A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi and the application in phytoremediation of secondary effluent

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi was developed. The suitable conditions for pellet formation by filamentous fungi were determined. Among the strains tested, Trichoderma reesei QM 9414 showed superior pellet forming ability. Its pellets were used to harvest oleaginous microalga Scenedesmus sp. With increasing volume ratio of fungal pellets to microalgae culture up to 1:2, >94% of microalgal cells were rapidly harvested within 10 min. The ratio of fungal pellets could manipulate both harvesting time and initial concentration of microalgal cells in the pellets. The microalgae–fungal pellets were successfully used as immobilized cells for effective phytoremediation of secondary effluent from seafood processing plants under nonsterile condition. The chemical oxygen demand, total nitrogen, and total phosphorus removal were >74%, >44%, and >93%, respectively. The scanning electron microscopy showed that the microalgal cells were not only entrapped in the pellets but also got attached to the fungal hyphae with sticky exopolysaccharides, possibly secreted by the fungi. The extracted lipids from the pellets were mainly composed of C16–C18 (>83%) with their suitability as biodiesel feedstocks. This study has shown the promising strategy to rapidly harvest and immobilize microalgal cells and the possible application in phytoremediation of industrial effluent.     

Colorimetric protein determination in microalgae (Chlorophyta): association of milling and SDS treatment for total protein extraction

The use of colorimetric methods for protein quantification in microalgae is hindered by their elevated amounts of membrane‐embedded intracellular proteins. In this work, the protein content of three species of microalgae was determined by the Lowry method after the cells were dried, ball‐milled, and treated with the detergent sodium dodecyl sulfate (SDS). Results demonstrated that the association of milling and SDS treatment resulted in a 3‐ to 7‐fold increase in protein quantification. Milling promoted microalgal disaggregation and cell wall disruption enabling access of the SDS detergent to the microalgal intracellular membrane proteins and their efficient solubilization and quantification.     

微藻高效基因枪转化方法的建立

基因枪法是外源基因导入微藻细胞的重要手段。然而,发展至今,微藻细胞基因枪转化效率一直偏低（10~50个转化子/μg DNA）,高价低效的转化方法阻碍了基于高通量转化子的基因功能分析。为了提高基因枪的转化效率,本研究以三角褐指藻为材料,从抗生素选择培养基的改良,微载体的选择、制备、包埋、点膜和轰击参数的优化,以及受体细胞的处理等方面进行了系统研究。结果显示,采用50%海水盐度f/2培养基可以提高博来霉素的效价,f/2固体培养基中2216E营养物质的加入能缩短1/3的平板筛选时间。微载体制备应选择对金（钨）粉没有吸附作用的离心管,制备量/管应少于3.5 mg。微载体轰击量每次大约为0.75 mg,过量将会造成一个轰击死亡圈,过少将导致轰击成本上升。当轰击间距A为6.35 mm,间距B为11 mm,间距C为6 cm时,可以获得最多的转化细胞。109个受体细胞铺成较厚的多细胞层能显著提高转化效率。经过上述优化与改进,本研究将现有文献报道的转化效率提高了4.7~30倍,达到295 ± 60个转化子/μg DNA。该方法除适用于三角褐指藻外,也可广泛应用于其他微藻（杜氏盐藻、小球藻）的基因枪转化研究,可以为微藻基因工程研究提供快速,高效和可靠的操作技术。     

Optimal control for micro‐algae on a raceway model

We apply numerical optimal control methods to an existing algae growth model with the aim to determine the best performance of the model under known conditions using a variety of decision variables. We transform the system of differential algebraic equations in the existing model to a system of ordinary differential equations which introduces dynamics for average light intensity and chlorophyll. In addition, we allow for variable nitrogen concentration of the inflow as well as variable initial nitrogen concentration of the raceway. Our main focus is on optimizing of the production of lipids. We calculate both open and closed loop optimal controllers and test their robustness. Finally, we also consider raceway depth as a decision variable. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:107–119, 2018     

