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\begin{abstract}  The influence of resource availability on planktonic and biofilm microbial  community membership is poorly understood. Heterotrophic bacteria derive some to all of their organic carbon (C) from photoautotrophs while simultaneously  competing with photoautotrophs  for inorganic nutrients such as phosphorus (P) or nitrogen (N). Therefore, C inputs have the potential to shift the competitive balance of aquatic microbial communities by increasing the resource space available to heterotrophic bacteria heterotrophs  (more C) while decreasing the resource space available to algae photoautotrophs  (less mineral nutrients due to increased competition from osmotrophic heterotrophs). To test how resource dynamics affect membership of planktonic communities and assembly of biofilm communities we amended a series of flow-through mesocosms with C and P to alter the availability of C among treatments. Each mesocosm was fed with unfiltered seawater and incubated with sterile glass substrate sterilized microscope slides as surfaces  for biofilm formation. We used 454 pyrosequencing of bacterial 16S and 23S plastid genes to ask how resource driven shifts in the  pool size of each community affected characterize biofilm and planktonic  community membership and structure. The highest C treatment had the lowest planktonic algal abundance yet photoautotroph abundance,  highest planktonic bacterial abundance heterotroph abundance,  and highest biofilm biomass. Biofilm communities had higher alpha diversity (i.e. richness)  than the planktonic communities in all mesocosms. Bacterioplankton Heterotroph plankton  and biofilm membership was distinct in all but the highest C treatment where heterotroph  biofilm and planktonic plankton  communitiesincreasingly  resembled each other over time. after 17 days.  Unlike the bacteria, algal heterotrophs,  photoautotroph  biofilm and plankton communities displayed distinct microbial membership and structure in all treatments including the highest C treatment. Our results suggest that even though resource amendments affect community membership, microbial lifestyle (biofilm or planktonic) places a significantly stronger constraint on community assembly and membership. \tiny  \keyFont{ \section{Keywords:} microbial ecology, 16S, 23S, planktonic,  diff --git a/Discussion.tex b/Discussion.tex  index 811422b..b7f0a51 100644  --- a/Discussion.tex  +++ b/Discussion.tex 
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\section{Discussion} \subsection{Biomass Pool Size} The goal of this study was to evaluate how changes in available C affected the biomass pool size,  membership and structure of planktonic and biofilm communities. Our results suggest that C subsidies increased bacterial biomass in both plankton and biofilm communities as predicted. Carbon subsidies C input  also resulted in decreased photoautotroph biomass in the plankton community, but there was no significant change in biofilm  photoautotroph biomass of the biofilm communities among between  resource treatments. Although the DOC concentration in the highest C treatment was significantly higher than the other treatments the concentrations we measured were in the range of those reported in natural marine ecosystems \citep{Mopper1980} and it is has been noted that glucose concentrations in coastal marine ecosystems may fluctuate over several orders of magnitude \citep{Alonso2006}. The changes in the biomass pool size that did occur were consistent with changing relationships (commensal to competitive) between the autotrophic and heterotrophic components of the plankton communities but not necessarily of the biofilm communities. While we recognize that other mechanisms may drive the shift in biomass pool size of these two components of the microbial community (e.g. increased grazing pressure on the algae with C additions, or production of secondary metabolites by the bacteria that inhibit algal growth) previous studies \cite{Stets_2008,Cotner_2002} and the data reported here suggest that altered nutrient competition is the most parsimonious explanation for this shift in biomass pool size. \subsection{Biofilm and Plankton Alpha and Beta Diversity} Beyond changes in the biomass pool size of each community community,  we explored how shifts in resource C affected a) the membership and structure of each community, and b) the recruitment of plankton during biofilm community assembly. Intuitively, shifts in planktonic community composition should alter the  available pool or microorganisms that can be recruited into a biofilm. For  example, if planktonic diversity increases, the number of potential   taxa that can be recruited to the biofilm should also increase, potentially  increasing diversity within the biofilm. Similarly, a decrease in  mineral nutrients available to photoautotrophs should decrease photoautotroph  pool size, potentially decreasing photoautotroph diversity and therefore  candidate photoautotroph taxa that are available for biofilm formation. In  addition, C in excess of resource requirements may increase the production of  extra cellular polysaccharides (EPS) by planktonic cells thus increasing the  probability that planktonic cells are incorporated into a biofilm by adhesion.  