Dr Han L. Golterman,a Limnologist: a tribute on the occasion of his 65th birthday and retirement

The green algae D. tertiolecta, the flagellate I. galbana and the diatom C. gracilis were grown in batch cultures. The organisms were analysed for lipid class composition at the logarithmic and stationary growth phases using the Chromarod-Iatroscan thin layer chromatography with flame ionization detection (TLC-FID) system.There were major differences in lipid class production among the organisms investigated, but few differences in lipid class distribution between log phase and stationary phase cultures of D. tertiolecta and I. galbana. C. gracilis displayed the general trend exhibited in diatom metabolism, which can be characterized by an increase in triacylglycerol synthesis in situations of stress.     

The presence of glycollate oxidase and dehydrogenase in Dunaliella primolecta

Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O₂-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O₂ uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm³, while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm³.     

Protein turnover in relation to maintenance metabolism at low photon flux in two marine microalgae

Acclimation to very low photon fluxes involves adjusting a suite of physiological characteristics that collectively elicit a physiological response. Facilitating such changes is pro‐tein turnover. Dunaliella tertiolecta (Butcher) and Phaeodactylum tricornutum (Bohlin) were grown in turbidostats at a range of photon fluxes between 2 and 300 µmol photons m^?2 s^?1. The kinetics of pulse‐chase labelling of the protein with ³H showed that (1) two protein pools were present, one of which turned‐over rapidly (hours), and a second which turned over more slowly (days); and (2) protein turnover rates were slower in P. tricornutum than in D. tertiolecta. Phaeodactylum tricornutum had a lower maintenance coefficient for protein turnover than D. tertiolecta, and correspondingly a smaller proportion of its respiratory demands (30%) were associated with protein turnover than in D. tertiolecta (36%). There appears to be a correlation between lower metabolic activity, requiring lower protein concentrations, and an associated decreased cost of maintenance processes in P. tricornutum compared to D. tertiolecta. Differences between protein turnover rates and maintenance metabolic costs may be one of the photo‐acclimation strategies that determine which photon niches microalgae can successfully exploit.     

Photoacclimation and Photoprotection of Juvenile Sporophytes of Macrocystis pyrifera (Laminariales,Phaeophyceae) Under High-light Conditions During Short-term Shallow-water Cultivation1

This study was designed to understand better if and how juvenile sporophytes of Macrocystis pyrifera can photoacclimate to high-light conditions when transplanted from 10 to 3 meters over 7 d. Acclimation of adult sporophytes to light regimes in the bathymetric gradient has been extensively documented. It primarily depends on photoacclimation and translocation of resources among blades. Among other physiological differences, juvenile sporophytes of M. pyrifera lack the structural complexity shown by adults. As such, juveniles may primarily depend on their photoacclimation capacities to maintain productivity and even avoid mortality under changing light regimes. However, little is known about how these mechanisms operate in young individuals. The capacity of sporophytes to photoacclimate was assessed by examining changes in their photosynthetic performance, pigment content, and bio-optical properties of the blade. Sporophytes nutritional status and oxidative damage were also determined. Results showed that juvenile sporophytes transplanted to shallow water were able to regulate light harvesting by reducing pigment concentration, and thus, absorptance and photosynthetic efficiency. Also, shallow-water sporophytes notably enhanced the dissipation of light energy as heat (NPQ) as a photoprotective mechanism. Generally, these adjustments allowed sporophytes to manage the absorption and utilization of light energy, hence reducing the potential for photo-oxidative damage. Furthermore, no substantial changes were found in the internal reserves (i.e., soluble carbohydrates and nitrogen) of these sporophytes. To our knowledge, these results are the first to provide robust evidence of photoprotective and photoacclimation strategies in juveniles of M. pyrifera, allowing them to restrict or avoid photodamage during shallow-water cultivation.     

