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Read Erika Greipel´s 2016 (Albitech Ltd.) abstract here!

Erika Greipel Bioengineer M.Sc., researcher Head of laboratory at Albitech Biotechnological Ltd. E-mail: greipel.era@gmail.com Phone: 00 36 20 424 8668

Measurement of intra- and extracellular cytokinin content of algae cultures and application of Scenedesmus sp. cultures for plant growth promotion – resulted the new foliar fertilizer of Albitech Ltd.

Erika Greipel¹, Péter Futó¹, Zsuzsanna Bencsik¹, Attila Bencsik¹, Annamária Kósa², Péter Nyitrai³, Prof. Béla Böddi², József Kutasi¹

¹: Albitech Biotechnological Ltd.
²: Eötvös Loránd Science University Department of Plant Anatomy
³: Eötvös Lóránd Science University Department of Plant Physiology and Molecular Biology

Abstract

Foliar fertilization is a widespread procedure in agriculture for applying inorganic fertilizers or other substances on the foliar surface of plants in order to enhance crop yield. Microalgae suspensions has been used for a long time for the purpose of phytostimulation, owing to their hormone-producing ability. In our work cytokinin content of Scenedesmus sp. BEA D01_12, Chlorella minutissima K2012 41 cultures were investigated. Prolification of algae was stimulated by the addition of Azospirillum brasilense NCAIM 001411 bacterial culture in a low concentration. Scenedesmus sp. cultures were also tested in greenhouse pot trials and small pot field studies. According to the results the algae suspension was effective on fruit, vegetables and cereal crops as well. Cucumber, tomato, apple, sweet pepper and sunflower have shown a significant increase of crop yield. Increased the number of flowers in sweet pepper and tomato, the root weight of sweet pepper, plant height and the number of seeds in maize and the oil content of the seeds in sunflower. In horticulture significant increase in the number of leaves and flowers was observed.

About the author

Born in Budapest (Hungary) and graduated B.Sc. and than M.Sc. degree in Bioengineering at Budapest University of Technology and Economics. She worked on the topic of Heterocypris incongruens immobilization test further development and application of micro-pollutants contaminated testing samples of liquid and solid phase and Isolation of filamentous fungi with cellulase activity from soil samples, perform various enzyme activity assays after shake flask fermentations at the University. She took part a 6 week-long traineeship at the Fermentation plant of Gedeon Richter Plc. (pharmaceutical industry).

Beside M.Sc. courses in 2010 she started to work at Albitech Biotechnological Ltd., she coordinates the inoculum preparation and other manufacturing operations of microalgae-based foliar fertilizer formulation and quality control, moreover she is carrying out research activities with different microalgal and soil bacterial strains. Since 2014 she is PhD. student at Eötvös Lóránd Science University where she study the interactions between

Scenedesmus rubescens green microalga and Azospirillum brasilense soil bacterium.

About the company

Albitech Biotechnological Ltd. is a dinamically developing Hungarian enterprise established in 2007 aiming at microalgae research and development other microalgae based products for agricultural or other purposes.

The fact that algae are suitable for large-scale biomass production could lead to a breakthrough in the biotechnological industry. Our goal is to introduce a new branch of the plant biotechnological industry in Hungary to be utilized in agricultural production by the screening and controlled, industrial-scale culturing of new algal strains capable of producing bioactive compounds. The first fruit of research was the microalgae based Algafix® phytostimulator, a growth-promoting spray-on which was has been authorized in 2010 and produced continuously in the photobioreactor plant at the company.

Several microalgae strains show phytohormone-like activity. Intensive auxin- and cytokinin-like plant growth hormone production has been observed in green algae species, recently. In agriculture, algae have been used as organic fertilizers or soil conditioners, however, interest has recently grown in their antimicrobial, anti-fungal and plant growth regulating (PGR) properties. The concentration of phytohormones in algae biomass range between 0.3 – 4,0 μg / kg wet weight which approximates that of higher-order plants. Industrial scale culturing of saltwater algae is not economical in Hungary, due to the treatment of large volumes of industrial wastewater with high salt concentrations. Our research confirmed that in optimized conditions, freshwater algae can produce and excrete plant growth promoting hormones, antimicrobial or antifungal substances in higher concentrations than saltwater algae and contain vitamins and trace-elements vital for plant health.

