Biodiversity Data Journal :
Research Article
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Corresponding author: Yolanda del Carmen Pérez-Luna (yperez@upchiapas.edu.mx)
Academic editor: Anne Thessen
Received: 19 Jul 2018 | Accepted: 05 Sep 2018 | Published: 26 Sep 2018
© 2018 Yazmin Sánchez Roque, Yolanda Pérez-Luna, Joel Moreira Acosta, Neín Farrera Vázquez, Roberto Berrones Hernández, Sergio Saldaña Trinidad, Joseph Pathiyamattom
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Sánchez Roque Y, Pérez-Luna Y, Moreira Acosta J, Farrera Vázquez N, Berrones Hernández R, Saldaña Trinidad S, Pathiyamattom J (2018) Evaluation of the population dynamics of microalgae isolated from the state of Chiapas, Mexico with respect to the nutritional quality of water. Biodiversity Data Journal 6: e28496. https://doi.org/10.3897/BDJ.6.e28496
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As Chiapas state, México, counts on an extensive hydrography with diverse nutrimental and climatic characteristics, it therefore allows isolating and identifying microalgae with bioenergetics potential.
For this purpose, samples from 8 locations were collected, corresponding to 6 rivers, a wastewater and a springwater. The isolation of microalgae was developed for 4 weeks with 12:12 light/dark cycles. We demonstrated that the most efficient means for the isolation of microalgae of the hydrographic areas evaluated was the medium BG11 with 80.53% effectiveness. Of the microalgal consortium identified, 90% are composed of microalgae belonging to the class Chlorophycear. It was shown that another factor favouring the richness of morphotypes identified in the Santo Domingo River is associated with adequate concentrations of macroelements such as nitrates, nitrites, ammonium, phosphorus, sodium, potassium, magnesium and calcium at concentrations of 0.03 mg/l, 0.0006 mg/l, 0.08 mg/l, 0.03 mg/l, 62.93 mg/l, 5.46 mg/l, 34.52 mg/l and 48.78 mg/l respectively and microelements such as copper, zinc, iron, andmanganese at concentrations less than 0.2 mg/l in all microelements. The identified morphotypes, according to literature, have lipid contents ranging from 2 to 90%; this is of biotechnological importance for the production of biodiesel.
Hydrographic areas, isolation, microalgae
As the human population increases, world energy demand and dependence on fossil fuels have continued to increase. As a result, global carbon emissions, including greenhouse gases, have increased to contribute to global warming, so the need to move towards sustainable alternatives to fossil fuel use is not only necessary to addressClimate change, but also to address the depletion of world energies (
Biofuels generated from algae in particular have been identified as an exceptional source of carbon and renewable energy (
The success and economic viability of a microalgae-based biofuel industry will depend on a number of factors, including the selection of resistant strains that exhibit exceptional growth rates, lipid profiles suitable for biodiesel production and tolerance to a wide range of environmental parameters (
Water samples used to isolate microalgae were collected aseptically from sites that appeared to contain algal bloom. About eight different water samples were collected from different locations in Chiapas, Mexico. Three samples of water of 1 litre per area were obtained from 6 rivers, 1 spring water and 1 filtering gallery of wastewater, each sample area was evaluated in triplicate, transported in coolers with 1 litre flasks per sample; the locations are presented in Table
Distribution of sample collection of the different hydrographic zones of the state of Chiapas, Mexico.