STATE TRANSITIONS AND NONPHOTOCHEMICAL QUENCHING DURING A NUTRIENT‐INDUCED FLUORESCENCE TRANSIENT IN PHOSPHORUS‐STARVED DUNALIELLA TERTIOLECTA1

Assessments of nutrient‐limitation in microalgae using chl a fluorescence have revealed that nitrogen and phosphorus depletion can be detected as a change in chl a fluorescence signal when nutrient‐starved algae are resupplied with the limiting nutrient. This photokinetic phenomenon is known as a nutrient‐induced fluorescence transient, or NIFT. Cultures of the unicellular marine chlorophyte Dunaliella tertiolecta Butcher were grown under phosphate starvation to investigate the photophysiological mechanism behind the NIFT response. A combination of low temperature (77 K) fluorescence, photosynthetic inhibitors, and nonphotochemical quenching analyses were used to determine that the NIFT response is associated with changes in energy distribution between PSI and PSII and light‐stress‐induced nonphotochemical quenching (NPQ). Previous studies point to state transitions as the likely mechanism behind the NIFT response; however, our results show that state transitions are not solely responsible for this phenomenon. This study shows that an interaction of at least two physiological processes is involved in the rapid quenching of chl a fluorescence observed in P‐starved D. tertiolecta: (1) state transitions to provide the nutrient‐deficient cell with metabolic energy for inorganic phosphate (P_i)‐uptake and (2) energy‐dependent quenching to allow the nutrient‐stressed cell to avoid photodamage from excess light energy during nutrient uptake.     

Effect of Ammonia and Nitrate on Growth, Photosynthesis, and Ribulosediphosphate Carboxylase Content of Dunaliella tertiolecta

When the marine Chlorophycean flagellate Dunaliella tertiolecta Butcher was grown with short photoperiods of bright light, the use of ammonia rather than nitrate as a nitrogen source led to a 30 % reduction of the doubling time of cell matter. The cell cycle (onset of light to completion of cell division) was shortened by about 10% only. Ammonia-grown cells possessed a greater capacity for photosynthetic oxygen evolution at light saturation than did nitrate-grown cells; their content of ribulosediphosphate carboxylase was likewise greater. The faster growth of Dunaliella tertiolecta with ammonia may be partly a consequence of a general increase in net protein synthesis resulting in a greater content of photosynthetic enzymes.     

Thermophilic,anaerobic co-digestion of microalgal biomass and cellulose for H<Subscript>2</Subscript> production

Microalgal biomass has been a focus in the sustainable energy field, especially biodiesel production. The purpose of this study was to assess the feasibility of treating microalgal biomass and cellulose by anaerobic digestion for H₂ production. A microbial consortium, TC60, known to degrade cellulose and other plant polymers, was enriched on a mixture of cellulose and green microalgal biomass of Dunaliella tertiolecta, a marine species, or Chlorella vulgaris, a freshwater species. After five enrichment steps at 60°C, hydrogen yields increased at least 10% under all conditions. Anaerobic digestion of D. tertiolecta and cellulose by TC60 produced 7.7 mmol H₂/g volatile solids (VS) which were higher than the levels (2.9–4.2 mmol/g VS) obtained with cellulose and C. vulgaris biomass. Both microalgal slurries contained satellite prokaryotes. The C. vulgaris slurry, without TC60 inoculation, generated H₂ levels on par with that of TC60 on cellulose alone. The biomass-fed anaerobic digestion resulted in large shifts in short chain fatty acid concentrations and increased ammonium levels. Growth and H₂ production increased when TC60 was grown on a combination of D. tertiolecta and cellulose due to nutrients released from algal cells via lysis. The results indicated that satellite heterotrophs from C. vulgaris produced H₂ but the Chlorella biomass was not substantially degraded by TC60. To date, this is the first study to examine H₂ production by anaerobic digestion of microalgal biomass. The results indicate that H₂ production is feasible but higher yields could be achieved by optimization of the bioprocess conditions including biomass pretreatment.     

