Pulse Amplitude Modulation Fluorometry Research Articles

Photoacclimation and photoregulation strategies of Corallina (Corallinales, Rhodophyta) across the NE Atlantic

ABSTRACTThis study characterizes the photoacclimation and photoregulation mechanisms that allow calcified macroalgae of the genus Corallina (Corallinales, Rhodophyta) to dominate rock pool habitats across the NE Atlantic despite the highly variable irradiance regimes experienced. Rapid light curves (RLCs) were performed with pulse amplitude modulation (PAM) fluorometry in situ across a full seasonal cycle in the UK intertidal with C. officinalis and C. caespitosa. Latitudinal comparisons were performed across the full extent of C. officinalis’ range in the NE Atlantic (Iceland–northern Spain), and for C. caespitosa in northern Spain. Ex situ RLCs with dark recovery were further employed to assess the optimal, as compared with actual, photophysiology across seasons and latitudes. Corallina species were shown to photoacclimate at seasonal timescales to changing irradiance, increasing light-harvesting during low-light autumn/winter periods and protecting photosystems during high-light summer conditions. Seasonal photoacclimation was achieved through alteration in the number of photosystem (PS) units (PSII and light harvesting antennae) over time. Non-photochemical quenching (NPQ) served as an important photoregulation mechanism utilized by Corallina to prevent or minimize photoinhibition over shorter time scales (seconds–hours), though the efficiency of NPQ was dependent on the seasonal-acclimated state. With increasing latitude the efficiency of photoregulation decreased, representing potential differential photoadaptation of Corallina across species ranges in the NE Atlantic. In contrast, highly conserved inter-specific patterns in photophysiological responses to irradiance were apparent. This study demonstrates the photophysiological mechanisms allowing Corallina to optimize use of the variable irradiance conditions apparent in rock pool environments, when and how they are employed, and their limitations.

Photoheterotrophy of photosynthetic bacteria ( Rhodopseudomonas palustris ) growing on oil palm and soybean cooking oils

Abstract Cooking oil wastes are environmentally damaging and costly to deal with. Photoheterotrophic growth of Rhodopseudomonas palustris (photosynthetic bacteria) (CGA 009) was used to decompose different types of cooking oil (palm and soybean oils; cooked and uncooked palm oil) under anaerobic and microaerobic conditions. Photosynthesis was measured in R. palustris using PAM technology (Pulse Amplitude Modulation Fluorometry): Optimum irradiance ≈ 284 μ mol photon m−2 s−1 and maximum photosynthetic electron transport rate (ETR) ≈ 341 μ mol e − g−1 BChl a s−1. R. palustris grew photoheterotrophically on both cooking oils as the sole carbon source grown anaerobically and microaerobically in PM media with a NH3-nitrogen source. R. palustris grew well on used palm oil. R. palustris produces small volumes of hydrogen gas from biodegradation of oil but only under nitrogen fixing conditions (anaerobic: no ammonia or organic nitrogen). N-fixing cells grew very slowly. Cooking oil waste residues are also produced at the production stage (Palm Oil Milling Effluent, POME) and in recycling them for reuse or to produce biodiesel. Hence, biodegradation of cooking oil wastes is an important issue at the production, domestic consumption and recycling stages of their use. A two-stage digestion using an anaerobic yeast first stage, followed by a R. palustris digestion was feasible. Two Line Capsule: The photosynthetic bacterium R. palustris grows photoheterotrophically on Palm Oil and Soybean Oil. Photosynthetic rates were measured using PAM technology.

