Transient state characteristics of changes in light conditions for the cyanobacterium Oscillatoria agardhii

The cyanobacterium Oscillatoria agardhii was subjected to changes in irradiance and to changes in light period. During transient states parameters as growth rate, pigment contents, photosynthetic activities and pool sizes of carbohydrate and proteins were followed. The changes in pigments and photosynthesis were similar for irradiance transitions and transitions in light period length. Carbohydrates served for the supply of carbon and energy during adaptation to low light conditions until a basal level of 125 g · mg dry wt^-1 was reached. After transfer to high light conditions excess carbon fixation led to the storage of carbohydrate reserve polymers up to 600 g · mg dry wt^-1. During adaptation to longer light periods cells showed an overcapacity for carbohydrate accumulation even in the presence of a high carbohydrate content at the start of the light period. A model for the feed back repression of photosynthesis related to carbohydrate accumulation was presented. In all cases protein synthesis was directly maximized under the given conditions. Growth rate defined as specific rate of change in carbon showed the fastest response after a shift in light conditions. It was concluded that adaptation of O. agardhii to changes in light conditions was directed to the optimization of growth. The observation that carbohydrate is used to supply carbon and/or energy during adaptation leads to the conclusion that changes on survival in low light depend on carbohydrate level, the efficiency of its conversion in cell material and the maintenance requirements. Such a survival strategy enables cyanobacteria to cope succesfully with light limiting conditions.Abbreviations HL high irradiance Em^-2s^-1 - LL low irradiance Em^-2s^-1 - L/D light-dark cycle h - specific growth rate h^-1 - _e specific maintenance coefficient h^-1 - _max maximal specific growth rate h^-1 - _c specific rate of change of carbon h^-1 - _protein specific rate of change of protein h^-1 - Chl a chlorophyll a - CPC C-phycocyanin - dry wt dry weight mg - light utilization efficiency nmol O₂ · mg dry wt^-1 · min^-1/Em^-1 s^-1 - P _max photosynthetic capacity nmol O₂ · mg dry wt^-1 · min^-1 - PSI photosystem I - PS II photosystem II - RC II reaction center of PS II - PQ plastoquinone     

Transient states of geosmin,pigments, carbohydrates and proteins in continuous cultures of Oscillatoria brevis induced by changes in nitrogen supply

Transitions in the growth limiting factor from light (I) to nitrogen (N) and vice versa caused changes in geosmin production, protein and carbohydrate content, and the synthesis of pigments such as chlorophyll a (Chl a), phycobiliproteins (PBPs), and -carotene of the cyanobacterium Oscillatoria brevis. Following IN transition the first 150h, the decrease in protein content was compensated for by an increase of carbohydrates, and thereby, a constant biomass level was maintained in this period. Thereafter, biimass dropped to 15% of its initial level. A decrease in geosmin and pigment content was observed during transition from IN-limited growth. However, geosmin increased relative to phytol (Chl a) and -carotene which may indicate that a lowered demand for phytol and -carotene during N-limited growth allows isoprenoid precursors to be directed to geosmin rather than to pigment synthesis. Synthesis of Chl a and -carotene at the expense of geosmin was suggested for the observed start of increase in geosmin production only at the time that Chl a and -carotene had reached their I-limited steady state. Transition from nitrogen to light limited growth caused an acceleration of metabolism shown by a rapid decrease in carbohydrate content accompanied by an increase in protein content. The growth rate of the organisms temporarily exceeded the dilution rate of the culture and the biomass level increased 6-fold. Due to the only modest changes in geosmin production (2-fold) compared to changes in biomass level (6-fold) during I-or N-limited growth, environmental factors seem to have limited effect on geosmin production.Abbreviations Chl a chlorophyll a - dry wt dry weight; - I-limited light-limited - N-limited nitrogen-limited - PBP phycobiliprotein This research was performed at the Department of Microbiology, University of Amsterdam, with finacial support provided by the Royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

Acclimation of the diatom Stephanodiscus neoastraea and the cyanobacterium Planktothrix agardhii to simulated natural light fluctuations

