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
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.
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
ALVARENGA DO, FIORE MF, VARANI AM. 2017. A metagenomic approach to cyanobacterial genomics. Front Microbiol 8: 809.
ANANYA AK, AHMAD IZ. 2014. Cyanobacteria “the blue green algae” and its novel applications: A brief review. IJIAS 7: 251–261.
ANDREWS S. 2010. FastQC: A quality control for high throughput sequence data. Retrieved from httpp://www.bioinformatics.babraham.ac.uk/projects/ fastqc
BALAJI S, KALAIVANI T, SHALINI M, GOPALAKRISHNAN M, MUHAMMAD MAR, RAJASEKARAN C. 2016. Sorption sites of microalgae possess metal binding ability towards Cr (VI) from tannery effluents – A kinetic and characterization study. Desalin Water Treat 57: 31.
BERGMAN B, RAI AN, RASMUSSEN U. 2007. Cyanobacterial associations. In: Elmerich C, Newton WE eds. Associative and endophytic nitrogen-fixing bacteria and cyanobacterial associations. New York: Springer. p. 257–301.
BOLGER AM, LOHSE M, USADEL B. 2014. Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, btu170.
BUCHFINK B, XIE C, HUSON DH. 2015. Fast and sensitive protein alignment using DIAMOND. Nat Methods 12: 59–60.
CASTENHOLZ RW. 2015. Portrait of a geothermal spring, Hunter’s hot springs, Oregon. Life 5: 332–347.
CAWIS RMM. 2018. NTFMC closes small-scale mining operations in Itogon. Retrieved from https://pia.gov.ph/news/articles/1013819
CHATURVEDI V, CHANDRAVANSHI M, RAHANGDALE M, VERMA P. 2013. An integrated approach of using polystyrene foam as an attachment system for growth of mixed culture of cyanobacteria with concomitant treatment of copper mine waste water. J Waste Manage p. 1–7.
CHEN L, ZHU Y, SONG Z, WANG J, ZHANG W. 2014. An orphan response regulator Sll0649 involved in cadmium tolerance and metal homeostasis in photosynthetic Synechocystis sp. PCC6803. J Proteomics 103: 87–102.
CIRÉS S, CASERO MC, QUESADA A. 2017. Toxicity at the edge of life: A review on cyanobacterial toxins from extreme environments. Mar Drugs 15(7).
CLIMATE-DATA.ORG. 2019. Climate Graph/Weather by Month: Itogon. Retrieved from https://en.climate-data.org/asia/philippines/benguet/itogon-20187/#climate-graph
BENGUET CORPORATION. 2018. Company Profile. Retrieved from http://benguetcorp.com/home/about-us/
COURTNEY R. 2013. Mine tailings composition in a historic site: implications for ecological restoration. Environ Geochem Health 35: 79–88.
DADHEECH PK, GLÖCKNER G, CASPER P, KOTUT K. 2013. Cyanobacterial diversity in the hot spring, pelagic and benthic habitats of a tropical soda lake. FEMS Microbiol Ecol 85(2): 389–401.
DEBNATH M, MANDAL NC, RAY S. 2009. The study of cyanobacterial flora from geothermal springs of Bakreswar West Bengal, India. Algae 24(4): 185–193.
DE GUZMAN MA, CAO EP. 2010. Cadmium-binding ability of the blue-green algae Hapalosiphon welwitschii under controlled conditions. Philipp Sci Lett 3(1): 76–86.
DHAL PK, SAR P. 2014. Microbial communities in uranium mine tailings and mine water sediment from Jaduguda U mine, India: A culture-independent analysis. J Environ Sci Health A Tox Hazard Subst Environ Eng 49: 694–709.
DHAMOTHARAN R, MURUGESAN S, YOGONANDAM M. 2008. Bioremediation of tannery effluent using cyanobacteria. Biosci Biotechnol Res Asia 5(1).
DOUZERY EJP, SNELL EA, BAPTESTE E, DELSUC F, PHILLIPE H. 2004. The timing of eukaryotic evolution: Does a molecular clock reconcile proteins and fossils? PNAS 101(43): 15386–15391.
ESCOBAR-ZEPEDA A, GODOY-LOZANO EE, RAGGI L, SEGOVIA L, MERINO E, GUTIERREZ-RIOS RM, JUAREZ K, LICEA-NAVARRO AF, PARDO-LOPEZ S, SANCHEZ-FLORES A. 2018. Analysis of sequencing technologies for taxonomic annotation: Defining standards for progressive metagenomics. Sci Rep 8: 12034.