Desiccation effect on the PSII photochemical efficiency of cultivated Japanese Caulerpa lentillifera under the shipping package environment

In Japan, fresh cultivated green alga, Caulerpa lentillifera (Caulerpaceae, Bryopsidales), are packed into polystyrene containers without seawater. They can stay fresh for 1 week inside the container under room temperature, suggesting a high tolerance to desiccation. This tolerance was evaluated in the present study by employing methods based on pulse amplitude modulation (PAM) fluorometry. The chronological change of the photochemical efficiency of photosystem II (PSII) of C. lentillifera in response to desiccation under conditions experienced during the commercial shipping was examined. Desiccation experiments were carried out for 2, 4, 8, and 12 days at 20 °C. Maximum quantum yields (Fv/Fm) were measured at the initial state, after each desiccation period, and after 10-min and 3, 6, and 24-h re-immersion in seawater. Fv/Fm of samples after a 2-day desiccation remained relatively high (0.78 ± 0.04 standard deviation (SD)), while those after 4- and 8-day desiccation periods decreased and yet were considered active with values greater than 0.40 following rehydration. Samples under the 12-day desiccation had the greatest decline in Fv/Fm, (0.10 ± 0.10), along with 72% critical water loss. Failure of the seaweed to recover from desiccation stress further indicates deactivation of the photosystem, which is perhaps associated with multiple cellular alterations that result at the end in a dysfunctional alga. The packaging system that makes use of polystyrene containers for thermal insulation and an absorbent sheet for moisture control may have provided an optimal environment for the seaweed to extend its “freshness” up to 1 week by maintaining photochemical efficiency.

The physiology of Chlorella vulgaris grown in conventional and biodigested treated vinasses

Conventional and biodigested vinasses are organic and nutrient rich residues from the sugar cane alcohol industry. Their disposal in running waters and soils are controlled due to their eutrophication potential. Using it as algal culture medium, lower costs and residue remediation can be achieved. Here, we report on the physiology of Chlorella vulgaris grown in 60% conventional and 80% biodigested treated vinasses in 6days experiments. Cell densities, chlorophyll a concentrations and pulse amplitude modulated (PAM) fluorometry were used for culture monitoring. Maximum and operational quantum yields, photochemical (qP) and non-photochemical quenchings (qN and NPQ) and rapid light response curves were determined and efficiency of light use (α) and light saturation (IK) obtained for all treatments. The results showed that C. vulgaris grew better in vinasse (1.5–1.6d−1) than in controls (0.9–1.2d−1). PAM fluorometry showed that, despite the increased algal growth in vinasses, photosynthesis was higher in controls than in the residues, supporting that C. vulgaris used organic carbon as energy source. We suggest that the high competitiveness of the microalgae was due to its ability to grow mixotrophically in the residue even though heterotrophic contaminants were present. A reduction of the vinasses eutrophication potential was obtained after the microorganisms' growth. This study is a contribution to the knowledge of microalgae photosynthetic physiology in organic rich residues, information that can help improve algal biomass production in residues at the same time that performs its remediation.

A four state parametric model for the kinetics of the non-photochemical quenching in Photosystem II

The phenomenon of non-photochemical quenching (NPQ) was studied in spinach chloroplasts using pulse amplitude modulated (PAM) fluorometry. We present a new analysis method which describes the observed fluorescence quantum yield as the sum of the product of four different states of PSII and their corresponding quantum yields. These four distinct states are PSII in the quenched or unquenched state, and with its reaction center either open or closed depending upon the reduction of the QA site. With this method we can describe the dynamics of the NPQ induction and recovery as well as quantify the percentage of photoinactivated RC throughout the measurement. We show that after one cycle of quenching followed by a period of recovery, approximately 8–9% of the RC are photoinactivated, after two cycles of illumination this number becomes 15-17%. The recovery from the quenching appeared with rates of (50s)−1 and (1h)−1. The new analysis method presented here is flexible, allowing it to be applied to any type of PAM fluorometry protocol. The method allows to quantitatively compare qualitatively different PAM curves on the basis of statistically relevant fitting parameters and to quantify quenching dynamics and photoinactivation. Moreover, the results presented here demonstrate that the analysis of a single PAM fluorometry quenching experiment can already provide information on the relative quantum yield of the four different states of PSII for the intact chloroplasts - something no other form of spectroscopy could provide in a single measurement.

