European Biomass Conference and Exhibition 2017 ...

25th European Biomass Conference and Exhibition, 12-15 June 2017, Stockholm, Sweden

MICROALGAL S PEC IES PROS PECTING FROM DIFFERENT ECOLOGICAL HABITATS IN ALGERIA Lylia Hamel1*, Douadi Khelif2, Daniel Grzebyk3, UM R 9190 M ARBEC M arine Biodiversity, Exploitation and Conservation, IFREM ER, CNRS, IRD, Université M ontpellier, Station M arine IFREM ER, avenue Jean M onnet, 34200 Sète, France 2 Laboratory of Genetic Biochemistry and Plant Biotechnology, Faculty of Nature and Life Sciences, Université Constantine 1, Constantine, Algeria 3 UM R 9190 M ARBEC M arine Biodiversity, Exploitation and Conservation, IFREM ER, CNRS, IRD, Université M ontpellier, CC 093, Place Eugène Bataillon , 34095 M ontpellier, France 1* [email protected] / [email protected] / Phone: +33622657181 1

ABSTRACT: There are hundreds of thousands to millions of microalgal species, most of which have not been isolated or characterized. Organisms isolated from extreme environments (extremophiles) often possess unique physiological responses that enable their survival in stressful conditions such as extreme light, temperatures or pH, nutrient limitation or high salinity. Algeria hosts the largest desert in the world, with a surface of 450000 km2 and is submitted to a large range of climatic environments from Saharan arid to temperate climates, where the high diversity of microalgae strains remains almost completely undiscovered. The present study reports the pigment characterization of 17 microalgae species isolated from a large longitudinal range of different ecological habitats in Algeria. Algal isolation was performed using a three different selective media (3NBBM , BG11 and diatom medium) under standardized growth conditions. Identification was done using microscopic observations, supported by screening and identification of several 18S DNA and 16S DNA sequencings. Chlorophycean and Cyanophycean were the most dominant of all the isolated algal groups. The highest number of isolated strains was hosted by the Afilal habitat located in the national park of Hoggar in the heart of the Sahara, and Temekrest experimenting intense solar radiation and a large range of temperatures. This study highlights the importance adaptation mechanisms developed by some rare strains isolated in natural guelta under saharan climate, such as Carteria crucifera or Pachycladella umbrina, or cyanobacterial species such as Synechocystis salina. These study highlights the assessment of some species have attracted interest in the field of algal biotechnology, and represent a step toward the development of strains suitable for exteremophiles arid area at the industrial scale. Keywords: Carteria Crucifera, Pachycladella umbrina LH2, ecological habitat, phylogeny, biomass mixture

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M icroalgae and cyanobacteria can be grown in nonarable land and require far less land than terrestrial crops, thus not competing with agriculture and not compromising food production and supply. These microorganisms can also grow in a wide variety of environmental conditions and also in low quality waters, reducing the requirements for freshwater [6].

INTRODUCTION

By some estimates, there are hundreds of thousands to millions of microalgal species, most of which have not been isolated or characterized [1]. Organisms isolated from extreme environments (extremophiles) often possess unique physiological responses that enable their survival in stressful conditions such as extreme light, temperatures or pH, nutrient limitation or high salinity [2]. Additionally, microalgal strains within the same species can exhibit different physiological activities and metabolite profiles depending on habitat and physicochemical condition range, i.e., phenotypic plasticity within a single genotype. It is therefore expected that strains belonging to the same, or closely related, species but isolated from very different environments will demonstrate different physiological responses or produce different levels of biomolecules [3]. The Algerian desertic climate is an extreme environment that covers nearly 2 × 10 6 km2 and experiences intense solar rad iation and large range of temperatures, the coldest month is January (−10 to 3 °C), the hottest months are July and August (45–56 °C). Some microalgal strains isolated from the Sahara have been investigated for their ability to produce hydrogen [4], and Fatty Acid and some carotenoids like astaxanthin and canthaxanthin [5]. In the present study, we have isolated microalgal strains from different temperate, arid, Saharan and desertic climates in Algeria, where the diversity of microalgae remains largely undiscovered. All the isolated strains were identified at the genus or species level and were analyzed and discussed in relation to the phylum or initial habitat characteristic.

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MATERIALS AND M ETHODS

M icroalgae was sampled on November 2014 from a large latitudinal range, from 18 to 2012 m of altitudes and from varying temperate climate in the region of Bejaia (M ezaia Lake) on the M editerranean side, Sidi Rached lake from the sub saharan arid climate and in the south sahara, in tamanrasset city, with the saharan climate with freshwater temekrest native gueltat. The degree of salinity, of anthropisation, length of photoperiod, temperature and climates are widely varied. Our study reports the assessment profile of 17 microalgae species isolated from these habitats (Table 1). Algal isolation was performed using a different selective medium (3NBBM , BG11 and diatom medium) with a nearest average content of salinity and under standardized growth conditions (26°C, 12:12 photoperiod at 120 μE m-2 s-1) in batch and non-axenic culture collection; serial dilutions followed by plating and regular microscopic observations. Identification was done using microscopic observations, supported by screening and identification of several 18S DNA, 16S DNA, ITS1, ITS2 (the internal transcribed spacer) and 28S rRNA sequencings analysis.

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TCCTGCCAGTAGTCATATGC-3’), and the Chlorophycea etargeting primer CHL-1747-19-R (reverse,5'-CCRATCCGAAMACTTCACC-3') designed in this study. The PCR reactions were run in 50µL volume as follows: denaturing at 95°C for 10 s, 30 cycles including 10s at 95°C, 10s annealing at 55°C and 2 min elongation at 68°C, and a final elongation at 68°C for 5 min. Amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen, Germany). The purified amplicons were sequenced using the BigDye Terminator V3.1 Cycle Sequencing Kit with the 18S-F PCR primer and three internal sequencing primers; the reactions were run on a ABI 3130 XLGenetic Analyzer (Applied Biosystems, USA). The sequencing chromatograms were checked by eye and the ambiguities were cleared. The amplicon sequence was reconstructe dusing the CAP contig assembly program in the BioEdit program [13]. The obtained 1664 nucleotide-long sequence was deposited in GenBank under the accession number ZZ000000.