GHOSH S, DAS AP. 2018. Metagenomics insights into the microbial diversity in manganese-contaminated mine tailings and their role in biogeochemical cycling of manganese. Sci Rep 8: 8257.
GOSWAMI S, SYIEM MB, PAKSHIRAJAN K. 2015. Cadmium removal by Anabaena doliolum Ind1 isolated from a coal mining area in Meghalaya, India: Associated physical and structural alterations. Environ Eng Res 20(1): 41–50.
GUREVICH A, SAVELEV V, VYAHHI N, TESLER G. 2013. QUAST: Quality assessment tool for genome assemblies. Bioinformatics 29(8): 1072–1075.
HATTINGH JM, van DEVENTER PW. 2004. The effect of the chemical properties of tailings and water application on the establishment of a vegetative cover on gold tailings dams [report no. 899/1/04]. Pretoria, South Africa: Pretoria Water Research Commission.
HAZARIKA J, PAKSHIRAJAN K, SYIEM MB. 2015. Bioremoval of Cu (II), Zn (II), Pb (II) and Cd(II) by Nostoc muscorum isolated from a coal mining site. J Appl Phycol 27: 1525–1534.
HUCKLE JW, MORBY AP, TURNER JS, ROBINSON NJ. 1993. Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions. Mol Microbiol 7: 177–187.
HUERTA-CEPAS J, FORSLUND K, CUELHO LP, SZKLARCZYK D, JENSEN LJ, von MERING C, BORK P. 2017. Fast genome-wide functional annotation through orthology assignment by eggNOG-maper. Mol Biol Evol 34(8): 2115–2122.
HUERTA-CEPAS J, FORSLUND K, CUELHO LP, SZKLARCZYK D, COOK H, HELLER D, WALTER MC, RATTEI T, MENDE DR, SUNAGAWA S, KUHN M, JENSEN LJ, von MERING C, BORK P. 2016. eggNOG-maper 4.5: A hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucl Acids Res 44(D1): D286–D293.
HUERTAS MJ, MAURY LL, LAMIA JG, SANCHEZ-RIEGO AM, FLORENCIO FJ. 2014. Metals in cyanobacteria: Analysis of the copper, nickel, cobalt and arsenic homeostasis mechanisms. Life 4: 865–886.
HYATT D, CHEN JL, LOCASCIO PF, LAND ML, LARIMER FW, HAUSER LJ. 2010. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinf 8(11): 119.
ILLUMINA. 2015. Retrieved from http//www.illumina.com/ on 05 Jan 2018.
JAO IKC. 2004. Isolation, characterization and rapid screening of cyanobacteria and microalgae from some mining areas for bioremediation of metal-polluted waters [MS Thesis]. Quezon City, Philippines: University of the Philippines Diliman – Institute of Biology. 98p.
KOLBE DL, EDDY SR. 2011. Fast filtering for RNA homology search. Bioinformatics 27: 3102–3109.
KUENEN JG, JONGENSEN BB, REVSBECH NP. 1986. Oxygen microprofiles of trickling filter biofilms. Water Res 20: 1589–1598.
KUMAR, HD, BISARIA GP, BHANDARI LM, RANA BC, SHARMA V. 1974. Ecological studies on algae isolated from the effluents of an oil refinery, a fertilizer factory, a brewery. Indian J Environ Health 14: 247–255.
LAGESEN K, HALLIN P, RODLAND EA, STAERFELDT HH, ROGNES T, USSERY DW. 2007. RNAmmer: Consistent and rapid annotation of ribosomal RNA genes. Nucleic acids Res 35(9): 3100–3108.
LASLETT D, CANBACK B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32: 11–16.
LIU X, HU W, HUANG X, DENG, H. 2015. Highly effective biosorption of Sr(II) from low level radioactive wastewater. Water Sci Technol 71(11): 1727–1733.
MENDEZ MO, MAIER RM. 2008. Phytoremediation of mine tailings in temperate and arid environments. Rev Environ Sci Biotechnol 7(1): 47–59.
