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  1. Introduction

Duckweed species are part of the sub family Lemnoideae family Araceae, order Alismatales, subclass Monocotiledoneae, class Angiospermeae, filum Plantae. The subfamily is now raised to the Lemnaeace family. It includes the genus Lemna, Lemna (with the four species, L. minor, L. trisulca, L. gibba, L. minuta – invasive species newly appearing on the Danube Delta Nebun of North America), Spirodela with one species, S. polirrhiza), Wolffia (with a species, W. arrhyza) and Wolfiella.

They live in slow flowing waters, ponds, freshwater lakes and lack in marine waters. Being a floating plant, the mesophile of the plant is composed of a special tissue called aerenchim, full of air filled spaces for the plants to float always. The plants give up vegetal stems specific to angiosperms, being an evolutionary regression to the algae, with the vegetable stem reduced to a vegetal as algae (Wolffia arrhyza), even without roots. The color of talus varies from chloroplasts to leucoplasts (white-yellowish) or carotenoid pigments to the middle of the summer, the plants becoming yellow-brown or yellow-orange, especially in the case of L. gibba in European flora. The reproduction occurs throughout the plant season, is asexuated, and is based on the division of the plant, thanks to the rapid development of meristemic tissues with a role in asexual reproduction, which leads to the growth and division of the talus or pseudotal of the plant in two. Occasionally reproducing and sexing.

Flowers have two stamen with petiole, they have the form of a spadix type, characteristic of the Araeeae family. Wolffia, with a diameter of 0.3 mm, is the smallest upper plant or angiosperm in the world. Through sexual reproduction, occasionally a floating fruit called the tricusse (L. Elias, 1986). It develops more strongly in heavily anthropogenic ecosystems in nature, from where they can be used as biofilters in industrial aquaculture systems.

It is also a good source of protein (Landesman Louis, 2012, Appenroth KJ, Sveek, 2017) and can be used as food for humans, namely obtaining valuable nutritional supplements for humans (De Benkelaar, Mirsthe F., Iurriann J., Fisher, Arnouts RH, 2019). For example, in Denmark, for example, recently made (2018) food preparations from Lemna. The molecular phylogenetic classification confirmed that the species belong to the Araceae family, classical taxonomy (Lidia I. Cabreza, Gerardo A, Salazar, et al., Pp. 1153-1165, 2013). Spirodela derives from the Arabidopsis thalianae ancestral genus, being the most primitive base genus from which Wolfiella and Wolffia derive, then Lemna (Wong W., Kerstatter Randall a., Toud P., 2011).

In the USA, through research project DOE (2009), research facilities have been created to demonstrate the possibility of obtaining new biofuels by ecological methods, such as renewable energy production (bioethanol), at Rutger University and the State University of Carolina . Efficiency is given by growth rates and low costs in line with the Convention on Climate Change, thus stopping global warming.

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Fig.1. The ethanol content of fresh (blue) and dry (red) (M. Kesaano, Sustainable Management of Duckweed Biomass Grown for Nutrient Control in Municipal Wastewater All Graduate Theses and Dissertations, Paper 879. (2011).)

Duckweed is an effective bioremediator eliminating pathogenic bacteria, N, P and other excess compounds in wastewater of any type (John Cross, 2011, Kribb Wayne, 2001-2004). It is used for the ecological reconstruction of wetlands, depolarizing for large wetland complexes. Also, Lemna species are considered to be very good bioaccumulators, even hyperaccumulators tolerant to radionuclides, hydrocarbons, organic and inorganic solvents, to which all species of the Lemnaceae family are resistant, metabolizing them faster than any other plant. L. minor is the most cultivated plant, but also the most common in Europe and has spread to other continents. Plants are 1-8 cm long, 0.6 to 5 mm wide and the flower has one egg and two stamens. Birds contribute to the spread of the species found in Africa, Asia, Europe and North America, except for the Arctic and Subarctic climate, Australia and South America, but it has recently been introduced.

1.1. Ecological living conditions.

For Lemna minor crops, under optimum conditions the pH value should be between 6.5-8 and 6-33ºC. Under these conditions, growth is very fast. At 6-7ºC the resistance forms or the turbines, which descend to the bottom of the standing waters, are produced. In spring, plant growth and lifting of turbons are restarted. Other optimal conditions would be the lack of competition with weak algae and wind, otherwise it causes waves that can overturn the plants. Another factor in the growth of Lemna minor is rainwater. The plant needs in excess of N, P and K, which must be in the concentration of 20-30 mg / l increasing the protein concentration.

