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Phagotrophic algae can consume bacteria that are the predominant microorganisms present in the waste activated sludge (WAS) generated from municipal wastewater treatment processes. In this study, we developed a combined ultrasonication-phagotrophic algal process for WAS conversion. The ultrasonic pretreatment released small volatile solids (VS) including bacteria from WAS flocs. A phagotrophic alga Ochromonas danica then grew by consuming more than 80% of the released VS, with approximately 30% (w/w) algal cell yield. The process reduced the overall WAS VS by 42.4% in 1 day, comparing very favorably with the 27% reduction in 10 days by aerobic digestion. For stabilizing the solids remaining from the ultrasonic step, the total oxygen uptake required was 65%-92% lower than that for the original WAS, indicating substantially reduced aeration cost. Overall, this novel process enhanced the WAS digestion at lower energy requirements and produced microalgae for other potential uses. © 2021 Water Environment Federation PRACTITIONER POINTS: At least 80% of released VS from WAS can be processed by phagotrophic algae. Significant amounts of algae can be produced from WAS. Ultrasonication-phagotrophic algal process can make sludge management more sustainable.

Application of plant hormones is an effective strategy to further improve the accumulation of microalgae biomass. In this study, the IAA (indole-3-acetic acid) production capacity of symbiotic bacteria isolated from the culture system of Ochromonas and Chlorella was detected and compared, with the bacterial community structure revealed. The results demonstrated that Algoriphagus, Porphyrobacter, Roseococcus, and Rhizobium were the dominant bacterial genera both in Ochromonas and Chlorella system, distributed in Proteobacteria and Bacteroidetes. Sixteen of 40 strains classified into 15 genera isolated from the two genera of microalgae could produce IAA, with Agrobacterium and Rhizobium as the main representatives with highest IAA production level (22.53–34.18 mg/L in 30 h). And through determination of changes of chlorophyll content in the co-culture system of microalgae and the supernatant of IAA high-yielding bacteria suspension from medium with or without tryptophan (synthetic precursor of IAA), Rhizobium (C-3) and Agrobacterium (O-16, C-2) were proved to promote the growth of Ochromonas and Chlorella through secreting IAA. The biomass yield of Ochromonas and Chlorella could be respectively increased by 78.98% and 72.73% through the co-culture with Agrobacterium (C-2), by 68.15% and 69.93% for Rhizobium (C-3) and by 80.89% and 65.73% for Agrobacterium (O-16). Moreover, compared with the application of exogenous IAA to improve the microalgae biomass, it is more efficient and economical to construct algae bacteria symbiosis system using IAA secretory bacteria commonly existed in microalgae culture system. In addition, the growth promoting effect of IAA secretory bacteria on microalgae was different among the species of algae.

Mixotrophy usually is considered with respect to the advantages gained and the associated trade-offs of this form of nutrition, compared to specialized competitors, strict photoautotrophs and heterotrophs. However, we currently have an incomplete understanding of the functional diversity of mixotrophs and the factors controlling niche differentiation in different mixotrophic species. Here we experimentally studied the light-dependent niche differentiation in two chrysophyte species. We show that the newly isolated Ochromonas sp. is an obligate phototroph and possibly an obligate mixotroph. In contrast, Poterioochromonas malhamensis is a facultative mixotroph; photosynthesis and heterotrophy in this species represent substitutable routes of resource acquisition. We further hypothesize that the variable plasticity in the considered traits of the here tested species may result in different niche differentiation with regard to a vertical light gradient. Ochromonas sp. should perform better in stable stratified surface water layers, where light is available, but prey abundances might be low. However, P. malhamensis should be able to also successfully grow in deeper water layers, benefiting from higher bacterial production. This study represents a first step towards understanding competition between mixotrophs engaging in different physiological strategies, and consequently their potential co-occurrence due to niche differentiation.

Driven by global warming and eutrophication, outbreaks of cyanobacterial blooms have severely impacted ecosystem stability and water safety. Of the organisms used to control cyanobacteria, protozoa can highly resist cyanotoxins, efficiently control cyanobacterial populations, and show considerably different feeding strategies from those of metazoans. Thus, protozoa have great potential to control harmful cyanobacteria and improve phytoplankton composition in eutrophic waters. To evaluate the actual effects of protozoa in controlling cyanobacteria and improving the phytoplankton community structure in the field, an in situ microcosm study was performed using a flagellate Ochromonas gloeopara that ingests Microcystis. Results showed that adding Ochromonas reduced the cyanobacterial populations and increased the chlorophyte and diatom proportions. Furthermore, the species richness and diversity of the phytoplankton community were enhanced in microcosms with Ochromonas. Additionally, there was a gradual increase in the chlorophyte population in the unicellular Microcystis control, while Ochromonas addition significantly accelerated the replacement of dominant species. This study was the first to show the practical effects of protozoa on controlling cyanobacteria in the field, highlighting that a reduction in in situ cyanobacteria via protozoa can improve the phytoplankton community structure, dredge the toxic cyanobacteria-dominated microbial food web, and mitigate harmful cyanobacteria risks in fresh waters.

Cyanobacterial blooms are increasingly threatening the aquatic ecosystem functioning as a result of the global warming and eutrophication. The "top-down" control of cyanobacteria from consumers like the protozoans shows great potential because of the effectiveness and environment-friendliness. To reveal how the nutrition availability and elevated temperature affect the cyanobacteria removal through protozoans grazing, we grew the toxic Microcystis aeruginosa and the mixotrophic Ochromonas gloeopara in monocultures and cocultures at environmentally relevant nitrogen levels (0.5-8.0 mg L

The chrysophycean genus Lepidochromonas Kristiansen was established to accommodate a single species, Ochromonas diademifera E. Takah. characterized by cells covered by both plate-like scales and crown-shaped scales. Subsequently Lepidochromonas has been synonymized with Paraphysomonas De Saedeleer. Currently, the genus Paraphysomonas is restricted to the species with nail-like spine scales and species with basket-like scales have been removed to a newly described genus Clathromonas Scoble & Caval.-Sm. referred to the monotypic family Clathromonadidae Scoble & Caval.-Sm. Although both Clathromonas and Clathromonadidae are valid under the International Code of Zoological Nomenclature they are illegitimate names according to the rules of the International Code of Nomenclature for algae, fungi and plants. Here we reinstate the genus Lepidochromonas as it has priority over Clathromonas, and we propose 30 new nomenclatural combinations. The formal description of a new family Lepidochromonadaceae fam. nov. is also provided.

