J Appl Phycol (2013) 25:1039–1045 DOI 10.1007/s10811-012-9909-y Microbial diversity and diazotrophy associated with the freshwater non-heterocyst forming cyanobacterium Lyngbya robusta Jason N. Woodhouse & Sarah E. Ongley & Mark V. Brown & Brett A. Neilan Received: 18 June 2012 /Revised and accepted: 12 September 2012 /Published online: 25 September 2012 # Springer Science+Business Media Dordrecht 2012 Abstract Lake Atitlan, Guatemala, a freshwater lake in Introduction South America, experiences annually recurring blooms comprised of the planktic filamentous cyanobacterium Cyanobacteria are well established as organisms that dem- Lyngbya robusta. Previous physiochemical characterisation onstrate a wide array of morphological, physiological and of the bloom identified diurnal nitrogenase activity typical chemical diversity (Tan 2007). Globally, cyanobacteria pose of non-heterocystous cyanobacteria, in addition to the low- a number of threats to both humans and the environment level detection of the cyanotoxins cylindrospermopsin and through the formation of toxic blooms (Eiler and Bertilsson saxitoxin. A molecular approach, combining deep sequenc- 2004; Rinta-Kanto et al. 2005; Pearson et al. 2008). The ing of the 16S rRNA and nifH genes, was applied to a increasing occurrence and, in some instances, intensity of cyanobacteria-dominated sample collected during the exten- these toxic blooms have been noted over the last 10 years. sive 2009 bloom. Lyngbya accounted for over 60 % of the Blooms composed of toxic cyanobacteria, such as Microcystis, total 16S rRNA sequences with the only other cyanobacterial Anabaena and Cylindrospermopsin, receive much attention, species detected being the picophytoplankton Synechococcus. often due to their proximity to urban areas and drinking The remaining bacterial population was comprised of organ- water supplies, with a number of molecular assays available isms typical of other eutrophic freshwater bodies, to monitor their proliferation (Pearson and Neilan 2008; although the proportionate abundances were atypical. Al-Tebrineh et al. 2012). Blooms comprised of organisms, An obligate anaerobe Opitutus, not typically found in such as Oscillatoria, Planktothrix and Lyngbya, filamen- freshwater systems, was identified within the community tous forms lacking heterocysts, are being increasingly which suggests it may have a role in enhancing nitrogen reported as a consequence of increasing anthropogenic fixation. Primary nitrogen fixation was attributed to contamination (Sivonen et al. 1990; Prakash et al. 2009). Lyngbya, with other putative nitrogen fixers, Desulfovibrio, Cyanobacterial blooms are complex microbial assemb- Clostridium and Methylomonas, present at very low lages, comprising many representatives from characterised abundance. phyla (Pope and Patel 2008; Li et al. 2011; Wilhelm et al. 2011). The morphological features of organisms, such as Keywords Cyanobacteria . Freshwater . Nitrogenase . Lyngbya and Oscillatoria, specifically the arrangement of Bloom the thallus and production of exopolysaccharide mucilage, allow for the formation of associative microbial assemblages analogous to biofilms (Zehr et al. 1995;Reidetal.2000; : : : Omoregie et al. 2004; Burke et al. 2011). Cyanobacteria- J. N. Woodhouse S. E. Ongley M. V. Brown B. A. Neilan (*) dominated microbial mats, which are common in aquatic School of Biotechnology and Biomolecular Sciences, systems, have also been characterised (Zehr et al. 1995; The University of New South Wales, Sydney, New South Wales 2052, Australia Ferris et al. 1996; Steppe et al. 1996; Ward et al. 1998; e-mail: [email protected] Neilan et al. 2002; Dupraz and Visscher 2005; Steppe and 1040 J Appl Phycol (2013) 25:1039–1045 Paerl 2005). In particular, several studies have demonstrat- Within a few hours of sampling, the samples were air dried ed interactions between cyanobacteria and their associated in a nearby facility under sterile conditions before being microbial community with respect to carbon, nitrogen and transported to the laboratory. Samples were hydrated with sulphur cycling, often exhibiting diurnal patterns (Fründ sterile phosphate-buffered saline (pH 7.0) prior to micro- and Cohen 1992; Steppe et al. 1996; Teske et al. 1998; scopic analysis. Microscopic inspection was performed with Visscher et al. 1998; Steppe and Paerl 2005). the use of an Olympus BX51 fixed stage fluorescence In aquatic systems where blooms comprising planktic microscope. Bloom material was stained with 300 nM organisms generally dominate, the precise nature of these 4′,6-diamidino-2-phenylindole (DAPI) and observed under microbial associations is not understood. Bloom initiation, bright field and using two fluorescence filter sets. maintenance and subsequent