The effluent with ammonia-rich water and other minerals is preferred by Lemna minor plants when grown in the lagoons of urine purification, washing waters, and swine fever (swine) with excess ammonia. In these decanting basins the best mineralization occurs if the water of the basins or urine lagoons is diluted by mixing with 80% pure water and 20% is direct drainage. Total nitrogen Kjeldahl N (54 mg / l, ammonium 31 mg / l, total phosphorus 16 mg / l) is obtained by diluting to 20% of the initial medium with 80% water by gradual addition. Potassium (K) and Phosphorus (P) waters can favor the growth of the Lemna minor species, but increased attention should be paid to nitrogen adjustment and correction (Bergmann, B. A., page 13-20, 2000).

Due to the fact that Lemna minor is a tolerant species for maximum temperature rise (it grows better between 15-33ºC and only after 33-36ºC there is a decrease in growth and productivity, more significant above 36ºC) these systems of fish aquaculture or purification of liquid waste from swine farms and fish farms lead to a very large increase in plant biomass.

In this way, water is extracted from excess organic nutrients by continuously rinsing with the clean water resulting from the washing of the farm. The Lemna minor species has been successfully used to treat and purify city and industrial waters, for example Devils Lake, North Dakota, U.S.A. (Chang J., 825-5511, 2002b). Plant harvesting can be used as an agricultural amendment or fertilizer. If used in scrubbing technology or there are no toxic substances, heavy metals, the production of lobster obtained is used directly as feed for broiler chickens but especially ducks and geese. This tradition is common in China, Thailand, Indonesia, to feed the palmipeds with dill in the sewage treatment basins, even human.

In parallel, in these ponds or lakes where the excrement is discharged, complete mineralization is achieved not only with Lemna but also with other species of monocellular green algae, aerobic bacteria and cyanobacteria on the surface and anaerobic in and on the substrate. All enumerated taxa complement the scheme for the purification and absorption of nutrients from elestee and politrofe, hypertrophic, excess nutrients, pour them to the quality of water acceptable for the life of fish and other hydrobonds. The increase in biomass of Lemna minor floating populations shows a production of 28.5 g / 1 m 2 and 104.03 tons / year, accounting for 85% of the nitrogen and phosphorus contained in North Carolina lakes (Cheng J., 1003-1010 , 2002 b, Akter M. et al., 251-261, 2011, Bergmann BA, 263-269, 2000).

Floating populations of Lemna minor develop very well, and in the anaerobic environment they even activate their development and absorption of nutrients through the root and bottom of the plant in contact with water. The absorption of nutrients from the anaerobic environment takes place in the absence of total air until the total purification and extraction of N, P, K (Caicedo J.R., 83-89, 2000, Rodrigo A., 98-104, 2012).

The recommended anaerobic treatment is over the 100 mg / liter aquaculture liquid medium, respectively Kjieldahl N and 50 mg / l P total, at which the highest rate of increase in biomass production and productivity of the Lemna minor floating populations ( Sasmaz M., Obek E., Sasmaz A., 246-253, 2015, Akter N., Chodbury SD, SD, SD Akter Y., Khatum MA, 252-261).

It has been shown that Lemna minor produces more starch / water surface acres than maize cultivated with many maintenance, growth, fertilization, pest and pest treatments, irrigation than maize on the same surface (Jay Cheng, 2018, Carolina University North). Duckweed was grown in small ponds for organic waste disposal of urban and industrial waste, where large silt stocks were spread after purification of these waters in riparian lakes of thousands and tens of thousands of hectares. It has been shown that Lemna minor has an enormous appetite for human and animal excreta, which it converts into starch by rapid conversion, which is then converted into ethanol at low industrial costs.

Lemna minor is an excellent bioremediator, concentrating heavy metals by absorption and detoxification, transforming them into non-toxic chemical compounds (Pb, Zn, Fr, Cd,). The dry line contains 25-45% of proteins (depending on growth conditions), 4.4% of lipids and 8-10% of fiber, calculated as dry vegetable biomass. It is used in the US to feed cattle as well as the bioremediation of heavy metal polluted waters with excess organic substances as a test body for environmental studies or as a system of genetic expression and testing for the industrial production of drugs and other biopharmaceuticals made by biotechnology. When it becomes invasive it is harvested as food for the Cyprinidae (Carpina carpio, Chinese carp) and Cichlydae (Tilapia nilotica) family.

