmol Aphanizomenon-dominated blooms and C2 H4 liter 1 h 1). It decreased at dusk, the two autumn, Anabaena-dominated but not proportionately with illumina- blooms. The most convenient way of con- tion. Most unexpected at the time of the sidering the results is to separate them experiments was the nocturnal fixation into those of spring and autumn. activity which persisted all night, gener- Spring Aphanizomenon blooms—In ally at moderately high levels. Relatively Nitrogen fixation. 4. 331

400 600

r 1 1.0 200

200 0.5 z —I 0 0

400 1.0 b >< 0.5

c.1 Z 600

200 200

2400 0600 1200 1800 2400 . 0600

Fig. 1. Diel variations in N2 fixation (as ethylene produced in gmolliter'h), chlorophyll a, and sunlight at height of three spring Aphanizomenon blooms. a-26 June 1970; b-16 June 1971; 0-21-22 June 1972. Solid bars are N 2 fixation and open bars are chlorophyll a, both expressed per unit of surface area for mixed water layer. Line represents solar radiation.

few samples (70) were incubated in total well. Efficiency of N2 fixation was re- darkness, so the actual rates observed are markably consistent day and night and best viewed as preliminary. In 1971 and year to year (Table 2). Most values were 1972 fixation from 0100 to 0300 hours was between 2 and 4 gmol C2 H4 h 1 mg' a third to half that found during the 14 h Chl a and efficiency was highest in the of daylight. There is little possibility that afternoon. The constancy of nitrogenase such findings of dark ethylene reduction activity was not directly due to the pres- were due to methodological error, since ence of a constant number of heterocysts the technique is quite standardized. Bot- because, if expressed as a percentage of tles containing algae but no acetylene ad- vegetative cells, heterocysts amounted to dition never produced any detectable 1.5% in 1970 and 1971 and only 0.5% in

ethylene (peak height, 0.1). Nocturnal N 2 1972. This represented photic zone av- ig fixation added considerably to the annual erage heterocyst concentrations per mil- nitrogen income of the lake because this liliter of about 5 x 102 (1970), 102 (1971), bloom was long, lasting 2-3 months and 102 (1972). 'Home and Goldman 1972; Home 1975a), When depth as well as time was con- and the dark values were consistent. sidered, the patterns were surprisingly The rate of N2 fixation expressed per simple. Isopleths of N 2 fixation and chlo- unit of N2-fixing algae gives a measure of rophyll are shown in Fig. 2. Thermal efficiency. In this spring bloom, chloro- stratification occurred on all occasions, phyll a represented Aphanizomenon lasting from around noon until the onset 332 Home

Table 2. Diel variations in efficiency of N 2 fixation (as amol C 2H4 h 1 mg 1 Chi a for whole water column) in spring and autumn blooms 1970-1972. Column numbers represent time periods.

0600- Dark 0900 1900 2400 Dark hours hours hours Dawn fi

Jun '70 - - 0.6 2.05 2.95 2.15 1.52 - - -

Jun '71 - - 1.92 2.72 4.1 3.14 4.1 2.35 - - Jun '72 - 2.1 1.13 2.92 1.94 2.44 2.22 1.4 2.45 8.6