Each of these mechanisms suggest that an increase in labile C to the system  should result in increased alpha diversity in both bacterioplankton and  bacterial biofilm communities while decreasing alpha diversity within both  planktonic and biofilm photoautotroph communities.  We highlight three key results that we find important for understandingthe assembly of  aquatic biofilms. biofilm assembly.  First, biofilm community richness was higher than exceeded  planktonic community richness (Figure~\ref{fig:rarefaction}) in all mesocosms. Second, for the control, C:P = 10 and C:P = 100 resource treatments the membership and structure of the bacterial biofilm and plankton communities were more similar within a lifestyle (plankton versus biofilm) than within a resource treatment. However, for the bacteria in the highest C treatment (C:P = 500) both membership and structure of biofilm and planktonic communities at day 17 were more similar to each other than to communities from other treatments (Figure~\ref{fig:pcoa}). Third, C subsides acted differently on the photoautotroph and bacterial communities. Specifically while the highest level of C subsidies (C:P = 500) resulted in a merging of membership in the bacterioplankton and bacterial biofilm communities the same merging of membership was not observed for the photoautotroph biofilm and plankton communities which had distinct membership in all treatments. % Fakesubsubsection:We propose two potential  We propose two potential mechanisms that could result in for  the increased diversity of the biofilm communities relative to the planktonic communities. community diversity.  First, it is possible that the planktonic community composition of our flow through incubators was dynamic in time. In this case the biofilm community would represent a temporally integrated sample of the planktonic organisms moving through the reactor resulting in higher apparent alpha diversity (i.e. mass effects would be the dominant assembly mechanism). Second, the biofilm environment may disproportionately enrich for the least abundant members of the  of the planktonic community. In this case it is probable that the biofilm would  incorporate the most abundant members from the planktonic community (i.e. mass 
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nutrient and pH gradients that are not present in the planktonic environment.  In this case the more diverse habitat would be able to support a more diverse  community due to an abundance of additional environmental habitats (i.e.  niches). We evaluated the first mechanism by comparing membership among the plankton samples taken 9 days apart (t=8 and t=17). While bacterioplankton communities were not identical between the time points (Figure~\ref{fig:pcoa}), communities within a treatment were more similar to each other between timepoints than any other bacterioplankton community (treatment or timepoint). In addition, the control and two lowest C treatments (C:P=10 and C:P=100) separated completely from biofilm communities in principle coordinate space (Bray-Curtis distance metric). This suggests that the biofilm community was not integrating variable bacterioplankton community membership, but rather was at least in part selecting for a community that was composed of distinct populations when compared to the most abundant members of the plankton community. As noted above, in the highest C treatment (C:P = 500) the bacterial biofilm and plankton community membership had significant overlap at the final timepoint (Figure~\ref{fig:pcoa}). However, bacterioplankton membership for the highest C treatment among timepoints (8 and 17 days) were also qualitatively as similar to each other as any other community. Thus, variable planktonic community composition among timepoints would not explain the higher diversity observed in the biofilm compared to the planktonic community. Rather, two results point to enrichment of planktonic community members within the biofilm as the mechanism for higher diversity in the biofilm compared to the plankton. First, the increasing similarity between the plankton and the biofilm communities over time in the highest resource C treatment suggests that \textit{in situ} resource conditions were sufficient to alter the relative abundance of the populations within each community. Second, an analysis of the OTU relative abundance in biofilm and planktonic libraries where OTUs are sorted by planktonic sample rank (Figure~\ref{fig:rank_abund}) shows that the least abundant members of the plankton community were routinely highly abundant within the biofilm community. This was true for both photoautotroph and bacterial communities, at all treatment levels and both timepoints. While we did not (could not) specifically measure niche diversity within the biofilm communities our results suggest that the biofilm habitat selected for unique members of the photoautotroph and bacterioplanktonic community that were invery  low abundance in the planktonic habitat but readily became major constituents of the biofilm community. % Fakesubsubsection:Very few Fakesubsubsection:few  studies have Very few Few  studies have simultaneously evaluated the relationship among membership and/or diversity of the plankton and the biofilm community from complex  environmental microbial communities. One notable study looked at planktonic  community composition and biofilm formation on glass beads placed for three 