Carotenoid Production Process Using Green Microalgae of the Dunaliella Genus: Model-Based Analysis of Interspecies Variability

The engineering of photosynthetic bioprocesses is associated with many hurdles due to limited mechanistic knowledge and inherent biological variability. Because of their ability to accumulate high amounts of β-carotene, green microalgae of the Dunaliella genus are of high commercial relevance for the production of food, feed, and high-value fine chemicals. This work aims at investigating the interspecies differences between two industrially relevant Dunaliella species, namely D. salina and D. parva. A systematic work flow composed of experiments and mathematical modeling was developed and applied to both species. The approach combining flow cytometry and pulse amplitude modulation (PAM) fluorometry with biochemical methods enabled a coherent view on the metabolism during the adaptational stress response of Dunaliella under carotenogenic conditions. The experimental data was used to formulate a dynamic-kinetic reactor model that covered the effects of light and nutrient availability on biomass growth, inte...

Primary productivity as a control over soil microbial diversity along environmental gradients in a polar desert ecosystem.

Primary production is the fundamental source of energy to foodwebs and ecosystems, and is thus an important constraint on soil communities. This coupling is particularly evident in polar terrestrial ecosystems where biological diversity and activity is tightly constrained by edaphic gradients of productivity (e.g., soil moisture, organic carbon availability) and geochemical severity (e.g., pH, electrical conductivity). In the McMurdo Dry Valleys of Antarctica, environmental gradients determine numerous properties of soil communities and yet relatively few estimates of gross or net primary productivity (GPP, NPP) exist for this region. Here we describe a survey utilizing pulse amplitude modulation (PAM) fluorometry to estimate rates of GPP across a broad environmental gradient along with belowground microbial diversity and decomposition. PAM estimates of GPP ranged from an average of 0.27 μmol O2/m2/s in the most arid soils to an average of 6.97 μmol O2/m2/s in the most productive soils, the latter equivalent to 217 g C/m2/y in annual NPP assuming a 60 day growing season. A diversity index of four carbon-acquiring enzyme activities also increased with soil productivity, suggesting that the diversity of organic substrates in mesic environments may be an additional driver of microbial diversity. Overall, soil productivity was a stronger predictor of microbial diversity and enzymatic activity than any estimate of geochemical severity. These results highlight the fundamental role of environmental gradients to control community diversity and the dynamics of ecosystem-scale carbon pools in arid systems.

Wilted cucumber plants infected by Fusarium oxysporum f. sp. cucumerinum do not suffer from water shortage.

Fusarium wilt is primarily a soil-borne disease and results in yield loss and quality decline in cucumber (Cucumis sativus). The main symptom of fusarium wilt is the wilting of entire plant, which could be caused by a fungal toxin(s) or blockage of water transport. To investigate whether this wilt arises from water shortage, the physiological responses of hydroponically grown cucumber plants subjected to water stress using polyethylene glycol (PEG, 6000) were compared with those of plants infected with Fusarium oxysporum f. sp. cucumerinum (FOC). Parameters reflecting plant water status were measured 8d after the start of treatment. Leaf gas exchange parameters and temperature were measured with a LI-COR portable open photosynthesis system and by thermal imaging. Chlorophyll fluorescence and chloroplast structures were assessed by imaging pulse amplitude-modulated fluorometry and transmission electron microscopy, respectively. Cucumber water balance was altered after FOC infection, with decreased water absorption and hydraulic conductivity. However, the responses of cucumber leaves to FOC and PEG differed in leaf regions. Under water stress, measures of lipid peroxidation (malondialdehyde) and chlorophyll fluorescence indicated that the leaf edge was more seriously injured, with a higher leaf temperature and disrupted leaf water status compared with the centre. Here, abscisic acid (ABA) and proline were negatively correlated with water potential. In contrast, under FOC infection, membrane damage and a higher temperature were observed in the leaf centre while ABA and proline did not vary with water potential. Cytologically, FOC-infected cucumber leaves exhibited circular chloroplasts and swelled starch grains in the leaf centre, in which they again differed from PEG-stressed cucumber leaves. This study illustrates the non-causal relationship between fusarium wilt and water transport blockage. Although leaf wilt occurred in both water stress and FOC infection, the physiological responses were different, especially in leaf spatial distribution.