Disgusting fishy odor problems have become a major concern in drinking water quality, and are commonly related to algal proliferation in source water. Unlike the typical musty/earthy odorants 2-methylisoborneol (MIB) and geosmin, identification of the corresponding fishy odorants is still a big challenge. In this study, two species of fishy-odor-producing algae, Ochromonas sp. and Cryptomonas ovate, were cultured to explore the odor production characteristics and typical odorants. When algae were ruptured in the stationary and decline phases, fishy odor intensities of 4 to 8 characterized by FPA were produced. However, some frequently reported aldehydes that could cause fishy odor, including n-hexanal, 2-octenal, heptanal, 2,4-heptanal and 2,4-decadienal, were not detected in either of the cultured algae. The possible fishy odor-causing compounds were further identified by combining gas chromatography-olfactometry (GC-O/MS) and comprehensive two-dimensional gas chromatography (GC × GC-TOFMS) using retention indices (RIs). From GC-O/MS analysis, twelve and six olfactometry peaks with various odor characteristics were identified in Ochromonas sp. and Cryptomonas ovate, respectively, of which three and two olfactometry peaks showed fishy odor characteristics. 2-Nonenal, 2,4-octadienal, fluorene and 2-tetradecanone were identified as fishy odorants in Ochromonas sp., and 1-octen-3-ol, 6-methyl-5-hepten-2-one, 1-octen-3-one, 2-nonenal and 2,4-octadienal were identified in Cryptomonas ovate. Other identified compounds, including butyl butanoate (fragrant odor), ionone (fragrant odor), bis (2-chloroisopropyl) ether (chemical odor) etc., did not show fishy features. Therefore, the fishy odor might be a synthetic and comprehensive odor, which resulted from the combination of different odorants and their synergistic effects. The results of this study will be helpful for understanding fishy odor problems, which will provide support for fishy odor management and control in the drinking water industry.

Fishy odor has become one of the most often encountered aesthetic water quality problems in drinking water. While fresh water algae living in colder water can produce offensive fishy odors, their environmental behaviors remain poorly understood. In this study, two chrysophyte species (Synura uvella and Ochromonas sp.), which are often associated with fishy odor events, were selected to investigate the effect of temperature (8, 16, and 24 °C) and light intensity (10, 41, and 185 μmol photons m−2 s−1) on algal growth and odorant production. Five polyunsaturated aldehyde derivatives, including 2,4-heptadienal, 2-octenal, 2,4-octadienal, 2,4-decadienal, and 2,4,7-dectridienal, were identified as fishy/cod liver oil/fatty/rancid descriptors in the cultures of the two algae based on gas chromatography-olfactometry-mass spectrometry and comprehensive two-dimensional gas chromatography mass spectrometry. While biomass yield increased with the increase in temperature for both species, significantly higher odorant yields (production of odorants per cell) were obtained at 8 °C. The total odorant production and cell yield of the odorants decreased with the increase in light intensity from 10 to 185 μmol photons m−2 s−1. The biodegradation half-lives for the released odorants were 6–10 h at 8 °C and 2–4 h at 24 °C, whereas the volatilization half-lives were 36–97 d at 8 °C and 6–17 d at 24 °C, suggesting that temperature-dependent biodegradation was an important factor controlling the fate of fishy compounds in aquatic environments. The results of this study will help clarify why most fishy odor events occur in cooler seasons, and provide knowledge related to cold water persistence for the management of fishy odor problems associated with algae.

The first step in the evolution of complex multicellular organisms involves single cells forming a cooperative group. Consequently, to understand multicellularity, we need to understand the costs and benefits associated with multicellular group formation. We found that in the facultatively multicellular algae Chlorella sorokiniana: (1) the presence of the flagellate Ochromonas danica or the crustacean Daphnia magna leads to the formation of multicellular groups; (2) the formation of multicellular groups reduces predation by O. danica, but not by the larger predator D. magna; (3) under conditions of relatively low light intensity, where competition for light is greater, multicellular groups grow slower than single cells; (4) in the absence of live predators, the proportion of cells in multicellular groups decreases at a rate that does not vary with light intensity. These results can explain why, in cases such as this algae species, multicellular group formation is facultative, in response to the presence of predators.

Removal of harmful cyanobacteria is an extremely urgent task in global lake management and protection. Conventional measures are insufficient for simultaneously removing cyanobacteria and hazardous cyanotoxin, efficient and environmental-friendly measures are therefore particularly needed. Herbivorous protozoa have great potentials in controlling algae, however, large-sized colonial Microcystis is inedible for protozoa, which is a central problem to be solved. Therefore, in present study, a measure of protozoa grazing assisted by ultrasound was investigated in laboratory scale for eliminating harmful colonial Microcystis. The results showed that with ultrasound power and time increasing, the proportion of unicellular Microcystis increased significantly. With Ochromonas addition, approximately 80% of colonial Microcystis and microcystin was removed on day 4 under ultrasound power of 100 W for 15 min, while Ochromonas only reduced Microcystis by less than 20% without assistance of ultrasound. Moreover, when directly exposed to low-intensity ultrasound, Ochromonas showed strong resistance to ultrasound and were not inhibited in grazing Microcystis. Overall, ultrasound increases edible food for protozoa via collapsing Microcystis colonies and assists Ochromonas to remove Microcystis, thus intermittently collapsing colonial Microcystis using low-intensity ultrasound can significantly improve the removal efficiency of Microcystis by protozoa grazing, which provided a new insight in controlling harmful colonial Microcystis.

