Culture conditions and the development of the photosynthetic mechanism; influence of light intensity on photosynthetic characteristics of Chlorella

1. Chlorella pyrenoidosa has been grown in a continuous-culture apparatus under various light intensities provided by incandescent lamps, other conditions of culture being maintained constant. Light intensity curves for cells immersed in the No. 11 Warburg buffer and in Knop''s solution + 4.4 per cent CO₂ at a saturating light intensity were determined as characteristics of the photosynthetic mechanism. These characteristics were referred to the centrifuged cell volume as an index of quantity of cellular material. 2. Cells grown at intensities in the range of about 35 f.-c. develop a capacity for a high rate of photosynthesis (c.mm. O₂/hour/c.mm. cells). At culture intensities above or below this range the cells produced have a lower capacity for photosynthesis. A similar effect is observed for rate of photosynthesis per unit dry weight or rate per unit cell nitrogen. 3. The rate of photosynthesis per cell or rate per unit chlorophyll shows no maximum at any light intensity of culture but increases continuously throughout the range of light intensities studied. 4. Maximum rate of growth is attained at a light intensity of about 100 f.-c. The hypothesis is advanced that at culture intensities above that needed to give maximum rate of growth (100 f.-c.) a mechanism is developed which opposes the photosynthetic process and removes the photosynthetic products. 5. The low capacity for photosynthesis shown by cells grown at culture intensities below 35 f.-c. finds no immediate explanation. 6. The shape of the light intensity curve is markedly affected by the light intensity at which the cells have been cultured. Cells grown at lower intensities give light intensity curves approaching the Blackman type with a short transitional region between light limitation and light saturation.     

Estimation of oxygen evolution by marine phytoplankton from measurement of the efficiency of Photosystem II electron flow

The relation between photosynthetic oxygen evolution and Photosystem II electron transport was investigated for the marine algae t Phaeodactylum tricornutum, Dunaliella tertiolecta, Tetraselmis sp., t Isochrysis sp. and t Rhodomonas sp.. The rate of Photosystem II electron transport was estimated from the incident photon flux density and the quantum efficiency of Photosystem II electron transport as determined by chlorophyll fluorescence. The relation between the estimated rate of Photosystem II electron transport and the rate of oxygen evolution was investigated by varying the ambient light intensity. At limiting light intensities a linear relation was found in all species. At intensities approaching light saturation, the relation was found to deviate from linearity. The slope of the line in the light-limited range is species dependent and related to differences in absorption cross-section of Photosystem II. The observed non-linearity at high irradiances is not caused by photorespiration but probably by a Mehler-type of oxygen reduction. The relationship could be modelled by including a redox-state dependent oxygen uptake. In the diatom t Phaeodactylum tricornutum, the photochemical efficiency of dark adapted open Photosystem II centers was found to be temperature-dependent with an optimum near 10°C.     

Interactions between Glucose and Inorganic Carbon Metabolism in Chlorella vulgaris Strain UAM 101

Chlorella vulgaris strain UAM 101 has been isolated from the effluent of a sugar refinery. This alga requires glucose to achieve maximal growth rate even under light saturating conditions. The growth rate of cultures grown on light + CO₂ + glucose (3.16 per day) reaches the sum of those grown on light + CO₂ (1.95 per day) and on dark + glucose (1.20 per day). Unlike other Chlorella strains, uptake of glucose (about 2 micromoles per milligram dry weight per hour) was induced to the same extent in the light and dark and was not photosensitive. The rate of dark respiration was not affected by light and was strongly stimulated by the presence of glucose (up to about 40% in 4 hours). The rate of photosynthetic O₂ evolution was measured as a function of the CO₂ concentration. These experiments were conducted with cells which experienced different concentrations of CO₂ or glucose during growth. The maximal photosynthetic rate was inhibited severely by growing the cells in the presence of glucose. A rather small difference in the apparent photosynthetic affinity for extracellular inorganic carbon (from 10-30 micromolar) was found between cells grown under low and high CO₂. Growth with glucose induced a reduction in the apparent affinity (45 micromolar) even though cells had not been provided with CO₂. Experiments performed at different pH values indicate CO₂ as the major carbon species taken from the medium by Chlorella vulgaris UAM 101.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