Modeling the impact of high temperatures on microalgal viability and photosynthetic activity

BackgroundCulture collapse due to high temperatures can significantly impact the profitability of outdoor algal cultivation systems. The objective of this study was to model for the first time the impact of high temperatures on algal activity and viability.ResultsViability measurements on Dunaliella salina cultures were based on cytometry with two fluorescent markers (erythrosine and fluorescein di-acetate), and photosynthetic activity was measured by Pulse Amplitude Modulation (PAM) fluorometry. Kinetic studies revealed that viability and activity losses during exposure to high temperatures could be described by a Weibull model. Both mortality and activity were shown to be functions of the thermal dose received by the algae, defined as the product of duration of exposure to high temperatures and an exponential function of temperature. Simulations at five climatic locations revealed that culture collapse due to high temperatures could impact productivity of D. salina in non-temperature-controlled outdoor photobioreactors by 35 and 40% in arid and Mediterranean climates, respectively.ConclusionsThe model developed in this study can be used to forecast the impact of high temperatures on algal biofuel productivity. When coupled with models predicting the temperature of outdoor cultivation systems, this model can also be used to select the best combination of location, system geometry, and algal species to minimize the risks of culture collapse and therefore maximize biofuel productivity.

Toxicity estimates for diuron and atrazine for the tropical marine cnidarian Exaiptasia pallida and in-hospite Symbiodinium spp. using PAM chlorophyll-a fluorometry

Effective ecotoxicological risk assessments for herbicides in tropical marine environments are restricted by a lack of toxicity data, sensitive test methods and endpoints for relevant species, and this requires rectification. The symbiotic sea anemone Exaiptasia pallida is a suitable test species, representing the phylum Cnidaria and allowing for assessments of toxicological responses of both the animal host and in-hospite Symbiodinium spp. Pulse amplitude modulated (PAM) chlorophyll-a fluorometry is recognised as a valuable ecotoxicological tool, and here newly-developed test methods are presented using PAM fluorometry to measure herbicide effects on photosynthetic efficiency of in-hospite Symbiodinium spp. Additionally, measurements on healthy laboratory-reared E. pallida provide baseline data demonstrating the normal effective quantum yield (EQY) and the maximum electron transport rate (ETRm) for Symbiodinium spp. in the absence of herbicide stress. Concentration-dependant reductions in the EQY and ETRm occurred during diuron and atrazine exposures; a mean 48-h EC50 (effective concentration; 50%) of 8μg/L of diuron was estimated, however atrazine elicited a much lower toxicity. Twelve-day exposures to 10–200μg/L diuron showed that the greatest EQY effect occurred during the first 48h, with little subsequent change. However, longer exposures to the lowest diuron treatment (1μg/L) showed the lowest EQYs after 96h followed by recovery to control levels within 12d. Furthermore, asexual reproduction was inhibited during 12-d exposures to diuron, and 12-d EC50 values of 100 and 132μg/L were estimated to inhibit successful reproduction of pedal lacerates and juveniles by 50% respectively. This study provides much needed data contributions to species sensitivity curves for development of diuron and atrazine water quality guidelines in tropical marine environments.

The plastoquinone pool of Nannochloropsis oceanica is not completely reduced during bright light pulses

The lipid-producing model alga Nannochloropsis oceanica has a distinct photosynthetic machinery. This organism possesses chlorophyll a as its only chlorophyll species, and has a high ratio of PSI to PSII. This high ratio of PSI to PSII may affect the redox state of the plastoquinone pool during exposure to light, and consequently may play a role in activating photoprotection mechanisms. We utilized pulse-amplitude modulated fluorometry to investigate the redox state of the plastoquinone pool during and after bright light pulses. Our data indicate that even very intense (5910 μmol photons s-1m-2 of blue light having a wavelength of 440 nm) light pulses of 0.8 second duration are not sufficient to completely reduce the plastoquinone pool in Nannochloropsis. In order to achieve extensive reduction of the plastoquinone pool by bright light pulses, anaerobic conditions or an inhibitor of the photosynthetic electron transport chain has to be utilized. The implication of this finding for the application of the widely used saturating pulse method in algae is discussed.

