Buoyancy regulation in Microcystis aeruginosa grown at different temperatures

Abstract The buoyancy regulation in light-limited cultures of the gas vacuolate cyanobacterium Microcystis aeruginosa AK1 was studied at three temperatures, 15, 20 and 28°C. At the two highest temperatures the organism remained buoyant during the entire light period, whereas at the lowest temperature the buoyancy was reduced at the start of the light period. With this temperature the buoyancy was lost during the light period. This reduced buoyancy was caused by an increase in ballast and a decrease in the gas vesicle volume. Buoyancy changes during a transient state with slow changes in temperatures, i.e., 1°C per day, were caused by changes in polysaccharide ballast. The gas vesicle volume showed no significant change during the transient state.
The maximal photosynthetic rate was dependent upon the growth and incubation temperature, whereas the light harvesting efficiency was independent of the temperature. The results are discussed in an ecological context.     

REGULATION OF GAS VESICLE CONTENT AND BUOYANCY IN LIGHT-OR PHOSPHATE-LIMITED CULTURES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYTA)1

Measurements of the gas vesicle space in steady-state light or phosphate-limited cultures of Aphanizomenon flos-aquae Ralfs, strain 7905 showed that gas vesicle content decreased as energy-limited growth rate increased but was the same at several phosphate-limited growth rates. Upon a decrease in growth irradiance, gas vesicle content did increase in phosphate-limited cultures, but the cultures remained nonbuoyant as long as P was limiting. Buoyant, energy-limited cultures lost their buoyancy in less than 2 h when exposed to higher irradiances. The primary mechanism for buoyancy loss was the accumulation of polysaccharide as ballast. Collapse of gas vesicles by turgor pressure played a minor role in the loss of buoyancy. When cultures were exposed to higher irradiances, cells continued to synthesize gas vesicles at the same rate as before the shift for at least 1 generation time. The amount of ballast required to make individual filaments in the population sink varied 4-fold. This variation appears to be due to differences in gas vesicle content among individual filaments.     

REGULATION OF GAS VESICLE CONTENT AND BUOYANCY IN LIGHT- OR PHOSPHATE-LIMITED CULTURES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYTA)1

Measurements of the gas vesicle space in steady-state light or phosphate-limited cultures of Aphanizomenon flos-aquae Ralfs, strain 7905 showed that gas vesicle content decreased as energy-limited growth rate increased hut was the same at several phosphate-limited growth rates. Upon a decrease in growth irradiance, gas vesicle content did increase in phosphate-limited cultures, hut the cultures remained nonbuoyant as long as P was limiting. Buoyant, energy-limited cultures lost their buoyancy in less than 2 h when exposed to higher irradiances. The primary mechanism for buoyancy loss was the accumulation of polysaccharide as ballast. Collapse of gas vesicles by turgor pressure played a minor role in the loss of buoyancy. When cultures were exposed to higher irradiances, cells continued to synthesize gas vesicles at the same rate as before the shift for at least 1 generation time. The amount of ballast required to make individual filaments in the population sink varied 4-fold. This variation appears to be due to differences in gas vesicle content among individual filaments.     

DOES CARBOHYDRATE CONTENT AFFECT THE SINKING RATES OF MARINE DIATOMS?1

We tested the hypothesis that positive relationships between sinking rate and irradiance were due to increases in cell density caused by accumulations of carbohydrate. In semicontinuous batch cultures of Thalassiosira weissflogii (Gru.) Fryxell el Hasle and Ditylum brightwellii (t. West) Grunow in Van Huerk, carbohydrate content was varied by growing cells under diel cycles of high or low light. Sinking rate was measured at the end of the light period and the end of the dark period, on live and heat-killed cells. No positive correlations were found between sinking rate (which varied from – 0.060 to 0.13 m·d^?1) and carbohydrate content (which varied from 10 to 950 pg · cell^?1), indicating that accumulations of carbohydrate did not significantly affect sinking rate. There were no large diel variations in the sinking rate of T. weissflogii, but sinking rates of D. brightwellii grown under high light ranged from being negative (i.e. cells were floating) at the end of the light period to positive at the end of the dark period. This is the first report of positive buoyancy in vegetative D. brightwellii, a phenomenon that may only occur in D. brightwellii grown under diel cycles.     