Studies on plant embryonic development, biochemistry, photosynthesis, toxicity, genome studies, drug-induced motility are being studied. Researchers in the field of the environment use it in the ecological reconstruction of water through the implementation of water purification applications and patents. Major projects were carried out with the Lintiţa genome project (2009) to increase plant production and productivity (New Jersey State University). The Rutgers Institute of Genomics at Rutgers University is working in an international partnership with other Universities – University of Jena, Germany, Institute of Integrative Biology of Switzerland, Kyoto University, Japan, Oregon State University, University of California – coordinating the DOE World Program – the Genome Institute with four national labels; Lavrence Berkley, Laurence Livermore, Los Alamos, OAK Ridge, and Pacific North-West, Genomic Institute at Stanford University.

  1. Conclusions regarding the investigations carried out by the EcoPlate Biolog method on the intimate biological filtration mechanisms of the Lemna species, studied comparatively in natural conditions and in captive-controlled crops.

The purpose of this research was to characterize the phenotypic biodiversity of aquatic community communities free of aquatic … and microbial communities associated with aquatic species: Lemna trisulca, L. minor, Wolffia arrhiza and Spyrodella polyrrhiza.

In this study, a total of 8 samples were harvested and analyzed, including 4 samples of water collected from pools and 4 samples of aquatic plants represented by Lemna trisulca, L. minor, Wolffia arrhiza and Spyrodella polyrrhiza .

Research activities included the characterization of the physiological profiles of aquatic microbial communities and microbial communities associated with Lemna trisulca, L. minor, Wolffia arrhiza and Spyrodella polyrrhiza aquatic plants based on the degree of use of carbon sources in the EcoPlates Biologist system.

The results obtained at different time periods (24 h, 72 h, 96 h, 192 h and 216 h) indicated that the microbial communities in the analyzed aquatic basins exhibit less metabolic activity compared to the metabolic activity of microbial communities associated with aquatic plants. At 24 h, the lowest values ​​of the AWCD parameter were recorded between 0.98 and 2.21. The lowest value in microbial activity was observed for the water samples taken from the water basins of the L. trisulca and Spyrodella polyrrhiza species, and the largest sample of water taken from the L. minor species basin. Absorbance readings at 450 nm indicated an increase in the metabolic activity of microbial populations in the analyzed aquatic basins over 96 hours. The highest values ​​of metabolic activity were observed at 144 h of incubation for water samples from the aquatic basins of Lemna and Spyrodella polyrrhiza species and 168 h of incubation for water samples from the aquatic basins of the Wolffia arrhiza species.

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Fig 2. AWCD values ​​for microbial communities in water samples.

            The analysis of the metabolic diversity parameter based on the determination of the number of C sources used showed that the microbial associated populations of the plants have a larger spectrum of use of the carbon sources compared to the free microbial populations in the water basins (Figure 3). Thus, the values ​​of metabolic diversity for microbial communities associated with aquatic plant species ranged between 83.87% and 90.32%, while for the microbial communities in the water samples from the pools they ranged between 54.83% and 64.51%.

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Fig. 3. The appearance of the Ecoplates Biologist plates inoculated with the water sample from the aquatic basin of the Lemna minor species (left) and the microbial suspension obtained by removing microorganisms adhering to the surface of the Lemna minor species.

          Investigation of the physiological profiles of the aquatic microorganisms communities in suspension and associated of different aquatic plant species was accomplished by cultivating water samples studied in the presence of 31 substrates other than C at different time periods which allowed the characterization of metabolic responses and study the diversity of microbial communities.

The temporal dynamics analysis of the AWCD parameter, which describes the average use of C sources by microbial communities, has shown an increase in time of metabolic activity of microbial populations from both water samples and those associated with aquatic plants of L. trisulca, L. minor , Wolffia arrhiza and Spyrodella polyrrhiza, reaching a maximum of 144 hours after incubation.

