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Chuck Pepe-Ranney added Material and Methods.tex
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Experimental Design - We placed test tube racks in one smaller (185 L) and 3 larger (370L) flow-through mesocosms. Each mesocosm had an adjustable flow rate that resulted in a residence time of approximately 12h. Irregular variation in inflow rate meant that flow rate varied around that target, throughout the day however regular monitoring ensured that the entire volume of each system was flushed approximately two times per day. To provide a surface for biofilm formation we attached coverslips to glass slides using nail polish and then attached each slide to the test tube racks using office-style binder clips. Twice daily 10 ml of 37 mM KPO4 and 1, 5 and 50 ml of 3.7M glucose were added to each of 3 mesocosms to achieve target C:P resource amendments of 10, 100 and 500 respectively. The control mesocosm did not receive any C or P amendments.
DOC and Chlorophyll Measurements- To assess the efficacy of the carbon additions we sampled each mesocosm twice daily during the first week of the experiment to evaluate dissolved organic carbon (DOC) content. Six days after the initiation of the experiment we collected plankton on filters to evaluate planktonic Chl a and bacterial abundance. Once it was clear (day 8) that pool size of each community had been altered we filtered plankton onto 0.2 m filters and harvested coverslips to assess bacterial and algal community composition (16S and 23S rDNA). In addition all mesoscosms were analyzed a second time (day 17) to assess community composition of both the plankton and biofilm communities.
Samples for dissolved organic carbon (DOC) analysis were collected in acid washed 50 mL falcon tubes after filtration through a 0.2 polycarbonate membrane filter attached to a 60 mL syringe. Syringes and filters were first flushed with the control sample to prevent leaching of carbon from the syringe or the filter into the sample. Samples were then frozen and analyzed for organic carbon content with a Shimadzu 500 TOC analyzer (Wetzel and Likens 2000).
Planktonic Chl a was collected on GF/F filters (Whatman) while biofilm Chl a was gently removed biomass from the complete area of each coverslip before extraction with 90-95% acetone for ~ 32 hours at -20C using a Turner 10-AU fluorometer (Wetzel and Likens 2000).
We analyzed bacterial abundance of the planktonic samples using Dapi staining and direct visualization on a Zeis Axio epifluorescence microscope after the methods of Porter and Feig (1980). Briefly, 1-3 mL of water was filtered through a 0.2 m black polycarbonate membrane filter and post stained with a combination of Dapi and Citifluor mountant media (Ted Pella Redding, Ca) to a final concentration of 1g ml-1.
DNA extraction For plankton, cells were collected by filtering between 20 – 30 mL of water onto a 0.2 m pore-size polycarbonate filter. For biofilm communities, biomass from the entire coverslip area of three separate slides were collected and combined in an eppendorf tube by gentle scrapping the slip surface with an ethanol rinsed and flamed razor blade. DNA was extracted using a Mobio Power Soil DNA isolation kit.
PCR: The 16SS rRNA gene was amplified using bacterial specific primers that targeted the ??? region of the SSU rRNA gene. The specific primers used were 5'-GAGTTTGATCNTGGCTCAG-3' (28F ???) and 5'-GTNTTACNGCGGCKGCTG-3' (519R ???), respectively. Each primer included a barcode and adapter sequence for 454 amplicon sequencing on the FLX-Ti platform (Roche). The PCR program for 16S amplification was as follows: NEED TO TRACK THIS DOWN. To assess the diversity of the algal comuunity, the 23S region of the plastid genome was targeted (REF). The specific primer pair used was NAME and NAME (5'-GGACAGAAAGACCCTATGAA-3' and 5'-TCAGCCTGTTATCCCTAGAG-3', respectively) (REF). The plastid genome 23S sequence has proved to yield reliable phylogenies for algae (REF). The PCR program for amplification of plastid 23S sequences was as follows: NEED TO TRACK THIS DOWN.
PCR products were pooled in equimass amounts. Mass was determined using ???. Multiplexed PCR product pools were sequenced at the Research and Testing Sequencing Facility (address).