Recent experiments and thermodynamic arguments suggest that mitochondrial temperatures are higher than those of the cytoplasm. A "hot mitochondrion" calls for a closer examination of the energy balance that endows it with these claimed elevated temperatures. As a first step in this effort, we present here a semi-quantitative bookkeeping whereby, in one stroke, a formula is proposed that yields the rate of heat production in a typical mitochondrion and a formula for estimating the number of active ATP synthase molecules per mitochondrion. Scaling laws are shown to determine the number of active ATP synthase molecules in a mitochondrion and mitochondrial rate of heat production. Mitochondrial population of active ATP synthases and mitochondrial rate of heat production appear, both, to scale with cell volume. Four heterotrophic protozoa cell types are considered in this study. The studied cells, selected to cover a wide range of sizes (volumes) from ca. 100 μm3 to 1 million μm3, are estimated to exhibit a power per mitochondrion ranging from ca. 1 pW to 0.03 pW. The corresponding number of active ATP synthases per mitochondrion in these cells ranges from 5,000 to just about a hundred.

International audience; In the Arctic Ocean, the small green alga Micromonas polaris dominates picophytoplankton during the summer months but is also present in winter. It has been previously hypothesized to be phago-mixotrophic (capable of bacteria ingestion) based on laboratory and field experiments. Prey uptake was analyzed in several M. polaris strains isolated from different regions and depths of the Arctic Ocean and in Ochromonas triangulata, a known phago-mixotroph used as a control. Measuring ingestion of either fluorescent beads or fluorescently labeled bacteria by flow cytometry, we found no evidence of phago-mixotrophy in any M. polaris strain while O. triangulata was ingesting both beads and bacteria. In addition, in silico predictions revealed that members of the genus Micromonas lack a genetic signature of phagocytotic capacity.

The chlorosulfolipids are amphiphilic natural products with stereochemically complex patterns of chlorination and sulfation. Despite their role in toxic shellfish poisoning, potential pharmacological activities, and unknown biological roles, they remain understudied due to the difficulties in purifying them from natural sources. The structure of these molecules, with a charged sulfate group in the middle of the hydrophobic chain, appears incompatible with the conventional lipid bilayer structure. Questions about chlorosulfolipids remain unanswered partly due to the unavailability of structural analogues with which to conduct structure–function studies. We approach this problem by combining enantioselective total synthesis and membrane biophysics. Using a combination of Langmuir pressure–area isotherms of lipid monolayers, fluorescence imaging of vesicles, mass spectrometry imaging, natural product isolation, small-angle X-ray scattering, and cryogenic electron microscopy, we show that danicalipin A (1) likely inserts into lipid bilayers in the headgroup region and alters their structure and phase behavior. Specifically, danicalipin A (1) thins the bilayer and fluidizes it, allowing even saturated lipid to form fluid bilayers. Lipid monolayers show similar fluidizing upon insertion of danicalipin A (1). Furthermore, we show that the halogenation of the molecule is critical for its membrane activity, likely due to sterically controlled conformational changes. Synthetic unchlorinated and monochlorinated analogues do not thin and fluidize lipid bilayers to the same extent as the natural product. Overall, this study sheds light on how amphiphilic small molecules interact with lipid bilayers and the importance of stereochemistry and halogenation for this interaction.

Microplastics with different sizes exist widely in fresh waters, which may affect the interspecific dynamics between predator and prey. The flagellate Ochromonas gloeopara can efficiently eliminate Microcystis aeruginosa and degrade microcystins, which shows great potential for controlling harmful Microcystis. In order to evaluate the effects of microplastics on O. gloeopara eliminating Microcystis, we designed an experiment of O. gloeopara feeding on Microcystis under different sizes (0.07 and 3 μm) and concentrations (0, 0.4, 0.8, 1.6, and 2.0 mg L−1) of microplastics. The results showed that maximum abundance of M. aeruginosa decreased significantly with addition of microplastics, regardless of the size and concentration. O. gloeopara can ingest the microplastics and suffer from their adverse effects. The maximum abundance of O. gloeopara decreased with enhancing concentrations of 3 μm microplastics during the process of O. gloeopara eliminating M. aeruginosa, whereas 0.07 μm microplastics did not affect the growth of O. gloeopara obviously. During the period of exposure under microplastics, clearance rate of O. gloeopara on M. aeruginosa decreased with the increasing concentrations of microplastics. Specially, 3 μm microplastics had a stronger reduction on clearance rate of O. gloeopara. The time to M. aeruginosa extinction was prolonged with the increasing concentrations of microplastics in both sizes. Comparatively speaking, 3 μm microplastics had a stronger delayed effect on the removal of Microcystis. These findings suggest that microplastics can interfere with protozoa eliminating toxic Microcystis, which may aggravate their adverse impacts on aquatic ecosystem.

Driven by global warming and eutrophication, outbreaks of cyanobacterial blooms have severely impacted ecosystem stability and water safety. Of the organisms used to control cyanobacteria, protozoa can highly resist cyanotoxins, efficiently control cyanobacterial populations, and show considerably different feeding strategies from those of metazoans. Thus, protozoa have great potential to control harmful cyanobacteria and improve phytoplankton composition in eutrophic waters. To evaluate the actual effects of protozoa in controlling cyanobacteria and improving the phytoplankton community structure in the field, an

Grease balls collected from a municipal wastewater treatment plant were melt-screened and used for cultivation of microalga Ochromonas danica, which could phagocytize droplets and particles as food. After autoclaving, the waste grease (WG) separated into two (upper and lower) phases. O. danica grew well on both, accumulating 48-79% (w/w) intracellular lipids. Initial WG contained approximately 50:50 triglycerides and free fatty acids (FFAs); over time, almost only FFAs remained in the extracellular WG presumably due to hydrolysis by algal lipase. PUFAs, mainly C18:2n6, C18:3n3, C18:3n6, C20:4n6, and C22:5n6, were synthesized and enriched to up to 67% of intracellular FAs, from the original 15% PUFA content in WG. The study showed feasibility of converting wastewater-originated WG to PUFA-rich O. danica algae culture, possibly as aquaculture/animal feed. WG dispersion was identified as a major processing factor to further improve for optimal WG conversion rate and cell and FA yields.