Modelling time-series of photosynthesis and comparisons with the fluorescence yield in Chlorella vulgaris: a study of adaptation, inhibition and recovery

Time-series of ¹⁴C uptake and fluorescence yield (i.e., thefluorescence enhancement after addition of the photosyntheticinhibitor DCMU) were measured in Chorella vulgaris at variouslight intensities. Adaptation and recovery processes after alterationof the light intensity were also studied. At a constant lightintensity, both the rate of ¹⁴C uptake and the fluorescenceyield decreased with time. Comparison of time-series data of¹⁴C uptake at different light intensities showed that this phenomenonconsisted of several processes (i.e., at low light intensitiessmall changes in uptake rates were mainly due to photoadaptation,while at higher light intensities relatively larger changesoccurred, as result of photoinhibition). Transfer of an algalsample to low light intensities after a period of exposure toinhibiting light intensities resulted in an exponential recoveryof the ¹⁴C uptake rate with time, coupled with an exponentialrecovery of the fluorescence yield. A mechanistic model is presented,which describes the algal ¹⁴C uptake rate as a function of timeand light intensity. The model includes adaptation, inhibitionand recovery. Six parameters, characterising the algal suspension,have to be estimated from the results of one P versus I curveand one time-series ¹⁴C uptake, which includes a period of recovery.Using these parameters the model can predict the time-courseof ¹⁴C uptake at every constant light intensity, as well aswhen the light intensity is changed during the experiment. Whenapplied to a culture of C. vulgaris, the theoretical valuesclosely approach the actual measurements. The resemblance betweenthe measured time-series of fluorescence yield and the rateof ¹⁴C uptake indicates, that the changes in the rate of ¹⁴Cuptake are due to changes in the photosynthetic apparatus, ratherthan to changes of diffusion of ¹⁴C into the cell.     

Correlation of alpha-Linolenate to Photosynthetic O(2) Production in Chlorella

Photosynthetic oxygen evolution per milligram of chlorophyll in Chlorella vulgaris varies with the age of the culture. The rate of oxygen evolution is low in the starting cells, it rises to a maximum after 24 hours of growth and then declines to the initial low value after 72 to 90 hours. These changes in photosynthetic competence of chlorophyll in Chlorella are paralleled by changes in α-linolenate per milligram of chlorophyll. In general the magnitude of the photosynthetic competence of chlorophyll is directly proportional to the magnitude of the ratio of α-linolenate to chlorophyll, regardless of whether high ratios are due to high α-linolenates or low chlorophyll values. This relationship holds when the cultures are grown either under continuous or intermittent illumination.     

Photosynthetic enhancement at high light intensities

Enhancement of photosynthetic oxygen evolution by Chlorella pyrenoidosa (211-8b) decreased almost linearly as the rate of photosynthesis was increased. The observed decrease was predicted most accurately by a model assuming exponential rate versus intensity curves as opposed to a model assuming hyperbolic light curves. The decrease in enhancement was not changed significantly upon changing from 0.03% to 5% carbon dioxide.     

The development of photosynthesis in a greening mutant of chlorella and an analysis of the light saturation curve

Photosynthetic oxygen evolution considerably precedes the rise in chlorophyll during the greening of a yellow mutant of Chlorella vulgaris. Dark-grown cells required 20 times more light to saturate photosynthesis than light-grown or normal cells. The chlorophyll appears to add first to active reaction centers, then to fill in a more general antenna. The carotenoid pigments seem to add more randomly to the reaction centers. The shape of the light saturation curves can be explained with the assumption that an excitation in the antenna can reach several reaction centers. The efficiency of the total unit is constant during the greening process.     

Culture conditions and the development of the photosynthetic mechanism; influence of light intensity on cellular characteristics of Chlorella

1. Chlorella pyrenoidosa has been grown in a continuous-culture apparatus under various light intensities provided by incandescent lamps, other conditions of culture being maintained constant. The harvested cells were analyzed for cell number, dry weight, nitrogen, and chlorophyll per unit cell volume. 2. Cell nitrogen and cell volume are parallel measures of cellular material over the range of light intensity studied. 3. The dry weight per cell volume increases slowly with light intensity, showing about a 20 per cent variation. 4. Chlorophyll concentration and cell number show a concomitant decrease with increasing light intensity, varying in such a way that there are always about the same number of chlorophyll molecules per cell. It is considered that this phenomenon has bearing on the interpretation of data which has led to the theory of the photosynthetic unit.     