N. |
River |
Town |
Geographical coordinates |
1 |
Nandayapa |
Acala |
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2 |
Huixtla |
Huixtla |
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3 |
Novillero |
Tonalá |
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4 |
Sto Domingo |
Chiapa de Corzo |
|
5 |
Suchiate |
Suchiate |
|
6 |
Pijijiapan |
Pijijiapan |
|
7 |
Spring water Cahuaré |
Chiapa de Corzo |
|
8 |
Filter gallery of wastewater “La Chacona” |
Tuxtla Gutiérrez |
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The physical and chemical properties of the water samples were determined for soluble chemical analysis, each sample being filtered through 0.2 µm syringe filters and stored at −20°C for subsequent analysis. Nitrate, nitrite and phosphate were measured using a Metrohm 850 Professional Ion Chromatograph (IC) (Metrohm Inc., Switzerland) with a Metrosep A Supp 5-250 anion column (Metrohm Inc., Switzerland). For anion analysis, 3.2 mM Na2CO3, with 1.0 mM NaHCO3 eluent were pumped at 2.6 ml/min, with a 100 mM HNO3 suppressor solution and using a 20 µl sample loop. The determinations were corroborated by the Hach kits (Hach, Loveland, CO, USA), per standard methods 10127, 8039 and 8507 for the determination total phosphorus, nitrates and nitrites, respectively. Ammonium was measured using Hach kits (Hach, Loveland, CO, USA), as per standard methods 8155. pH was measured using a Fisher-Scientific probe (
Sample preparation consisted only of acidifying each water with 1% HNO3 (v/v) and adding 0.1% lanthanum chloride as a releasing reagent for calcium (Ca) and magnesium (Mg) and as an ionization suppressant for sodium (Na) and potassium (K). All analyses were carried out with the PerkinElmer PinAAcle 500 flame atomic absorption (AA) spectrometer. Measurements were made at a wavelength of 422.67, 324.75, 248.33, 285.21, 766.49, 589.00 and 213.86 nm for Ca, Cu, Fe, Mg, K, Na and Zn, respectively and a slit setting of 0.7 for Ca, Cu, Mg, K and Zn and 0.2 for Fe and Na. The visible range was used and the source current was set at 14 ma. A scale setting of 1 and an air flow rate of 2.5 l/min were employed. Before the start of each series of analyses, the gas (acetylene) flow rate was adjusted to give maximum absorbance while aspirating a standard solution. This value was usually 10 l/min. The aspiration rate was checked by using a stopwatch and graduated cylinder. Plugging of the aspirator was not excessive and, when it occurred, was rectified by aspirating 1:1 hydrochloric acid for 1 minute (
For the isolation process, 10 ml of water sample was transferred to a 500 ml conical flask containing 250 ml of sterilized BG11 medium and CHU medium (
All microalgae species were morphologically validated; the latin scientific name and class were confirmed in the database of AlgaeBase” (http://www.algaebase.org) and database of NCBI (https://www.ncbi.nlm.nih.gov/pubmed).
The dry cell weight (DCW) of microalgae biomass was also obtained by filtering 50 ml of aliquots of culture BG11 and CHU through a cellulose acetate membrane filter (0.45 µm pore size, 47 mm in diameter). Each loaded filter was dried at 105°C until the stability of weight was reached. The dry weight of the blank filter was subtracted from that of the loaded filter to obtain the microalgae dry cell weight (
After treatment, aliquots were collected in 50 ml vials and then analysed to determine the size frequency of algal cells per ml of suspension using a FlowCam (Fluid imaging Technologies). The sizes were expressed as equivalent length (EL), according to the shape of the microalgae identified. FlowCam is a continuous imaging flow cytometer designed to characterize particles that pass through a flow chamber. The FlowCam captures digital images of particles in a fluid stream using laser light detection, enabling the measurement of many cell parameters, such as length (
The statistical software used was the STATGRAPHICS PLUS (1999) for windows. For the first experiment, prior to statistical analysis, data were assessed for equality of variance and normality. The proportion of the size range of algae concentrate between 1 and 10 μm was transformed to Arcsin square root to improve the homogeneity of variance assumption (
All microalgae species were morphologically validated; the latin scientific name and class were confirmed in the database of AlgaeBase (http://www.algaebase.org) and database of NCBI (https://www.ncbi.nlm.nih.gov/pubmed).
Once the samples were taken, they were evaluated to identify the presence of microalgae, in such a way that microalgae and cyanobacteria that remain associated in consortiums were identified.
Physical and chemical analyses of water samples
In the physicochemical characterization of the hydrographic areas of the state of Chiapas, it was observed that the Santo Domingo River presented the highest concentrations of nitrates, nitrites and ammonium with 0.03, 0.006 and 0.08 mg/l, respectively. However, the filtration gallery of the wastewater “La Chacona” presented the lowest concentrations of nitrates, nitrites, ammonia and total phosphorus with 0.01, 0.002, <0.01 and 0.08 mg/l respectively (Table
Physicochemical characterization of the water of the different hydrographic zones of the state of Chiapas, Mexico.