Sep '70 - - 5.9 11.7 9.15 5.56 4.67 - - - Sep '71 0.39 0.33 1.32 2.05 1.4 1.5 * Cisl a not detected of darkness. This partially explains the surface and thus to gain maximum-energy_ changes in chlorophyll with depth, but for photosynthesis and the energetically since Aphanizomenon can regulate its costly N2-fixing process is dependent on buoyancy very effectively in Clear Lake its ability to avoid death from irradiation, the subsurface peaks were due more to and this in turn appears to be dependent gas vacuole collapse and synthesis than on both environmental and intracellular to turbulence. Nitrogen fixation exhibit- factors (Walsby pers. comm.). This notion ed much greater heterogeneity than did is considered further below. chlorophyll (Fig. 2). There was virtually The peaks in N2 fixation were caused no relationship between layers of chlo- not by a physical accumulation of Apha- rophyll and N 2 fixation in the spring nizomenon, which, was quite evenly dis- bloom, although this was not true in au- tributed (Table 3, Fig. 2D-F), but pre- tumn. In 1970, N2 fixation fell linearly sumably by a physiological condition from a maximum of 425 nmol . lit er_i. h_i allowing short-lived high rates. In the to <100 at 4 m, just below the photic zone longest diurnal observation, in 1972, the (Fig. 2A). The spring seasons of 1971 and subsurface nitrogenase activity peak at 2- 1972 were very different (Fig. 2B, C), il- 3 m was very consistent, forming in mid- lustrating my point that several diurnal morning, moving upward, disappearing studies should be made even if this takes altogether at night but forming again at several years to accomplish. In 1971 and the same depth next morning. 1972 there were distinct surface and sub- Many factors may play a role in deter- surface peaks which changed position in mining ephemeral changes in the depth- the water column and in intensity of time distribution of N2 fixation; the most nitrogenase activity. Subsurface peaks in important are light, mixing, oxygen and activity at 1 m (1971) and 2 m (1972) de- carbon dioxide, nutrients, pH, and tem- veloped in the early morning, moving perature. Incoming radiation was similar downward in the course of the day in on all occasions (Fig. 1) and only the 1971, but upward in 1972 when a surface amount of light-absorbing material influ- peak was recorded for the first time in enced the depth for optimal photosynthe- late afternoon (Fig. 2B, Q. Midsummer sis, which was normally between 10 and in the Coast Range of northern California 30% I. The photic zone was much shal- brings very intense radiation, even at lower in 1970 (Secchi depth, 85-108 cm) only 400 m above sea level, and on many than in 1971 (84-123 cm) or 1972 (100- occasions has been observed to kill sur- 325 cm), but chlorophyll alone cannot ex- face Aphanizomenon blooms, presum- plain the higher transparency of the 1972 ably by inducing photo-oxidative cellular season, when chlorophyll a was roughly destruction. The cells must strike a bal- twice that of 1971. ance between optimal and lethal light. The extent of mixing in the water col- The ability of a species to be near the umn was controlled by the depth of the

Nitrogen fixation. 4. 333

N2 FIXATION CHLOROPHYLL a

2400 0600 200 1800 2400 0600 2400 0600 1200 1800 2400 0600 ' I I I 0 Jil : I I 26 June 1970 1 1.0 /I III Uc

2.0

3.0 - A II 4.0 0.

1.0 150 25 I 2.0 F— I25 .25 3.0 125 20 I I6June 1971 I 4.01

do 1.00 F77 *&.

I'5( \1J 2.0- 00)1110 ) ~ ) 75 3.0 I t 2h2 June1972 j 4 .0 ______ 50 I I .III.lI I 2400 0600 12bo 1800 200 0600 2400 0600 1200 1800 2400 0600 TIME OF DAY Fig. 2. Isopleths of N2 fixation and chlorophyll a for three spring Aphanizomenon blooms, 1970-1972. Units same as Fig. 1. N2 fixation assayed as ethylene produced; isopleths are for every 25 nmol ethyl- eneliter'h'. Chlorophyll a isopleths every 5jgliter'. Horizontal line(s) approximates photosynthetic compensation point in1970 and 1971, but is below 4 m in 1972.

temporary thermocline produced in mid- highest and lowest chlorophyll values morning while the lake was calm. The varied by a factor of 5 and did not corre- exact depth of mixing changes with time spond to the intermediate chlorophyll jr and is difficult to establish, since small levels. Oxygen levels exceeded satura- relative increases at these high absolute tion in all diurnal observations (Table 4) temperatures may have produced strong but were not particularly high in spring. density gradients. Stratification lasted Significant CO2 depletion is not often ev- longer in the afternoon in 1972 than in ident in the moderately hard water of the previous years and the consequent Clear Lake and has never been detected lack of mixing in the upper meter prob- in our frequent routine analyses. Since ably inhibited photosynthesis and N 2 fix- spring 1972 pH values have hovered be- ation. Certainly self-shading was not in- tween 8 and 9 (Table 5), and CO 2 limi- volved, since the surface maxima for the tation has been transient or more likely

334 Home

C'] N C) CI * N • N * C) —co C) o c, cc C) C. '-I

00 C) * C) * CC * C) * C) CC co '© C CCNC'CICCC')C)CC_CC C O C']

C - * C' C) N C) CI IR i f) cl C) 0 CI C) - C) '' CC CI a —- -C

' _** - CCC) co CC )) CC C) - ']C'] In gN I_C C)..0 4)11)

•4)C) ' '*'-.'*' cq ' R g 0 .C) CC -i -I - CI

I_C) c.- EE- —-- )')ç C') co ' C C) CI (C 11) C) C - "1 CO N q C) 'CC'4 jC')C')C'CI gC_— >

4)4)

- —CM S -V S

CC > C) Cl. .11 C)C) C) C) C) C 4) 040 0 4)

CI C') 'C Nitrogen fixation. 4. 335

Table 4. Diel temperature ('C) and dissolved oxygen (mg'liter') profiles for fall and spring.