光照强度、温度、pH、盐度对小球藻(Chlorella)光合作用的影响

利用测定净光合放氧速率的方法研究了光照强度、温度、pH、盐度对小球藻(Chlorella sp.XQ-200419)和海洋小球藻(Chlorella marina NJ-016)光合作用的影响。小球藻(Chlorella sp.XQ-200419)的适宜光照强度范围为100～1600μmo·lm-2·s-1,光饱和点在500μmo·lm-2·s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为6.5～9.0,最适pH值为7.0;对盐度的适应范围较广,在0～0.6mol/L范围内,随着盐度的升高,净光合放氧速率有下降趋势。海洋小球藻(Chlorella marina NJ-016)的适宜光照强度范围为400～1600μmol·m-2·s-1,光饱和点在1400μmo·lm-2.s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为5.0～9.0,最适pH值为8.0;对盐度有很好的适应性,在0～0.6mol/L范围内,随着盐度升高,净光合放氧速率明显上升。小球藻和海洋小球藻的净光合放氧速率随光照强度、温度、pH值和盐度变化的规律,表明了两种小球藻的基本生理生态学特性:能适应较强的光照强度、较高的温度、中性偏碱的环境和较高的盐度。研究结果有助于小球藻培养条件的优化。两种小球藻对光照强度、温度、pH值和盐度变化的反应也有所不同:与小球藻(Chlorella sp.XQ-200419)相比,海洋小球藻(Chlorella marina NJ-016)对光照强度有更好的适应性,对pH值变化有更宽的适应范围,适当提高盐度对其光合作用有明显的促进作用。这表明海洋小球藻(Chlorella marina NJ-016)在快速生长繁殖方面具有更大的潜力,这一研究结果为筛选适合于大量培养的优良藻种提供了依据。     

Adaptation of the Photosynthetic Apparatus of Chlorella and Ankistrodesmus to Blue and Red Light

The mode of adaptation of the photosynthetic apparatus of three unicellular green algae, Ankistrodesmus braunii, Chlorella fusca and Chlorella saccharophila to red and blue light are documented by the fluence-rate curves of photosynthetic oxygen evolution. For all three algae tested photosynthetic capacity, respiration and light compensation point were higher for cells grown under red light, while the chlorophyll content increased in blue light-grown cells. Blue light-adapted cells have a lower chlorophyll a to chlorophyll b ratio and more chlorophyll in the light-harvesting system than red light-adapted cells, as shown in the electrophoretic profile of the pigment-protein complexes. It is concluded that the action of red light resembles that of high levels of white light, while blue light causes the same effects as low levels of white light. In agreement with previous publications these findings indicate that the mode of adaptation to different light qualities is ubiquitous in unicellular green algae.     

EFFECTS OF ENVIRONMENTAL FACTORS ON PHOTOSYNTHESIS PATTERNS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE). II. EFFECT OF OXYGEN1

Oxygen inhibited the rate of light-saturated photosynthesis of the marine diatom Phaeodactylum tricornutum Bohlin. However, inhibition could only be detected with O₂ concentrations approaching 100%. Atmospheric concentrations of O₂ (21%) had little effect on photosynthesis. In this, Phaeodactylum more closely resembles the so-called C-4 plants which show low rates of photorespiration. The results presented here agree with others in showing increased O₂ inhibition at reduced bicarbonate concentrations. The biochemical mechanism of photorespiration in Phaeodactylum appears to be similar to that reported for other photosynthetic systems. The activity of ribulose-1,5 diphosphate (RuDP) carboxylase in cell-free extracts was also inhibited, by oxygen. Inhibition by O₂ was optimal at pH 9.2 as was the RuDP-dependent O₂ uptake. RuDP carboxylase/oxygenase ratios decreased with increasing pH and were greater in cells grown at lower light intensities. Carboxylase levels were less affected by the light intensity for growth than were the levels of the oxygenase. Short-term incorporation of NaHCO₃-¹⁴C by cells grown at high light intensities showed increased labelling of glycolate and glycine plus serine under O₂ compared with nitrogen. There was a concomitant decrease in the radioactivity found in phosphoglyeric acid (PGA) and sugar phosphates in the presence of O₂. The effects of O₂ on the short-term pattern of photosynthesis were less marked when the alga was previously grown at low light intensities.     

