Bernd M. A. Kroon

Application of light‐emitting diodes in bioreactors: Flashing light effects and energy economy in algal culture (Chlorella pyrenoidosa)

Light‐emitting diodes (LEDs) were used as the sole light source in continuous culture of the green alga Chlorella pyrenoidosa. The LEDs applied show a peak emission at 659 nm with a half‐power bandwidth of 30 nm. Selection of this wavelength range, which is optimal for excitation of chlorophylls a and b in their “red” absorption bands makes all photons emitted potentially suitable for photosynthesis. No need for additional supply of blue light was found. A standardized panel with 2 LEDs cm−2 fully covered one side of the culture vessel. At standard voltage in continuous operation the light output of the diode panel appeared more than sufficient to reach maximal growth. Flash operation (5‐μs pulse duration) enables potential use of higher operating voltages which may render up to three times more light output. Flat airlift fermentor‐type continuous culture devices were used to estimate steady state growth rates of Chlorella pyrenoidosa as a function of the light flux (μmol photons · m−2 · s−1) and the flashing frequency of the light‐emitting diodes (which determines the duration of the dark “off” time between the 5‐μs “on” pulses). At the fixed voltage and turbidostat setting applied a 20‐kHz frequency, which equals dark periods of 45 μs, still permitted the maximum growth rate to become nearly reached. Lower frequencies fell short of sustaining the maximal growth rate. However, the light flux decrease resulting from lowering of the flash frequency appeared to reduce the observed growth rates less than in the case of a similar flux decrease with light originating from LEDs in continuous operation. Flash application also showed reduction of the quantum requirement for oxygen evolution at defined frequencies. The frequency domain of interest was between 2 and 14 kHz. LEDs may open interesting new perspectives for studies on optimization of mixing in mass algal culture via the possibility of separation of interests in the role of modulation on light energy conversion and saturation of nutrient supply. Use of flashing LEDs in indoor algal culture yielded a major gain in energy economy in comparison to luminescent light sources.

Impact of ultraviolet-B radiation on photosystem II activity and its relationship to the inhibition of carbon fixation rates for Antarctic ice algae communities

One goal of the Icecolors 1993 study was to determine whether or not photosystem II (PSII) was a major target site for photoinhibition by ultraviolet‐B radiation (QUVB, 280–320 nm) in natural communities. Second, the degree to which QUVBinhibition of PSII could account for QUVBeffects on whole cell rates of carbon fixation in phytoplankton was assessed. On 1 October, 1993, at Palmer Station (Antarctica), dense samples of a frazil ice algal community were collected and maintained outdoors in the presence or a bsence of QUVBand l or ultraviolet‐A (QUVB, 320–400 nm) radiation. Samples were then collected at intervals over the day to track the time course of UV inhibition of primary production. The ice algae were assessed for changes in pigment composition and rates of carbon fixation. The quantum yield of PSII (ØIIc°) was measured by Pulse Amplitude Modulated fluorometry. Over the day, ØIIc° declined due to increasing time‐integrated dose exposure of QUVB. The QUVB‐driven inhibition of ØIIc° increased from 4% in the early morning hours to a maximum of 23% at the end of the day. The QUVB photoinhibition of PSII quantum yield did not recover by 6 h after sunset. In contrast, photoinhibition by QUVA and photosynthetically available radiation (QPAR, 400–700 nm) recovered during the late afternoon. Flourescence‐based estimates of carbon fixation rates were linearly correlated (P =0.002, r2=0.45) with measured carbon fixation. Fluorescence overestimated the observed QUVB inhibition in measured carbon fixation rates by 8% in the morning hours; however, the discrepancy increased during the afternoon. Therefore, researchers should be cautious in using fluorescence measurements to infer ultraviolet inhibition for rates of carbon fixation until there is a greater understanding of the coupling of carbon metabolism to PSII activity for natural populations. Despite these current limitations, fluorescence‐based technologies represent powerful tools for studying the impact of the ozone hole on natural populations on spatial/ temporal scales not possible using conventional productivity techniques.