Resource competition theory predicts that when two species compete for a single, finite resource, the better competitor should exclude the other. However, in some cases, weaker competitors can persist through intraguild predation, that is, by eating their stronger competitor. Mixotrophs, species that meet their carbon demand by combining photosynthesis and phagotrophic heterotrophy, may function as intraguild predators when they consume the phototrophs with which they compete for light. Thus, theory predicts that mixotrophy may allow for coexistence of two species on a single limiting resource. We tested this prediction by developing a new mathematical model for a unicellular mixotroph and phytoplankter that compete for light, and comparing the model's predictions with a laboratory experimental system. We find that, like other intraguild predators, mixotrophs can persist when an ecosystem is sufficiently productive (i.e., the supply of the limiting resource, light, is relatively high), or when species interactions are strong (i.e., attack rates and conversion efficiencies are high). Both our mathematical and laboratory models show that, depending upon the environment and species traits, a variety of equilibrium outcomes, ranging from competitive exclusion to coexistence, are possible.

Ochromonas triangulata, the type species for a genus with over 125 taxa, was collected for only the second time, again from the type locality. Cell morphology, cell division, palmelloid stage and cyst structure generally agreed with the original description. Molecular phylogenetic analysis based on the 18S rRNA gene revealed 13 clades of Ochromonas-like flagellates as well as the clade represented by our O. triangulata strain and the nearly identical strain RCC-21/AC025. We also conducted a concatenated analysis using the 18S rRNA and the rbcL genes, and we recovered the same 14 clades. One clade, containing strains CCAP 933/27 and CCMP1861, previously named Ochromonas tuberculata, was re-identified as Chrysastrella paradoxa and Chrysastrella breviappendiculata, respectively. One clade included the Poterioochromonas strains but we were unable to convincingly connect species names to the strains because authentic strains were unknown or not examined. Organisms in the clade that included the well-...

Mixotrophs account for a high proportion (occasionally up to 80%) of the phytoplankton biomass. Chrysophyte is one major component of mixotrophs. Because of their possible toxicity and linkage between microbial community and higher trophic levels, the effect of mixotrophic golden algae on potential grazers received much attention. The present study investigated the effect of Ochromonas gloeopara at different proportions in diet (combined with Scenedesmus obliquus) on the life history of Daphnia similoides sinensis. Results showed that osmotrophically grown O. gloeopara in light produced fish toxins and hemolysins, and negatively influenced the survival and reproduction of D. similoides sinensis. The mortality of the cladoceran increased as the proportion of O. gloeopara in food increased. The D. similoides sinensis could not reproduce throughout the life when Ochromonas comprised above 35%. When fed foods containing 15% of Ochromonas, the time to first brood of D. similoides sinensis was prolonged, together with the reduced number of offspring in first brood and total number of broods. Replacement by 100% S. obliquus delayed the time to death, but did not improve the reproduction of Daphnia. The present study indicated the strong inhibitory effect of O. gloeopara on D. similoides sinensis, and underlined the importance of evaluating its ecological role in aquatic ecosystems.

Cadmium (Cd) is one of the predominant anthropogenic pollutants in aquatic systems. As Cd has negative effects on species at all trophic levels, the community composition in aquatic habitats can be changed as a result of Cd stress. The response of mixotrophic protists to environmental stressors is particularly important as they act as both producers and consumers in complex planktonic communities. In this study, we used mixotrophic Ochromonas gloeopara to study its growth and photosynthetic responses to Cd, and specially focused on the effects of initial Cd concentrations and nutrient levels on its capacity to remove Cd. Results showed that when Cd concentration reached 0.5 mg L−1, the growth rate and carrying capacity were significantly inhibited, whereas the photosynthesis was markedly decreased when Cd concentration reached 0.15 mg L−1. Moreover, under Cd concentration 0.15, 0.5, 0.9, 1.6, and 2.0 mg L−1, the removal efficiencies of Cd by O. gloeopara were 83.2%, 77.7%, 74.6%, 70.1%, and 68.8%, respectively. The increase of nitrogen did not cause significant effect on the removal capacity of Cd by O. gloeopara, but increased concentration of phosphorus significantly enhanced the removal capacity of Cd. Our findings indicated that the mixotrophic O. gloeopara has strong tolerance and capacity to remove Cd, and increasing concentration of phosphorus can increase its removal capacity, suggesting that O. gloeopara has great potential application value in mitigating Cd pollution in waters.

Ochromonas sp. (Chrysophyceae) occupies different trophic levels depending on its prevalent nutritional strategy in the field. Ochromonas sp. can utilize inorganic and organic carbon to grow photoautotrophically, mixotrophically, and heterotrophically. To examine how exogenous organic carbon influences population growth and photosynthesis in Ochromonas sp., we cultured Ochromonas sp. in media with different glucose concentrations (0, 50, 100, and 150 mg L−1) and two treatment modes (single addition of glucose at the beginning and fed-batch mode with repeated addition every 12 h) for 15 days. Results showed that (1) compared to photoautotrophic growth, adding glucose significantly enhanced the growth rate and carrying capacity of Ochromonas sp. in a concentration-dependent manner; (2) photosynthesis also increased with glucose addition during the exponential growth stage; (3) repeated glucose addition caused higher biomass and photosynthetic performances than single addition, and (4) with depletion of glucose and the population reaching a stationary stage, photosynthetic parameters decreased slightly but remained at a relatively stable level, which was similar to that of photoautotrophic growth. We conclude that in comparison with pure photoautotrophy, mixotrophic growth can result in higher population growth rate and final biomass in a shorter time, especially in fed-batch cultures, indicating that this would be a highly efficient way for mass cultivation of the flagellate Ochromonas.