          The conclusions from the comparative study are as follows:

  1. AWCD values ​​have shown a higher metabolic activity of microbial communities associated with aquatic plants compared to the metabolic activity of free, native microbial communities, or attached to nevium support (substrate, vessel walls) in the aquatic basins analyzed.
  2. The metabolic diversity of microbial populations in water samples taken from aquatic basins was lower than that of microbial populations associated with aquatic plants, suggesting a greater diversity of microbial communities adhering to the surface of aquatic plants compared to free, suspended water mass.
  3. It is clear and almost certain that we have a very wide variety of associations of photosynthetic microorganisms / aerobic bacteria / aquatic fungi / aerobic / anaerobic microorganisms living in areas with less oxygen, usually under the biodiversity of the roots and the lower part of the leaves which do not have direct contact with water and dissolved air. They live in probiotic activating relationships of increasing the production and productivity of vegetal and microbial biomass on floating floating plants because there are all favorable abiotic factors there (more oxygen, maximum illumination during daytime photoperiodation, agitation and permanent change of water gloss in in parallel with stirring, hydrogen sulphide, carbon dioxide, other toxic gases, atmospheric dissolution of as much oxygen as possible in the air-water interface, using surface tension and air-water exchange of classical water, the high favorable temperature which intensifies microbial processes specific to aerobic bacteria as well as photosynthetic physiological processes encountered or specific to photosynthetic cyanobacterial microorganisms, green monocellular algae such as Chlorella, yellow-golden algae – Phyllum Chrsophyta).

These conclusions, and especially the latter, will allow the design of tertiary filtration systems that will further activate photosynthetic and nutrient absorption processes at the highest speed to continually activate the speed of aquaculture water treatment.

Knowing now that this symbiosis exists and develops in the first 2-3 mm hydrobiologically specific of the category called pleuston (the level of floating plants that form a carpet at the surface of the water: Lemna minor, Lemna gibba, Lemna minuta, Wolffia arrhyza-wolfie, the smallest plant in the world, Spirodella polirrhyza) we can design biofilters according to the requirements the totally different ecologicals of these plants and associated symbiont microorganisms that have the same preference for light, temperature, oxygen, agitation and aeration, intense illumination (abiotic factors) and nutrient-producing biotic factors (fishfood, excrement and nutrient suppliers will be permanently cleaned by biofilters in recirculating and favored dynamic systems, both for nutrient feeding of photosynthetic hydrobiones – microorganisms and floating plants as well as water purification that must return extremely clean and sterilized in aquaculture basins of fish or other aqua animals animals called animal hobbies).

Of all the species of Lemna, the most odd is Lemna trisulca, which develops in the absence of too much light (especially in winter) in full floating stage as other species of cushion (for example Lemna minor).

The light intensity factor rather than the temperature is the one that leads to the abandonment of the submerged colony-like ecological form by division and agglomeration to the bottom of the water in aquatic ecosystems with 40-80 cm or slowly flowing ecosystems, but with direct sunlight and temperature constant water (14-28ºC).

And in this species, using the modern Eco Plates Bilog method, it has been demonstrated that the photosynthetic species of microorganisms develop on the whole surface of the individuals of Lemna trisulca in the water mass, the species being submerged and immersed during the summer forming huge colonies from the substrate to the surface of the water .

Curiously, only Lemna trisulca has no roots. But there are certainly allelopathic relationships that attract photosynthetic microorganisms / algae / saprophytic aquatic fungi / aerobic and fermentative anaerobic bacteria to form this microbial bio-organism all over their surface. More or less intense light that is not a problem for this species of lobster allows its water mass development, volumetric to the entire lentic or limbic aquatic ecosystem. So Lemna trisulca is less demanding at the intensity of light, while the other 4 species are extremely heliophytes (maximum amount of light to develop effectively and the maximum intensity of photosynthesis is at 33ºC). Fortunately, all Lemna species form this magnificent symbiosis that explains their tremendous power of purification.

  1. Because the trisculo Lemna develops in the mass of water is exactly what will allow us to realize an experimental module (based on the present results) of a tertiary biological recirculating filter with slow flowing water on the ecological principle of the continuous river equipped with multiple illumination systems (horizontal illumination , lateral, left, right and underneath the vessel, oblique, vertical) to allow the entire volume of filtration of the vessel to flow while the water is slowly recirculated so that as much of the tertiary biofilter as possible takes place the intensification of the processes filtration and absorption of nutrients, organic waste-excrements, toxic gases, ammonia, microorganisms molding and submerged duckweed. On the basis of this conclusion, we will achieve at the next year’s stage at least one prototype model with several more efficient and competitive variants, with a filtering role at the research facility set up by our association in the Frasin locality in Suceava County (see the Aquaterra de biosample of experimental scientific research).
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