THE EFFECT OF NUTRIENT LIMITATION AND ITS INTERACTION WITH LIGHT UPON THE PRODUCTS OF PHOTOSYNTHESIS IN MERISMOPEDIA TENUISSIMA (CYANOPHYCEAE)1

The metabolic fate of photosynthetically-fixed CO₂ was determined by labeling samples of Merismopedia tenuissima Lemmerman for 30 min with NaH¹⁴CO₃ and analyzing its incorporation into low molecular weight compounds, polysaccharide and protein. In N- and P-sufficient cultures, relative incorporation into protein increased as the irradiance used during the labeling period was decreased to 20 μE · m^-2 s^-1. This pattern was found for cells grown at irradiances of either 20 or 180 μE · m^-2· s^-1, although incorporation into protein was greater in cultures grown at the higher irradiance. In N-limited continuous cultures, relative incorporation into protein was low, independent of growth rate, and the same for samples tested at 20 or 180 μE · m^-2· s^-1 irradiance. In contrast, ¹⁴C incorporation into protein by P-limited cultures increased as growth rate increased, and at relative growth rates greater than 0.25, the incorporation was greater at 20 than at 180 μE · m^-2· s^-1. However, the total RNA content and maximum photosynthetic rate of the cultures was the same at all growth rates tested. The interaction between nutrient concentration and light intensity was studied by growing-limited continuous cultures at the same dilution rate, but different irradiances. Relative incorporation into protein was highest in cultures grown at 20 μE · m^-2· s^-1, in which the relative growth rate was 0.4. These results suggest that photosynthetic carbon metabolism may respond to relative growth rate μ/μ_max rather than to growth rate directly.     

Diel Buoyancy Changes by the Cyanobacterium Aphanizomenon ovalisporum from a Shallow Reservoir

In the summer of 1999, a bloom (11 100 filaments ml^–1)of the gas vacuolate cyanobacteriumAphanizomenon ovalisporumdeveloped in a shallow (1.7 m deep) reservoir containing nutrient-enrichedwater from Lake Kinneret (Israel). During 4 days, A. ovalisporumshowed a marked diel periodicity in buoyancy: the proportionof floating filaments fluctuated between 76–84% from middayto evening and 94–98% at the end of the night, in bothsurface and bottom samples. Buoyant filaments were present throughoutthe water column, presumably due to wind-driven vertical mixing.Aphanizomenonfilaments collected from the reservoir were maintained undermean photon irradiances of 15 (LL), 150 (ML) and 1100 (HL) µmolm^–2 s^–1 in a computer-controlled set-up, which simulatedthe diel light changes at different depths in the reservoir.In the LL cultures, filament buoyancy showed no diel fluctuationpatterns during the 4 days of incubation, but ML and HL culturesshowed regular diel changes, with a higher proportion of filamentsfloating at the end of the night than during midday–evening.There was no evidence for either turgor-driven collapse of gasvesicles or dilution of gas vesicles by cell growth by any ofthe treatments. Gas vesicles of A. ovalisporum had a relativelylow mean critical pressure (p_c of 0.57 MPa), but the daytimerise in turgor pressure was too small to cause gas vesicle collapse.The observed diel buoyancy changes may be explained by accumulationof carbohydrate ballast during the day and decrease during thenight.     

Effects of macronutrients upon buoyancy regulation by metalimnetic Oscillatoria agardhii in Deming Lake, Minnesota

Gas-vacuolate filaments of Oscillatoria agardhii form a metalimneticlayer in Oeming Lake, Minnesota. The environmental factors whichaffect buoyancy and the physiological processes which mediatechanges in buoyancy were determined. Buoyant filaments losttheir buoyancy in a few hours when incubated at light intensitiesabove those found in situ ({small tilde}15 µnol photonsm^–2 s^–1, or 1% of the surface value). The rate ofbuoyancy loss was accelerated by the addition of 10 µMphosphate at irradiances >200mol photons m^–2 s^–1.The effect of nutrient additions on buoyancy was also investigatedover a longer time period by incubating metalimnetic samplesin situ. The samples were deployed for 6 days at a depth wherethe irradiance was 8% of the surface value. As found in short-termexperiments, the addition of phosphate resulted in the largestdecrease in buoyancy. However, the addition of ammonia in additionto phosphate attenuated the buoyancy loss on day 2, and on day6 the filaments in these treatments were almost completely buoyant.The physiological status of the filaments in these treatmentswas assayed by analysis of elemental ratios of C, N and P, andby measurement of cellular chlorophyll, polysaccharide and protein.In addition, the cellular content of gas vesicles was determined.The construction of ballast balance sheets from these data indicatedthat changes in buoyancy were primarily due to differences inthe amount of polysaccharide ballast in the cells. However,in another set of in situ experiments, the increase in measuredballast molecules did not explain the observed loss of buoyancy.We hypothesized that another, undetected ballast-providing moleculehad accumulated in the cells.     

