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Metagenomic Analysis Reveals the Presence of Heavy Metal Response Genes from Cyanobacteria Thriving in Balatoc Mines, Benguet Province, Philippines

Libertine Rose S. Sanchez1 and Ernelea P Cao1,2

1Institute of Biology (IB), College of Science (CS)
University of the Philippines (UP) Diliman, Quezon City 1101 Philippines
2Natural Sciences Research Institute (NSRI), CS, UP Diliman, Quezon City 1101 Philippines

*Corresponding Author: This email address is being protected from spambots. You need JavaScript enabled to view it.

 

 


ABSTRACT

Tailing ponds of mining sites heavily contaminated with metals is a serious problem in many parts of the world. Metagenomic sequencing and bioinformatics analysis of water samples from the Balatoc mine tailings – an abandoned mining site in Itogon, Benguet, Philippines – revealed microbial communities, particularly cyanobacteria consortia that implied their ability to survive in metal-stressed environments. Thus, their presence can be further investigated for applications in bioremediation. Surface water samples were collected from three sampling points in the Balatoc mine tailings. Physicochemical properties of the samples were also determined. Genomic DNA was extracted from all water samples and subjected to shotgun sequencing using Illumina NextSeq2500 2 x 150 paired ends. Thirty-eight (38) Gbases raw reads obtained from three data sets showed similar microbial assemblages using St. Petersburg genome assembler (SPAdes v3.10.1). Taxonomic assignments to contigs using CLAssfier based on reduced K-mers (CLARK) revealed the relative abundance of 97% Bacteria and 3% Archaea. All sampling sites were found to have relatively the same physicochemical properties. The abandoned Balatoc tailing site exhibited high temperature (31.50 ˚C), alkaline pH (8.42), and elevated levels of copper (Cu2+) (1.53 mg/L) and zinc (Zn2+) (0.077 mg/L). A CLARK v1.2.5 custom database of cyanobacteria was also used to determine the classification, taxonomic assignment, as well as the estimation of percentage relative abundance of the cyanobacteria. Taxonomic assignments of all metadata revealed a dominant cyanobacterium, classified as Leptolyngbya sp., which comprises about 3% of the assembled contigs. Prokka v1.12 was used for annotation and protein-coding sequences (CDS) were evaluated for gene ontology (GO) using the evolutionary genealogy of genes-Non-supervised Orthologous Groups (eggNOG) Mapper v4.5. The genes conferring stress-response to metal ions Cu2+, Zn2+, lead (Pb2+), and cadmium (Cd2+) are reported to be involved in efflux/transport functions and heavy metal resistance that can be major attributes of Leptolyngbya sp. for survival to extreme metal conditions.

 


INTRODUCTION

Cyanobacteria are a group of diverse Gram-negative, oxygenic, photoautotrophic prokaryotes. They have high adaptability; hence, they are widely distributed in extreme environments like radioactive wastewater and polluted watersheds (Liu et al. 2015, Smucker et al. 2018); mine tailings (Orlekowsky et al. 2013. Dhal and Sar 2014, Goswami et al. 2015, Hazarika et al. 2015, Seiderer et al. 2017, McCutcheon et al. 2017, Sibanda et al. 2019); geothermal habitats (Debnath et al. 2009, Ward et al. 2012, Dadheech et al. 2013, Roy et al. 2014, 2017, Castenholz 2015); ice-capped lakes (Singh and Elster 2007, Cirés et al. 2017); and Arctic and Antarctic rocks (Whitton and Potts 2000, Strunecky et al. 2012). They are also reportedly found in industrial effluents (Vijayakumar 2005, 2012; Vijayakumar et al. 2005, 2007) such as those found in an oil refinery, fertilizer factory, brewery (Kumar et al. 1974), tannery (Dhamotharan et al. 2008), and even in the dye industry (Vijayakumar et al. 2005). Cyanobacteria have a high multiplication rate and metal absorption capacity (Vijayakumar 2012). They are commonly regarded as blue-green algae, which have a superficial resemblance to green algae due to chlorophyll a (chlora) pigments and phycocyanin, with some species proven to be agents for bioremediation (Ananya and Ahmad 2014). Studies conducted by Kuenen et al. 1986 on cyanobacteria revealed that these photosynthetic prokaryotes provide a favorable condition for the removal of heavy metals from the environment since their interior pH is almost two units higher than the surrounding liquid. Cyanobacteria forming mats, such as   Phormidium and Oscillatoria, were reported to absorb hexavalent chromium (Cr6+) from tannery effluents (Balaji et al. 2016) and copper from mine wastewater (Chaturvedi et al. 2013). An immobilized cyanobacterium Anabaena doliolum exhibited an increased uptake of Fe3+ and Cu2+ than that of the free-living cells (Rai and Mallic 1992). Cyanobacteria are able to protect cells in the presence of excessive metals by sequestering them (Huertas et al. 2014). They have been found to possess several mechanisms for metal homeostasis, as well as proteins that can be used to assess their metal bioremediation potential. . . . read more

 

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