Bacterial and phytoplankton responses to nutrient amendments in a boreal lake at different taxonomic resolutions
Nutrient manipulation experiments were conducted in Lake Alinen Mustajärvi during the 2009 open water period, at the beginning of May, July and September. Water for the experiments was taken from the upper 1.5 m of the water column where the natural DOC concentration is around 10 mg C L-1, but during the period of the experiments it had been elevated to around 12 mg C L-1 by monthly additions of cane sugar as part of a parallel project. Each experiment had four treatments (control, +N, +P and +NP) with three replicates for each sampling day. Nutrient additions were made only at the beginning of each experiment with the target rise in concentrations being 0.35 mg L-1 for N and 0.05 mg L-1 for P. These relatively high additions were necessary to ensure nutrient availability relative to labile DOC throughout the experiments. The nutrient sources used were NH4NO3 for nitrogen and Na3PO4 for phosphorus. The water for each experiment was taken with a 30-cm-long acrylic tube sampler (Limnos vol 2 L). Water was sieved with a 50 µm mesh to remove larger zooplankton and mixed thoroughly prior to and after nutrient amendments. 2 L replicates were measured into polypropylene bags, which were then sealed and incubated in situ at 0.5 m depth, approximating the effective light climate of the mixed layer of the water column. Each experiment lasted for seven days and sampling was conducted at the start and on days four and seven; here results are mainly reported from day seven.    For DNA extraction 100 ml of water from each sample was freeze dried with an Alpha 1-4 LD plus (Christ, Osterode, Germany). The DNA extraction procedure was modified from protocol described by Griffiths et al. (2000). Briefly, 0.4 mg of freeze-dried material was homogenized with glass beads in mixture of phenol-chloroform-isoamylalcohol (PCIAA; 25:24:1) and hexadecyltrimethyl-ammonium bromide. After 5 min incubation on ice to allow humic acids dissolve into PCIAA, tubes were centrifuged. The upper aqueous phase was then re-extracted with chloroform-isoamylalcohol (24:1), precipitated with polyethylene glycol and dissolved in 50 µL of TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA). DNA concentrations were measured with PicoGreen in a Qubit fluorometer (Invitrogen Corporation, Carlsbad, USA).  Amplification of bacterial 16S rRNA genes (E. coli positions 341 to 805) was conducted using general bacteria primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHV GGGTATCTAATCC-3’) (Herlemann et al, 2011). Primer 341F carried a 454FLX adaptor B at the 5 ´end and primer 805R carried a 5 bp molecular barcode specific for each sample followed by a 454FLX adaptor A at the 5 ´end. PCR and amplicon processing prior to sequencing was performed as described in Eiler et al. (2011), except for purification of PCR products with Agencourt AMPure XP purification system (Beckman Coulter, Danvers, USA) and amplicon quantification with PicoGreen in a Qubit fluorometer.   Equal concentrations of amplicons were sequenced from each sample with Titanium chemistry from adaptor A, using a 454 GS-FLX system (454 Life Sciences, Branford, CT) at the Institute of Biotechnology hosted by the University of Helsinki, Finland. The resulting reads carried the sample-specific molecular barcode and covered the entire V4 region of the 16S rRNA gene as well as flanking regions. barcode ATATA ne1_d0    barcode ATACT ne1_d4_C    barcode ATAGC ne1_d4_N    barcode ATCAT ne1_d4_P    barcode ATCTC ne1_d4_NP    barcode ATCGA ne1_d7_C    barcode ATGAC ne1_d7_N    barcode ATGTG ne1_d7_P    barcode ATGCA ne1_d7_NP    barcode ACATC ne2_d0    barcode ACACA ne2_d4_C    barcode ACAGT ne2_d4_N    barcode ACTAT ne2_d4_P    barcode ACTCG ne2_d4_NP    barcode ACTGA ne2_d7_C    barcode ACGAG ne2_d7_N    barcode ACGTA ne2_d7_P    barcode ACGCT ne2_d7_NP  barcode ACGTA ne3_d0  barcode AGTCA ne3_d7_C  barcode AGCAG ne3_d7_N  barcode TGTCT ne3_d7_P  barcode CACTC ne3_d7_Np