Fall 1971 • ,.. . Spring 1972

2030 ', 1130;- 1230 2300 Depth - (m) Temp DO -. Temp DO Temp DO Temp DO o 29 20 26 • 18. 26 9.6 24.2 ' 9.9

0.5 28 20 26 :.. 16' 25 9.9 24.2 10.3 1.0 27 8.9 25 6.9 .' 24 12 24.2 10.4 1.5 25.5 - 23.5 . 2.8 23 9.7 24.1 10.4 2.0 24 - 23 1.1 22.5 8.9 24 10.2 3.0 23 - 22.5 0.4 22.5 8.8 23.7 9.7 4.0 Bottom 22 8.5 23.1 9.1 6.0 22 4.8 22.1 5.7 8.0 20 2.5 21.3 2.6 absent in spring. Lake temperatures dur- ently occurs in Lake George, Uganda ing the spring diurnals varied from 20°C (Ganf and Viner 1973). The only conclu- at the deeper points to 26°C at the surface sions to be drawn are that all inorganic near noon. Nitrogen fixation does not nitrogen species were always in short have a very large Q 10 over this range supply and phosphate was not (Table 1). (Fogg and Stewart 1968; Home unpubi.), The most facinating feature of the so the subsurface patches are unlikely to spring diel studies was the occurrence of be due to simple temperature effects. nocturnal N2 fixation (Fig. 1). Figure 2B Variations in major nutrients were mea- and C illustrate the rapid breakdown of sured during one diel study only, since the surface and subsurface peaks of N 2 N2 fixation itself is not affected rapidly by fixation with the onset of dusk to produce small changes (Home et al. 1979). Never- a uniform distribution of activity with theless it was surprising to find no over- depth. This was to be expected, since all night accumulation of scarce nutrients the N2 fixation would be accomplished by such as nitrate or ammonium, as appar- use of stored energy evenly spread

Table 5. Diel variations in major physical variables at surface of lake in spring

0600- 0930- 1230- 1500- 1800- 2200- 0200- 0600- variable' 1700 2030 0800 1030 1330 1700 2000 2400 0400 1800 Spring Secchi - - 90 85 85 108 - - - - 1970 Spring Temp - - 21 21.5 23 25 22 - - - 1971 Secchi - - 104 103 123 120 84 - - - 1%I - - >300 275 275 250 Spring Temp - - 23 26 26 23 24.4 24.2 23.8 23.8 1972 DO - - 9.4 8.6 9.6 11.2 11.9 9.9 9.7 9.0 pH - - - 8.4 - 8.5 - 8.8 - - Secchi - - 325 175 100 138 150 0 0 - Turb - - - 5.2 - 3.7 - 3.0 - - Fall Temp - - 19.5 20 21 24.5 21.5 1970 Secchi - - 20 69 62 68 67 - - - Fall Temp 29 28 24.5 23 26 30 32 - - - 1971 DO >20 >20 13 16.2 17.7 18.3 >20 pH 10 9.4 9.4 9.3 9.3 9.7 9.7 - - - Secchi 40 0 45 34 39 50 45 - - - Turb 42 17 22 27 26 18 20 - - - 'Secehi depth, cm; temperature, 'C; 1% 1,, cm; DO, mgIiterr ; turbidity, JTU 336 Home

10 3

300 500 1.0

z 0 0 0 C) I-

>< E U- 3 600 CO C'l 10 3 3 z p.., 400

500 1 10 200 fo.s

1800 2400 0600 1200 1800 Fig. 3. As Fig 1, but for two autumn Anabaena blooms. a-30 September 1970; b-14 September 1971.