EFFECT OF LIGHT QUALITY AND INTENSITY ON GLYCEROL CONTENT IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) AND THE RELATIONSHIP TO CELL GROWTH/OSMOREGULATION1

Dunaliella tertiolecta Butcher was grown at two intensities (33, 150μEin · m^?2· s^?1) of blue light and white light at 0.25, 0.50 and 1.00 M NaCl. Growth rates were used as an indication of the relative osmoregulatory ability of cells in the various treatments. There was no significant effect on growth rate due to various NaCl molarities. No significant difference in growth rate was found between blue- and white-light cultures at the high intensity, the average growth constant being 2.07 divisions/day. However, at the low intensity illumination, blue light produced a significant increase in growth rate; 1.42 vs. 0.93 divisions/day for blue light and white light grown cells respectively. The average glycerol content of exponentially dividing cells grown at 0.25, 0.50 and 1.00 M NaCl was 0.12, 0.41 and 1.12 mg/10⁸ cells, respectively, as measured by gas chromatography. The intracellular glycerol content was significantly reduced by blue light at both light intensities and at each NaCl molarity. However, high light intensity reduced cellular glycerol content more than the reduction effected by blue light. Glycerol accumulated in the medium throughout culture growth. Intracellular glycerol content also increased with cellular aging reaching 2.72 mg/10⁸ cells in stationary phase, low intensity 1.00 M NaCl cultures. A negative correlation between glycerol content and growth rate was found. Total inhibition of glycerol production could not be obtained by treatment with blue light. However, this negative correlation possibly indicates that D. tertiolecta expends energy producing an excess amount of glycerol over that required for osmoregulation, leading to a reduction in the growth rate for the organism.     

SALINITY ADAPTATION BY DUNALIELLA TERTIOLECTA. I. INCREASES IN CARBONIC ANHYDRASE ACTIVITY AND EVIDENCE FOR A LIGHT-DEPENDENT Na+/H+ EXCHANGE1,2

When Dunaliella tertiolecta, previously adapted to medium containing 0.5 M NaCl, is transferred to higher salinities, there is a lag in growth, suggesting an adaptation period. Since there is no significant difference in the Na⁺ content of cells grown between 0.5 and 3.5 M NaCl, a mechanism for Na⁺ extrusion or exclusion is indicated. Increasing the salinity of cell suspensions stimulates an incorporation of H⁺ by the cells, suggesting an H⁺/Na⁺ exchange. Cells adapted to higher salinities have, increased carbonic anhydrase activity, suggesting that increased CO₂ or HCO₃^? transport may be required at higher salinities. Growth, of D. tertiolecta at salinities above 2.5 M requires continuous illumination; therefore a light-driven H⁺/Na⁺ exchange accompanied by a HCO₃^? influx is proposed.     

Does growth under elevated CO2 moderate photoacclimation in rice?

Acclimation of plant photosynthesis to light irradiance (photoacclimation) involves adjustments in levels of pigments and proteins and larger scale changes in leaf morphology. To investigate the impact of rising atmospheric CO₂ on crop physiology, we hypothesize that elevated CO₂ interacts with photoacclimation in rice (Oryza sativa). Rice was grown under high light (HL: 700 µmol m^?2 s^?1), low light (LL: 200 µmol m^?2 s^?1), ambient CO₂ (400 µl l^?1) and elevated CO₂ (1000 µl l^?1). Leaf six was measured throughout. Obscuring meristem tissue during development did not alter leaf thickness indicating that mature leaves are responsible for sensing light during photoacclimation. Elevated CO₂ raised growth chamber photosynthesis and increased tiller formation at both light levels, while it increased leaf length under LL but not under HL. Elevated CO₂ always resulted in increased leaf growth rate and tiller production. Changes in leaf thickness, leaf area, Rubisco content, stem and leaf starch, sucrose and fructose content were all dominated by irradiance and unaffected by CO₂. However, stomata responded differently; they were significantly smaller in LL grown plants compared to HL but this effect was significantly suppressed under elevated CO₂. Stomatal density was lower under LL, but this required elevated CO₂ and the magnitude was adaxial or abaxial surface‐dependent. We conclude that photoacclimation in rice involves a systemic signal. Furthermore, extra carbohydrate produced under elevated CO₂ is utilized in enhancing leaf and tiller growth and does not enhance or inhibit any feature of photoacclimation with the exception of stomatal morphology.     