Technology-wise the axenic algal strains are selected and maintained by classic microbiological methods. The identified algal cell lines are preserved and cultured via closed-system sterile photofermentation. According to our new protocol the Scendesmus sp. BEA D01_12 is cultured in air lift photobioreactors. Production of industrial-scale volumes for agricultural use is implemented via scaling up. Laboratory-scale volumes of shaken liquid cultures are produced which go through culturing in several 2-10 l volume photobioreactors to reach 30 and 200 litre photobioreactors. Adequate light intensity is provided by high-power light sources to ensure proper growth rate. The production capacity has quadrupled in the past 4 years. A new technology developed in the last few years makes it possible to achieve a five-fold increase in the alga cell count.

In Albitech Ltd. there are currently six people working in our laboratories: Dr. József Kutasi PhD., co-owner and head of research, Attila Bencsik, co-owner and head of informatics and technology, two bioengineers: Erika Greipel MSc. (PhD-student at Eötvös Lórand Sicence University), Péter Futó MSc., an administrator, a laboratory and a fermentation assistant. Our young researchers have participated in both national and international conferences with oral and poster presentations. In the framework of granted research projects, our company has hosted bioengineering undergraduates from Budapest Technical University for traineeship and thesis work.

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INTRODUCTION
Invasive dreissenid species (D. polymorpha, D. r. bugensis) was introduced from Danube River to Lake Balaton – the largest shallow
lake in Central Europe – supposedly by ship transport (Sebestyén, 1932; Balogh et al., 2009).

The dreissenid-mediated impacts on aquatic environments are of special interest because they can create and/or modify habitats, and promote numerous direct and indirect system-wide effects; hence they are considered as ecosystem-engineers (Jones et al., 1994, 1997; Karatayev et al., 2002; Vanderploeg et al., 2002; Gonalez et al., 2008).

Dreissena species as dominant freshwater filter feeders playing intermedier role in the food chain. Their biomass (dry mass without shell) reaches around 22 t/year in Lake Balaton (Balogh, 2008), where dreissenids are main food sources for carp, bream and roach (Bíró, 1997). We found that the protein content of the soft tissue was high enough (55-60% dw, Balogh and Serfőző, 2017) to count with this animal as a potential protein source alternative provided by freshwater ecosystem service for feeding developing fishes in intensive fish farming.

The aim of this study was to investigated the type and quantity of algae that yields intensive growing, higher weight and therefore more energy source biomacromolecules of dreissenids, which is beneficial in fish bait composition. Monocultures of those algae were used, which can be found with high number in the natural environment of dreissenids and can be easily cultured in laboratory conditions.

MATERIALS AND METHODS
Algae-mussel laboratory experiment were carried out to know the optimal algae concentration which dreissenids consume daily, and using this optimal concentration we measured the growth of the animals in a mid-term (3 months) microcosm study (Fig. 1a.). We used four kind of algae species (Chlorella sp., Scenedesmus sp., Neochloris sp., Phaeodactylum sp.) and an algae-mix, containing the mixture of them (Fig. 1i.). The lake water filled with natural plankton represent the control. In the microcosm study, the weight, length, width and the shell area of the mussels (10 pieces per cup, in 5-10 parallel) were measured (Fig. 1d-h.).

The shell length and area increment were determined by using Image J softwer. We collected animals living in the western eutrophic (Keszthely) and eastern oligotrophic (Siófok) bays of Lake Balaton (Fig. 1j). We followed the algae concentration by flow cytometry (Fig. 1b.). We measured (Fig. 1c.) glycogen (total carbohydrate) content according to Van Handel (1965), adapted to mussels by De Zwaan and Zande (1972). Protein was measured with BCA method (ThermoScientific), and the fatty acids by the phosphovaniline method (Nalepa et al., 2014).

To analyse differences in used algae species (transformed in case of need), we used a GLM ANOVA (different letters means significant differences).

RESULTS
We found that optimal algae concentration for mussel growth was 1-3×107 ind/L/day.
There were significant differences on the growth (weight, size) parameters of the mussels depending on the algae species used to feed them (Fig. 2a., b). Reduction of the weight was found in control samples (Fig. 2a).