Functional and structural characteristics of the photosynthetic apparatus were studied in the diatom Stephanodiscus neoastraea and the cyanobacterium Planktothrix agardhii which were grown semi-continuously under constant irradiance or under simulated natural light fluctuations. The light fluctuations consisted of 24 oscillations of exponentially increasing and decreasing irradiance over a 12-h light period. Maximum irradiance was 1100 μmol photons m⁻² s⁻¹ with the ratio of maximum to minimum intensities being 100, simulating Langmuir circulations with a ratio of euphotic to mixing depth of 1. S. neoastraea acclimated to the light fluctuations by doubling the number and halving the size of photosynthetic units (PS II) while the amount of chlorophylls and carotenoids remained unchanged. The chlorophyll-specific maximum photosynthetic rate was enhanced while the slope of the photosynthesis versus irradiance curves was not influenced by the light fluctuations. Acclimation of P. agardhii was mainly characterized by an increase in chlorophyll content. Both photosystems showed only little changes in number and size. Maximum photosynthetic rate, saturating irradiance and initial slope of the photosynthesis versus irradiance curves did not vary. Although both high and low light were contained in the fluctuating light, an analogy to low or high light acclimation was not found for the diatom nor for the cyanobacterium acclimated to light fluctuations. We suggest that the acclimation to fluctuating light is a response type outside the known scheme of low and high light acclimation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO₂ of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CAP chloramphenicol     

Isolation and characterization of phycoerythrocyanin and chromatic adaptation of the thermophilic cyanobacterium Mastigocladus laminosus

The thermophilic cyanobacterium Mastigocladus laminosus exhibits chromatic adaptation: In green light the production of the phycobiliprotein phycoerythrocyanin is enhanced drastically.Phycoerythrocyanin was characterised with respect to its molecular weight, isoelectric point, absorption spectra and size of its aggregates. The two subunits of the protein were separated and characterised according to these criteria. Their chromophore contents, amino acid compositions and N-terminal amino acid sequences were also determined. The sequences were compared with those of allophycocyanin and C-phycocyanin from Mastigocladus laminosus.Abbreviations PEC phycoerythrocyanin - -PEC -subunit of PEC - -PEC -subunit of PEC - PE phycoerythrin - PC phycocyanin - APC allophycocyanin - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - chl a chlorophyll a     

Effect of oxygen on the cyanobacterium Oscillatoria limnetica

Oscillatoria limnetica grown photoautotrophically under aerobic or anaerobic conditions contained a single superoxide dismutase (SOD) of identical electrophoretic mobility in both cases. Its activity was cyanide resistant and H₂O₂ sensitive, implicating Fe-SOD. The enzyme level was high in aerobically and low in anaerobically growing cells. Anaerobically grown cells were more sensitive than aerobic to photooxidation, as expressed by bleaching of phycocyanin and disintegration of the trichomes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - SOD superoxide dismutase     

Temporal separation of nitrogen fixation and photosynthesis in the filamentous,non-heterocystous cyanobacterium Oscillatoria sp.

When growing in laternating light-dark cycles, nitrogenase activity (acetylene reduction) in the filamentous, non-heterocystous cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) is predominantly present during the dark period. Dark respiration followed the same pattern as nitrogenase. Maximum activities of nitrogenase and respiration appeared at the same time and were 3.6 mol C₂H₄ and 1.4 mg O₂ mg Chl a ^-1·h^-1, respectively. Cultures, adapted to light-dark cycles, but transferred to continuous light, retained their reciprocal rhythm of oxygenic photosynthesis and nitrogen fixation. Moreover, even in the light, oxygen uptake was observed at the same rate as in the dark. Oxygen uptake and nitrogenase activity coincided. However, nitrogenase activity in the light was 6 times as high (22 mol C₂H₄ mg Chl a ^-1·h^-1) as compared to the dark activity. Although some overlap was observed in which both oxygen evolution and nitrogenase activity occurred simultaneously, it was concluded that in Oscillatoria nitrogen fixation and photosynthesis are separated temporary. If present, light covered the energy demand of nitrogenase and respiration very probably fulfilled a protective function.     

Effects of light on nitrate-limited Oscillatoria agardhii in chemostat cultures

The effects of the average light irradiance (I) on growth and nitrate uptake kinetics of the cyanobacterium Oscillatoria agardhii, in nitrate-limited chemostat cultures, were studied. Light was nonsaturating for I <9.4 Wm^–2, for all growth rates () studied. However, was throughout limited by the availability of nitrate. Under light-saturating conditions the kinetics of nitrate-limited growth could be adequately described by both the Monod and Droop equations. Under light-non-saturating conditions the internal nitrogen content (Q) was a function of both and I, for which new formulas were derived. The high uptake capacity (V _max) of nitrate-limited cells was independent of , but was significantly increased for cells growing at I <9.4 Wm^–2. The half-saturation constant for nitrate uptake (K _s ^u ) increased with increasing , but was independent of the prevailing light conditions. The effects of light during nitrate-limited growth were associated with the regulation in the nitrogen-containing pigments.The results reported herein have important consequences for the use of Q, K _s ^u and V _max values as indicators of nutrient-deficiency of natural populations.     