Microscopic green algae and cyanobacteria in high-frequency intermittent light

The effects of fluctuations in the irradiance onScenedesmus quadricauda, Chlorella vulgaris andSynechococcus elongatus were studied in dilute cultures using arrays of red light emitting diodes. The growth rate and the rate of photoinhibition were compared using intermittent and equivalent continuous light regimes in small-size (30 ml) bioreactors. The CO₂ dependent photosynthetic oxygen evolution rates in the intermittent and continuous light regimes were compared for different light/dark ratios and different mean irradiances. The kinetics of the electron transfer reactions were investigated using a double-modulation fluorometer. The rates of photosynthetic oxygen evolution normalized to equal mean irradiance were lower or equal in the intermittent light compared to the maximum rate found in the equivalent optimal continuous light regime. In contrast, the growth rates in the intermittent light can be higher than the growth rate in the equivalent continuous light. Photoinhibition is presented as an example of a physiological process affecting the growth rate that occurs at different rates in the intermittent and equivalent continuous lights. The difference in the dynamics of the redox state of the plastoquinone pool is proposed to be responsible for the low photoinhibition rates observed in the intermittent light.     

The photosynthetic unit in chlorella measured by repetitive short flashes

Apparent size of the photosynthetic unit in Chlorella pyrenoidosa was estimated by the method of Emerson and Arnold: rate of oxygen evolution was measured under repetitive saturating flashes of about 10-microsecond duration separated by dark periods of 0.033 to 0.100 second. Cells used were taken from six steady state cultures maintained at different light intensities. Cell characteristics included a variation in chlorophyll content from 1 to 5%. Apparent size of the photosynthetic unit varied systematically with chlorophyll content in the range of 1560 to 2350 chlorophylls per O₂ per flash. Values for unit size showed no unusual statistical distribution and were not changed significantly by addition of low level background light at 645 or 705 nanometers. Maximal rate of unit turnover, calculated from light-saturated rate and unit size, varied inversely with chlorophyll content in the range of 70 to 180 per second.     

The effect of low temperatures on fatty acid biosynthesis in plants

1. Of three systems, bulb tissue, plant leaf tissue and intact green algal (Chlorella vulgaris) cells, only the former shows an increase in rate of formation of unsaturated fatty acids with decrease in temperature. 2. In bulb tissue the oxygen concentration is rate-limiting for synthesis of unsaturated fatty acids at temperatures down to 10°. 3. At elevated oxygen concentrations the formation of unsaturated fatty acids in bulb tissue increases with temperature. 4. The failure of photosynthetic tissues to respond to either lower temperatures or increased oxygen concentrations in the presence of light is attributed to photosynthetic production of excess of oxygen. This is supported by the fact that in the dark a potentiating oxygen effect on the formation of unsaturated fatty acids can be demonstrated. 5. The HCO₃⁻ ion concentration has a small effect on the formation of unsaturated fatty acids. 6. Elevated content of unsaturated acids at lower temperatures in plants is attributed to increases in oxygen concentration in solution.     

The Adaptation of Plankton Algae IV. Light Adaptation in Different Algal Species

Two types with regard to adaptation to different light intensities are described: tbe Chlorella type and the Cyclotella type. The Chlorella type is mostly found among the green algae, the Cyclotella type among the diatoms. The Chlorella type adapts to a new light intensity mainly by changing the pigment content. Therefore the cells adapted to a high light intensity have a lower chlorophyll a content per cell than cells adapted to a low light intensity. Light saturation is mostly rather low for cells adapted to low light intensities. The light-saturated rate of photosynthesisist mostly lower for cells adapted to a high light intensity than for cells adapted to a low light intensity. The actual photosynthesis is not much higher at a high light intensity than at a low one. The actual photosynthesis is the photosynthesis at the light intensity where the cells are grown. - The Cyclotella type adapts only by changing the light-saturated rate. The chlorophyll content is the same in cells grown at low and high light intensities. Light saturation for cells grown at a low light intensity is rather high. The light-saturated rate is much higher in the case examined at the high light intensity than at the low one. The actual photosynthesis is considerably higher for cells grown at the high light intensities than for cells grown at low light intensities.- The two adaptation types are not sharply separated since transition types occur.     