PHYSIOLOGICAL RESPONSES TO PHOSPHORUS LIMITATION IN BATCH AND STEADY-STATE CULTURES OF DUNALIELLA TERTIOLECTA (CHLOROPHYTA): A UNIQUE STRESS PROTEIN AS AN INDICATOR OF PHOSPHATE DEFICIENCY1

A protein unique to phosphorus stress observed in Dunaliella tertiolecta Butcher was studied in the context of phosphate-limited cell physiology and is a potential diagnostic indicator of phosphate deficiency in this alga. Cells were grown over a range of limited, steady-state growth rates and at maximum (replete) and zero (phosphate-starved) growth rates. The stress protein, absent in nutrient-replete cells, was produced under all steady-state phosphate-limited conditions and increased in abundance with increasing limitation (decreasing growth rate). Cellular carbon: phosphorus ratios and the maximum uptake rate of phosphate (V_m) increased with increasing limitation, whereas the ratio of chlorophyll a: carbon decreased. Alkaline phosphatase activity did not respond to limitation but was measurable in starved, stationary-phase cells. F_v/F_m, a measure of photochemical efficiency, was a nonlinear, saturating function of p, as commonly observed under N limitation. The maximum F_v/F_m of 0.64 was measured in nutrient-replete cells growing at μ_max, and a value of zero was measured in stationary-phase starved cells. When physiological parameters were compared, the P-stress protein abundance and F_v/F_m were the most sensitive indicators of the level of deficiency. The stress protein was not produced under N- or Fe-limited conditions. It is of high molecular weight (>200) and is associated with internal cell membranes. The stress protein has several characteristics that make it a potential diagnostic indicator: it is 1) unique to phosphorus limitation (i.e. absent under all other conditions), 2) present under limiting as well as starved conditions, 3) sensitive to the level of limitation, and 4) observable without time-course incubation of live samples.     

Towards the development of a nuclear transformation system for <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

A nuclear transformation system for the microalga Dunaliella tertiolecta was explored using electroporation. Plasmids incorporating the D. tertiolecta RbcS1 5′ and 3′ untranslated regions flanking the Streptoalloteichus hindustanus gene encoding bleomycin resistance (ble) were introduced into D. tertiolecta cells both transiently and stably. Southern hybridisation was used to examine the fate of the plasmid following electroporation and revealed that the DNA was entering the cells but was quickly degraded. Using the same procedure one stably transformed line was recovered.     

EFFECTS OF HARVEST STAGE AND LIGHT ON THE BIOCHEMICAL COMPOSITION OF THE DIATOM THALASSIOSIRA PSEUDONANA1

The marine diatom Thalassiosira pseudonana (Hustedt, clone 3H) Hasle and Heimdal was cultured under three different light regimes: 100 μmol photon · m^?2· s^?1 on 12:12 h light : dark (L:D) cycles; 50 μmol photon · m^?2· s^?2 on 24:0 h L:D; and 100 μmol photon · m^?2· s^?1 on 24:0 h L:D. It was harvested during logarithmic and stationary phases for analysis of biochemical composition. Across the different light regimes, protein (as % of organic weight) was highest in cells during logarithmic phase, whereas carbohydrate and lipid were highest during stationary phase. Carbohydrate concentrations were most affected by the different light regimes; cells grown under 12:12 h L:D contained 37–44% of the carbohydrate of cells grown under 24:0 h L:D. Cells in logarithmic phase had high proportions of polar lipids (79 to 89% of total lipid) and low triacylglycerol (≤10% of total lipid). Cells in stationary phase contained less polar lipid (48 to 57% of total lipid) and more triacylglycerol (22 to 45% of total lipid). The fatty acid composition of logarithmic phase cells grown under 24:0 h L:D were similar, but the 100 μmol photon · m^?2· s^?1 (12:12 h L:D) cells at the same stage contained a higher proportion of polyunsaturated fatty acids (PUFAs) and a lower proportion of saturated and monounsaturated fatty acids due to different levels of 16:0, 16:1(n-7), 16:4(n-1), 18:4(n-3), and 20:5(n-3). With the onset of stationary phase, cells grown at 100 μmol photon · m^?2· s^?1 (both 12:12 and 24:0 h L:D) increased in proportions of saturated and monounsaturated fatty adds and decreased in PUFAs. Concentrations (% organic or dry weight) of 14:0, 16:0, 16:1(n-7), 20:5(n-3), and 22:6(n-3) increased in cells of all cultures during stationary phase. The amino acid compositions of cells were similar irrespective of harvest stage and light regime. For mariculture, the recommended light regime for culturing T. pseudonana will depend on the nutritional requirements of the animal to which the alga is fed. For rapidly growing bivalve mollusc larvae, stationary-phase cultures grown under a 24:0 h L:D regime may provide more energy by virtue of their higher percentage of carbohydrate and high proportions and concentrations of energy-rich saturated fatty acids.     

