GRAZING AND GROWTH OF THE MIXOTROPHIC CHRYSOMONAD POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) FEEDING ON ALGAE

The growth and grazing characteristics of Poterioochromonas malhamensis (Pringsheim) Peterfi (= Ochromonas malhamensis Pringsheim) (ca. 8 μm) feeding on phytoplankton, including the cyanobacteria Synechococcus sp. (ca. 2 μm) and Microcystis viridis (A. Brown) Lemmermann (ca. 6 μm) and the green alga Chlorella pyrenoidosa Chick (ca. 13 μm), were investigated in laboratory experiments involving the following treatments: (1) light without added algal prey (autotrophy), (2) light with added algal prey (mixotrophy), and (3) dark with added algal prey (phagotrophy). There were significantly higher cell numbers under mixotrophic and phagotrophic growth than under autotrophic growth. With phytoplankton as food, growth rates under both mixotrophy and phagotrophy were about two or three times higher than those under autotrophy, indicating that the algal diets were readily able to support the population growth of P. malhamensis. There were no significant differences in growth rate between mixotrophic and phagotrophic cultures during exponential growth. The ingestion rate of P. malhamensis with algal prey was also similar under both continuous light and dark. Poterioochromonas malhamensis ingested on average 0.27 M. viridis cells·flagellate^{− 1} ·h^{− 1} and 0.18 C. pyrenoidosa cells·flagellate^{− 1} ·h^{− 1} in continuous light and 0.25 M. viridis cells·flagellate^{− 1} ·h^{− 1} and 0.18 C. pyrenoidosa cells·flagellate^{− 1} ·h^{− 1} in continuous dark during exponential growth. The results showed that light had no effect on the growth and ingestion rates of P. malhamensis for phagotrophy during exponential growth. However, phagotrophic populations of P. malhamensis were incapable of growth in continuous darkness for longer than 5 days. Populations of P. malhamensis showed no increase when prey was added again after 4 days in continuous darkness, indicating that light is necessary for sustained phagotrophic growth of P. malhamensis. The study suggests that P. malhamensis, which has strong tolerance for light, is light dependent for phagotrophy.     

Light-dependent phagotrophy in the freshwater mixotrophic chrysophyte Dinobryon cylindricum

The mixotrophic (bacterivorous), freshwater chrysophyte Dinobryon cylindricum was cultured under a variety of light regimes and in bacterized and axenic cultures to investigate the role of phototrophy and phagotrophy for the growth of this alga. D. cylindricum was found to be an obligate phototroph. The alga was unable to survive in continuous darkness even when cultures were supplemented with high concentrations of bacteria, and bacterivory ceased in cultures placed in the dark for a period longer than one day. Axenic growth of the alga was poor even in an optimal light regime. Live bacteria were required for sustained, vigorous growth of the alga in the light. Carbon (C), nitrogen (N), and phosphorus (P) budgets determined for the alga during growth in bacterized cultures indicated that bacterial biomass ingested by the alga may have contributed up to 25% of the organic carbon budget of the alga. Photosynthesis was the source of most (75%) of the organic carbon of the alga. D. cylindricum populations survived but did not grow when cultured in a continuous low light intensity (30 E m^–2 sec^–1), or in a light intensity of 150 E m^–2 sec^–1 for only two hours each day. Net efficiency of incorporation of bacterial C, N, and P into algal biomass under these two conditions was zero (i.e., no net algal population growth). We conclude that the primary function of bacterivorous behavior in D. cylindricum may be to provide essential growth factor(s) or major nutrients for photosynthetic growth, or to allow for the survival of individuals during periods of very low light intensity or short photoperiod. Offprint requests to: David A. Caron     

Phagotrophy of fluorescently labeled bacteria by an oceanic phytoplankter

Using fluorescently-labeled bacteria and detection by flow cytometry and epifluorescence microscopy, we demonstrate inducible mixotrophy in a marine photosynthetic flagellate, Ochromonas sp. (class Chrysophyceae). Phagotrophic uptake of bacteria increases under conditions of low or limiting light and nutrients, but deceases in periods of prolonged darkness; sustained phagotrophy may require light. In addition, this alga appears to discriminate between and preferentially ingest different types of bacteria. Although this clone is primarily photosynthetic, phagotrophy contributes to its nutrition, especially when light or nutrients limit photosynthesis.Correspondence to: M.D. Keller     