Sampling areas |
Nitrates (mg/l) |
Nitrites (mg/l) |
Ammonium (mg/l) |
Total phosphorus (mg/l) |
Suchiate river |
*0,02ª + 0.04 |
0,003ab + 0.01 |
0,04b + 0.01 |
0,18ª + 0.02 |
Nandayapa river |
0,02ª + 0.01** |
0,003ab + 0.01 |
< 0,01c + 0.00 |
< 0,02d + 0.00 |
Huixtla river |
0,02ª + 0.02 |
0,005ab + 0.02 |
< 0,01c + 0.00 |
0,12b + 0.01 |
Spring water Cahuaré |
0,03ª + 0.04 |
0,004ab +0.00 |
< 0,01c + 0.00 |
0,1bc + 0.01 |
Novillero river |
0,02ª + 0.01 |
0,005a + 0.01 |
< 0,01c + 0.00 |
0,1bc + 0.01 |
Wastewaters "La chacona" |
0,01ª + 0.00 |
0,002b + 0,00 |
< 0,01c + 0.00 |
0,08c + 0.02 |
Pijijiapan river |
0,02a + 0.01 |
0,002b + 0.00 |
0,04b + 0.01 |
0,04d + 0.02 |
Santo Domingo river |
0,03a + 0.02 |
0,006a + 0.00 |
0,08a + 0.02 |
0,03d + 0.01 |
* Mean values of three replicates;
** Means (±standard error) within each column, with no common superscript, differ significantly at P <0.05.
The Santo Domingo River presented the highest concentrations of nitrogen compared to the other hydrographic areas. This is related to the relative abundance in the identification of morphotypes, since it was the area with the highest number of morphotypes identified (29%).
Analysis of minerals in the water samples with an Atomic Absorption Spectrometer
In the evaluation of minerals, the 8 hydrographic zones of the analyzed Chiapas state showed concentrations lower than 0.2 mg/l of microelements such as copper, zinc, iron and manganese. However, the Santo Domingo river showed the highest concentrations of sodium, potassium, magnesium and calcium with 62.93, 5.46, 34.52 and 48.78 mg/l, respectively. On the other hand, the Pijijiapan river showed the lowest concentrations of sodium, potassium, magnesium and calcium with 8.87, 0.05, 0.81 and 6.59 mg/l, respectively (Table
Evaluation of the concentration of macroelements and microelements of water samples from the different hydrographic zones of the state of Chiapas, Mexico.
Sampling areas |
Microelements |
Macroelements |
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Copper (mg/l) |
Zinc (mg/l) |
Iron (mg/l) |
Manganese (mg/l) |
Sodium (mg/l) |
Potassium (mg/l) |
Magnesium (mg/l) |
Calcium (mg/l) |
|
Suchiate river |
< 0.2a* + 0.01 |
< 0.2a + 0.01 |
< 0.2a + 0.00 |
< 0.2a + 0.01 |
17,09d + 0.11 |
2,77ab + 0.01 |
5,89d + 0.02 |
14,36c + 0.11 |
Nandayapa river |
< 0.2a + 0.01 |
< 0.2a + 0.01 |
< 0.2a + 0.01 |
< 0.2a + 0.11 |
57,87b + 0.12 |
4,13a + 0.12 |
21,45b + 0.16 |
51,06a + 0.15 |
Huixtla river |
< 0.2a + 0.03** |
< 0.2a + 0.00 |
< 0.2a + 0.01 |
< 0.2a + 0.00 |
23,71c + 0.03 |
2,76ab + 0.11 |
13,74c + 0.04 |
13,78c + 0.12 |
Spring water Cahuaré |
< 0.2a + 0.02 |
< 0.2a + 0.01 |
< 0.2a + 0.02 |
< 0.2a + 0.02 |
17,06d + 0.17 |
2,41ab + 0.10 |
14,97c + 0.09 |
45,62a + 0.13 |
Novillero river |
< 0.2a + 0.01 |
< 0.2a + 0.02 |
< 0.2a + 0.01 |
< 0.2a + 0.01 |
9,07e + 0.14 |
0,92b + 0.01 |
1,63e + 0.02 |
15,06c + 0.09 |
Wastewaters "La chacona" |
< 0.2a + 0.01 |
< 0.2a + 0.01 |
< 0.2a + 0.00 |
< 0.2a + 0.00 |
10,82e + 0.08 |
1,69b + 0.11 |
13,51c + 0.11 |
40,50b + 0.12 |
Pijijiapan river |
< 0.2a + 0.00 |
< 0.2a + 0.03 |
< 0.2a + 0.03 |
< 0.2a + 0.02 |
8,87e + 0.07 |
0,05c + 0.09 |
0,81e + 0.01 |
6,59d + 0.09 |
Santo Domingo river |
< 0.2a + 0.01 |
< 0.2a + 0.02 |
< 0.2a + 0.00 |
< 0.2a + 0.00 |
62,93a + 0.14 |
5,46a + 0.14 |
34,52a + 0.12 |
48,78a + 0.11 |
*Mean values of three replicates.