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reported that planktonic diversity was significantly higher relative to biofilm  diversity (the opposite of what we found in our study). Given the differences  in the source of the planktonic community among studies, this result is not  surprising. While biofilm communities were established on glass beads in \citet{22237539} and glass slides (this study) over a similar time period (~21  days, \citet{22237539} and ~17 days this study) the origin of the planktonic  community in each study wasvery  different. The \citet{22237539} study was conducted in three boreal streams during snow melt when connectivity between  the terrestrial and aquatic habitats was high and potentially highly variable  depending on how hydrologic pathways differed among precipitation events. In 
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few studies that attempt to compare biofilm community composition and the  overlying planktonic community \citep{Besemer_2007, 22237539, Jackson_2001,  Lyautey_2005}. Those studies illustrate community composition among the two  habitats are unique withvery  few taxa found in both. This is consistent with our findings in this experimental system with a natural marine planktonic  source community. In addition, our study also evaluated photoautotroph  community composition which showed a similar result suggesting that both the 
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throughput culture collection' and is a prefix for strains cultured  under low nutrient conditions \citep{Cho_2004, Connon_2002}.  \subsection{Conclusion} In summary this study shows that changes in low resolution community level dynamics are concurrent with changes in the underlying constituent populations that compose them. We found that autotrophic pools and heterotrophic pools responded differently to amendments of labile C as hypothesized. Notably while C amendments altered both pool size and membership of the bacterial communities  we did not see similar dynamics within the photoautotroph communities.  Planktonic photoautotrophs decreased in response to C amendments presumably in  response to increased competition for mineral nutrients from a larger bacterial community, however there was not a similar decrease in biofilm photoautotroph community. In addition membership of the photoautotroph communities between the plankton and biofilm lifestyles did not become more similar in the photoautotrophs as it did for the bacteria in the highest C treatment. Consistent with a growing body of work our results suggest that complex environmental biofilms are a unique microbial community that form from taxa (both heterotrophs and autotrophs alike) that are found in low abundance in the neighboring communities. This membership was affected by resource amendments for heterotrophic but not autotrophic microbes and then only in the most extreme resource environment. This suggests that lifestyle is a major division among environmental microorganisms and although biofilm forming microbes must travel in planktonic form at some point, reproductive success and metabolic contributions to biogeochemical processes comes from those taxa primarily if not exclusively while they are part of a biofilm. Our results point to lifestyle (planktonic or biofilm) as an important trait that explains a portion of the exceptional diversity found in snapshots used to characterize environmental microbial communities in space and time. diff --git a/Introduction.tex b/Introduction.tex  index 29ac4d5..dbec28d 100644  --- a/Introduction.tex  +++ b/Introduction.tex 
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than the exception for microbial lifestyle in many environments. Large and  small-scale architectural features of biofilms play an important role in their  ecology and influence their role in localized biogeochemical cycles  \citep{17170748}. While fluid mechanics have been shown to be important drivers  of impact  biofilm structure and assembly \citep{hoedl_2011,19571890, 14647381}, it is less clear how other abiotic factors such as resource availability affect biofilm assembly. Aquatic biofilms are initiated initiate  with seed propagules that  originate within from  the planktonic community \citep{hoedl_2011, 22120588}. Thus, how resource amendments influence planktonic communities has the potential to influence the formation of microbial biofilms during community assembly. % Fakesubsubsection:In a crude sense, biofilm and planktonic microbial  In a crude sense, biofilm and planktonic microbial communities can be broken divide  into two key groups: phototrophic oxygenic phototrophs including  eukaryotes and cyanobacteria (hereafter "photoautotrophs") and bacteria and archaea. "photoautotrophs"), and, heterotrophs.  