One pulse, one light curve: Fast characterization of the light response of microphytobenthos biofilms using chlorophyll fluorescence

Research on microphytobenthos (MPB) photosynthesis has increasingly relied on pulse amplitude modulation fluorometry, often through the generation of light response curves of the relative electron transport of PSII (rETR), generated through the sequential exposure to a range of actinic irradiances. However, fast, vertical phototactic responses by the motile diatoms that dominate these biofilms occur during the generation of sequential light curves. Light-induced vertical migration is known to confound the characterization of the inherent physiological response of the cells forming the biofilm. The alternative approach of shortening the duration of the light curve protocol prevents the reaching of a physiological steady state and results in light curves heavily dependent on initial light conditions. This study tested the application to MPB of a recently introduced method allowing for the fast generation of non-sequential light curves, based on steady state and temporally independent measurements (“single-pulse light curves,” SPLC). This method was compared to the commonly used sequential protocols regarding its ability to (1) measure a steady-state light response, (2) minimize the effects of vertical migration, and (3) minimize the effects of photoacclimation state induced by initial conditions. The results indicate that the SPLC method can be optimized by applying one light exposure period of just 60 s prior to the generation of the light curve, resulting in a clearly advantageous alternative to the sequential protocols. It effectively allows characterizing the steady-state light response of MPB samples while avoiding the effects of light-induced vertical migration and of the initial light conditions on light curve parameters.

Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde-3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that α-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.

Seasonal dynamics influencing coastal primary production and phytoplankton communities along the southern Myanmar coast

Myanmar is tenth among the world’s fish-producing countries and third in ASEAN (Association of Southeast Asian Nations). To understand the mechanisms underlying the high production, oceanographic and phytoplankton surveys, including primary productivity measurements based on pulse amplitude modulation fluorometry, were conducted near an active fishing ground near Myeik City. Three surveys, one in each of the representative seasons and covering the characteristic coastal environments, showed well-defined seasonality in primary production and phytoplankton occurrence. End of the dry season was the most productive, with productivity of 2.59 ± 1.56 g C m−2 day−1 and high concentration of chlorophyll a (3.14 ± 2.64 µg L−1). In this season, the phytoplankton population was dominated by high densities of the diatoms Bellerochea horologicalis and Chaetoceros curvisetus, whereas primary productivity was low at the onset of the dry season, 1.36 ± 0.77 g C m−2 day−1. However, this low primary production might be compensated by activation of microbial food chains originating from high dissolved organic carbon. The rainy season exhibited the lowest production, 6.6% of the end of the dry season, due to the extensive discharge of turbid water from the rivers which lowered euphotic layer depth and resulted in an unusually high diffuse attenuation coefficient of 2.30 ± 1.03 m−1. This incident of turbid water may be related to soil erosion from deforestation and mangrove deterioration. This research reveals the seasonal trend in Myanmar’s coastal productivity and its relationship to the tropical monsoon climate as well as emphasizing the importance of tropical coastal environments to the sustainability of the fisheries.

Significance of structural variation in thylakoid membranes in maintaining functional photosystems during reproductive growth.

Structural variation in the stroma-grana (SG) arrangement of the thylakoid membranes, such as changes in the thickness of the grana stacks and in the ratio between grana and inter-grana thylakoid, is often observed. Broadly, such alterations are considered acclimation to changes in growth and the environment. However, the relation of thylakoid morphology to plant growth and photosynthesis remains obscure. Here, we report changes in the thylakoid during leaf development under a fixed light condition. Histological studies on the chloroplasts of fresh green Arabidopsis leaves have shown that characteristically shaped thylakoid membranes lacking the inter-grana region, referred to hereafter as isolated-grana (IG), occurred adjacent to highly ordered, large grana layers. This morphology was restored to conventional SG thylakoid membranes with the removal of bolting stems from reproductive plants. Statistical analysis showed a negative correlation between the incidences of IG-type chloroplasts in mesophyll cells and the rates of leaf growth. Fluorescence parameters calculated from pulse-amplitude modulated fluorometry measurements and CO2 assimilation data showed that the IG thylakoids had a photosynthetic ability that was equivalent to that of the SG thylakoids under moderate light. However, clear differences were observed in the chlorophyll a/b ratio. The IG thylakoids were apparently an acclimated phenotype to the internal condition of source leaves. The idea is supported by the fact that the life span of the IG thylakoids increased significantly in the later developing leaves. In conclusion, the heterogeneous state of thylakoid membranes is likely important in maintaining photosynthesis during the reproductive phase of growth.