The viability of Scenedesmus bicellularis cells immobilized on alginate screens following nutrient starvation in air at 100% relative humidity

The viability of algal cells immobilized on screens and starved in a water-saturated air stream was studied at the laboratory scale. This new process for wastewater biotreatment has been developed using immobilized cells, which were starved in air, to obtain a high rate of nutrient removal. A unicellular green microalgae, Scenedesmus bicellularis, was isolated from secondary decantation tanks at an urban wastewater treatment plant and grown in a synthetic medium for 12 days. The cells were then concentrated by centrifugation and immobilized on alginate screens. The screens were then inserted in a photochamber saturated at 100% relative humidity and subjected to a photoperiod of 16 h in the light and 8 h in the dark, with an illumination of 150 muE m(-2) s(-1) provided by fluorescent lamps. After 48 h of nutrient starvation, the immobilized cells were used for the removal of ammonium and orthophosphate from a synthetic secondary wastewater effluent in a plexiglass reactor. During the sequential operation of starvation followed by incubation in the presence of nutrients, fast growth of viable cells in the gel matrix was obtained and there was no appreciable decay of chlorophyll a or cell activity. When these immobilized and starved cells were incubated in wastewater, ammonium (NH(4) (+)) and orthophosphate (PO(4) (3-)) ions were quickly taken up from medium. After three successive 2-h exposures to wastewater, immobilized algal cells were freed by dissolving the Ca-alginate with phosphate as 0.2 M Na(3)PO(4) and resuspended in fresh culture medium. Results indicate that free cells transferred to rich medium remained viable, but the growth rate revealed that the viable cells decreased their culturability. (c) 1995 John Wiley & Sons, Inc.     

The microbial loop in a humic lake: seasonal and vertical variations in the structure of the different communities

Seasonal and vertical variations of the main microbial communities (heterotrophic bacteria, autotrophic picoplankton, auto- and heterotrophic nanoflagellates, ciliated protozoa and microalgae) and auto- and heterotrophic activities were estimated in a brown-colored humic and moderately acid lake in central France, the lake of Vassivière. The results demonstrated the dominant role of light in the vertical distribution of autotrophic and mixotrophic microorganisms which are confined to the 0–5 m layer during thermal stratification. The bacterial biomass was high throughout the water column probably because of the great availability of dissolved organic matter. Consequently, the predatory microzooplankton and particularly the various trophic groups of ciliated protozoa, were distributed in the water column according to the vertical distribution of the particular food resources (detritus, bacteria, algae). However, despite the great abundance of algae and bacteria, biomass of flagellated and ciliated protozoa was relatively weak. Most of the phytoplanktonic biomass was filamentous (Diatoms) or colonial (Cyanobacteria) and therefore almost probably difficult to ingest for algivorous microzooplankton. Regarding the low abundance of bacterivorous protozoa, the relation with the special physicochemical properties of this lake is discussed.

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Résumé Les variabilités saisonnière et verticale de l'abondance et de la biomasse des principles communautés de la boucle microbienne (bactéries hétérotrophes, picoplancton autotrophe, protozoaires flagellés auto- et hétérotrophes, protozoaires ciliés, microalgues et microcyanobactéries), et des activités auto- et hétérotrophes, ont été étudiées dans un lac à caractère humique et modérément acide du Massif Central Français, le lac de Vassivière.Le dénombrement des communautés de microorganismes a été réalisé en microscopie inversée et à épifluorescence après mise en oeuvre des fixations et des colorations adéquates. Les activités photosynthétique, photo- et chemohétérotrophes ont été mesurées à partir de l'assimilation de NaH¹⁴CO₃ et d'un mélange d'acides aminés tritiés grâce à une technique de double marquage. Les résultats obtenus mettent en évidence le rôle prépondérant du facteur lumineux dans la répartition verticale des microorganismes autotrophes et mixotrophes, dont l'essentiel de la biomasse est confiné dans la zone 0–5 m en période de stratification, alors que la biomasse bactérienne est élevée sur l'ensemble de la colonne d'eau en raison, sans doute, de la grande disponibilité en matiére organique dissoute. Consécutivement, le microzooplancton prédateur, et notamment les différents types trophiques de protozoaires ciliés, se répartit dans la colonne d'eau selon la distribution verticale des ressources nutritives particularies. Cependant, compte-tenu de l'abondance bactérienne et algae, la biomasse des protozoaires flagellés et ciliésest relativement peu importante. Concernant les espèces algivores, l'essentiel de la biomasse phytoplanctonique est de nature filamenteuse (Diatomées) ou coloniale (Cyanobactéries) est est donc sans doute difficilement ingérable pour le microzooplancton. Enfin, les relations entre les caractéristiques physico-chimiques spécifiques de ce lac et le faible développement des protozoaires bactérivores sont discutées.