through the population following thermal the pattern was not repeated. The rela- destratification by late afternoon winds. tionship between maximal nitrogenase The spring 1972 diel study was carried activity, heterocysts ; and vegetative cells out in slightly deeper water than in pre- is shown in Table 3. During the day the vious springs or in any autumn when the number of heterocysts corresponded well lake was shallower: the algae had be- with the depth of maximum nitrogenase tween 6 and 8 m in which to move. The activity. By contrast nocturnal fixation diel movements of active heterocyst- showed no relationship of heterocysts bearing Aphanizomenon populations, il- with maxinium activity, implying that the lustrated in Table 3, did not follow the vegetative cells were contributing some generalized pattern proposed for blue- fixed nitrogen. green algae in lakes of this type by Reyn- Autumn Anabaena blooms—Ex- olds and Walsby (1975), in that photic pressed per unit area, there was little dif- zone biomass increased during the day ference between the diel patterns of N 2 and decreased at night. This finding is fixation of Anabaena and those of Apha- examined in further detail below. In gen- nizomenon. Fixation rose rapidly in mid- eral the diel movement of heterocysts fol- morning and remained relatively con- lowed N2 fixation patterns for the first day stant until dusk (Fig. 3). Quantities of N 2 of the experiment, but on the next day fixed were also similar in both spring and Nitrogen fixation. 4. 337

N2 FIXATION CHLOROPHYLL a BOO 2400 0600 1200 1800 1 1800 10 0600 1200 1800 0 ooI3O SeP 19701 1.0 OR

50 2.0 E 3.0 A-

0 0. LLI o .o . 30

2.0

III'' 14 Sep1971 D

4.01 . III I II I I I 1800 2400 0600 1200 1800 1800 2400 0600 1200 1800 TIME OF DAY Fig. 4. As Fig. 2, but for two autumn A,2abaena l)looms of 1970 and 1971; N2 fixation isopleths every 100 nmolliter'h and chlorophyll a isopleths at 50 g1iter' intervals.