Enhancement models at high light intensities

The separate package and spillover models of photosynthetic enhancement are generalized and extended to light saturation assuming hyperbolic or exponential rate versus intensity curves. The available data are more consistent with the exponential model at high light intensities. Two significant conclusions follow: (a) the decreases in enhancement observed at high light intensities are accounted for by the curvatures of the light intensity curves for photosynthesis, and (b) nonlinearity of rate versus intensity curves may be sufficient to reduce enhancement greatly even when the intensity curves appear linear.     

Adaptation to quantum flux by the emerson photosynthetic unit

The size of the Emerson photosynthetic unit was measured in Chlorella pyrenoidosa strain no. 252 grown at light intensities between 50 and 1000 foot candles. The Emerson photosynthetic unit changed from a minimum size of 1970 molecules chlorophyll a + b/O₂ per flash in cells grown at 1000 foot candles to a maximum size of 3150 molecules chlorophyll a + b/O₂ per flash for cells grown at 50 foot candles. The size changes were interpreted as a partial adaptation where the trapping center antenna responded to changes in incident light intensity. Light-induced changes in chlorophyll content and size of the Emerson photosynthetic unit were directly related.     

Photosynthetic Apparatus Formation during the Cell Cycle of Chlorella

Synchronous cell division in cultures of Chlorella vulgaris Beijerinck was induced by intermittent illumination: 9 hours light, 6 hours darkness. The rate of photosynthetic O₂ evolution per cell increases 4-fold in a one-step manner at the beginning of the light period, to the same extent as the increase in cell number. Over the division cycle, the following accumulation times during the light period were found: chlorophyll a, between 2 and 8 hours, chlorophyll b, between 5 and 8 hours, reaction centers of photosystems I and II, between 2 and 6 hours; and cytochrome f, between 2.5 and 5 hours. Cytochrome f accumulation is closely followed by an increase in amplitude of the rapid phase in light-induced absorption increase at 520 nanometers and in intensity of the delayed light emission. Enhancement of the delayed fluorescence yield per flash under continuous illumination (caused by the establishment of the pH difference across the thylakoid membrane) is maximal by the first hour of the light period.     

The Kok Effect in Chlamydomonas reinhardi

A Haxo-Blinks rate-measuring oxygen electrode together with a modulated light source gave an average current signal (change in net O₂ exchange) and a modulated current signal (photosynthetic O₂ evolution). Using this apparatus, net O₂ exchange and photosynthetic O₂ evolution at low intensities have been studied in the green alga, Chlamydomonas reinhardi. At both 645 nm and 695 nm, the curves of net O₂ exchange as a function of light intensity were steeper at lowest intensities than about compensation, indicative of the Kok effect. The effect was greater at 695 nm than at 645 nm. The corresponding curves of photosynthetic O₂ evolution, on the other hand, showed no Kok effect; here, the slope was lowest at lowest intensity. The absence of the Kok effect in O₂ evolution, together with its sensitivity to monofluoroacetic acid, show that it is due to an interaction of photosynthesis and respiration. The effect was exaggerated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In the presence of concentrations of this inhibitor sufficient to inhibit O₂ evolution completely, a light-induced change in net O₂ exchange remained. This was interpreted as a system I dependent depression of respiratory O₂ uptake. The Kok effect remained undiminished in concentrations of carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol which partially uncoupled either oxidative phosphorylation alone or both oxidative and photosynthetic phosphorylations. The above results can be explained within a model of the Kok effect in which O₂ uptake is depressed by diversion of reductant away from respiratory electron transport and into photosystem I. The same photodepression of O₂ uptake also appears to account for a transient in net O₂ exchange seen in several algae upon turning off the light.