The survival of the mussels were up to 85% at the end of the three-month experiment. The highest survival rate reached with the mussels feeded by the algae-mix.

Protein content was significantly higher in case of feeding the mussels with Chlorella sp. or with Phaeodactylum sp. compared with those mussels kept under Lake Balaton water obtained from the oligotrophic part. On the other hand, high biomacromolecule values of the experimental samples were exceeded that of the samples taken from the eutrophic part of the lake (Fig. 3a).

Carbohydrate data showed significant emergence only in case of feeding with Scenedesmus sp., Phaeodactylum sp. (Fig. 3b), similarly to fatty acids (Fig. 3c).

CONCLUSION
Scenedesmus sp., Neochloris sp. and the algae-mix ensure the best food sources for the mussels. All growth parameters (weight, lengths) as well the body protein content were higher at the mussels feeded by these algae species. Absolute values of all energy source materials measured were the highest in the case of nourishing the mussels with these algae.

High biomacromolecule values of the experimental samples were exceeded the value that of the samples taken from the eutrophic part of the lake. Thus, the mussel body content indicates the trophic state of the lake, showing the gradiently declining trophic state along
the longitudinal axis from the eutrophic Western to the oligo-mesotrophic Eastern basins (Padisák, 1994, 2001; Padisák et al., 1998; Tátrai et al., 2000; Vörös et al., 2000). In the experiment, the mussel growing dynamics were similar to that found earlier in the field (Balogh et al., 2019), and in the substrata implanted into the lake (Balogh et al., unpublished).

In conclusion, considering natural conditions, amount of biomacromolecules could be enriched in dreissenids fed on selective algae monocultures.

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The post POSSIBLE APPLICATION OF INVASIVE DREISSENIDS AS PROTEIN SOURCE ALTERNATIVE IN INTENSIVE FRESHWATER FISH FARMING PROJECTS: GROWING DYNAMICS AND NUTRIENT VALUE OF MUSSELS IN SITU AND FEEDING WITH ALGAE appeared first on Microalgal fermentation - Photobioreactor design and construction - Albitech Ltd..

¹ Research Department, Agricultural Research and Development Station of Braila, Sos. Vizirului, km. 9, Braila, Romania.

² Agrochemical Laboratory, BRAICOOP Agricultural Cooperative, No. 38, Imparatul Traian Street, Braila, Romania

³ Agriculture Department, “Dunarea de Jos” University of Galati, Engineering and Agronomy Faculty of Braila, No. 29, Calea Calarasilor, Braila, Romania

Abstract

Experience with bio-fertilizers included two sub-experiences,with maize and sunflower. Placing of experiences was performed by
the method of Latin,rectangle in three repetitions, on Braila Plain, Romania, in 2014 year. The biological products used were Bactofil and
Algafix, in four doses (V2 – Bactofil 285g / ha + Algafix 1.5 l / ha; V3 – Bactofil 500g / ha + Algafix 2 l / ha; V4 – Bactofil 571g / ha +
Algafix 2.5 l / ha; V5 – Bactofil 642,8g / ha + Algafix 3 l / ha). The control was variant without fertilizers. Biometric measurements were
performed throughout the growing period and at harvest were determined elements of productivity and average production to determine
optimal doses for increased production in terms of quantity and quality.

Keywords: bio-fertilizers, doses, corn, sunflower, yield.

INTRODUCTION

The importance of biological fertilization increases continuously to practice sustainable agriculture and decrease environmental pollution. The specific consumption of nutrients for the species studied is very big: corn consumed 27.5 kg N, P2O5 and 16.5 kg K2O 12.5kg and sunflower consumed 36.5kg N, P2O5 and 50.0 kg 17.5kg K2O per ton of main and secondary crop (Davidescu, 1999). In case of using organic products, these doses decreased significantly (Trifan, 2015). Maize and sunflower are very important in world agriculture, but requires knowledge about growing areas of hybrids and varieties, about fertilization and the sowing and harmonization of ecological factors with rational use of technological measures specific to each culture, while also improving the physic, chemical and biological parameters of soil and implementation of incentive income for farmers (Rinchita, 2008).