     

Population growth dynamics of the rotifer Brachionus plicatilis cultured in non-limiting food condition

Population growth parameters in batch culture of Brachionus plicatilis under a continuous supply of freeze-dried microalgae powder have been determined. Two B. plicatilis strains (L- and S-types) and four microalgae species (Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloris oculata and Tetraselmis suecica) have been tested, establishing the dynamics of growth at different daily food rations. Cultures showed a short lag phase, an exponential growth phase, a long post-exponential growth phase and long decline with episodic increases. In both rotifer strains, the best growth was obtained with Nannochloropsis oculata and the poorest with Nannochloris oculata.     

DNA:ATP RATIOS IN MARINE MICROALGAE AND BACTERIA: IMPLICATIONS FOR GROWTH RATE ESTIMATES BASED ON RATES OF DNA SYNTHESIS1

DNA: ATP and carbon: DNA (C:DNA) ratios were measured in a total of 14 species of marine microalgae and bacteria. Comparison of several DNA assay methods with results obtained with cultures uniformly labeled with ³³P indicated that by far the most accurate results were obtained using diaminobenzoic acid (DABA) or diphen-ylamine, with DABA having the highest precision. Both the Hoechst and DAPI methods seriously underestimated DNA concentrations in algal cultures. Average DNA: ATP ratios in the algal and bacterial cultures were I7 and 34 by weight, respectively, with almost all values lying in the range of 10–40. DNA: ATP ratios in the microalgae showed no correlation with growth conditions but varied by about a factor of 3 among species. C:DNA ratios for individual species of microalgae and bacteria ranged from 21 to 155 by weight and averaged 50 for the microalgae and bacteria taken together. Growth rates of microalgal species grown in cyclostats were estimated to within 8% of dilution rates when calculated from the uptake of ³H-adenine and the DNA: ATP ratio of the species. Use of the ³H-adenine method for estimating microalgal growth rates in the field may thus be a useful tool for investigating the physiology of microalgae in nature.     

Seasonal changes in alkaline phosphatase activity of bacteria and microalgae in Lake Pavin (Massif Central,France)

Seasonal changes in alkalinephosphatase activity of bacteria and microalgae werestudied in the dimictic Lake Pavin (Massif Central,France), to test whetherthis activity is primarily algal or bacterial andwhether the APA presents seasonal variations coupledwith abiotic and biotic variables. Samples werecollected at different depths from May 1992 to May1993. The specific phosphatase activities wereanalysed spectrophotometrically with p-NitrophenylPhosphate (p-NPP) as substrate and were related to theprotein concentrations. No correlation was foundbetween alkaline phosphatase activity and solublereactive phosphorus (SRP) concentrations across anannual cycle. The specific activities of the smallclass (0.2–1.2 m) were the highest and thecontribution of this picoplanktonic size class(0.2–1.2 m) increased with depth. In addition, thelinear correlations between alkaline phosphataseactivity and protein concentration seemed to indicatethat most of these enzymes are constitutive. However,it cannot be excluded that the high phosphorusconcentrations repress APA.Finally, the measure of APA does not seem to be avalid quantitative test of the deficiency ofphosphorus for aquatic microorganisms.