Effects of prey abundance and light intensity on the mixotrophic chrysophyte Poterioochromonas malhamensis from a mesotrophic lake

1. Previous studies of mixotrophy in the flagellate Poterioochromonas malhamensis (Chrysophyceae) were performed on strains that had been in culture for > 30 years. This study aims to compare mixotrophy in a cultured strain with one recently isolated from a mesotrophic lake (Lacawac) in Pennsylvania, U.S.A. 2. P. malhamensis from the lake exhibited a nutritional flexibility similar to that of the culture strain, growing phototrophically but inefficiently in comparison to other nutritional modes (growth rate (μ) = 0.015 h^?1). Supplementing an inorganic salts medium with 1 mM glucose resulted in a doubling of μ to 0.035 h^?1 and 0.033 h^?1 in the light and the dark, respectively. Addition of an algal prey, Nannochloris, to the inorganic salts medium increased growth to rates similar to those observed with glucose. Maximum growth of the lake strain, 0.095 h^?1, was achieved when bacteria was supplied as food. During growth on bacteria, cellular chlorophyll a (Chl a) decreased from 140 fg cell^?1 to 10 fg cell^?1 over 22 h when cultured either in the light or dark. In illuminated cultures, cell-specific Chl a concentration recovered to 185 fg cell^?1 after bacteria became limiting. 3. In contrast to the cultured strain, however, the lake isolate exhibited an inverse relationship between light intensity and ingestion rate. Calculated grazing rates, based upon the ingestion of fluorescently labeled bacteria, were 3.2, 5.2 and 9.4 bacteria flagellate^?1 h^?1, for P. malhamensis incubated in high light, low light and darkness, respectively. Phagotrophy is thus influenced by a light regime in this predominately heterotrophic mixotroph.     

Temperature‐dependent phagotrophy and phototrophy in a mixotrophic chrysophyte

The roles of temperature and light on grazing and photosynthesis were examined for Dinobryon sociale, a common freshwater mixotrophic alga. Photosynthetic rate was determined for D. sociale adapted to temperatures of 8, 12, 16, and 20°C under photosynthetically active radiation light irradiances of 25, 66, and 130 μmol photons · m^?2 · s^?1, with concurrent measurement of bacterial ingestion at all temperatures under medium and high light (66 and 130 μmol photons · m^?2 · s^?1). Rates of ingestion and photosynthesis increased with temperature to a maximum at 16°C under the two higher light regimes, and declined at 20°C. Although both light and temperature had a marked effect on photosynthesis, there was no significant difference in bacterivory at medium and high irradiances at any given temperature. At the lowest light condition (25 μmol photons · m^?2 · s^?1), photosynthesis remained low and relatively stable at all temperatures. D. sociale acquired the majority of carbon from photosynthesis, although the low photosynthetic rate without a concurrent decline in feeding rate at 8°C suggested 20%–30% of the carbon budget could be attributed to bacterivory at low temperatures. Grazing experiments in nutrient‐modified media revealed that this mixotroph had increased ingestion rates when either dissolved nitrogen or phosphorus was decreased. This work increases our understanding of environmental effects on mixotrophic nutrition. Although the influence of abiotic factors on phagotrophy and phototrophy in pure heterotrophs and phototrophs has been well studied, much less is known for mixotrophic organisms.     

LIGHT AND ELECTRON MICROSCOPY OF GRAZING BY POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) ON A RANGE OF PHYTOPLANKTON TAXA1