fall, although the efficiency of fixation per similar patterns of biomass, both produc- unit biomass by Anabaena was consid- ing distinct surface and subsurface max- erably greater than by Aphanizomenon ima which broke down overnight (cf. in 1970 and somewhat less in 1971. Figs. 2 and 4). Anabaena was generally Isopleths of N2 fixation with depth and nearer the surface—a position best ex- time showed that it was confined to the plained by the turbidity of the water. Sec- upper layers, i.e. the shallow photic zone, chi disk depths were 20-70 cm in 1970 except at night (Fig. 4A, B). Another dis- and 34-45 cm in 1971 when turbidity was similarity from the spring blooms was a measured by nephelometry at 17-30 reasonably good correlation between al- JTU. The photosynthetic compensation gal biomass and N2 fixation (Fig. 4). Sur- point in September was at about 2 m in face fixation (0-0.5 m) dominated quan- 1970 and 1 m in 1971, and all Anabaena titatively, in contrast to the situation in biomass peaks were above these depths spring. Once again this was probably be- (Fig. 4C, D). For the spring Aphanizo- cause autumn sunlight was less lethal. menon bloom the compensation depth As with Aphanizomenon blooms, the was 3 to 4 m, with subsurface chlorophyll variations in efficiency were not due to maxima correspondingly deeper. The di- differences in heterocysts. Anabaena minished clarity in fall was not wholly Ii populations normally had 5 times as due to algae, the biomass of which many heterocysts as Aphanizomenon reached similar levels in fall and spring, h (Home and Goldman 1972; Home 1975b). but to sediments stirred up by winds at In 1970 there were about 4% heterocysts the edges of the lake. It is not evident in Anabaena populations, in 1971 about why late summer sediments should have 6%. been more prone to suspension than Anabaena and Aphanizomenon showed those of spring. Suspended sediment is 338 Home transported clockwise round the lake in man 1972), nocturnal N 2 fixation is im- autumn and mixes with both deep and portant for the whole lake's ecology. edge waters (Wrigley and Home 1974). Anabaena seems less able to make use of Another possible reason for reduced darkness for N2 fixation, although the re- water clarity is the presence of a substan- sults are not totally conclusive. Three tial population of small flagellates and questions spring to mind concerning noc- cryptomonads amongst the Anabaena turnal N 2 fixation in lakes: Why does it bloom. Aphanizomenon blooms were al- occur at all? Why does it occur at night, most unialgal. Small phytoplankton con- but not in the aphotic zone during the tribute more to light absorption than do day or in the sediments? How does the large clumps of blue-green algae (Home dark fixation stragegy of Aphanizomenon and Blank unpubl.), so although chloro- and Anabaena relate to that used by phyll concentrations were similar in stream Nostoc? spring and autumn biotic light absorption Blue-green algae are an ancient group was not. and lack many complex enzymatic induc- Nocturnal N2 fixation by Anabaena was tion and repressor systems found in other apparently less important than that by microorganisms (Carr 1973). Even though Aphanizomenon, although there were in- N2 fixation is energy demanding, it is not dications of some quite high Anabaena always directly harnessed to photosyn- activity after dusk. thesis (Home and Fogg 1970; Home Lake temperatures, higher in autumn 1975b). Unlike photosynthesis, N 2 fixa- than in spring and showing more diel tion should fall slowly to insignificance variation, had the net effect of producing after sunset—the length of time for which stronger thermal stratification (Tables 4 it continues being related by the amount and 5), unless clouds prevented the daily of energy stored in the glycogen pool pro- replenishment of surface heat. During duced during the preceding day. This these diel experiments there were no seems to be the case for terrestrial algae, clouds (Fig. 3); algae were redistributed lichens, and attached stream Nostoc apparently either by buoyancy regulation where daily light regimes can be mea- (Walsby 1972) or by wind breakup of sured (Henriksson 1971; Home 1975b). loose Anabaena aggregations up to 5 cm In lakes the vertical mixing process pre- across. Winds did not affect thermal or vents knowledge of the exact prior light algal stratification even at 1700 hours history of the algae. However, most (Fig. 4B, D). planktonic blue-green algae will have energy available for N 2 fixation at sunset. Discussion Three factors will determine the amounts Although indisputable positive dark N2 fixed: the stored energy available, the fixation by aquatic algae was unknown in need for nitrogen, and the dissolved oxy- 1970, it has since been shown in lakes gen levels during the night. (Duong 1972; Vanderhoef et al. 1975; Nitrogen stresses are severe in Clear Burns and Peterson 1978), in a stream Lake in both