**Means (±standard error) within each column, with no common superscript, differ significantly at P <0.05.
Isolation, purification, identification and determination of size of microalgae
In our study, more than twenty-one isolates were isolated from the collected water samples, but only thirteen axenic microalgae isolates were selected and sub-cultured on slants on its specific isolation media (BG11) and kept in a refrigerator for further investigation due to their purity. The isolation of microalgae was developed with the use of BG11 and CHU culture media from which, after a period of 90 days with a 12/12 photoperiod, it was demonstrated that the most efficient means for the isolation of microalgae was the medium BG11 with an 80.53% effectiveness. The CHU medium was, however, efficient at 19.46%, showing a statistically significant difference with α= 0.05 (Fig.
Relative abundance of the morphotypes and classes of microalgae identified in the different hydrographic zones evaluated in the state of Chiapas. SR: Suchiate river; NAR: Nandayapa river; HR: Huixtla river; SWC: Spring water Cahuaré; NR: Novillero river; WC: Wastewaters "La chacona"; PR: Pijijiapan river; SDR: Santo Domingo river.
Microalgae identified in the different hydrographic zones of the state of Chiapas using a FlowCam coupled to a microscope (100x). Suchiate river: a) Monoraphidium contortum; b) Neospongiococcum gelatinosum; c) Desmodesmus serratus; Nandayapa river: d) Raphidonema nivale; e) Nephrocytium lunatum; Huixtla river: f) Asterococcus superbus; g) Chlorococcum echinozygotum; Spring water Cahuaré: h) Monoraphidium contortum; i) Scenedesmus quadricauda; j) Monoraphidium griffithii; Novillero river: k) Chlorococcum echinozygotum; l) Leptolyngbya sp.; m) Microspora floccosa; Wastewaters "La chacona": n) Asterococcus superbus; Pijijiapan river: ñ) Chlorococcum echinozygotum; Santo Domingo river: o) Oscillatoria brevis; p) Monoraphidium contortum; q) Chlorococcum echinozygotum; r) Desmodesmus serratus; s) Closteriopsis acicularis; t) Hydrodictyon reticulatum.
Evaluation of microalgal biomass production in BG11 and CHU medium after a period of 90 days with a 12/12 photoperiod for the different hydrographical areas of the state of Chiapas: SR: Suchiate river; NAR: Nandayapa river; HR: Huixtla river; SWC: Spring water Cahuaré; NR: Novillero river; WC: Wastewaters "La chacona"; PR: Pijijiapan river; SDR: Santo Domingo river. Bars are + one standard deviation. A means values of three replicates. The means followed by the same letter are not significantly different (P-value <0.05).
In Fig.
Of the water samples analyed, 13 microalgae morphotypes were isolated. Of the microalgal consortia identified, 90.47% of a total of 21 microorganisms are composed of microalgae belonging to the class Chlorophyceae and9.52% correspond to the class Cyanophyceae, as shown in Fig.