McCUTCHEON JM, TURVEY CC, WILSON SA, HAMILTON JL, SOUTHAM G. 2017. Experimental deployment of microbial mineral carbonation at an asbestos mine: Potential applications to carbon storage and tailings stabilization. Minerals 7(10): 191.
McHARDY AC, RIGOUTSOS I. 2007. What’s in the mix: Phylogenetic classification of metagenome sequence samples. Curr Opin Microbiol 10(5): 499–503.
MORBY AP, TURNER JS, HUCKLE JW, ROBINSON NJ. 1993. SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: Identification of a Zn-inhibited DNA-protein complex. Nucleic Acids Res 21(4): 921–925.
MORTAZAVI R, ATTIYA S, ARIYA PA. 2015. Arctic microbial and next generation sequencing approach for bacteria in snow and frost flowers: Selected identification, abundance and freezing nucleation. Atmos Chem Phys 15: 6183–6204.
MOYNAHAN OS, ZABINSKI CA, GANNON JE. 2002. Microbial community structure and carbon-utilization diversity in mine tailings revegetation study. Restor Ecol 10: 77–87.
NURK S, MELESHKO D, KOROBEYNIKOV A, PEVZNER PA. 2017. metaSPAdes: A new versatile metagenomic assembler. Genome Res 27: 824–834.
OLAFSON RW. 1984. Prokaryotic metallothionein. Int J Pept Protein Res 24: 303–308.
OLAFSON RW, ABEL K, SIRE RG. 1979. Prokaryotic metallothionein: Preliminary characterization of a blue-green algae heavy metal binding protein. Biochem Biophys Res Commun 89: 36–43.
OLAFSON RW, LOYA S, SIM RG. 1980. Physiological parameters of prokaryotic metallothionein induction. Biochem Biophys Res Commun 95: 1495–1503.
ORLEKOWSKY T, VENTER A, van WYK F, LEVANTES A. 2013. Cyanobacteria and algae of goldmine tailings in Northwest province of South Africa. Nova Hedwigia 97: 281–294.
OUNIT R, LONARDI S. 2016. Higher classification sensitivity of short metagenomic reads with CLARK-S. Bioinformatics 32(24): 3823–3825.
OUNIT R, WANAMAKER S, CLOSE TJ, LONARDI S. 2015. CLARK: fast and accurate classification of metagenomic and genomic sequences using discriminative k-mers. BMC Genomics 16: 236.
PEREIRA S, MICHELETTI E, ZILLE A, SANTOS A, MORADAS-FERREIRA P, TAMAGNINI P, PHILIPPIS RD. 2011. Using extracellular polymeric substances (EPS)-producing cyanobacteria for the bioremediation of heavy metals: Do cations compete for EPS functional groups and also accumulate inside the cell? Micro Bio 157: 451–458.
PETERSEN TN, BRUNAK S, von HEIJNE G, NIELSEN H. 2011. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat Methods 8: 785–786.
RAI LC, MALLIC N. 1992. Removal and assessment of toxicity of Cu and Fe to Anabaena doliolum and Chlorella vulgaris using free and immobilized cells. World J Microbiol Biotechnol 8: 110–114.
ROBINSON NJ, GUPTA A, FORDHAM-SKELTON AP, CROY RRD, WHITTON BA, HUCKLE JW. 1990. Prokaryotic metallothionein gene characterization and expression: Chromosome crawling by ligation mediated PCR. Proc R Soc London, Ser B 242(1305): 241–247.
RODRIGUEZ-ESPELETA N. 2005. Monophyly of primary photosynthetic eukaryotes: Green plants, red algae and glaucophytes. Curr Biol 15: 1325–1330.
ROY S, DEBNATH M, RAY, S. 2014. The study of cyanobacterial flora of the geothermal springs at Panifala, West Bengal, India. Phykos 44(1): 1–8.
ROY S, GOPE M, RAY S. 2017. Cyanobacterial flora from Sidpur geothermal spring, Jharkhand, India – First report. Phykos 47(1): 76–87.
RZYMSKI P, KLIMASZYK P, MARSZELEWSKI W, BOROWIAK D, MLECZEK M, NOWINSKI K, PIUS B, NIEDZIELSKI P, PONIEDZIALEK B. 2017. The chemistry and toxicity of discharge waters from copper mine tailing impoundment in the valley of the Apuseni mountains in Romania. Environ Sci Pollut Res Int 24(26): 21445–21458.