Grazing of fluorescent latex beads, bacteria, and various species of phytoplankton by Poterioochromonas malhamensis (Pringsheim) Peterfi (about 8.0 μm in diameter) was surveyed. The alga ingested fluorescent beads and various live or killed and nomnotile or motile organisms including bacteria, blue-green algae, green algae, diatoms, and chrysomonads. The size range of grazed prey was from 0.1 to 6.0 μm for latex beads and from 1.0 μm (bacteria) to about 21 μm (Carteria inverse) for organisms. As many as 17 latex beads (2.0 μm) or more than 10 Microcystis cells (5–6 μm) were ingested by a single P. malhamensis cell. Following such grazing, the cell increased in volume by up to about 30-fold. The range of cell volume of ingested prey was from 0.52 μm³ (bacteria) to about 3178 μm³(Carteria inversa). This study demonstrates for the first time that P. malhamensis is capable of grazing algae 2–3 times larger in diameter than its own cell and of grazing intact motile algae. Poterioochromonas malhamensis is an omnivorous grazer. Food vacuole formation and digestion processes were examined. The membrane that was derived from the plasma membrane and surrounded the prey disappeared sometime after ingestion. The food vacuole was then formed by successive fusion of numerous homogeneous vesicles accumulated around the prey. The prey was enclosed in a single membrane-bound food vacuole and then digested.     

A classification of mixotrophic protists based on their behaviour

1. Mixotrophs are organisms which combine phototrophy and heterotrophy; such nutritional behaviour is widespread among protists. This ability to combine multiple modes of nutrition varies between species and is not related to their taxonomic grouping. A classification of mixotrophic protists, based on their behaviour, is proposed, dividing them into four groups.
2. Group A includes protists whose primary mode of nutrition is heterotrophy and where phototrophy is employed only when prey concentrations limit heterotrophic growth. In groups B, C and D phototrophy is the dominant mode of nutrition. In group B phagotrophy supplements growth when light is limiting, therefore ingestion of prey is inversely proportional to light intensity; in group C phagotrophy provides essential substances for growth and ingestion is proportional to light intensity; and group D includes those who have very low ingestion rates, ingesting prey only, for example, for cell maintenance during prolonged dark periods.
3. This classification is aimed towards predicting the impact of any particular mixotrophic protist on the aquatic food web, and how this impact may vary depending on the environmental conditions. A model representation of the four groups is discussed.     

A classification of mixotrophic protists based on their behaviour

1. Mixotrophs are organisms which combine phototrophy and heterotrophy; such nutritional behaviour is widespread among protists. This ability to combine multiple modes of nutrition varies between species and is not related to their taxonomic grouping. A classification of mixotrophic protists, based on their behaviour, is proposed, dividing them into four groups.
2. Group A includes protists whose primary mode of nutrition is heterotrophy and where phototrophy is employed only when prey concentrations limit heterotrophic growth. In groups B, C and D phototrophy is the dominant mode of nutrition. In group B phagotrophy supplements growth when light is limiting, therefore ingestion of prey is inversely proportional to light intensity; in group C phagotrophy provides essential substances for growth and ingestion is proportional to light intensity; and group D includes those who have very low ingestion rates, ingesting prey only, for example, for cell maintenance during prolonged dark periods.
3. This classification is aimed towards predicting the impact of any particular mixotrophic protist on the aquatic food web, and how this impact may vary depending on the environmental conditions. A model representation of the four groups is discussed.     

Interactions between metazoans,autotrophs, mixotrophs and bacterioplankton in nutrient‐depleted high DOC environments: a long‐term experiment

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Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d-1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.     

Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca

The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10^-9 to 10^-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10^-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10^-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP^* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP^* N⁶-2-O-dibutyryl-adenosine-35-monophosphate     

Significance of algal extracellular products to bacteria in lakes and in cultures

In simulated diurnal experiments withChlorella pyrenoidosa andPseudomonas fluorescens, bacterial growth was virtually confined to the daylight period and occurred at the expense of glycolate, the predominant extracellular product of the alga. Both glycolate levels and¹⁴C-DOC excretion rates were much lower in mixed algal-bacterial than in axenicChlorella cultures.This close coupling of bacterial growth to algal photosynthesis and extracellular release was also observed in Jack's Lake, but not in Lake Erie. Experimental enrichment with lake water particulates >30m suppressed the daytime growth of bacteria in Jack's Lake, but increased it dramatically in Lake Erie. Daylight doubling times for bacteria in lakewater ranged from 2 to 19 days. In mixed culture withChlorella, Pseudomonas had a doubling time of about 2 hours in the light.     