INDUCTION OF SPECIFIC PROTEINS IN EUKARYOTIC ALGAE GROWN UNDER IRON-, PHOSPHORUS-, OR NITROGEN-DEFICIENT CONDITIONS1

What limits phytoplankton growth in nature? The answer is elusive because of methodological problems associated with bottle incubations and nutrient addition experiments. We are investigating the possibility that antibodies to proteins repressed by a specific nutrient can be used as probes to indicate which nutrient limits photosynthetic carbon fixation in the ocean. The diatom Phaeodactylum tricornutum Bohlin and the chlorophyte Dunaliella tertiolecta Butcher were grown in batch cultures in artificial seawater and f/2 nutrient lacking either phosphorus, iron, or nitrogen. Chlorosis was induced by nutrient limitation in both species with the exception of phosphorus-limited D. tertiolecta. The synthesis and appearance of specific proteins were followed by labeling with ¹⁴C-bicarbonate. Nutrient limitation in general leads to a decrease in the quantum efficiency of photosystem II, suggesting that deficiency of any nutrient affects the photosynthetic apparatus to some degree: however, the effect of nitrogen and iron limitation on quantum efficiency is more severe than that of phosphorus. A crude fractionation of the soluble and membrane proteins demonstrated that the large proteins induced under limitation by phosphorus and iron were associated with the membranes. However, small iron-repressible proteins were located in the soluble fraction. Isolation with anion-exchange chromatography and N-terminal sequencing of iron-repressible, 23-kDa Proteins from D. tertiolecta, P. tricornutum, and Chaetoceros gracilis revealed that these small soluble proteins have strong homology with the N-terminal sequence of flavodoxins from Azotobacter and Clostridium. The identity of the flavodoxin from D. tertiolecta was confirmed by immunodetection using antiflavodoxin raised against Chlorella. Flavodoxin was detected only under iron deprivation and was absent from nitrogen-and phosphorus-limited algae. Flavodoxin is a prime candidate for a molecular probe of iron limitation in the ocean. The requirements to confirm its utility in nature are discussed.     

Effects of accumulation of 3-O-Methylglucose on levels of intracellular glycerol inDunaliella tertiolecta

Regulation of the concentration of osmotic solute was studied inDunaliella tertiolecta grown at an external salinity ranging between 0.5 and 1.5 mol/L NaCl. The total solute content of the cells was increased by applying 3-O-methylglucose (8 mmol/L), which was not metabolized, but accumulated at concentrations ranging between 7.5 and 12.5 μmol per mg dry mass within 2 h after its addition to the medium. 3-O-Methylglucose uptake resulted in a decreased concentration of glycerol, the solute mainly responsible for adaptation ofD. tertiolecta to high external salinity. 3-O-Methylglucose had no direct effect on the pathway of glycerol synthesis or degradation after external salinity increased or decreased, respectively. Thus, 3-O-methylglucose had no direct effects on glycerol metabolism, and it can bo assumed that it acts solely as an inert osmotic solute with the cells. 3-O-Methylglueose accumulation increased the respiration rate, as expected from an active transport.     