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Abstract

Foliar fertilization is a widespread procedure in agriculture for applying inorganic fertilizers or other substances on the foliar surface of plants in order to enhance crop yield. The efficiency of foliar fertilization can be 8-20% better than solid fertilizers. Certain axenic, high-concentration algal cultures have a growth promoting effect on field cultivations, due to nitrogen-fixation and production of growth-promoting hormone-like substances. These concentrated suspension of microalgae and/or cyanobacteria – or its extract – efficiently enhance growth of plants by increasing plant height and leaf surface, improving germination capacity of seeds, or even increasing the clorophyll content of leaves, or the starch, oil and protein concentration of the fruit.

Our product, Algafix® is a liquid culture of microalgae, containing phytohormones, macro- and microelements. For home gardens, greenhouses, agricultural and horticultural use for mono- and dycotyledon cultivars. It contains Scenedesmus rubescens/obtusiusculus, a freshwater green microalga isolated from the Lake Balaton, Hungary. Between it’s plant hormone producing capacity it produses different lipids and molecules with antioxidant capacity which can delay the scenescence of leaves. About agricultural experiments it can be used for cereals, sunflower, corn, vegetables, fruit-trees, grape and also for indoor plants. According to the patent HU 1200251 Scenedesmus strains are cultivated together with the (plant growth promotion rhizobacteriae) Azospirillum brasilense NCAIM (P) 001411(National Collection of Agricultural and Industrial Microorganisms )reached surprisingly higher algae cell number and more effective plant stimulating properties. These bacteria are able to produce auxins, in the first line indole-3-acetic acid whitch can stimulate the proliferation and hormone production of algae. In this way cultivated microalga culture has 10⁸cell/ml cell concentration, stimulate the germination, has antimicrobial and antifungal activity, furthermore in case of field plants stimulate the stem elongation and blossoming and increase the yield.

The cytokinine and gibberellin-like plant hormon production of axenic cultures of Scenedesmus sp. BEA(Banco Espanol de Algas) D01_12(Algafix®,  Patent HU 1200251) Chlorella minutissima K 2012 (Albitech collection) and Botryococcus braunii green  alga strains were investigated. In case of S. rubescens/obtusiusculus we found kinetin production an order of magnitude higher than in case of the other two strains. Trans-zeatin-riboside whitch is one of the most effective natural cytokinin was found in evapored extracts of B. braunii, C. minutissima and in S. obtusiusculus cultures as well. B. braunii cultures haven’t investigated in this field previously. It seems that the S. obtusiusculus, the strain of Algafix® foliar fertilizer is really suitable to use for regulating plants. These results can serve as a good basis for the development of algae-based foliar fertilizer products and for a better understanding of their mode of action.

Erika  Greipel, Zsuzsanna Bencsik, Péter Futó, József Kutasi:

Preparated  Cladophora  cellulose,  as a  substrate  of  cellulase fermentation

Abstract

Some filamentous algae collected from the sea are used for paper production. Moreover, it can serve as a raw material of cellulose production as a more efficient substrate than plant cellulose.  Moreover algae are currently exlioited by the food industry for a range of ingradients. The filamentous freshwater green algae Cladophora contents the cellwall cellulose mostly in the crystalline form. In contrast to the plant cell wall cellulose in this case the Iα whitch is more accessible to the enzymes is present in 63% while the Iβ form in 37%. Plants have reverse ratio making it difficult to process the fermentation plant cellulose. Cladophora cellulose forms gel structures at cellulose concentrations as low as 0.2% (w/v), whereas the lower threshold for the commercially available analog is around 1.5% (w/v) solids contents (Mihranyan 2011). It’s powder  could be a useful alternative to commercially available dispersible cellulose grades when very low quantities of stabilizing agent are required.

With the extracted cellulose from Chladophora the cellulase enzyme induction in soil bacteria were investigated for facilitating and to enhance the production of enzymes. The bacterial digestion was performed on dried and milled, but untreated, Cladophora fibers as well. The success of the experiment can be explained by that the algae cell wall doesn’t contain lignin, which would inhibite the access of cellulase enzymes to the untreated material.