Substantial energy is reserved in waste activated sludge (WAS) organics but much of it is difficult to recover because the solid organics require long time to solubilize. In this work we introduced the new approach of recovering WAS organics into the biomass of phagotrophic algae. Phagotrophic algae have the unique ability to grow by ingesting insoluble organic particles including microbial cells. This phagotrophic ability renders the solubilization of WAS organics unnecessary and makes this approach remarkably fast. The approach consists of two stages: a short anaerobic digestion treatment followed by the algal growth on treated WAS. The short anaerobic digestion was exploited to release discrete bacteria from WAS flocs. Phagotrophic algae could then grow rapidly with the released bacteria as well as the solubilized nutrients in the treated WAS. The results showed that WAS organics could be quickly consumed by phagotrophic algae. Among all studied conditions the highest WAS volatile solids (VS) reduction was achieved with 72 h anaerobic digestion and 24 h algal growth. In this optimal process, 28% of WAS VS was reduced, and 41% and 20% of the reduced VS were converted into algal biomass and lipids, respectively. In comparison, only 18% WAS VS were reduced after the same time of aerobic digestion without algae addition. Through this approach, the amount of WAS organics requiring further treatment for final disposal is significantly reduced. With the production of significant amounts of algal biomass and lipids, WAS treatment is expected to be more economical and sustainable in material recycling.

Many halogenases interchangeably incorporate chlorine and bromine into organic molecules. On the basis of an unsubstantiated report that the alga Ochromonas danica, a prodigious producer of chlorosulfolipids, was able to produce bromosulfolipids, we have investigated the promiscuity of its halogenases toward bromine incorporation. We have found that bromosulfolipids are produced with the exact positional and stereochemical selectivity as in the chlorosulfolipid danicalipin A when this alga is grown under modified conditions containing excess bromide ion.

The Deepwater Horizon oil spill released millions of barrels of crude oil into the Gulf of Mexico, and saw widespread use of the chemical dispersant Corexit. We assessed the role of traits, such as cell size, cell wall, motility, and mixotrophy on the growth and photosynthetic response of 15 phytoplankton taxa to oil and Corexit. We collected growth and photosynthetic data on five algal cultures. These responses could be separated into resistant (Tetraselmis astigmatica, Ochromonas sp., Heterocapsa pygmaea) and sensitive (Micromonas pusilla, Prorocentrum minimum). We combined this data with 10 species previously studied and found that cell size is most important in determining the biomass response to oil, whereas motility/mixotrophy is more important in the dispersed oil. Our analysis accounted for a third of the variance observed, so further work is needed to identify other factors that contribute to oil resistance.

Toxic Microcystis bloom is a tough environment problem worldwide. Microcystin is highly toxic and is an easily accumulated secondary metabolite of toxic Microcystis that threatens water safety. Biodegradation of microcystin by protozoan grazing is a promising and efficient biological method, but the mechanism in this process is still unclear. The present study aimed to identify potential pathways involved in resisting and degrading microcystin in flagellates through transcriptomic analyses. A total of 999 unigenes were significantly differentially expressed between treatments with flagellates Ochromonas fed on microcystin-producing Microcystis and microcystin-free Microcystis. These dysregulated genes were strongly associated with translation, carbohydrate metabolism, phagosome, and energy metabolism. Upregulated genes encoding peroxiredoxin, serine/threonine-protein phosphatase, glutathione S-transferase (GST), HSP70, and O-GlcNAc transferase were involved in resisting microcystin. In addition, genes encoding cathepsin and GST and genes related to inducing reactive oxygen species (ROS) were all upregulated, which highly probably linked with degrading microcystin in flagellates. The results of this study provided a better understanding of transcriptomic responses of flagellates to toxic Microcystis as well as highlighted a potential mechanism of biodegrading microcystin by flagellate Ochromonas, which served as a strong theoretical support for control of toxic microalgae by protozoans.

In this study waste activated sludge (WAS) was sonicated to release bacteria-sized volatile solids (VS) from flocs, after initial pH adjustment to 10 for higher energy efficiency. The released VS supported growth of phagotrophic alga Ochromonas danica. Initial-rate growth experiments confirmed the Monod-type kinetics but the specific cell growth rate, μ, correlated with the prey-to-predator ratio, i.e., the ratio of (fed VS concentration)-to-(initial O. danica concentration), significantly better than with the VS alone, as the typical Monod dependency on soluble substrates. The best-fit kinetics had the following parameters: μmax = 0.198 h−1 and KM = 1.056 (g-VS/g-algae). Post-sonication reflocculation could render particles too large to ingest by O. danica; therefore, pH and VS effects on reflocculation were investigated. Batch cultivations were then conducted in fermentors at pH 5, under nonsterile conditions. Algae number reached 8.86 × 1010 L−1 after 20 h, corresponding to ∼2.3 g/L dry-weight and volumetric algae productivity of 2.8 g/L-day. VS reduction was 38%, giving an O. danica VS yield of 44.5%. The ultrasonication-algae process can be used to produce algae while achieving at least partial WAS treatment.

A substantial amount of organic matter is wasted in current wastewater treatment processes. To reclaim the value of organic matter, a two-stage continuous-flow open process has been developed by utilizing the capability of phagotrophic algae in ingesting bacterial cells. In this process, wastewater is first pumped into a bacteria tank to grow bacterial cells, and then the effluent containing grown bacteria cells is fed to an algae tank to grow phagotrophic algae. The operation conditions such as dilution rate, pH, and dissolved oxygen level were comprehensively investigated and optimized with long-term tests. Results show that phagotrophic algae can be stably cultivated with wastewater organics through this open process without costly chemical/physical sterilization. The produced phagotrophic algae had high lipid content and can be potentially used as biofuel feedstock.

Background Ochromonas is a genus of mixotrophic chrysophytes that is found ubiquitously in many aquatic environments. Species in this genus can be important consumers of bacteria but vary in their ability to perform photosynthesis. We studied the effect of light and bacteria on growth and gene expression of a predominantly phagotrophic Ochromonas species. Axenic cultures of Ochromonas sp. were fed with heat-killed bacteria (HKB) and grown in constant light or darkness. RNA was extracted from cultures in the light or in the dark with HKB present (Light + HKB; Dark + HKB), and in the light after HKB were depleted (Light + depleted HKB). Results There were no significant differences in the growth or bacterial ingestion rates between algae grown in light or dark conditions. The availability of light led to a differential expression of only 8% of genes in the transcriptome. A number of genes associated with photosynthesis, phagotrophy, and tetrapyrrole synthesis was upregulated in the Light + HKB treatment compared to Dark + HKB. Conversely, the comparison between the Light + HKB and Light + depleted HKB treatments revealed that the presence of HKB led to differential expression of 59% of genes, including the majority of genes involved in major carbon and nitrogen metabolic pathways. Genes coding for unidirectional enzymes for the utilization of glucose were upregulated in the presence of HKB, implying increased glycolytic activities during phagotrophy. Algae without HKB upregulated their expression of genes coding for ammonium transporters, implying uptake of inorganic nitrogen from the culture medium when prey were unavailable. Conclusions Transcriptomic results agreed with previous observations that light had minimal effect on the population growth of Ochromonas sp. However, light led to the upregulation of a number of phototrophy- and phagotrophy-related genes, while the availability of bacterial prey led to prominent changes in major carbon and nitrogen metabolic pathways. Our study demonstrated the potential of transcriptomic approaches to improve our understanding of the trophic physiologies of complex mixotrophs, and revealed responses in Ochromonas sp. not apparent from traditional culture studies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3549-1) contains supplementary material, which is available to authorized users.