Photosynthesis of Oil Palm (Elaeis guineensis)

Oil Palm (Elaeis guineensis) plantations cover more than 107,000km2 in SE-Asia but there is little information on its photosynthetic characteristics. Photosynthesis in seedling, juvenile and adult Oil Palm (6 months, 1–2y and >5y old, respectively) were measured using blue-diode Pulse Amplitude Modulation Fluorometry (PAM). The Absorptance Factor of the leaves (Abt465nm) was measured using a Reflectance Absorptance Transmission (RAT) meter to calculate actual, rather than relative Electron Transport Rates (ETR). Midday measurements in juvenile and adult Oil Palm tree were: maximum quantum yield of Photosystem II (Ymax) in juveniles was about 0.63, Optimum irradiance (Eopt) was ≈300μmolphotonm−2s−1. The maximum ETR (ETRmax) of juvenile plants was 24.06±1.69μmole−m−2s−1: based on 4e−/O2 this is equivalent to a maximum Photosynthetic Oxygen Evolution Rate (POERmax) of 89.21±6.25μmol O2 mg Chl a−1h−1. Adult Oil Palm plants have a Ymax similar to juveniles but Eopt was ≈600μmolphotonm−2s−1. The ETRmax of adult plants was 58.97±2.07μmole−m−2s−1: or POERmax=305.3±10.7μmol O2 mg Chl a−1h−1. Oil Palm is a classic sun plant with high photosynthetic rates at high irradiances. Photosynthetic efficiencies (α0) were high for C3 sun plants: α0 in adult plants (0.272±0.018e− photon−1) was higher than juvenile plants (0.204±0.034e− photon−1) expressed on a leaf surface area basis and on a Chl a basis (1.406±0.093 vs. 0.757 ± 0.124 O2 photon−1m2g Chl a−1). In juvenile and adult plants Eopt, POERmax and α0 tend to maximize about midday. In seedlings, Eopt and POERmax are highest in the early morning but maximum α0 is at about midday. Oil Palms strategically optimize photosynthesis for high irradiances and manipulate their photosynthesis vs. irradiance characteristics during the course of daylight.

Isolation and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis (Chlorophyceae) produced by dielectric barrier discharge plasma

:Haematococcus pluvialis has potential commercial application for its ability to accumulate astaxanthin under stress conditions. To improve astaxanthin productivity, nonthermal plasma can be used to produce mutant algal strains with enhanced production of astaxanthin. In this work, atmospheric pressure argon dielectric barrier discharge (DBD) was used for mutation induction. Several new algal strains with increased growth rate and higher astaxanthin accumulation were obtained. For analysis of the DBD-treated algal mutants, genetic variation was examined by random amplification of polymorphic DNA, and the pigment pattern in response to inhibitors (diphenylamine and nicotine) of carotenoid biosynthetic pathway was characterized by high-performance liquid chromatography. Furthermore, photosynthetic activity and gene expression were also examined by pulse amplitude-modulated fluorometry and quantitative real-time polymerase chain reaction, respectively. Our results confirmed that the enhanced astaxanthin accumulation in the mutants was associated with increased expression levels of lycopene cyclase, which is involved in the conversion of lycopene to β-carotene. As such, this work demonstrates a successful example for the effective application of DBD in the mutation breeding of astaxanthin-hyperproducing algal strains.

A radiative transfer modeling approach for accurate interpretation of PAM fluorometry experiments in suspended algal cultures.