Strains screening and growth conditions:

Table 1: Growth condition of Culture collection of 17 strains maintained under standardized condition (26 °C of continuous temperature and 12:12 of imposed photoperiod).

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RESULTS AND DISCUSSION Figure1:(a) carteria crucifera)(x40)(x100)(x100) (b) navicula sp(x100) - (c) acutodesmus obliquus(x100) (d) spirogyra sp(x40) - (e)Synechocystis salina (x40) -. (f) Anabaena sp(x40) - (g)Chlorelle sp(x100) (h) oscillatoria sp (x40).

The strain Carteria crucifera belonging to the Chlorophyta Phylum, and Chlamydomonadacea family, was formerly classified among Chlorococcales [7]. Carteria is a genus of unicellular, quadriflagellate green algae with worldwide distribution [8] . Approximativelly 60 species have been described, based on morphological differences (Ettl1979). The phylogeny of Carteria was studied on the basis of rRNA and organismal data [9]. Cells elongate-ovoid to ellipsoidal, with two contractille vacuoles at the base of the flagella. Chloroplast Hshaped in longitudinal section, with longitudinal fissures on the surface with flagella and a similar morphology to Chlamydomonas sp,Flagellar length equal to or slightly shorter than cell length. Cells 13-27 um long and 8-17 um wide. Distribution: Europe [6] [7] North America [10], Japan [11]. The molecular approach allowed us to identify a new strain, Pachycladella umbrina LH2 from the chlorophyceans families, the LH2 and three of the chlamydomonadaceae family strains made a consistent group. This group belongs to a clade closely related to Pachycladella umbrina (DQ009775) [12]. Cultured LH2 cells were collected by centrifugation and genomic DNA extraction was performed as described in Caporaso G. (2012). The PCR reactions were performed in a M astercycler Ep Gradient S thermal cycler (Eppendorf), using the PrimeSTAR GXL DNA Polymerase Kit (Takara Bio Inc., Japan) containing a high-fidelity enzyme.The 18S rDNA amplicons were generated using the eukaryotic primer 18S-F (forward,5’-

3.1 M olecular phylogenetic analysis The selection of reference sequences was done using the web interface NCBI BLA ST [14] both using M OLEBLAST and BLASTX similarity analyses [15] against the GenBank Nucleotide database. Alignments were generated with Clustal X 2.1 [16] and whenever it was necessary, manual corrections were done using the BioEdit program [17]. Phylogenetic analyses were performed using the online application Phylogeny.fr [18] implemented on the ATGC South of France BioinformaticsPlatform(http://www.atgc-montpellier.fr/) using the ―A-la-Carte‖ mode. M aximum likelihood analysis is performed using PHYM L 3.0 [19] with the following default settings: HKY85 substitution model, 4 categories of substitution rates, and with the Gamma distribution parameter, the proportion of invariable sites and the transition/transversion ratio estimated by the program.

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users, or marine and freshwater, aiming to improve both biomass productivities and the resilience of the consortium [22].Our prospecting analysis highlights some original habitats hosting the most of species : the region of Afilal with the Saharan climate at 23° 8' 58'' N 5°46' 2'' E in the region of Afilal National Park hosts: the chlorophycean Carteria crucifera , the diatoms Naviculas sp, the cyanobateria Anabaena sp and Spirogyra sp with the highest altitude 2012 m. Temekrest habitat at 22°7’85’N 5°72’48’’E, hosting the chlorophycean acutodesmus obliquus, the cyanobacteria nostoc sp and chrococcus sp with the 1367m of altitude and M enia habitat at 30°45’39’’91’’N 2°88’85’’71’’E hosting acutodesmus obliquus ,chlorella sp,synechocystis salina with 382 m of altitude.This study represent a step toward the development of strains suitable for extremophiles arid area at the industrial scale.

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[2]

Figure 2: M olecular phylogenic pachycladella umbrina LH2

analysis

of [3]

This species was isolated from a guelta resulting of a 4-month crystalline lake evaporation in cristallin lake at 23°02’33’’ N 5°.42’15’’E with 1535 of altitude.

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CONCLUSIONS [4]

Cultivation of microalgae and cyanobacteria has been the focus of several research studies worldwide, due to the huge biotechnological potential of these photosynthetic microorganisms. However, production of these microorganisms is still not economically viable. One possible alternative to improve the economic feasibility of the process is the use of consortia between microalgae and/or cyanobacteria [20]. However, several studies refer to the use of polycultures for single applications [21] have studied biomass and lipid productivities in single and mixed cultures of Chlorococcum sp., Scenedesmus sp., Chlorella sp. and Phaeodactylum tricornutum, concluding that mixed cultures were more productive than single ones in terms of biomass and lipids. Additionally, the study of a microalgal consortium composed by the freshwater microalgae Chlorella sp. and Scenedesmus sp. resulted in more effective removal of nitrogen and phosphorus from the culture medium when compared to single cultures of these microorganisms.because it is very difficult to select the microorganisms integrating the consortia. One possible alternative is to combine, for example, photoautotrophs and mixotrophs, ammonia and nitrate

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ACKNOWLEDGEM ENTS

This activity was possible thanks to UMR 9190 MARBEC; University of Montpellier. financed by: PROFAS and UM R 9190 MARBEC.

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