It is important to mention that the areas with the highest microalgae specific richness were the Santo Domingo River of the municipality of Chiapa de Corzo with 6 identified morphotypes corresponding to 28.57%, followed by the river Suchiate of the municipality of Suchiate with 3 identified morphotypes corresponding to 14.28% of the relative abundance (Fig.
According to morphological examination under a microscope based on cell shapes, fourteen microalgal isolates were identified as Monoraphidium contortum, Neospongiococcum gelatinosum, Desmodesmus serratus, Raphidonema nivale, Nephrocytium lunatum, Asterococcus superbus, Chlorococcum echinozygotum, Scenedesmus quadricauda, Monoraphidium griffithii, Leptolyngbya sp., Microspora floccosa, Oscillatoria brevis, Closteriopsis acicularis and Hydrodictyon reticulatum. The aforementioned morphotypes are shown in Fig.
As can be seen in Fig.
Of the 14 identified morphotypes, the measurements were between 10 μm to 150 μm in length. The identified morphotypes of larger size were Monoraphidium griffithii and Microspora floccosa with 50 and 150 μm, respectively, both corresponding to the class Chlorophyceae (Table
Analysis of the morphotypes identified in the hydrographic zones of the state of Chiapas according to the environment of isolation, class, size and morphological characteristics.
N. |
Name |
General environment |
Size (μm) |
Characteristics |
References |
CLOROPHYCEAE | |||||
1 |
Monoraphidium contortum ID: 307511 |
Sweet water |
40 |
It has a fusiform body, narrow towards the extremities, with a sharp point, is sigmoid and also contains two parietal chloroplasts |
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2 |
Neospongiococcum gelatinosum ID: 1158268 |
Land |
21 |
Multinucleate species, formed by a rigid wall, is characterized by being individualistic and staying inside a gelatinous sphere |
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3 |
Desmodesmus serratus ID: 91204 |
Sweet water |
26 |
Cenobios 2-4-8 linear cells. Ovoid cells with 1-4 teeth at the ends that are rounded or truncated, external convex walls |
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4 |
Raphidonema nivale ID: 155715 |
Freshwater/terrestrial species |
20 |
A filamentous structure, forming a green, non-greenish cenocito approximately 0.5 μm thick with a polar tip |
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5 |
Nephrocytium lunatum ID: 1662585 |
Sweet water |
31 |
Colonies of 2-4 cells, embedded within a gelatinous sheath; cell body in the shape of a crescent, contains a single plate as a chloroplast with a pyrenoid |
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6 |
Asterococcus superbus ID: 269637 |
Sweet water |
25 |
The body is spherical and ellipsoidal. It has a smooth and transparent cell wall containing a single star-shaped chloroplast, a single nucleus and two contractile vacuoles |
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7 |
Chlorococcum echinozygotum ID: 48000 |
A terrestrial species |
15 |
Almost spherical cells. Membrane not thickened in anterior papilla. Chloroplast with a large pyrenoid. Large eye patch. |
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8 |
Scenedesmus quadricauda ID: 3089 |
Sweet water |
15 |
Colonial individual, consisting of 4, 8 or 12 cells. The central cells are elongated and without appendages, the terminals bulge in the centre and present two spines that project towards the outside. |
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9 |
Monoraphidium griffithii ID: 307514 |
Sweet water |
50 |
Spindle cells, straight, more than 12 times longer than wide (50-72 x 1.5-4.5 μm), attenuated towards the ends and terminated at a short point. Clearly constricted parietal chloroplast in the centre and without pyrene |
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10 |
Hydrodictyon reticulatum ID: 3107 |
Fresh and wastewater |
30 |
It has a transparent net shape with a green sack-like shape with thousands of cylindrical oblong-oval cells, with thick and angled walls |
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11 |
Microspora floccosa ID: 1603044 |
Sweet water |
150 |
Green H-shaped row, formed by cylindrical cells, the ends of the filaments have an adhesive disc with which they are fixed, chloroplasts do not enclose pyrenoids |
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12 |
Closteriopsis acicularis ID: 82138 |
Sweet water |
10 |
Single-celled stems elongated and pointed tips, central or parietal chloroplasts in the form of band, formed by multiple pyrenoids of 2 to 14. |
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CYANOPHYCEAE |
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13 |
Oscillatoria brevis ID: 177969 |
Sweet water |
70 |
Each filament consists of trichomes that are composed of rows of cells, formed by fragments called hormogonias. The tip of the tricoma oscillates like a pendulum, of green brown colour. |
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14 |
Leptolyngbya sp. ID: 47254 |
Fresh and wastewater |
62 |
Long, solitary or coiled filaments in groups and thin mats formed by generally colourless facultative pods attached to or slightly distant from the trichomes which are composed of rounded apical cells |
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The algae were identified through the morphological analysis based on the general features of the freshwater algae that occur most frequently and are described by
Determination of the microalgal biomass in BG11 and CHU medium
After a period of 90 days with a 12/12 photoperiod, the evaluation of the BG11 and CHU medium was developed to determine the effectiveness of both media in the production of microalgal biomass that allowed the identification of the existing morphotypes of microalgae. For that effect, greater microalgal biomass production was demonstrated in the BG11 medium from 1.4 to 10 g while the CHU medium showed a production of 1 to 1.4 g of microalgal biomass, so that a significant statistical difference between the two media was observed. Therefore, the BG11 medium is the better medium for the isolation and biomass production (Fig.