spring and autumn, but due (Home 1975b), and in wet soils (Hen- to release of ammonium from the sedi- riksson 1971; Jones 1974). Some previous ments in summer (Lallatin 1972; Home 24-h dark bottle [15N]N2 studies may 1975a), which alleviates the nitrogen show dark N2 fixation, since dark values stress in autumn, the spring depletion is were sometimes almost as high as light most extreme (Table 1). This may ac- 1; ones (Home and Fogg 1970). Nocturnal count for greater nocturnal fixation in fixation in the spring Aphanizomenon spring. bloom accounts for about a third of the Two factors involving oxygen favor 24-h total of N 2 fixed, and since the spring nocturnal N 2 fixation in the spring bloom contributes half of the lake's an- Aphanizomenon bloom over that of the nual nitrogen income (Home and Gold- autumn Anabaena population: the lower Nitrogen fixation. 4. 339 nocturnal oxygen concentrations in lected below the photic zone may have spring, and the colony shape and size of been illuminated shortly before and will the two genera. The spring bloom does have quantities of photosynthetic oxygen not raise dissolved oxygen levels as high nearby. It takes several hours to synthe- as does the dense fall bloom (Tables 4 size very much nitrogenase, so the apho- and 5). High afternoon oxygen levels per- tic zone N2 fixation is akin to the early sist through much of the night in the fall postdusk N2 fixation, i.e. a tailing off of bloom, even though there is some de- the daytime processes. crease. Oxygen, even at normal concen- A conceptual problem with the inter- trations, inhibits N2 fixation in blue-green pretation of diel curves of N 2 fixation is algae partially by inactivating the 0 2-la- variation in rates of nitrogenase synthe- bile nitrogenase enzyme (Stewart and sis. If oxygen-stable precursors of nitro- Pearson 1970). Aphanizomenon in Clear genase could be stored, many of the vari- Lake grows in two colony forms, one ations in diel curves of N 2 fixation by transient, the other relatively permanent. phytoplankton and periphyton could be The permanent form is the "flake" explained. This biochemical problem is (O'Flaherty and Phinney 1970) consist- in need of study. ing of upward of several hundred tn- Nostoc growing in balls up to several chomes, each of a hundred or more cells centimeters in diameter firmly attached (Home 1975a). Only very vigorous shak- to rocks in streams shows a strong after- ing in a closed vessel will destroy this noon depression in N2 fixation (Home form. The ephemeral form consists of 1975b), similar to that found for both N 2 thousands of flakes in a ball often several fixation and photosynthesis in some lakes centimeters in diameter, which is easily (Ganf and Home 1975; Home and Viner broken by agitation of the surrounding 1971). This depression is strikingly ab- water. Anabaena forms much smaller, sent from the daylight curves of both durable colonies, often of coiled fila- Aphanizomenon and Anabaena blooms. ments, and produces much smaller, tem- In most cases N 2 fixation for these species porary balls less frequently. At night the quickly reaches high values after day- center of a large colony of respiring cells break and rates of activity fluctuate may be depleted in oxygen, which would around this level until after sunset (Figs. both enhance N2 fixation in the hetero- 1, 3). Photorespiration was proposed as a cysts and enable N 2 fixation in the vege- major mechanism of the afternoon tative cells. This was postulated for the depression for Nostoc (Home 1975b) and similarly shaped colonies of the marine for very dense equatorial phytoplankton- cyanophyte Trichodesinium (Carpenter ic blue-green algae (in Lake George, and Price 1976). By analogy with Trich- Uganda: Ganf and Home 1975). In Clear odesmium, vegetative cells of Aphanizo- Lake the relatively lower populations menon could fix measurable amounts of (200-500 mgm 2 Chl a as against 800 in N2 overnight. A further indication of veg- Lake George) and moderate chemical etative fixation is the lack of relationship hardness apparently prevented photores- between heterocyst number and maxi- piration from robbing the nitrogenase mum nitrogenase activity at night but system of photosynthetically generated close correspondence by day (Table 3). energy. The absence of depressed after- Levels of fixation by night were low but noon N2 fixation would decrease nitrogen significant. stress and is also the most probable rea- Algae in darkness (below the photic son for the lack of a nocturnal peak, along zone) should fix N2 during the day, using with the general blurring of the overall both heterocyst and vegetative nitrogen- picture by nocturnal water mixing. ase; this was not observed in Clear Lake A generally favorable climate for other because the mixed depth normally ex- biological processes, especially photo- ceeds that of the photic zone. Algae col- synthesis, may explain why the algae in 340 Home