Acclimation of Arabidopsis thaliana to the light environment: changes in photosynthetic function

Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was grown under light regimes of differing spectral qualities, which results in differences in the stoichiometries of the two photosynthetic reaction centres. The acclimative value of these changes was investigated by assessing photosynthetic function in these plants when exposed to two spectrally distinct actinic lights. Plants grown in an environment enriched in far-red light were better able to make efficient use of non-saturating levels of actinic light enriched in long-wavelength red light. Simultaneous measurements of chlorophyll fluorescence and absorption changes at 820 nm indicated that differences between plants grown under alternative light regimes can be ascribed to imbalances in excitation of photosystems I and II (PSI, PSII). Measurements of chlorophyll fluorescence emission and excitation spectra at 77 K provided strong evidence that there was little or no difference in the composition or function of PSI or PSII between the two sets of plants, implying that changes in photosynthetic stoichiometry are primarily responsible for the observed differences in photosynthetic function.Abbreviations Chl chlorophyll - FR far-red light - HF highirradiance FR-enriched light (400 mol·m^–2·s^–1, RFR = 0.72) - HW high-irradiance white light (400 mol·m^–2 1·1 s^–1RFR = 1.40) - LHCI, LHCII light-harvesting complex of PSI, PSII - qO quenching of dark-level chlorophyll fluorescence - qN non-photochemical quenching of variable chlorophyll fluorescence - qP photochemical quenching of variable chlorophyll fluorescence - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Dr. Sasha Ruban for assistance with the 77 K fluorescence measurements and for helpful discussions. This work was supported by Natural Environment Research Council Grant GR3/7571A.     

Immunological characterization of nitrogenase in the filamentous non-heterocystous cyanobacterium Oscillatoria limosa

The filamentous non-heterocystous cyanobacterium Oscillatoria limosa was subjected to Western blot analyses using two antisera raised against the small subunit (Fe-protein) of the nitrogenase complex. Two polypeptides were recognized in nitrogen-fixing cultures irrespective of the antiserum used while no bands were detectable in nitrate-grown cultures. The apparent molecular weights of the two polypeptides were approximately 40.5 and 39.5 kDa respectively, with the former, probably an inactive form, dominating. In situ immunogold electron microscopy was used to reveal the cellular and subcellular localization on the Fe-protein. All cells of the trichomes of nitrogen-fixing O. limosa showed a dense label. The label was homogeneously distributed throughout the cytoplasm including the thylakoid area. Nitrate-grown cultures contained a very low label.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis This study was supported by the Swedish Natural Science Research Counsil and the M. and M. Wallenberg Fund (to B. Bergman). We are grateful to Dr. S. Nordlund (University of Stockholm, Sweden) for providing us with the antiserum of Rhodospirillum rubrum nitrogenase and to Drs. S. Reich and P. Böger (University of Konstanz, FRG) for the antiserum of Anabaena variabilis. Skilful technical assistence by K. Östlund and E. Danielsson is gratefully acknowledged. We would also like to thank M. Villbrandt (University of Oldenburg, FRG) for providing cultures of Oscillatoria limosa and Dr. P. Lindblad for valuable discussions and suggestions.To whom correspondence should be addressed.     

Loss of sulfide adaptation ability in a thermophilic Oscillatoria

A spontaneous variant incapable of anoxygenic photosynthesis was derived from a fully competent strain of Oscillatoria amphigramulata which was originally isolated from a high sulfide-containing hot spring of New Zealand. Although the variant (Oa-2) acquired a slight ability to photosynthesize in the presence of 0.3–0.4 mM sulfide, this was only after a 24 h exposure to sulfide and represented oxygenic photosynthesis only. Unlike the parent strain, the incompetent variant never grew in the presence of sulfide >0.05 mM, nor was there any relief of the inhibition by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] of CO₂ photoincorporation when sulfide was present. The variant strain has retained all of these characteristics over a 4 year period with monthyl transfers in non-sulfide medium. The wild type, under identical conditions, has retained all of its competence with respect to sulfide.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Isolation and biliprotein characterization of phycobilisomes from the thermophilic cyanobacterium Mastigocladus laminosus Cohn

A method for the effective isolation of functionally intact phycobilisomes from the thermophilic cyanobacterium M. laminosus is presented, using an unconventional high buffer molarity for stabilizing the aggregates and introducing a DNAse treatment of the disrupted cells to obtain sharp banding of the phycobilisomes in the linear sucrose density gradients.The structural integrity of the isolated phycobilisomes is demonstrated by a fluorescence emission maximum at 673 nm of aggregated allophycocyanin and by electron microscopy.Besides C-phycocyanin and allophycocyanin, phycoerythrocyanin is a constituent pigment of the phycobilisomes. These pigments indicated in the absorption spectrum of phycobilisomes with a maximum at 610 nm and two shoulders at 650 and 580 nm, respectively, were characterized by spectral data and isoelectric points.     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.     