The results of a numerical study on the simulation of pulse amplitude modulated (PAM) fluorometry within dense suspensions of photosynthetic microorganisms are presented. The Monte Carlo method was used to solve the radiative transfer equation in an algae-filled cuvette, taking into account absorption, anisotropic scattering, and fluorescence, as well as Fresnel reflections at interfaces. This method was used to simulate the transport of excitation and fluorescence light in a common laboratory fluorometer. In this fluorometer, detected fluorescence originates from a multitude of locations within the algal suspension, which can be exposed to very different fluence rates. The fluorescence-weighted fluence rate is reported, which is the local fluence rate of actinic light, averaged over all locations from which detected fluorescence originated. A methodology is reported for recovering the fluorescence-weighted fluence rate as a function of the transmittance of measuring light and actinic light through the sample, which are easily measured with common laboratory fluorometers. The fluorescence-weighted fluence rate can in turn be used as a correction factor for recovering intrinsic physiological parameters, such as the functional cross section of Photosystem II, from apparent (experimental) values. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1601-1615, 2016.

Arsenic toxicity in the water weed Wolffia arrhiza measured using Pulse Amplitude Modulation Fluorometry (PAM) measurements of photosynthesis

Accumulation of arsenic in plants is a serious South-east Asian environmental problem. Photosynthesis in the small aquatic angiosperm Wolffia arrhiza is very sensitive to arsenic toxicity, particularly in water below pH 7 where arsenite (As (OH)3) (AsIII) is the dominant form; at pH >7 AsO42− (As(V) predominates). A blue-diode PAM (Pulse Amplitude Fluorometer) machine was used to monitor photosynthesis in Wolffia. Maximum gross photosynthesis (Pgmax) and not maximum yield (Ymax) is the most reliable indicator of arsenic toxicity. The toxicity of arsenite As(III) and arsenate (H2AsO42−) As(V) vary with pH. As(V) was less toxic than As(III) at both pH 5 and pH 8 but both forms of arsenic were toxic (>90% inhibition) at below 0.1molm−3 when incubated in arsenic for 24h. Arsenite toxicity was apparent after 1h based on Pgmax and gradually increased over 7h but there was no apparent effect on Ymax or photosynthetic efficiency (α0).

A Bacterial Pathogen Displaying Temperature-Enhanced Virulence of the Microalga Emiliania huxleyi.

Emiliania huxleyi is a globally abundant microalga that plays a significant role in biogeochemical cycles. Over the next century, sea surface temperatures are predicted to increase drastically, which will likely have significant effects on the survival and ecology of E. huxleyi. In a warming ocean, this microalga may become increasingly vulnerable to pathogens, particularly those with temperature-dependent virulence. Ruegeria is a genus of Rhodobacteraceae whose population size tracks that of E. huxleyi throughout the alga’s bloom–bust lifecycle. A representative of this genus, Ruegeria sp. R11, is known to cause bleaching disease in a red macroalga at elevated temperatures. To investigate if the pathogenicity of R11 extends to microalgae, it was co-cultured with several cell types of E. huxleyi near the alga’s optimum (18°C), and at an elevated temperature (25°C) known to induce virulence in R11. The algal populations were monitored using flow cytometry and pulse-amplitude modulated fluorometry. Cultures of algae without bacteria remained healthy at 18°C, but lower cell counts in control cultures at 25°C indicated some stress at the elevated temperature. Both the C (coccolith-bearing) and S (scale-bearing swarming) cell types of E. huxleyi experienced a rapid decline resulting in apparent death when co-cultured with R11 at 25°C, but had no effect on N (naked) cell type at either temperature. R11 had no initial negative impact on C and S type E. huxleyi population size or health at 18°C, but caused death in older co-cultures. This differential effect of R11 on its host at 18 and 25°C suggest it is a temperature-enhanced opportunistic pathogen of E. huxleyi. We also detected caspase-like activity in dying C type cells co-cultured with R11, which suggests that programmed cell death plays a role in the death of E. huxleyi triggered by R11 – a mechanism induced by viruses (EhVs) and implicated in E. huxleyi bloom collapse. Given that E. huxleyi has recently been shown to have acquired resistance against EhVs at elevated temperature, bacterial pathogens with temperature-dependent virulence, such as R11, may become much more important in the ecology of E. huxleyi in a warming climate.