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¹ Agricultural Research and Development Station of Braila, Km. 9 Vizirului Rood, 810008,
Braila County, Romania
² ”Dunarea de Jos” University of Galati, Engineering and Agriculture Faculty of Braila,
29 Calarasilor Str., 810017, Braila County, Romania

Corresponding author email: dtrifan2015@gmail.com

Abstract

Foliar microbiological fertilizer Algafix is represented by a mixture of living green algae species
Scenedesmus obtisausculus who are able to assimilate different hormones and antioxidants that are
transmitted through the leaves of the plant, stimulating growth and resistance to biotic and abiotic stress,
and having an important influence for increased production. This paper presents an experience of
establishing optimal Algafix dosage of winter rape and spring barley, with four graduations or doses of 1.5
l/ha, 2 l/ha, 2.5 l/ha and 3 l/ha, which were compared with the control – untreated variant. We watched both
morphological and physiological differences on the characteristics of plants in the experimental variants,
and production quality indexes obtained, and the correlations established between doses of biofertilizers and
measured parameters. In the same time, we studied how the doses of biofertilizers influence the water and
mineral elements root absorption by plants grown in the two species, as well as economic efficiency
calculation. Compared with untreated control, all variants have obtained positive differences, most
production being registered variant V4 (dose of 2.5 l/ha), both in winter rape and spring barley cultures.

Key words: microbiological fertilizers, spring barley, rape.

INTRODUCTION

Currently, put increasing emphasis on the transition to a sustainable organic agriculture, especially when the products are used directly in human nutrition. On the other hand, it is very important to avoid environmental pollution, to use less fertilizers and gradually to proceed with both biological products for crop fertilization and for phytosanitarytreats (Leopa, 2013). Also, farmers can easily use the remaining waste for composting farm and obtain organic fertilizer, using various design patterns, depending on the size of farms (Trifan, 2013; Anghelache, 2012). Rape culture is autumn consuming nutrients, increase production is directly proportional to the applied fertilizers. Due to the short period of vegetation, spring barley is not pretentious for fertilization using the residual effect of previous crop, apply fertilizers. To see how to behave crop to biological fertilizer treatments compared with chemical fertilization was achieved with various doses of biofertilizers experience. Algafix is a foliar fertilizer microbiological green alga of the genus Scenedesmus  living in water suspension obtained by photofermentation technology, macro and trace elements, plant hormones culture removed from algae. Fractional application of chemical fertilizers provide plant supply throughout the growing season, enabling increased production of grains and based by algae fertilizer application, ensure soil nutrient absorption increase in the critical stages of plant nutrition (Trifan, 2014).

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Detection of cytokinine and gibberellin-like plant hormones in Scenedesmus obtusiusculus Chlorella minutissima and Botryococcus braunii cultures

Erika Greipel, Zsuzsanna Bencsik, József Kutasi

Albitech Ltd, 1045, Berlini út 47-49

Abstract

Foliar fertilization is a widespread procedure in agriculture for applying inorganic fertilizers or other substances on the foliar surface of plants in order to enhance crop yield. The efficiency of foliar fertilization can be 8-20% better than solid fertilizers. Suspension of microalgae cultures has been used for long as foliar fertilizer, and according to recent research, it utilizes the hormone-producing capacity of algae. Numerous scientific studies have been published about algal  cultures or extracts containing different types of plant hormones and their effect on plant growth and stimulation of germination. In our work, the cytokinine and gibberellin-like plant hormon production of axenic cultures of Scenedesmus obtusiusculus BEA(Banco Espanol de Algas) D01_12, Chlorella minutissima K 2012(Albitech collection) and Botryococcus braunii UTEX 2629 green alga strains were investigated. Both of these types of plant hormones significantly affect cell division. The gibberellins – as diterpenoids – stimulate cell division and cell growth so they promote the developing of leaves and the progress of blossoming. Additionally, these hormones induce the de novo syntesis of hydrolitic enzymes during the germination of seeds. Cytokinines delay senescence of leaves and they have morphogenetical effects as well. Detection of the mentioned plant hormones was conducted via thin layer chromatography following organic solvent extraction and evaporation (Tien et. al., 1979). Kinetin was present in evapored extracts of C. minutissima, S. obtusiusculus and B. braunii cultures as well. These evapored extract samples were observed also by HPLC-PDA. (Gupta et. al., 2011, Stirk et. al. 2004). The amount of kinetin was approximately 0,8 – 1,0 µg/ml (400 ng/ml in the algae liquid culture) and other cytokinin compontents were detected next to the kinetin in case of S. obtusiusculus.  In the extracts of C. minutissima and Botryococcus braunii were approximately 0,1 – 0,2 µg kinetin/ml (40-80 ng/ml in the algae liquid culture). The citokinin-like activity of liquid cultures was also measured by cucumber cotyledon growing test. Samples – obtained by evaporation of S. obtusiusculus, a C. minutissima és a B. braunii cultures on low temperature – showed effects on the weight of cucumber cotyledons after 3 days of incubation time. The gibberellin-like activity was proved also by measuring amylase production of barley endosperm (Jones and Varner, 1966). Presence of gibberellin-like substances was detected in C. minutissima cultures. These results can serve as a good basis for the development of algae-based foliar fertilizer products and for a better understanding of their mode of action.