Modelling of growth conditions for cyanobacterium Spirulina platensis in microcosms

The influence of cultivation conditions on the growth of the cyanobacterium Spirulina platensis was investigated by using two types of photobioreactors. In a rotative photobioreactor the doubling time (t _d) was 3.54 days. The better value found for t _d in an aerated photobioreactor by changing the initial nitrogen concentration (NaNO₃) at 0.003, 0.015, 0.030 and 0.060M was 2.5 days. A factorial experimental design was performed in order to estimate the contributions of initial nitrogen concentration, inoculum and cultivation time as well as their interactions. All three factors and their interactions proved to be significant in influencing the cellular concentration of S. platensis.     

Fluence rate and wavelength dependence of photobleaching in the cyanobacterium Anabaena variabilis

The fluence rate dependence of the photobleaching in the cyanobacterium Anabaena variabilis was studied under physiological conditions. According to the in-vivo absorption spectra measured every day during the 5 d exposition the phycobiliproteins are more sensitive to high fluence rates than chlorophyll a. The carotenoids are least sensitive, so that a relative, but not an absolute increase in the carotenoid content occurred. At very high fluence rates exceeding about 50 Wm^-2 white light the organisms were photokilled after 5 d of irradiation. Measurements of the nitrate concentrations during the experiments have shown that nitrate was not the limiting factor in these experiments. Analysis of the photobleaching kinetics at 13.5 Wm^-2 white light revealed that after about 8 d the contents of all the pigments studied have reached a new, constant level. After exposure of the photobleached cyanobacteria to low irradiances repigmentation occurred. Thus, photobleaching is a light adaptation process and not simply a photodamage phenomenon. Studying the wavelength dependence of photobleaching at a constant photon fluence rate of 4·10^-8 mol cm^-2 s^-1 we found that the photobleaching of both phycobiliproteins and chlorophyll a was exclusively caused by wavelengths absorbed by the phycobiliproteins, mainly phycoerythrocaynin, and red light absorbed by short wavelength chlorophyll. Wavelengths <520 nm were ineffective.     

Response of the estuarine cyanobacterium <Emphasis Type="Italic">Lyngbya aestuarii</Emphasis> to UV-B radiation

Growth response and changes in the spectral properties of methanolic extract of an estuarine cyanobacterium, Lyngbya aestuarii Agardh, to UV-B radiation were studied. Increase in growth accompanied by increase in chlorophyll a, protein and carbohydrate content was observed up to 12 h of UV-B irradiation followed by a decline with further increase in the duration of UV exposure. Carotenoid content progressively increased with the UV-B dose. The organism synthesized, to a significant extent, mycosporine amino acid-like substances (MAAs) upon UV-B exposure. The cells in the trichome became coiled followed by formation of small bundles as a response to UV-B radiation. SDS protein profile of the UV irradiated cells showed repression of 20 and 22 kDa proteins. However, irradiation with UV-B for 6–24 h led to overproduction of 84, 73, 60, 46, 40, 37 KDa proteins, possibly conferring protection to the organism from UV-B. UV irradiated cells cultured in florescent light for up to 7 days showed revival from UV damage of the pigments and macromolecular contents, suggesting existence of a repair mechanism in the organism.     

Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Q_y transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Q_y transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.Abbreviations APC - allophycocyanin - Chl a - chlorophyll a - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LD - linear dichroism - LD/A - linear dichroism divided by absorbance - LHC - light-harvesting complex - PBS - phycobilisome - PC - phycocyanin - PS - Photosystem     

Toxicity of the blue-green alga Oscillatoria agardhii to the mosquito Aedes aegypti and the shrimp Artemia salina

The cyanobacterium Oscillatoria agardhii 27, which does not produce mammalian neuro- or hepatotoxins, was highly toxic to the larval stages of the yellow fever mosquito Aedes aegypti: its 24-h LC₅₀ values against fourth-and second-instar larvae of A. aegypti were 8.7 and 6.1 g live cells/ml, respectively. The toxin was water-soluble and was partially purified but the chemical nature of the toxic compound(s) is still unknown. Aqueous solutions were also toxic to the newborn larvae of the brine shrimp, Artemia salina, used for the bioassay. The toxic activity of these solutions decreased markedly on heating to 90°C for 15 min.J. Kiviranta is with the Department of Pharmacy, P.O. Box 15, FIN-00014 University of Helsinki, Helsinki, Finland; A. Abdel-Hameed is usually with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt, but is presently with the Department of Applied Chemistry and Microbiology, P.O. Box 27, Viikki, Building B, FIN-00014 University of Helsinki, Helsinki, Finland.