VARIABILITY OF PHOTOSYSTEM II QUANTUM YIELD AND RELATED PROCESSES IN CHLORELLA PYRENOIDOSA (CHLOROPHYTA) ACCLIMATED TO AN OSCILLATING LIGHT REGIME SIMULATING A MIXED PHOTIC ZONE1

The regulation of photosystem II (PSII)‐related parameters was characterized for Chlorella pyrenoidosa Chick grown in continuous nutrient‐replete culture and acclimated to a light field simulating fluctuations of a natural environment. The maximum quantum yield for charge separation at PSII showed a light‐driven decrease during the first half of the photoperiod when incident irradiance (integrated hourly) was increasing to a midday maximum, This change was accompanied by a decrease in the effective cross‐section for all PSIIs and the connectivity between PSIIs, while the cross‐section for inactive centers increased. Furthermore, a decrease in the number of active PSII centers and the amount of QB bound to D1 became evident. During the second half of the day, as irradiance declined toward the end of the photoperiod, all the observed changes in PSII photochemistry were reversed. The low quantum yield values at the middle of the photoperiod returned to 85% of the value at the end of the photoperiod by the addition of far‐red light, suggesting that a State I–II–I transition took place during the day. Measured rates of oxygen production correlated well with fluorescence‐derived estimates of photosynthesis. The overall results suggest that PSII photochemistry is optimized to minimize the susceptibility to photoinhibition and to allow balanced growth under fluctuating irradiation and that the impact of these alterations on primary production can be adequately monitored through bio‐optical measurements alone.

From electron to biomass: A mechanistic model to describe phytoplankton photosynthesis and steady-state growth rates.

A system of differential equations was presented which describes the rate of linear and cyclic electron flow through photosystems II and I. The system describes the rate of photochemistry in terms of electrons generated that are available for cellular metabolism, and results in a realistic description of photosynthesis as a function of irradiance without implicit assumptions for the relationship. The system allows a concise and detailed simulation of fluorescence kinetics. The derivation of the general degree of reduction (γx) and its application to translate the rates of photochemistry (or measured fluorescence yields) to steady‐state rates of carbon fixation and growth was shown. The efficiency of light‐limited photosynthesis (α) was shown to depend on the cellular ratio of carbon to nitrogen. For any given antenna size, α increases with nitrate as N‐source, and decreases with ammonium as N‐source, if the cellular carbon to nitrogen ratio of the phytoplankton increases. Cyclic electron transport around photosystem I increases the ratio of ATP generated relative to linear electron (e−) flow. The increase of ATP/e− is larger under extreme light‐limiting conditions. The long‐known fact that protein synthesis saturates at lower light intensities than carbonate synthesis was explained in terms of the decrease of ATP/e− with increasing irradiance and the higher ATP demand of protein synthesis.

An algal cyclostat with computer-controlled dynamic light regime

The aim of this paper is to provide detailed information concerning the hardware and software needed to design and construct a system for the continuous growth of algae, employing a computer-controlled variable dynamic light regime. The light intensity is controlled by adjusting the angular displacement of slats of a Venetian blind with the help of a stepper motor. The software developed allows the simulation of nature-related light flux regimes occurring in stratified water columns, mixed water columns with optically clear water, and mixed water columns with optically dense water. The light period, circulation time and attenuation coefficient can be chosen freely. Different light regimes with equal total light doses can be generated with the help of mathematical equations developed for this purpose. The software developed is of the Terminate and Stay Resident (TSR) type and operates in the background, thus allowing the computer to be used for other tasks. The system can be used to investigate algal growth and photosynthesis in relation to a dynamic light regime. The characteristics of the light regimes applied during the Vth GAP workshop are presented. The results of these experiments will be described in subsequent papers.

Influence of medium frequency light/dark cycles of equal duration on the photosynthesis and respiration of Chlorella pyrenoidosa

Chlorella pyrenoidosa was grown in three continuous cultures each receiving a different light regime during the light period of a diurnal cycle. Hourly samples taken during the light period were subjected to medium frequency light/dark oscillations of equal duration, ranging from 3 to 240 seconds. The oxygen consumption and production of each sample were measured with an oxygen electrode in a small oxygen chamber. Although the light/dark cycles had little overall influence on photo synthetic activity, the microalgae appeared to adapt to the light regime to which they were subjected. Large differences were found between the maximum chlorophyll-specific production rates (P max B ), the chlorophyll-specific production rates (PB) and the respiration rates between the cultures and treated subsamples. Respiration rates increased during the light period, whilst PB either increased, or had a mid light period minimum or maximum. The culture which received an hourly light oscillation during the light period had the highest P max B and lowest respiration rates, and it is suggested that these algae react as in nature, whereas either a sinusoidal or a block light pattern is ‘unnatural’. The latter light regime is commonly used in laboratory studies.