Recently, microalgae have gained a lot of attention because of their ability to produce fatty acids in their surrounding environments. The present paper describes the influence of organic carbon on the different fatty acid pools including esterified fatty acids, intracellular free fatty acids and extracellular free fatty acids in Ochromonas danica. It also throws light on the ability of O. danica to secrete free fatty acids in the growth medium under photoautotrophic and mixotrophic conditions. Biomass production of photoautotrophically grown O. danica was higher than that of mixotrophically grown, where a cellular biomass formation of 1.8 g L(-1) was observed under photoautotrophic condition which was about five folds higher than that under mixotrophic conditions. Contrary, the esterified fatty acid content reached up to 99 mg g(-1) CDW under photoautotrophic conditions at the late exponential phase, while during mixotrophic conditions a maximum of 212 mg g(-1) CDW was observed at the stationary phase. Furthermore, O. danica cells grown under mixotrophic conditions showed higher intracellular free fatty acid and extracellular free fatty acid contents (up to 51 and 20 mg g(-1) CDW, respectively) than cells grown under photoautotrophic conditions (up to 26 and 4 mg g(-1) CDW, respectively). The intra- and extracellular free fatty acids consisted of a high proportion of polyunsaturated fatty acids, mainly C18:2n-6, C18:3n-3 and C20:4n-6.

There is an extensive literature on chrysophyte stomatocysts from diverse habitats and their use as paleolimnetic indicators of past environmental conditions in lakes. The majority of these cysts are unidentified, and more laboratory studies are needed to link stomatocyst morphotypes to vegetative stages. A laboratory procedure that resulted in stomatocyst production in the chrysophyte alga Ochromonas pinguis (Chrysophyceae) was tested to determine if the protocol would also stimulate the production of stomatocysts in six other chrysophytes. Chrysolepidomonas dendrolepidota, Chrysosaccus cf. sphaericus, Dermatochrysis sp., Synura cf. americana, Synura cf. macropora and a second species of Ochromonas all produced stomatocysts within two to three weeks. Morphology of the stomatocyst was described using scanning electron microscopy (SEM). This was the first report of stomatocysts from Chrysosaccus and the first SEM description of O. pinguis cysts. These autecological studies provide a greater linkage between sediment stomatocysts and their biological origin, strengthening the interpretive value of these microfossils and broadening our understanding of chrysophyte biogeography.

For many years Chrysastrella Chodat has been a cyst genus but recently it has been re-instated and re-defined. Currently, Chrysastrella contains Ochromonas-like flagellates including O. tuberculata Hibberd which was synonymized with the type species, Chrysastrella paradoxa Chodat. A mass development of stomatocysts, which we identified as Ch. paradoxa, was observed in a bog on the territory of “Yugyd va” National Park (the Subpolar Urals, Russia). The morphology of stomatocysts was studied by scanning electron microscopy and compared with previously described similar morphotypes. Stomatocysts #80 Hansen, #1 Wołowski & Płachno and #4 Bai & Chen seem to be identical to Chrysastrella paradoxa stomatocysts and we propose to reduce them to synonymy.

1,3-β-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-β-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-β-glucans.

The bioaccumulation kinetics of thioglycolic acid stabilized CdTe quantum dots (TGA-CdTe-QDs) in a freshwater alga Ochromonas danica was comprehensively investigated. Their photoluminescence (PL) was determined by flow cytometry. Its cellular intensity increased hyperbolically with exposure time suggesting real internalization of TGA-CdTe-QDs. This hypothesis was evidenced by the nanoparticle uptake experiment with heat-killed or cold-treated cells and by their localization in the vacuoles. TGA-CdTe-QD accumulation could further be well simulated by a biokinetic model used previously for conventional pollutants. Moreover, macropinocytosis was the main route for their internalization. As limited by their diffusion from the bulk medium to the cell surface, TGA-CdTe-QD uptake rate increased proportionally with their ambient concentration. Quick elimination in the PL of cellular TGA-CdTe-QDs was also observed. Such diminishment resulted mainly from their surface modification by vacuolar biomolecules, considering that these nanoparticles remained mostly undissolved and their expulsion out of the cells was slow. Despite the significant uptake of TGA-CdTe-QDs, they had no direct acute effects on O. danica. Overall, the above research shed new light on nanoparticle bioaccumulation study and would further improve our understanding about their environmental behavior, effects and fate.

This study aimed to investigate the influence of temperature on the ability of the mixotrophic flagellate Ochromonas to eliminate a toxic Microcystis population and degrade microcystins. We exposed Microcystis to cultures with or without Ochromonas YZ1 at 20, 25, and 30 °C for 10 days. Results showed that increased temperature promoted the growth of Ochromonas YZ1 and Microcystis, with the latter achieving high abundance without grazing. With increased temperature, Ochromonas YZ1 clearance rate increased, and Microcystis populations were earlier eliminated. Importantly, Ochromonas YZ1 degraded both intracellular and extracellular microcystins by grazing effects. The reduction ratios of Microcystis abundances and microcystins were both approximately 100% after 6 days at high temperature. In addition, more microcystins were released outside at 20 °C than at the higher temperatures. Overall, this study showed that high temperature favors elimination of toxin-producing Microcystis and degradation of microcystins by mixotrophic Ochromonas.