The Burgundy-blood phenomenon: a model of buoyancy change explains autumnal waterblooms by Planktothrix rubescens in Lake Zürich

Buoyancy changes of the cyanobacterium Planktothrix rubescens- the Burgundy-blood alga - were modelled from its buoyancy response to light and irradiance changes in Lake Zürich during autumnal mixing. The daily insolation received by filaments at fixed depths and circulating to different depths was calculated from the measured light attenuation and surface irradiance. The active mixing depth, za5, was determined from the vertical turbulent diffusion coefficient, Kz, calculated from the wind speed, heat flux and temperature gradients. The fixed depth resulting in filament buoyancy, zn, decreased from 13 to 2 m between August and December 1998; the critical depth for buoyancy, zq, to which filaments must be circulated to become buoyant, decreased from >60 m in the summer to <10 m in winter. When za5 first exceeded zn, in September, P. rubescens was mixed into the epilimnion. In October, zq > za5: circulating filaments would have lost buoyancy in the high insolation. Often in November and December, after deeper mixing and lower insolation, za5 > zq: filaments would have become buoyant but would have floated to the lake surface (the Burgundy-blood phenomenon) only under subsequent calm conditions, when Kz was low. The model explains the Burgundy-blood phenomenon in deeper lakes; waterblooms near shallow leeward shores arise from populations floating up in deeper regions of the lake.     

The influence of light and nutrients on buoyancy, filament aggregation and flotation of Anabaena circinalis

At Chaffey Dam, New South Wales, Australia, Anabaena circinalis filaments accumulated at the surface as diurnal surface layer thermal stratification developed. Previously buoyant, homogeneously distributed colonies accumulated in the top 2 m, but a proportion lost buoyancy. Similarly, a percentage of A.circinalis suspended in bottles lost buoyancy at depths experiencing >30% surface irradiance (Io). Nutrient addition reduced the proportion of filaments that lost buoyancy following a full day of high irradiance. The greatest axial linear dimension (GALD) was measured for A.circinalis deployed in bottles at three depths in the reservoir. GALD increased in samples exposed to 1 and 30% Io by the following day. The rank order of GALD from smallest to largest grouped samples exposed to 70, 30 and 1% Io, suggesting that increasing GALD is a function of irradiance. The increased GALD of biomass units was attributed to aggregation of filaments in low light. The enlargement of biomass units increased the mean floating velocity, supporting the theory that filament aggregation may be a strategy, utilized by light-limited filaments, to increase light exposure. High irradiance increased the carbohydrate content of cells and decreased the floating velocity of filaments.      

Effects of light intensity on the growth and buoyancy of detachedElodea nuttallii (Planch.) St. John during winter

The influences of light intensity on the growth and buoyancy of detachedElodea muttallii (Planch.) St. John during winter were examined under controlled experimental light conditions. Light was controlled by mesh-screens at five levels ranging between 0.3 and 51% of the aerial full sunlight in an outdoor pond. Growth of detached segments was compared with respect to shoot and root lengths, dry weight and starch content in tissues. Buoyancy of segments at each light level was evaluated by percentage frequency of floating segments. Critical light intensity for the winter growth was estimated as ca. 4.5% of the aerial full sunlight. Most segments at light levels lower than 4.5% had been floating in water since the early period of the experiment, while all segments at light levels higher than 17% had been sinking to the bottom until water temperature became higher than 10 C. The data on segment buoyancy and tissue analysis for starch content showed an inverse relationship between percentage frequency of floating segments and starch content in tissues. These results suggest that detached segments in nature could escape from the photosynthetically unsuitable regions by reduced specific gravity caused by the consumption of starch, and establish themselves only if they could arrive at a safe-site where light conditions are sufficient to accumulate photosynthate.     