Modelling microalgal productivity in a High Rate Algal Pond based on wavelength dependent optical properties

A model is presented to predict algal biomass concentration and productivity in a High Rate Algal Pond (HRAP) at all possible combinations of incident photon flux density (PFD), pond depth and hydraulic retention time (HRT). The total extinction coefficientkt and the absorption coefficient ka of algal biomass were measured at 1 nm intervals. Thekt values were used to calculate the underwater light climate, which included the spectral narrowing of the photon flux density with increasing depth. The number of quanta absorbed (QA) from the photosynthetic available radiation (PAR) was calculated using theka/kt ratio and incident PFD at 1 nm intervals. Algal oxygen production is related to QA by the quantum requirement (QR), which was determined fromka,and the slope of the photosynthesis versus irradiance curve (α). Based on this calculation we propose a new concept: the compensating absorption rate (CAR), which represents the rate of photon absorption necessary to balance oxygen consuming processes. The model calculated productivities using literature data on HRT, pond depth and incident PFD, that compared well with the actual measured productivities. To achieve optimal HRAP productivities under fluctuating climatological conditions, we propose a pond management strategy based on model simulations.

The effect of dynamic light regimes on Chlorella

The patterns of diurnal variations in pigmentation and optical cross-section were compared for two cyclostat cultures of Chlorella pyrenoidosa, where the dynamics of the photoperiod differed. Populations were light-limited, nutrient rich and growing on an 8:16 light-dark (LD) cycle. One light regime was an 8 h sine function of the light period (sinusoidal culture), while the second had an 1 h sine function super-imposed on the 8 hour sine function (oscillating sinusoidal culture). Hourly samples were taken throughout a 12 h period including the light period. Determinations were made of chlorophyll (Chl) a and b abundance, in vivo absorption spectra, cell number and volume and used to derive both cell-specific (σcell) and optical chlorophyll specific (σchl) cross sections, as well as the absorption efficiency, Q, of the cells. The results indicate that C. pyrenoidosa is capable of adapting to dynamics in light intensity within an 8 h photoperiod. The sinusoidal culture showed a constant decrease in the Chl a/b ratio of 28 % while the total Chl content per cell increased slightly and σchl and Q remained constant, suggesting coordinated changes in reaction centers and light harvesting complexes. Over the oscillating photoperiod, however, the second culture displayed a diurnal variation in Chl a/b ratio, a 20% increase in σchl and an apparent oscillation in Q. These observations suggest that an oscillating photoperiod promoted the capability of Chl molecules to collect light and that the fractional area of all Chl molecules exposed to the photon flux is inversely related to the photon flux.

The effect of dynamic light regimes on Chlorella. II: Minimum quantum requirement and photosynthesis-irradiance parameters

Comparisons were made of photosynthesis in three light limited cyclostat cultures (LD = 8:16, dilution rate 0.7 d−1) of Chlorella pyrenoidosa, differing only in the dynamics of irradiance supply: as a constant rate, i.e. a block culture; as a sine function of the light period, i.e. a sinusoidal culture; as an 8 h sine function superimposed by an 1 h sine function, i.e. an oscillating culture. The sinusoidal culture had a constant minimum quantum requirement for oxygen evolution (QR) of 10.8 over the photoperiod. The OR of the oscillating culture increased from 24 to 37 during the photoperiod. From changes in α and Pmax we suggest that: (1) photosynthetic units (PSU) of the block and sinusoidal sulture increased in number; (2) increasingly fewer chlorophyll molecules participated in oxygenic photosynthesis with a decreasing turnover time of the PSUs during an oscillating photoperiod. Values of Ik decreased slightly in the block culture, increased slightly in the sinusoidal culture and showed a twofold increase in the oscillating culture. From the ratio of in situ oxygen production (qO2) and Pmax we infer a balanced equilibrium between photosystem activity and electron transport capacity for the block and sinusoidal culture. We hypothesize that the qO2 values of the oscillating culture underestimated true oxygen production rates due to a nonlinear response at peak light intensities. The results show that a dynamical photoperiod provoked significantly different photosynthetic responses, even though the overall growth rate was unaffected.