Growth inhibition by high light intensities in algae from lakes undergoing acidification

Blooms ofChrysochromulina breviturrita Nich. (Prymnesiophyceae) have been found to be restricted to lakes above pH 5.5 even though the alga is able to tolerate pH 4.0 in laboratory culture. A possible explanation is the increased transparency in acidifying lakes and a sensitivity ofC. breviturrita to high light intensities. A comparison was made withMougeotia sp., a filamentous green alga which co-occurs in moderately acidic lakes and has a similar pH tolerance range. This alga forms dense, floating mats or amorphous clouds in the upper littoral zone, where it would be exposed to full sunlight irradiances. In cultures ofC. breviturrita, prolonged exposures to 1600 μE · m⁻² · s⁻¹ (I₀′) resulted in reductions in cell yield which were dependent age at the onset of exposure to high light intensity. Only cultures exposed to high light intensities during late stationary phase were able to recover to control levels and no recovery occurred if these cultures were nitrogen deficient.Mougeotia was more tolerant of both high light intensity and nitrogen limitation during the recovery period. The inability ofC. breviturrita to recover from the effects of high light intensity during nitrogen deprivation may be particularly important in small, stratified lakes which are undergoing acidification. The slow rate of vertical circulation, and increasing transparency, would prolong exposure of the alga to the high irradiance levels of nutrient-deficient epilimnetic waters. This suggests that the geographic distribution ofC. breviturrita may be explained in part by the increasing light intensities in lakes undergoing acidification.     

Environment-dependent metabolic investments in the mixotrophic chrysophyte Ochromonas

Mixotrophic protists combine photosynthesis and phagotrophy to obtain energy and nutrients. Because mixotrophs can act as either primary producers or consumers, they have a complex role in marine food webs and biogeochemical cycles. Many mixotrophs are also phenotypically plastic and can adjust their metabolic investments in response to resource availability. Thus, a single species's ecological role may vary with environmental conditions. Here, we quantified how light and food availability impacted the growth rates, energy acquisition rates, and metabolic investment strategies of eight strains of the mixotrophic chrysophyte, Ochromonas. All eight Ochromonas strains photoacclimated by decreasing chlorophyll content as light intensity increased. Some strains were obligate phototrophs that required light for growth, while other strains showed stronger metabolic responses to prey availability. When prey availability was high, all eight strains exhibited accelerated growth rates and decreased their investments in both photosynthesis and phagotrophy. Photosynthesis and phagotrophy generally produced additive benefits: In low-prey environments, Ochromonas growth rates increased to maximum, light-saturated rates with increasing light but increased further with the addition of abundant bacterial prey. The additive benefits observed between photosynthesis and phagotrophy in Ochromonas suggest that the two metabolic modes provide nonsubstitutable resources, which may explain why a tradeoff between phagotrophic and phototrophic investments emerged in some but not all strains.     

Factors Affecting the Mixotrophic Flagellate Poterioochromonas malhamensis Grazing on Chlorella Cells

Grazing behaviour between protozoa and phytoplankton exists widely in planktonic ecosystems. Poterioochromonas malhamensis is a well‐known and widespread mixotrophic flagellate, which is recognized to play an important role within marine and freshwater planktonic ecosystems and regarded as the greatest contamination threat for mass algal cultures of Chlorella. In this study, a comprehensive range of factors, including morphological characters, biochemical compositions, and specific growth rate of ten species or strains of Chlorella, were evaluated for their effect on the feeding ability of P. malhamensis, which was assessed by two parameters: the clearance rate of P. malhamensis on Chlorella spp. and the specific growth rate of P. malhamensis. The results showed that the clearance rate of P. malhamensis was negatively correlated with cell wall thickness and specific growth rate of Chlorella spp., while the specific growth rate of P. malhamensis was positively correlated with carbohydrate percentage and C/N ratio and negatively correlated with protein, lipid percentage, and nitrogen mass. In conclusion, the factors influencing feeding selectivity include not only the morphological character and chemical composition of Chlorella, but also its population dynamics. Our study provides useful insights into the key factors that affect the feeding selectivity of P. malhamensis and provides basic and constructive data to help in screening for grazing‐resistant microalgae.     