Clear Lake do not follow the classic pat- AND A. B. VINER. 1973. Ecological stability tern of pronounced sinking during the in a shallow equatorial lake (Lake George, day and rising overnight (Reynolds and Uganda). Proc. R. Soc. Lond. Ser. B 184: 321- 346. Walsby 1975). There were more Apha- HENRIKSSON, E. 1971. Algal nitrogen fixation in nizomenon cells in the photic zone at noon temperate regions. Plant Soil Spec. Vol., p. than at any other time (Table 3). How- 415-419. ever, the data on surface accumulations HORNE, A. J. 1975a. The ecology of Clear Lake phytoplankton. Clear Lake Algal Res. Unit of blue-green algae on which Reynolds' Spec. Rep. Lakeport, Calif 116 p. (1972) pattern was based were taken on 1975b. Algal nitrogen fixation in California very calm days. Under more normal con- streams: Diel cycles and nocturnal fixation. ditions of afternoon winds, or on larger Freshwater Biol. 5: 471-477. lakes, the pattern may shift in time. The AND C. J. CARMIGGELT. 1975. Algal nitrogen fixation in California streams: Seasonal speeds of rising and sinking of the algae cycles. Freshwater Biol. 5: 461-470. in Clear Lake relative to lake wind mix- AND G. E. Focc. 1970. Nitrogen fixation in ing, together with the need for energy for some English lakes. Proc. R. Soc. Lond. Ser. B N2 fixation, can account for a midday algal 175: 351-366. AND C. R. GOLDMAN. 1972. Nitrogen fixa- maximum. Alternatively, recruitment of tion in Clear Lake, California. 1. Seasonal vari- buoyant algae to the photic zone follow- ation and the role of heterocysts. Limnol. ing nocturnal deep mixing continued Oceanogr. 17: 678-692. well into the morning, whereas the av- J. C. SANDUSKY, AND C. J. CARMIGGELT. erage tendency to sink out was not evi- 1979. Nitrogen fixation in Clear Lake, Califor- nia. 3. Repetitive synoptic sampling of the dent until later in the day. It has been spring Aphanizomenon blooms. Limnol. suggested, however, that nitrogen stress Oceanogr. 24: 316-328. would interfere with buoyancy regula- AND A. B. VINER. 1971. Nitrogen fixation tion (Reynolds and Waisby 1975). and its significance in tropical Lake George, Uganda. Nature 232: 417-418. References JONES, K. 1974. NItrogen fixation in salt marsh. J. Ecol. 62: 553-565. Bususjs, R. H., AND R. B. PETERSON. 1978. Ni- LALLATIN, R. D. 1972. Alternative Eel River proj- trogen fixing blue-green algae: Their hydrogen ects and conveyance routes, Appendix C. Clear metabolism and activity in freshwater lakes. Lake water quality. Calif Dep. Water Resour. Ecol. Bull. 26: 28-40. Sacramento. 145 p. CARPENTER, E. J., AND C. C. PRICE. 1976. Marine O'FLAHERTY, L. M., AND H. K. PHINNEY. 1970. Oscillatoria (Trichodesmium): Explanation for Requirements for the maintenance and growth aerobic nitrogen fixation without heterocysts. of Aphanizomenon flos-aquae in culture. J. Science 191: 1278-1280. Phycol. 6: 95-97. CARR, N. G. 1973. Metabolic control and auto- REYNOLDS, C. S. 1972. Growth, gas vacuolation trophic physiology, p. 39-65. In N. G. Carr and and buoyancy in a natural population of a blue- B. A. Whitton [eds.], The biology of blue-green green alga. Freshwater Biol. 2: 87-106. algae. Univ. Calif. 1973. Growth and buoyancy of Microcystis DozIER, B. J. 1976. An autoradiographic study of aeruginosa Kütz emend. Elenkin in shallow eu- the relationship between photosynthetic rate trophic lake. Proc. R. Soc. Lond. Ser. B 184: and phytoplankton seasonal succession in Lake 29-50. Tahoe, California-Nevada. Ph.D. thesis, Univ. AND A. E. WALSBY. 1975. Water blooms. Calif., Davis. Biol. Rev. 50: 342-351. DUONG, T. P. 1972. Nitrogen fixation and produc- STEWART, W. D., AND H. W. PEARSON. 1970. Ef- tivity in a eutrophic, hard-water lake: In situ fects of aerobic and anaerobic conditions on and laboratory studies. Ph.D. thesis, Mich. growth and metabolism of blue-green algae. State Univ. Proc. R. Soc. Lond. Ser. B 175: 293-311. FOGG, A. E., AND W. D. STEWART. 1968. In situ STULL, E. A., E. DE AMEZAGA, AND C. R. GOLD- determinations of biological nitrogen fixation in MAN. 1973. The contribution of individual Antarctica. Brit. Antarct. Surv. Bull. 15: 39-40. species of algae to primary productivity in Cas- P. FAY, AND A. E. WALSBY. 1973. tle Lake, California. mt. Ver. Theor. Angew. The blue-green algae. Academic. Limnol. Verh. 18: 1776-1783. GANF, G. G., AND A. J. HORNE Diurnal stratifica- VANDERHOEF, L. N., P. J. LEIBSON, R. J. MUSIL, tion, photosynthesis and nitrogen fixation in a AND C.-Y. HUANG. 1975. Diurnal variation in shallow, equatorial lake (Lake George, Ugan- algal acetylene reduction (nitrogen fixation) in da). Freshwater Biol. 5: 13-39. situ. Plant Physiol. 55: 373-376.

Nitrogen fixation. 4. 341

WALSBY, A. E. 1972. Structure and function of gas , AND . 1975. Surface algal circulation vacuoles. Bacteriol. Rev. 36: 1-32. patterns in Clear Lake by remote sensing. WATT, W. D. 1971. Measuring the primary pro- NASA Tech. Memo. TMS-62. NASA-Ames, duction rates of individual phytoplankton Moffet Field, Calif. species in natural mixed populations. Deep-

WmGLEy,R. C., AND A. J. HORNE. 1974. Remote Submitted: 20 December 1976 sensing and lake eutrophication. Nature 250: Accepted: 20 September 1978 213-214.

41 This report was done with support from the Deprtment of Energy. Any cortclusiqns or opinions expressed in this report represent soicly those of the author(s) and not necessarily thpse of The Regents of the University of California. the Lawrence Berkeley Laboratory or the Department of Energy. Reference to a company or product name does not imply approval or recommendation of the product by the University of Californior the U.S. Department of Energy to the excll4sion of others that may bel suitable. tMth1 thlCJ)

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