INTERACTING EFFECTS OF LIGHT AND NUTRIENT LIMITATION ON THE GROWTH RATE OF SYNECHOCOCCUS LINEARIS (CYANOPHYCEAE)1

The blue-green alga Synechococcus linearis (Naeg.) Kom. was grown in P- and N-limited chemostats over a range of potentially limiting irradiances in order to determine the combined effects of light and nutrient limitation on some aspects of the composition and metabolism of this alga. Over a narrow range of low irradiances, simultaneous limitation of growth rate by light and either N or P was shown. This simultaneous limitation of growth rate by a nutrient and a physical factor can be explained by the ability of an increased supply of one to compensate in part for a decreased supply of the other. At all irradiances, the internal concentration of the limiting nutrient increased with increasing dilution rate, and the results could be fitted to the Droop relationship. With decreasing irradiance, the internal concentration of the limiting nutrient increased. There appeared to be little or no effect of light on the minimum internal concentration of P but that of N increased with decreasing light. Both chlorophyll a and biliprotein per unit particulate C increased with increasing dilution rate and decreasing irradiance. The critical N/P ratio increased with decreasing light as the N requirement of N-limited cells increased faster than did the P requirement of P-limited cells. The composition of exponentially growing cells in complete medium varied much less with light. Neither dilution rate nor irradiance during growth had a great effect on saturated rates of P or N uptake or alkaline phosphatase activity. Calculated assimilation ratios increased with light and dilution rate. The role of the flexibility of nutrient composition in adaptation to adverse conditions and the implications of the results for the use of physiological indicators of nutrient status are discussed.     

Modelling vertical migration of the cyanobacterium Microcystis

Computer models can be helpful tools to provide abetter understanding of the mechanisms responsible forthe complex movements of cyanobacteria resulting fromchanges in buoyancy and mixing of the water column ina lake. Kromkamp & Walsby (1990) developed a verticalmigration model for Oscillatoria, that wasbased on the experimentally determinedrelationship between the rates of density change andphoton irradiance in this cyanobacterium. To adaptthis model to Microcystis, we determinedrelated changes in carbohydrate content in cultures ofMicrocystis. Samples were incubated at variousconstant values of photon irradiance and then placedin the dark. The changes in carbohydrate content ofthe cells during these incubations were investigated.The relationship between the ratio of carbohydrate toprotein and cell density in Microcystis wasestablished to permit conversion of the rates ofcarbohydrate change to rates of density change. Byplotting the calculated rates of density changeagainst the values of photon irradiance experiencedduring the incubations, an irradiance-response curveof density change was established. The curve showed adistinct maximum at 278 µmol photons m^-2s^-1. At higher values of photon irradiance, therate of density change was strongly inhibited. Apositive linear correlation was found between celldensity and the rates of density decrease in the dark.The validity of the use of rate equations of densitychange, which are based on short-term incubations atconstant values of photon irradiance, to predictdensity changes in Microcystis in fluctuatinglight regimes was tested. This was accomplished bymeasuring the time course of change in carbohydratecontent of two continuous cultures of Microcystis, which were submitted to fluctuatinglight regimes, and comparing the results with thechanges in the carbohydrate contents of these culturespredicted by the rate equations of carbohydratechange. The results showed good agreement: the rateequations of density change were therefore introducedinto the model to simulate vertical migration of Microcystis. The model predicts that the maximummigration depth of Microcystis will increasewith colony size up to a maximum of 200 µm radius.The effect of colony size on the net increase in celldensity during the light period was also investigatedwith the model. It predicts that small colonies havea higher net increase in cell density than largecolonies, but are inhibited at high photon irradiancesat the surface.     