Response of growth and photosynthesis of marine red tide alga Heterosigma akashiwo to iron and iron stress condition

Heterosigma akashiwo, a red tide alga, was grown in Fe-deficient and Fe-replete batch cultures. Cell final yields and the growth rate were limited when Fe was below 10 nM and alleviated with 100 nM Fe. By comparison with the results under Fe-replete conditions, chlorophyll a-specific and cell-specific light saturated net photosynthetic capacity (P_m ^{chl a} and P_m ^cell), dark respiration rate (R_d ^{chl a} and R_d ^cell) and apparent photosynthetic efficiency (^{chl a} and ^cell) decreased proportionately, whereas the cells became light saturated at higher irradiance under Fe stress (Fe-limited conditions).     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Mixotrophic trade‐off under warming and UVR in a marine and a freshwater alga

Mixotrophic protists combine phagotrophy and phototrophy within a single cell. Greater phagotrophic activity could reinforce the bypass of carbon (C) flux through the bacteria‐mixotroph link and thus lead to a more efficient transfer of C and other nutrients to the top of the trophic web. Determining how foreseeable changes in temperature and UVR affect mixotrophic trade‐offs in favor of one or the other nutritional strategy, along the mixotrophic gradient, is key to understanding the fate of carbon and mineral nutrients in the aquatic ecosystem. Our two main hypotheses were: (i) that increased warming and UVR will divert metabolism toward phagotrophy, and (ii) that the magnitude of this shift will vary according to the organism's position along the mixotrophic gradient. To test these hypotheses, we used two protists (Isochrysis galbana and Chromulina sp.) located in different positions on the mixotrophic gradient, subjecting them to the action of temperature and of UVR and their interaction. Our results showed that the joint action of these two factors increased the primary production:bacterivory ratio and stoichiometric values (N:P ratio) close to Redfield's ratio. Therefore, temperature and UVR shifted the metabolism of both organisms toward greater phototrophy regardless of the original position of the organism on the mixotrophic gradient. Weaker phagotrophic activity could cause a less efficient transfer of C to the top of trophic webs.     

Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs)

Unrooted strawberry cv. `Akihime' shoots with three leaves obtained from standard mixotrophic cultures were cultured in the ``Culture Pack'-rockwool system with sugar-free MS medium under CO<sub>2</sub>-enriched condition. To examine the effect of superbright red and blue light-emitting diodes (LEDs) on in vitro growth of plantlets, these cultures were placed in an incubator, ``LED PACK', with either red LEDs, red LEDs1blue LEDs or blue LEDs light source. To clarify the optimum blue and red LED ratio, cultures were placed in ``LED PACK 3' under LED light source with either 100, 90, 80, or 70% red + 0, 10, 20, 30% blue, respectively, and also under standard heterotrophic conditions. To determine the effects of irradiation level, cultures were grown under 90% red LEDs + 10% blue LEDs at 45, 60 or 75 mol m^–2 s^–1 . Plantlet growth was best at 70% red + 30% blue LEDs. The optimal light intensity was 60 mol m^–2 s^–1. Growth after transfer to soil was also best after in vitro culture with plantlets produced were 70% red LEDs + 30% blue LEDs.     

An investigation of the heterotrophic culture of the green algaTetraselmis

The marine PrasinophyteTetraselmis may be cultured under both mixotrophic (photoheterotrophic) and heterotrophic conditions. The growth rate was slightly lower, and pigment levels and lipid composition were radically affected on heterotrophic culture in 1 L fermenters. Total chlorophyll levels of dark grown cultures were less than 1% of those observed in mixotrophically grown cells, the chlorophylla : b ratio also decreased as did the carotenoid content. In addition, the total amounts of lipids including polyunsaturated fatty-acids were also lower in heterotrophically cultured cells: 6.4 mg g^–1 (dried alga) and 0.35 mg g^–1 (dried alga); as compared to 37.1 mg g^–1 (dried alga) and 18.5 mg g^–1 (dried alga), for cells grown in the light. However, gross morphology and final yield (>16 g l^–1) were relatively unaffected. The algae produced were spray-dried and tested for their suitability as an aquaculture feed.Address for correspondence