Many halogenases interchangeably incorporate chlorine and bromine into organic molecules. On the basis of an unsubstantiated report that the alga Ochromonas danica, a prodigious producer of chlorosulfolipids, was able to produce bromosulfolipids, we have investigated the promiscuity of its halogenases toward bromine incorporation. We have found that bromosulfolipids are produced with the exact positional and stereochemical selectivity as in the chlorosulfolipid danicalipin A when this alga is grown under modified conditions containing excess bromide ion.

Microbial predator-prey interactions are one of the primary trophic interactions linking biogeochemical cycles to ecosystem dynamics. The mixotrophic flagellate Ochromonas danica was used as a model predator to investigate feeding trends when supplied with actively growing and non-growing bacteria representing a variety of phylogenetic groups. The rate at which bacteria were ingested and the subsequent growth rate (ecological fitness) of O. danica were determined for each type of prey in each growth state. O. danica preferred bacteria between 0.6 and 1.0 μm 3 . It was, in general, an indiscriminate predator in that it readily ingested growing and non-growing bacteria spanning a range of phylogenetic groups, with some notable exceptions. Bacilli/Actinobacteria were ingested at significantly lower rates than Alpha- and Gamma-proteobacteria. There were no significant differences among rates at which Proteobacteria (Alpha-, Beta-, and Gamma-) were ingested. The ecological fitness of O. danica could not be predicted from ingestion rates. Predation on Bacilli/Actinobacteria resulted in growth rates significantly lower than when the flagellate preyed upon Gammaproteobacteria. Betaproteobacteria were readily ingested but always resulted in a significant decline in predator fitness. The data reveal that different types of bacteria have different nutritional value to a consumer. If other flagellates respond similarly, then it would seem that trophic transfer efficiency and nutrient regeneration depend on the diet-breadth of predators.

Summary 1. Toxic compounds produced by many phytoplankton taxa are known to have negative effects on competitors (allelopathy), anti-predatory effects on grazers (mortality or impaired reproduction) or both. Although mixotrophs of the genus Ochromonas are known to be toxic to zooplankton, it has often been assumed in studies of plankton community processes that all flagellates in the size range of this taxon are edible to typical zooplankton grazers (i.e. cells ≤30 μm for Daphnia, ≤6 μm for rotifers). 2. We explored the toxicity of a species of Ochromonas to other planktonic taxa, including its competitors (two species of phytoplankton and protists) and consumers (two species of zooplankton). To test if mode of nutrition by this mixotroph influences its toxicity to other taxa, we exposed each test species to Ochromonas cultured in chemostats under four different nutritional regimes: osmotrophy (labile dissolved organic carbon) and phagotrophy (bacterial prey) in both light and dark conditions (i.e. with or without photosynthesis). 3. Filtrate from osmotrophically fed Ochromonas had a significant negative effect on the population growth rate of two obligate phototrophic phytoplankton, Cryptomonasozolini and Chlamydomonas reinhardtii. The protists Tetrahymena tetrahymena and Paramecium aurelia were also negatively affected by Ochromonas filtrate. Ochromonas cells were toxic to both the rotifer Brachionus calicyflorus and the cladoceran Daphnia pulicaria, with the toxic effects significantly more severe when fed at high cell densities (75 000 cells mL−1) than at low densities (7500 cells mL−1). Ochromonas cultured osmotrophically in the light was more toxic to the Daphnia than cells cultured under other conditions. In contrast, Ochromonas from all nutritional conditions was equally highly toxic to Brachionus. 4. Our findings support the view that Ochromonas can be toxic to other components of the food web with which it interacts. It is especially toxic to zooplankton that directly consume it, although the effect depends upon Ochromonas cell density and whether or not a good food source is simultaneously present. Our results call into question the common practice of pooling flagellates into a single ‘functional group’ included in an ‘edible phytoplankton’ category of cells

In this study, the concept of functional groups was applied for the first time to the seasonal dynamics of the phytoplankton community of Mudanjiang River (northeast China). The environmental variables as well as the phytoplankton communities were determined in 2007. The river showed strong seasonal fluctuations in water physical and chemical condition. A total of 11 phytoplankton functional groups (C, D, E, J, MP, P, T, TB, W1, X2 and Y) were identified that included 30 species. Groups C (Asterionella formosa and Cyclotella meneghiniana), MP (Cocconeis placentula, Cymatopleara solea, Diatoma vulgare, Ochromonas fragilis, Pinnularia major and Ulothrix variabilis), P (Fragilaria capucina, Fragilaris sp., Aulacoseira granulate and Aulacoseira granulate var. angustissima), TB (Aulacoseira varians and Navicula exigua, Navicula radiosa), X2 (Chlamydomonas sp.) and Y (Chroomonas acuta, Cryptomonas ovata, and Cryptomonas sp.) were the dominant groups during the study period. Mudanjiang River phytoplankton functi...

F-actin organization during the cell cycle was investigated in two stramenopile microalgae, Ochromonas danica (Chrysophyceae; UTEX LB1298) and Heterosigma akashiwo (Raphidophyceae; NIES-6) using FITC-phalloidin. In the interphase cell of O. danica, F-actin bundles were localized forming a network structure in the cortical region, which converged from the anterior region to the posterior, whereas in the interphase cell of H. akashiwo, F-actin bundles were observed forming a network structure in the cortical region without any polarity. In both O. danica and H. akashiwo, at the initial stage of mitosis the cortical F-actin disappeared, and then during cytokinesis assembly of an actin-based ring-like structure occurred in the cell cortex in the plane of cytokinesis. The ring-like structure initiated from aster-like structures was composed of F-actin in both O. danica and H. akashiwo. Different from animal cells, later stages of cytokinesis of O. danica seemed to be promoted by microtubules, although the early stages of cytokinesis progressed with a constriction of the ring-like structure, whereas cytokinesis of H. akashiwo was apparently completed by constriction of the cell mediated by the F-actin ring, as in animal cells.