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Juvenile plants are exposed to a series of stress factors, that can seriously affect crop yield. The most important of such factors are suboptimal weather conditions, poor soil quality, microbial infections and harmful chemicals (e.g. pesticide residues or the applied herbicide).

A key aspect in agricultural technologies is to minimize stress factors affecting the developing plants (optimal sowing time, appropriate plant protection) and to support plants in combatting inevitable stress factors. With the AlgaFix organic foliar fertilizer, such needs can be fully met, as our product improves the stress-tolerance and regenerative capacity of plants. The earlier it is applied, the reliable its effect is, since this year the risk of herbicide-related stress is higher than usual.

The AlgaFix AntiStress Jet is a device adapted for the treatment of developing plants. In Hungary, approximately 10,000 tons of herbicide is applied every year, which is a shockingly high quantity.

At least half of it could be excluded by applying weedkillers only on rows and the mechanical removal of weeds from inbetween the rows, by interrow cultivators. The excessive use of herbicides can lead to the contamination of drinking water (up to the magnitude of nanograms/l in the drinking water of Budapest).

Another concern is that herbicides cause severe stress to plants. This is manifested in reduced growth rate and yellow discoloration in the first 2 weeks after herbicide treatment. Of course, there are differences between herbicides and breeds/hybrids also show differences in their sensitivity. Suboptimal weather and soil conditions also reduce stress-tolerance. Certain weedkillers need at least 10-20 mm of rainfall after administration to take effect.

Last year it caused serious damage, as weeds, unaffected be the weedkiller, competed with the cultivars for moisture in the soil. During the 50 years of using chemical plant protection, the longest period of drought was experienced in the year 2012, which raises concerns over excessive herbicide residues in soils, a potential risk factor in the spring period. It is definitely worth finding out what are the prescriptions for certain second crop species.

Always follow instructions for deep ploughing and take soil pH into consideration. Plots are not homogenous and there is a high risk that the sowing can be at least partially damaged by herbicide residues.

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Research and development objectives

• Isolation and culturing of freshwater microalgae, extraction of natural oil content
• Collection of freshwater algal coatings and zooplankton, determination of oil content
• Investigation of the oil producing capacity of saltwater microalgae, optimisation of culturing conditions for maximal oil-biosynthesis
• Inducing mutations to obtain „oil-like” algal strains
• Concentration of oil fractions via HPLC, thin-layer and gas-chromatographic analyses of fractions
• Studies on the specific effects of lipid fractions
• Large-scale algal biomass production in photobioreactors at semi-industrial scale: technology development.

Development

Our first major development was the establishment and installation of a large-volume photobioreactor (Albitech, Foton). Pilot studies are in progress.

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Blue and green algae produce mainly C16-C18 unsaturated fatty acids, while in brown-, red and yellow-green algae and diatoms, the presence of C18-C22 unsaturated fatty acids is more frequent, in the form of mono-, di- and triglycerids.

Saltwater algae produce unsaturated fatty acids in a high amount. Bangia, Cladophora and Diatoma species isolated from algal coatings of rivers and lakes in Hungary were found to be producing EPA in concentrations approaching the capacity of saltwater species. In low environmental temperatures, higher EPA and DHA concentrations are found in algae, which makes early spring the ideal time of collection.

The possibility of contamination limits their use in human health prevention, which necessitates controlled culturing in industrial scale, the technology we aimed to develop.

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