EFFECT OF LIGHT HISTORY ON THE ULTRASTRUCTURE AND PHYSIOLOGY OF PROROCENTRUM MARIAE-LEBOURIAE (DINOPHYCEAE)1

Ultrastructural and physiological responses of Prorocentrum mariae-lebouriae (Parke & Ballantine) Faust are reported for cultures maintained at growth irradiances (I_g) ranging from 20.6 to 0.3 E m^?2.d^?1 and following downward shifts in light intensity. We tested the hypothesis that Prorocentrum grown under light regimes that elicit different responses in photosynthesis and pigmentation exhibit distinctive cell ultrastructures. Prorocentrum from high-light conditions had high saturation intensities for photosynthesis (I_k) and low levels of Chl a, Chl c and peridinin-cell^?1 These cultures were morphologically distinguished by a large starch volume fraction (V_v), small chloroplast V_v and fewer thylakoids lamella^?1. I_k values were lower and pigment concentrations higher in low-light treatments, and cells showed reduced starch V_v, large chloroplast V_v, and higher numbers of thylakoids · lamella ^?1. Cells grown under extremely low-light conditions appeared stressed as indicated by the absence of starch reserves and the presence of large vacuoles within the cytoplasm. Results for presence of large vacuoles within the cytoplasm. Results for quantiative electron microscopy, photosynthesis-irradiance (P-I) relations and cell pigmentation indicate that photoadaptation in P. mariae-lebouriae involves a strategy that encompasses changes in both the “size” and “number” of photosynthetic units.     

Production of Electricity and Butanol from Microalgal Biomass in Microbial Fuel Cells

Chlorella vulgaris (a freshwater microalga) and Dunaliella tertiolecta (a marine microalga) were grown for bulk harvest, and their biomass was tested as feedstock for electricity production in cubic two-chamber microbial fuel cells (MFCs) at 37°C. The anode inoculum was anaerobic consortium from a municipal sewage sludge digester, enriched separately for the two microalgal biomass feedstocks. After repeated subculturing of the two anaerobic enrichments, the maximum power density obtained in MFCs was higher from C. vulgaris (15.0 vs. 5.3 mW m^?2) while power generation was more sustained from D. tertiolecta (13 vs. 9.8 J g^-1 volatile solids). Anolytes of algal biomass-fed MFCs also contained substantial levels of butanol (8.7–16 mM with C. vulgaris and 2.5–7.0 mM with D. tertiolecta), which represents an additional form of utilizable energy. Carryover of salts from the marine D. tertiolecta biomass slurry resulted in gradual precipitation of Ca and Mg phosphates on the cathode side of the MFC. Polymerase chain reaction-denaturing gradient gel electrophoresis profiling and sequencing of bacterial communities demonstrated the presence of Wolinella succinogenes and Bacteroides and Synergistes spp. as well as numerous unknown bacteria in both enrichments. The D. tertiolecta enriched consortium contained also Geovibrio thiophilus and Desulfovibrio spp. Thus, the results indicate potential for combining fermentation and anaerobic respiration for bioenergy production from photosynthetic biomass.     

The salt relations of Dunaliella

Dunaliella tertiolecta (marine) and D. viridis (halophilic) were each trained by serial transfer to growth at salt concentrations previously regarded as the other's domain. D. viridis then had a salt optimum at 1.0–1.5 M sodium chloride whereas that for D. tertiolecta was less than 0–2 M. Nevertheless D. tertiolecta grew faster than the halophil at all salt concentrations up to 3.5 M, the highest at which they were compared.Both species accumulate glycerol, which is necessary for growth at elevated salinities and which responds in its content to water activity (a _w ) rather than specifically to salt concentration. Variation in glycerol content is a metabolic process which occurs in the dark from accumulated starch as well as photosynthetically. Regulation of glycerol content by a _w does not require protein synthesis. The NADP-specific glycerol dehydrogenase of each of the algae is likely to be directly involved in the regulation of glycerol content. Kinetic studies, together with those described in an earlier publication, show that the enzyme has regulatory properties, and that both glycerol and dihydroxyacetone act as effectors as well as reactants. A mechanism of the reaction is tentatively proposed.