deletions | additions       diff --git a/Materials_and_Methods.tex b/Materials_and_Methods.tex  index 5f5c4c0..367026c 100644  --- a/Materials_and_Methods.tex  +++ b/Materials_and_Methods.tex 
...
\section{Materials and Methods}  \subsubsection{Experimental Design}  We placed test tube racks in one smaller (185L, control) 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 KPO$_{4}$ 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. \subsubsection{DOC and Chlorophyll Measurements}  To assess the efficacy of the carbon C  additions we sampled each mesocosm twice daily during the first week of the experiment to evaluate dissolved organic carbon C  (DOC) content. After the initiation of the experiment we collected plankton on filters regularly to evaluate planktonic Chl \textit{a} and bacterial abundance. Once it was clear that pool size of each community had been altered (day 8) we filtered plankton onto 0.2 $\mu$m filters and harvested coverslips to assess bacterial and algal biofilm  community composition (16S and 23S rDNA). In addition all mesoscosms were analyzed for community composition a second time (day 17) to assess how community composition of both the plankton and biofilm communities had been altered over time. Control samples were only analyzed for community composition on day 17. Samples for dissolved organic carbon C  (DOC) analysis were collected in acid washed 50 mL falcon tubes after filtration through a 0.2 polycarbonate membrane filter (Millipore GTTP GTTP02500, Sigma Aldrich P9199) attached to a 60 mL syringe. Syringes and filters were first flushed multiple times with the control sample to prevent leaching of carbon C  from the syringe or the filter into the sample. Samples were then frozen and analyzed for organic carbon C  content with a Shimadzu 500 TOC analyzer \cite{Wetzel_2000}. \citep{Wetzel_2000}.  Biomass of all biofilm samples were measured by difference in pre-(without biofilm) and post-(with biofilm) weighed GF/F filters after oven drying overnight at 60C. For Chl \textit{a} analysis we collected plankton on GF/F filters (Whatman, Sigma Aldrich Cat. \# Z242489) by filtering between 500 mL and 1L from the water column of each mesocosm for each treatment. For biofilm samples, all biofilm was gently removed from the complete area of each coverslip (3 coverslips for each treatment per sampling event) before being placed in a test tube for extraction with 90-95\% acetone for ~ 32 hours at -20C and analyzed immediately after using a Turner 10-AU fluorometer \cite{Wetzel_2000}. \citep{Wetzel_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 from three separate water column samples through a 0.2 $\mu$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 1$\mu$ ml-1.  \textit{DNA 1$\mu$L  mL-1.   \subsubsection{DNA  extraction} For plankton, cells were collected by filtering between 20 – {\textendash}  30 mL of water onto a 0.2 $\mu$m pore-size polycarbonate filter (Whatman Nucleopore 28417598, Sigma-Aldrich cat\# WHA110656). For biofilm communities, biomass from the entire coverslip area of three separate slides was collected and combined in an eppendorf tube by gentle scrapping the slip surface with an ethanol rinsed and flamed razor blade. DNA from both the filter and the biofilm was extracted using a Mobio Power Soil DNA isolation kit (MoBio Cat. \# 12888). \subsubsection{PCR}  Samples were amplified for pyrosequencing using a forward and reverse fusion primer. The forward primer was constructed with (5’-3’) (5{'}-3{'})  the Roche A linker, an 8-10bp barcode, and the forward gene specific primer sequence. The reverse fusion primer was constructed with (5’-3’) (5{'}-3{'})  a biotin molecule, the Roche B linker and the reverse gene specific primer sequence. The gene specific primer pair for bacterial SSU rRNA genes was 27F/519R \cite{LANED.J.:1991}. \citep{LANED.J.:1991}.  The primer pair p23SrV\_f1/p23SrV\_r1 was used to target 23S rRNA genes on plastid genomes \cite{Sherwood_2007}. \citep{Sherwood_2007}.  Amplifications were performed in 25 ul reactions with Qiagen HotStar Taq master mix (Qiagen Inc, Valencia, California), 1ul of each 5uM primer, and 1ul of template. Reactions were performed on ABI Veriti thermocyclers (Applied Biosytems, Carlsbad, California) under the following thermal profile: 95$^{\circ}$C for 5 min, then 35 cycles of 94$^{\circ}$C for 30 sec, 54$^{\circ}$C for 40 sec, 72$^{\circ}$C for 1 min, followed by one cycle of 72$^{\circ}$C for 10 min and 4$^{\circ}$C hold. Amplification products were visualized with eGels (Life Technologies, Grand Island, New York). Products were then pooled equimolar and each pool was cleaned with Diffinity RapidTip (Diffinity Genomics, West Henrietta, New York), and size selected using Agencourt AMPure XP (BeckmanCoulter, Indianapolis, Indiana) following Roche 454 protocols (454 Life Sciences, Branford, Connecticut). Size selected pools were then quantified and 150 ng of DNA were hybridized to Dynabeads M-270 (Life Technologies) to create single stranded DNA following Roche 454 protocols (454 Life Sciences). Single stranded DNA was diluted and used in emPCR reactions, which were performed and subsequently enriched. Sequencing followed established manufacture protocols (454 Life Sciences).