This dichotomy, while somewhat  artificial (i.e. cyanobacteria are bacteria), has been shown to admittedly an abstraction, can  be a powerful paradigm for understanding community shifts across ecosystems of varying trophic state \citep{Cotner_2002}. Heterotrophic bacteria meet some to all of their organic carbon (C) requirements from photoautotroph produced C while simultaneously competing with photoautotrophs for limiting nutrients such as phosphorous (P) \citep{379}. The presence of external C inputs, such as terrigenous C leaching from the watershed \citep{Jansson_2008, Karlsson_2012} or C exudates derived from macrophytes \citep{Stets_2008, Stets_2008b}, can alleviate bacterioplankton reliance on photoautotroph derived C and shift the bacterioplankton-photoautotroph  relationship from commensal and competitive to strictly competitive \citep[see][Figure~\ref{fig:conceptual}]{Stets_2008}. Under Assuming  this mechanism mechanism,  increased C supply should increase the resource space available to the bacteria and lead to increased competition for mineral nutrients, decreasing nutrients available for photoautotrophs {\textendash} assuming that bacteria are superior competitors for limiting nutrients as has been observed \citep[see][Figure~\ref{fig:conceptual}]{COTNER_1992}. These dynamics should result in the increase in bacterial biomass relative to the photoautotroph biomass along a gradient of increasing labile C inputs. We refer to this differential allocation of limiting resources among components of the microbial community as niche partitioning, in reference to the n-dimensional resource space available to members of the microbial community. % Fakesubsubsection:While these gross level dynamics have been discussed  While these gross level dynamics have been discussed conceptually 
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on membership and structure of the photoautotroph and bacterial community has not been  directly evaluated in planktonic or biofilm communities. In addition, how  dynamics in planktonic communities are propagated to biofilms during community  assembly is not well understood. Intuitively, shifts in planktonic community  composition should alter the available pool that can be recruited into a  biofilm. For example, if bacterioplankton diversity increases, the number of  potential bacterial taxa that can be recruited to the biofilm should also  increase, potentially increasing bacterial diversity within the biofilm.  Similarly, a decrease in mineral nutrients available to photoautotrophs should  decrease photoautotroph pool size, potentially decreasing photoautotroph diversity and  therefore candidate photoautotroph taxa that are available for biofilm formation. In  addition, C in excess of resource requirements may increase the production of  extra cellular polysaccharides (EPS) by planktonic cells thus increasing the  probability that planktonic cells are incorporated into a biofilm by adhesion.  Each of these mechanisms suggest that an increase in labile C to the system  should result in increased alpha diversity in both bacterioplankton and  bacterial biofilm communities while decreasing alpha diversity within both  planktonic and biofilm photoautotroph communities. To evaluate these ideas we We  designed this study to test a) if C subsidies shifted the biomass balance between autotrophs and heterotrophs within the biofilm or its seed pool (the plankton) and b) measure how these putative changes in pool size altered membership and structure of the plankton communities and affected recruitment of plankton during biofilm community assembly. Specifically, we amended marine mesocosms with varying glucose  input and collected plankton and biofilm samples for community  characterization by DNA sequencing 16S rRNA genes and plastid 23S rRNA  genes. Additionally, we measured photoautotroph and bacterial biomass in  plankton and biofilm samples along the glucose amendment gradient.  diff --git a/Materials_and_Methods.tex b/Materials_and_Methods.tex  index 4df7cb2..54a36b0 100644  --- a/Materials_and_Methods.tex  +++ b/Materials_and_Methods.tex 
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\section{Materials and Methods} \subsubsection{Experimental Design} We placed test Test  tube racks were placed  in one smaller (185L, control) and 3 larger (370L) flow-through mesocosms. All mesocosms were fed directly with marine water from  an inflow source in Great Bay approximately 200 m from the shore. 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 goal of the resource amendments were to create a gradient of labile carbon among treatments. The same amount of P was added to each treated mesocosm to ensure that response to additions of C were not inhibited by extreme P limitation. The control mesocosm did not receive any C or P amendments. \subsubsection{DOC and Chlorophyll Measurements}  To assess the efficacy of the C additions we sampled each mesocosm twice 
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tube for extraction with 90-95\% acetone for ~ 32 hours at -20C and analyzed  immediately after using a Turner 10-AU fluorometer \citep{Wetzel_2000}.  We analyzed bacterial Bacterial  abundance of the planktonic samples was analyzed  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$\mu$L mL-1.   \subsubsection{DNA extraction} For plankton, cells were collected by filtering