It was observed that, in the Suchiate river and the Santo Domingo river, greater growth of microalgal biomass was demonstrated in the BG11 medium with 10 g and 9.2 g respectively after 90 days of inoculation (Fig.
In the present research work, 14 morphotypes were identified according to their qualitative and quantitative morphological characteristics (Table
The Santo Domingo River was the area with the highest number of morphotypes identified, the results showing the importance of nitrogen as explained by
Another important element for the production of microalgae is phosphorus because it is a macroelement that is found in smaller proportions than the rest in the biomass and are used by microalgae in the synthesis of enzymes, lipids and nucleic acids (
According to the results obtained in Table
The genera identified are part of the chlorophyce class whose abundance was evaluated by
Therefore, in the present research work, it is demonstrated that the nutrient richness of the different hydrographic areas allows the identification of the population dynamics of the microalgae in specific taxonomic groups, as was demonstrated by
Of the 13 species identified in the present study, 5 have been studied in order to identify fatty acids for the production of biodiesel, so that in Table 5, it can be seen that the lipid content in the identified microalgae of hydrographic areas of the state of Chiapas can be from 2% to 90%, according to studies developed by
Lipid content of five microalgae with potential for biodiesel production identified in the hydrographic zones of the state of Chiapas, Mexico.
Species of microalgae |
Lipid content (%) |
References |
Monoraphidium contortum |
2-20 |
|
Chlorococcum echinozygotum |
10-43 |
|
Scenedesmus quadricauda |
11-55 |
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Microspora floccosa |
04-90 |
|
Dunaliella salina |
09-47 |
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Observing the potential of Microspora floccosa to produce up to 90% of lipids, we can generate a relationship between the nutrients present in the medium from which they were isolated. Thus we observed that this morphotype comes from the Novillero river, which is characterized by low concentrations of nitrate, nitrite and ammonium with 0.02, 0.005 and <0.001 mg/l, respectively and the lowest concentration of sodium with 9.07 mg/l, so that this relationship benefits the synthesis of lipids in favour of the production of bioenergetics as demonstrated by
In the present study, the BG11 culture medium showed higher efficiency than the CHU medium in the production of microalgal biomass, the phenomenon observed being demonstrated by
The most important nutrients are those that represent sources of carbon and nitrogen in the culture medium (
At the metabolism level, carbon is vital for microalgae as an energy generator from the Calvin cycle (
It is important to mention that the identification of new morphotypes in different hydrographic space generates an area of scientific research to identify the biotechnological potential of microalgae associated with the high generation of lipids, proteins, pigments or as environmentally beneficial microorganisms involved in bioremediation processes. Thus the new isolations also allow the observation of the taxonomic evolution of microalgae classes, demonstrating the conservation, loss and/or appearance of biomolecules (
Fourteen morphotypes were isolated and identified according to the morphological classification of
The authors gratefully acknowledge to the 3IER, UPCH, for financial support.
The institution hosting were 3IER, UPCH, for financial support.