Regulation of growth and photosynthesis by Oscillatoria agardhii grown with a light/dark cycle

Abstract The cyanobacterium Oscillatoria agardhii was grown in turbidostat cultures with the light energy supply in either the continuous mode or in the pulsed mode (8/16 h light/dark (L/D) cycle). The light irradiance value used was sufficient to allow the maximal growth rate to be attained, when supplied continuously. Adaptation of O. agardhii to the L/D cycle was characterized by an increase in pigment content and photosynthetic performance, accompanied by a decrease in growth rate. This mode of adaptation resembled the adaptation of O. agardhii to continuous low light intensities. It is suggested that in this case the L/D cycle provokes this adaptation in order to allow the cells to accumulate carbohydrate rapidly during the light period. This was attributed to the storage of polyglucose, which served as a carbon and energy source for growth in the dark. The utilization of polyglucose in the dark was able to sustain the synthesis of all other cell components at the same rate as when cells were growing in the light. The growth yield in the dark, whilst metabolizing internally stored polyglucose, was 0.52 g cell C/g polyglucose C, or 0.62 g cell dry weight/g polyglucose. Although in the pulsed mode there is a 66% loss in light irradiance per 24 h when compared with a continuous light regime, the growth rate of the cyanobacteria grown in the pulsed mode was only 35% lower than the growth rate of a culture grown in continuous light. This can be explained by a high growth yield in the dark and by increased CO₂ fixation rates in the light of cells grown in the pulsed mode.     

Modeling of eicosapentaenoic acid (EPA) production from Phaeodactylum tricornutum cultures in tubular photobioreactors. Effects of dilution rate, tube diameter, and solar irradiance

A model for the prediction of eicosapentaenoic acid (EPA) productivity from Phaeodactylum tricornutum cultures that takes into account the existence of photolimitation and photoinhibition of growth under outdoor conditions is presented. The effects of the external irradiance on the culture surface, the average irradiance inside the culture, and the light regime at which the cells are exposed on pigments and EPA content are studied. The chlorophyll content decreases exponentially with the average irradiance, whereas the carotenoids content increases linearly with the external irradiance due to a higher extension of photoinhibition. A decrease in the fatty acid content of the biomass with irradiance on reactor surface is observed when photoinhibition becomes relevant. The average irradiance within the culture mainly influenced the fatty acid profile of the biomass. As the average irradiance becomes higher, percentages of saturated and monounsaturated fatty acids decrease, increasing the portion of EPA. By taking into account the different relationships among pigment and EPA content with the irradiance, the variation in EPA productivity over the year can be simulated as a function of average and external irradiance. For the two photobioreactors employed the maximum EPA productivity is attained in spring and fall (30 mg L(-1) day(-1) for tube diameter 0. 06 m and 50 mg L(-1) day(-1) for tube diameter 0.03 m). In winter, the biomass productivity is limited by low light availability although the EPA content is maximum. In summer, the biomass productivity is higher although the EPA content diminished by photoinhibition; the higher the dilution rate, the lower the minimum. Thus, the conditions that increase the biomass productivity and the polyunsaturated fatty acids content are in opposition, the optimum being reached by operating under photolimitation with high growth rates in order to produce a high proportion of polyunsaturated fatty acids.     

Buoyancy regulation and vertical migration by Oscillatoria rubescens in Crooked Lake,Indiana

Filaments of Oscillatoria rubescens stratified in the metalimnion of Crooked Lake, Indiana at depths of 6–9 m, where the incident light intensity averaged 2% of the surface intensity. Buoyancy (due to gas vesicles) was regulated in response to light intensity, and increased turgor pressure generated at high light intensity could contribute to the collapse of gas vesicles. Filaments exposed to irradiances of 20–50 µE m^-2 s^-1 had neutral buoyancy. As nutrient availability was increased (by resuspending filaments in nutrient-rich water from the hypolimnion or by preventing CaCO₃ precipitation with a calcium chelator), higher light intensities were necessary for buoyancy loss and increased turgor.

A series of traps were placed in the lake to intercept floating and sinking filaments. Migration activity (both floating and sinking) was greatest 1 m above the most dense concentration of O. rubescens. These results, together with vertical profiles of primary production, suggest that maximum production by O. rubescens occurred above the population maximum in the water column.     