decline have been shown to Observation of DAPI fluorescence was made with a U- depend to a large extent on the availability of nitrogen (N) MNUA2 (excitation 360–370 nm, emission 420–460 nm) and phosphorus (P) within the system (Levich 1996; mirror unit. Genomic DNA was extracted from 100 mg of Sañudo-Wilhelmy et al. 2001). Furthermore, it has been dried bloom material using the Soil DNA Extraction kit (MP demonstrated that certain ratios of N and P may select for Biosciences) according to manufacturer’s specifications. organisms capable of fixing atmospheric nitrogen over those which lack this physiology (Klausmeier et al. 2004). As the Ribosomal tag pyrosequencing incidence of Lyngbya/Oscillatoria/Planktothrix blooms increases worldwide, there is a need to further our under- Amplicon sequencing of partial 16S rRNA gene sequences standing of the role of nutrient availability during prolifer- was performed on extracted bloom DNA at the Research ation of these organisms. and Testing Laboratory (Lubbock, Texas). Primers 27f and Lake Atitlan in Guatemala has experienced three major 519r (Weisburg et al. 1991) were used to PCR amplify a Lyngbya robusta bloom incidences, with the largest reaching 500-bp product spanning the 16S rRNA variable regions its peak in November of 2009. The bloom of December 2008 V1–V3. Sequencing was performed using a Roche GS was the first reported global observation of the planktic L. FLX Titanium machine. Initial quality control measures robusta (Rejmánková et al. 2011). Preliminary investigations used to ensure sequence fidelity included the removal of indicated that bloom initiation occurred as a consequence of short sequences (<100 bp), any sequence with a discrepancy high levels of P and low N/P ratios that are indicative of to the 5′ primer or any sequence containing an unresolved nitrogen limitation. Diurnal patterns of nitrogen fixation indi- nucleotide (N). This provided ~10,000 reads upon which cated light-dependent nitrogen fixation in the presence of non- further quality control was performed using the Mothur pack- heterocystous cyanobacteria (Rejmánková et al. 2011). High age (Schloss et al. 2009). A 2 % pre-clustering step was rates of diurnal nitrogen fixation, oxygenic photosynthesis performed to remove other potential errors in sequence data. and the unique morphological characters of Lyngbya provide Chimeric sequences were identified and removed using the a specific niche for associated heterotrophic bacteria not found program chimera.slayer with minsnp 0 100. Sequences con- amongst freshwater cyanobacteria. taining long (>7) homopolymers and those that did not align In this study, molecular techniques were used to determine within the appropriate rRNA gene variable region were also the microbial composition of the December 2009 L. robusta- removed. The remaining 7,949 reads, representing 1,333 dominated bloom in an attempt to identify and understand the unique sequences, were taxonomically characterised using a role of the heterotrophic population associated with this highly curated database from the Ribosomal Database Project planktic freshwater filamentous cyanobacterium. In particular, (http://rdp.cme.msu.edu/). This dataset is publically available tagged amplicon sequencing was used to determine the iden- online at the MG-RAST server (http://metagenomics.anl.gov) tity and composition of microbial taxa associated with the L. under the project title “Lake_Aitlan_Bloom_16S”. The depth robusta bloom. Furthermore, taxa identified by this method of sampling of community members was examined using were correlated with the identification of genes encoding rarefaction analysis of operation taxonomic units (OTUs) bacterial nitrogenases to identify sources of primary and sec- defined by a similarity of >97 %. ondary nitrogen fixation and their relative compositions. Characterisation of the nifH-containing community Methods The dinitrogenase reductase gene, nifH, was amplified using Pfu polymerase (Promega) as previously described Sample collection, preparation and microscopy (Jungblut and Neilan 2009). The amplicons were A-tailed and cloned into pGEM-T-Easy vector (Promega) as per Cyanobacterial bloom material was collected from four dis- manufacturer’s instructions. This ligation was transformed tinct locations around Lake Atitlan on November 8, 2009. into NEB 5-alpha high efficiency chemically competent J Appl Phycol (2013) 25:1039–1045 1041 Escherichia coli (New England Biolabs). White colonies were picked and grown overnight with ampicillin selection, and the plasmids were extracted using a PureLink Quick Plasmid Miniprep kit (Invitrogen). Automated sequencing of the purified plasmid was performed with the vector- specific primer MPF (Jungblut and Neilan 2009), PRISM BigDye