Community-level selection is an important concept in evolutionary biology and has been predicted to arise in systems that are spatially structured. Here we develop an experimental model for spatially-structured bacterial communities based on coaggregating strains and test their relative fitness under a defined selection pressure. As selection we apply protozoan grazing in a defined, continuous culturing system. We demonstrate that a slow-growing bacterial strain Blastomonas natatoria 2.1, which forms coaggregates with Micrococcus luteus, can outcompete a fast-growing, closely related strain Blastomonas natatoria 2.8 under conditions of protozoan grazing. The competitive benefit provided by spatial structuring has implications for the evolution of natural bacterial communities in the environment.

We investigated the effect of pH on the competition of two closely related chrysomonad species, Poterioochromonas malhamensis originating from circumneutral Lake Constance, and Ochromonas sp. isolated from a highly acidic mining lake in Austria (pH ∼2.6). We performed pairwise growth experiments between these two species at four different pH ranging from 2.5 to 7.0. Heterotrophic bacteria served as food for both flagellates. Results were compared to growth rates measured earlier in single species experiments over the same pH range. We tested the hypothesis that the acidotolerant species benefits from competitive release under conditions of acid stress. The neutrophilic strain numerically dominated over the acidotolerant strain at pH 7.0, but was the inferior competitor at pH 2.5. At pH 3.5 and 5.0 both strains coexisted. Surprisingly, P. malhamensis prevailed over Ochromonas sp. under moderately acidic conditions, i.e. at the pH where growth rates of the latter peaked when grown alone. Since bacterial food was not limiting, resource competition is improbable. It appears more likely that P. malhamensis ingested cells of its slightly smaller competitor. Adverse effects mediated via allelopathy, either directly on the competing flagellate or indirectly by affecting its bacterial food, might also have affected the outcome of competition.

Strains of the green alga Chlamydomonas acidophila and two chrysomonads, Ochromonas spp., isolated from each of two similar acid mining lakes (AMLs) with extremely low pH (∼2.6) were investigated to consider a possible synergistic stress effect of low pH and unfavourable temperature. We measured flagellate growth rates over a combination of four pH (2.5, 3.5, 5.0 and 7.0) and three temperatures (10, 17.5 and 25°C) in the laboratory. Our hypothesis was that, under highly acidic conditions (pH 27°C. The lowest temperature tested reduced growth rates of all three chrysomonad strains significantly. Since all chrysomonads were tolerant to high temperature, growth rate of one Ochromonas spp. strain was measured exemplarily at 35°C. Only at this high temperature was the realized pH niche significantly narrowed. We also recorded significant intraspecific differences within the C. acidophila strains from the two AML, illustrating that the niche width of a species is broader than that of individual clones.

Ecological stoichiometry focuses on the balance between multiple nutrient elements in resources and in consumers of those resources. The major consumers of bacteria in aquatic food webs are heterotrophic and mixotrophic nanoflagellates. Despite the importance of this consumer-resource interaction to understanding nutrient dynamics in the aquatic food web, few data are available addressing the element stoichiometry of flagellate consumers. Ochromonas danica, a mixotrophic bacterivore, was used as a model organism to study the relationships among temperature, growth rate and element stoichiometry. Ochromonas danica was grown in chemostats at dilution rates ranging between 0.03 and 0.10 h(-1) and temperatures ranging between 15 and 28 °C. Cells accumulated elements as interactive functions of temperature and growth rate, with the highest element concentrations corresponding to cells grown at a low temperature and high growth rates. The highest concentrations of elements were associated with small cells. Temperature and growth rate affected the element stoichiometry (as C:N, C:P and N:P) of O. danica in a complex manner, but the growth rate had a greater effect on ratios than did temperature.

SUMMARY A cDNA library was constructed from the chrysophycean alga, Ochromonas danica E. G. Pringsheim. 5′-end sequencing of about 600 cDNA clones yielded 476 authentic expressed sequence tags (EST) of which 275 showed significant matches (E-value

Acidic mining lakes (pH

Two-dimensional (2-D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine-rich, epithelial growth factor (EGF)-like motifs, potentially involved in protein-protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up-regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.

The new type blue light (BL) receptor aureochrome (AUREO) was recently discovered in a stramenopile alga, Vaucheria (Takahashi et al. Proc Natl Acad Sci USA 104(49):19625-19630, 2007). AUREO has a bZIP (basic region/leucine zipper) and BL-sensing light-oxygen-voltage (LOV) domain and functions as a BL-activated transcription factor. It mediates BL-induced branching and regulates the development of the sex organ in V. frigida. Although AUREO sequences have previously been found in Fucus and some diatoms, here we report that AUREO orthologs are commonly conserved in photosynthetic stramenopiles. Five AUREO orthologs were isolated from three stramenopile genera (Fucus, Ochromonas, and Chattonella). By BLAST search, several AUREO sequences were also detected in genomes in Aureococcus anophagefferens (Pelagophyceae). However, AUREO was not found in heterotrophic stramenopiles or in closely related phyla, such as haptophytes and cryptophytes, or in green plants. Stramenopiles do not possess phototropin, the well-known BL receptor for phototropism of green plants. From comparative analysis of LOV domains, together with kinship analysis of AUREO bZIP domains, AUREO can be regarded as the BL receptor specific to phototrophic stramenopiles. The evolution of AUREO and the phylogeny of LOV domains in stramenopiles and green plants are discussed.

The possible effect of filtered cultures of flagellate Ochromonas sp. on colony formation in M. aeruginosa was investigated in this paper. The results show that filtered cultures of flagellates fed with M. aeruginosa could induce colony formation in M. aeruginosa. Furthermore, induction strength is clearly dependent on the concentration of flagellates and filtered cultures. However, no colonial M. aeruginosa was found in the treatments of filtered cultures of flagellates fed with Microcystis wesenbergii, filtered cultures of flagellate fed with Chlorella pyrenoidosa, and algae homogenates. This suggests that infochemicals released from flagellates fed with M. aeruginosa may be a trigger for colony formation in M. aeruginosa. The clearance rates of flagellates on algae were markedly decreased when they were cultivated with induced colonial M. aeruginosa. These indicate that colony formation in M. aeruginosa is a predator-induced defense which could reduce predation risk from flagellates (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)