Changes in the proportion of endogenous osmotic solutes accumulated by Chlorella emersonii in the light and dark

Abstract. The type of endogenous osmotic solute accumulated by Chlorella emersonii grown at high external osmotic pressure (π_ext) depended on the light/dark conditions: proline accumulated to high concentrations in cells in the light, while sucrose accumulated to high concentrations in the dark. These findings were made during the alternating light dark cycles used to obtain synchronized cultures, i.e. cultures containing cells at only one stage of development at any one time. Similar decreases in proline and increases in sucrose in the dark were found for cells previously grown in continuous light to obtain non-synchronized cultures, i.e. cultures containing cells at all stages of development.
In cultures synchronized at 200 mol m ⁻³ NaCl (π_ext= 1.01 MPa), recently divided 'daughter cells' at the beginning of the light periods contained 60 mol m⁻³ proline and 100mol m⁻³ sucrose, while mature cells towards the end of light periods contained 130 mol m proline and 20 mol m⁻³ sucrose. The changes in proline and sucrose which occurred in synchronized cultures were due mainly to light/dark conditions and to a much lesser extent to different stages of cell development. The proportion of proline to sucrose in daughter cells collected from non-synchronized cultures in continuous light was not different from the proportion in heterogeneous populations of cells.
Results are discussed in relation to the accumulations of two, rather than one, endogenous osmotic solute and to growth reductions of C. emersonii exposed to high external osmotic pressures.     

Interrelations of photosynthetic behaviour and buoyancy regulation in a natural population of a blue-green alga

The photosynthetic activity of Anabaena cirdnalis and associated changes in buoyancy were determined from prepared suspensions exposed in the natural light field of Crose Mere. The observations are related to variations in subsurface irradiance and temperature. Parallel experiments, aimed at trapping algal colonies undertaking controlled vertical movements within the lake system, are also described. Buoyancy loss and downward migration are clearly associated with specific photosynthetic rates: rates as low as 1.8 mg O₂ (mg chlorophyll a) h⁻¹ are shown to be sufficient to effect buoyancy loss, while movements in the lake tend towards a depth where rates of 5–7 mg O₂ (mg chlorophyll a)⁻¹ h⁻¹ are possible. These rates are significantly less than those possible at light saturation. The effect of increasing temperature is to depress the population in the light-gradient. The significance of this response is discussed in relation to the growth of natural populations of blue-green algae.     

Variations in chloroplast nucleoid number during the cell cycle in the algaScenedesmus quadricauda grown under different light conditions

Summary Synchronous cultures of the green algaScenedesmus quadricauda were grown at different mean irradiances (ranging from 15 Wm^–2 to 130Wm^–2). At each irradiance, the algae were exposed to illumination regimes which differed in light duration and dark intervals (222 to 240 hours). The cells from these cultures were sampled during their cycles, stained with DAPI and the number of nuclei and chloroplast nucleoids estimated.The nucleoids divided semisynchronously in steps which represented doublings in their number. For each doubling a constant amount of light energy (defined as the product of irradiance and light duration) had to be converted by the cells to become committed to this division. The times to the start of the nucleoid divisions were therefore inversely proportional to the irradiances applied and the final number of nucleoids was proportional to the light duration.Temporal relationships between nuclear and nucleoid divisions were also light dependent. Shortage of light energy caused delay in nucleoid division. The cell division rate was higher than the rate of nucleoid division and consequently, the cells tended to decrease their nucleoid number with decreasing irradiance. With increasing irradiance the start of nucleoid division was gradually shifted toward the beginning of the cell cycle. The rate of nucleoid division exceeded the rate of nuclear and cellular division, thus with increasing irradiance cells with increasing numbers of nucleoids were formed.Abbreviations DAPI 46-diamidino-2-phenylindole - pt-DNA chloroplast DNA     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN IN LIGHT/DARK CYCLES. II. PHASED PO4-LIMITED CULTURES1

The characteristics of phosphate uptake and photosynthetic capacity were studied in P-limited populations of Euglena gracilis Klebs (Z), using both P-limited batch cultures in stationary phase and cyclostat cultures grown on 14:10 LD. P uptake obeyed Michaelis-Menten kinetics between 0 and 150 μM PO₄ under both growth conditions. The value of V_max was 35% lower in the dark than in the light in the stationary phase cells. The value of K₈ was not affected by light conditions, and uptake was completely inhibited in the presence of 1 mm KCN. P uptake (at 2.0 μM PO₄) and photosynthetic capacity showed diel periodicity with peak rates occurring just before the beginning of the dark period for P uptake, and 8 h into the light period for photosynthetic capacity. V_max for P uptake increased by a factor of 1.5 over the light period, whereas K₈ remained constant at 1.4 μM PO₄. These patterns were displayed by both nondividing stationary phase cells and populations in which less than a third of the cells divided each day, indicating that the rhythmicity is not coupled to cell division.