SANCHEZ LRS, MAGBANUA FS, CAO EP. 2017. Isolation, characterization and rapid screening of copper-tolerant cyanobacteria consortia from mining sites and a strawberry farm in Benguet province, Philippines. J ISSAAS 23(2): 8–19.
SEEMANN T. 2014. Prokka: Rapid, prokaryotic genome annotation. Bioinformatics 30(14): 2068–2069.
SEIDERER T, VENTER A, van WYK F, LEVANETS A, JORDAAN A. 2017. Growth of soil algae and cyanobacteria on gold mine tailings material. S Afr J Sci 113(11/12).
SEMEDO-AGUIAR AP, PEREIRA-LEAL JB, LEITE RB. 2018. Microbial diversity and toxin risk in tropical freshwater reservoirs of Cape Verde. Toxins 10(5): 186.
SIBANDA T, SELVARAJAN R, MSAGATI T, VENKATACHALAM S, MEDDOWS-TAYLOR S. 2019. Defunct gold mine tailings are natural reservoir for unique bacterial communities revealed by high-throughput sequencing analysis. Sci Total Environ 650: 2199–2209.
SINGH SM, ELSTER J. 2007. Cyanobacteria in Antarctic lake environments: A mini review. In: Seckbach J ed. Algae and cyanobacteria in extreme environments. Dordrecht: Springer. p. 303–320.
SMUCKER N, SCHAEFFER B, BEAULIEU J, NIETCH C. 2018. Satellite-based monitoring of cyanobacteria blooms from 2002–2011 for 11 reservoirs with watersheds along an agricultural gradient. Society for Freshwater Science Annual Meeting; 20–24 May 2018; Detroit, MI, USA: Environmental Protection Agency.
SONG WH, THOMAS T. 2017. Binning refiner: improving genome bins through the combination of different binning programs. Chem Soc Rev 43(20): 6954–6981.
STEVENSON FJ, COLE MA. 1999. Cycles of Soil (Carbon, Nitrogen, Phosporus, Sulfur, Micronutrients). Hoboken: John Wiley and Sons Publishers. 427p.
STRUNECKY O, ELSTER J, KOMAREK J. 2012. Molecular clock evidence for survival of Antarctic cyanobacteria (Oscillatoriales, Phormidium autumnale) from Paleozoic times. FEMS Microbiol Ecol 82(2): 482–490.
TESSLER M, NEUMANN JS, AFSHINNEKOO E, PINEDA M, HERSCH R, VELHO LFM, SEGOVIA BT, LANSAC-TOHA FA, LEMKE M, DeSALLE R, MASON CE, BRUGLER MR. 2017. Large-scale differences in microbial biodiversity discovery between 16S amplicon and shotgun sequencing. Sci Rep 7: 6589.
TURNER JS, ROBINSON NJ. 1995. Cyanobacterial metallothioneins: Biochemistry and molecular genetics. J Ind Microbiol 14: 119–125.
VAN ROSSUM T, PYLATUK MM, OSACHOOF HL, GRIFFITHS EJ, LO R, QUACH M, PALMER R, LOWER N, BRINKMAN FSL, KENNEDY CJ. 2016. Microbiome analysis across a natural copper gradient at a proposed northern Canadian mine site. Front Environ Sci 3: 84.
VIJAYAKUMAR S, THAJUDDIN N, MANOHARAN C. 2005. Role of cyanobacteria in the treatment dye industry effluent. Pollution Research 24: 69–74.
VIJAYAKUMAR S, THAJUDDIN N, MANOHARAN C. 2007. Biodiversity of cyanobacteria in industrial effluents. Acta Botanica Malacitana 32: 27–34.
VIJAYAKUMAR S. 2005. Studies on cyanobacteria in industrial effluents – An environmental and molecular approach [Ph.D. dissertation]. Tamil Nadu, India: Bharathidasan University. 97p.
VIJAYAKUMAR S. 2012. Potential applications of cyanobacteria in industrial effluents – A review. J Bioremed Biodeg 3: 154.
WARD DM, CASTENHOLZ RW, MILLER SR. 2012. Cyanobacteria in geothermal habitats. In: Whitton B eds. Ecology of Cyanobacteria II. Dordrecht: Springer.
WHITTON BA, POTTS M. 2000. The ecology of Cyanobacteria: Their diversity in time and space. Kluwer Academic Publishers. p. 321–340