\section{Discussion} 
\subsection{Biomass Pool Size} The goal of this study was to evaluate how
changes in available C affected the biomass pool size and composition of
planktonic and biofilm communities. Our results suggest that C subsidies
increased heterotroph biomass in both plankton and biofilm communities.
C amendments also resulted in decreased photoautotroph biomass in the plankton
community, but there was no significant change in biofilm
photoautotroph biomass 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 photoautotrophs with C additions, or production of secondary
metabolites by the heterotrophs that inhibit algal growth) previous studies
\cite{Stets_2008,Cotner_2002} and the data reported here suggest that altered
nutrient competition among heterotrophic and photoautotrophic members of the
plankton is the most parsimonious explanation for the observed shift in biomass
pool size.

\subsection{Biofilm and Plankton Alpha and Beta Diversity} Beyond changes
in the biomass pool size of each community, we explored how shifts in
resource C affected the membership and structure of each community, and
the recruitment of plankton during biofilm community assembly.
Intuitively, shifts in planktonic community composition should alter the
available pool of 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
extracellular 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 heterotrophic plankton and
heterotrophic biofilm communities while decreasing alpha diversity within both
planktonic and biofilm photoautotroph communities. 

We highlight three key results that we find important for understanding
aquatic biofilm assembly. First, biofilm community richness 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 heterotroph 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 heterotroph communities. Specifically, while the highest
level of C subsidies (C:P = 500) resulted in a merging of membership in the
heterotroph plankton and heterotroph 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 for the increased richness of the biofilm
communities relative to the planktonic community richness. 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 the low abundance members 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
effects) but also select and enrich (i.e. species sorting) the least abundant
members of the planktonic community resulting in a higher level of detectable
alpha diversity. The second mechanism would result if the biofilm environment
represented a more diverse microhabitat including sharply delineated oxygen,
nutrient and pH gradients that are not present in the planktonic environment.
In this case the more diverse microhabitat 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 (day 8 and day 17). While heterotrophic
plankton communities were not identical between the time points
(Figure~\ref{fig:pcoa}), communities within a treatment were more similar to
each other than other heterotroph plankton communities regardless of time. 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
heterotroph biofilm and plankton community membership had significant overlap
at the final timepoint (Figure~\ref{fig:pcoa}). However, heterotrophic plankton
community composition 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 between each time point 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 heterotroph 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
heterotrophic community that were in low abundance in the planktonic habitat
but readily became major constituents of the biofilm community.

% Fakesubsubsection:few studies have
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
weeks in three boreal freshwater streams \citep{22237539}. While that study
system is markedly different than our study, the analyses and questions
addressed in each study were sufficiently similar to merit comparison.
\citet{22237539} concluded that the biofilm community membership was most
likely driven by species sorting over mass effects. This is consistent with
what we report here. However, in the \citet{22237539} study the authors
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 was 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
this study the source community was a marine intake located approximately 200
m from the shore during July when communities are more stable over the 17 day
period of the incubation. A separate study conducted in alpine and sub-alpine
streams clearly showed that stream plankton communities reflected localized
precipitation events and could be traced largely to soil source communities
from drainages within the watershed \citep{22626459}. While planktonic
communities in lake ecosystems can be linked to soil communities in the
watershed, as residence time of the system slows the relative influence of
species sorting increases. Thus, in headwater ecosystems stream plankton
communities can often be composed primarily of soil organisms \citep{22378536}.
In addition to the diverse source communities the \citet{22237539} study
sampled the plankton community at multiple timepoints and integrated the
samples before sequencing further increasing community richness as compared to
the current study where the plankton community was sampled and analyzed only at
two independent timepoints. Indeed, when we pool OTU counts from all planktonic
libraries and compare the rarefaction curve of the pooled planktonic libraries
(photoautotrophs and heterotrophs) against sample-wise biofilm libraries, we
found more total heterotroph and photoautotroph planktonic OTUs than in any
given single biofilm sample. It appears, however, that sample-wise heterotroph
biofilm rarefaction curves may exceed the integrated planktonic curve upon
extrapolation and most exceed the integrated planktonic curve at sampling
depths where data is present for the biofilm and pooled planktonic library
(Figure~\ref{fig:integ_rarefaction}). This result is consistent with our
conclusion that temporal heterogeneity in the plankton was not sufficient to
explain the higher diversity in the biofilm sample but would explain the
relative differences between planktonic and biofilm diversity found in
\citet{22237539} compared to this study.

% Fakesubsubsection:In addition, for this
In addition, for this study, it is important to note that biofilm community
richness peaked at the intermediate treatment (C:P = 100) and appeared to
decrease between each time point although with only two time points it was unclear how
pronounced the temporal effect was nor is it possible assess the statistical
significance of this effect (Figure~\ref{fig:rarefaction}). Since biomass of
the plankton and the biofilm increased with increasing C subsidies the
intermediate peak in OTU richness is consistent with a classic
productivity-diversity relationship that has been shown for many ecosystems and
communities both microbial and otherwise. However, as with other experiments
our experimental design did not allow us to tell whether resources drove
productivity that subsequently drove changes in diversity or whether resources
drove diversity which altered productivity. Rather, we note that as diversity
decreased in the highest C treatment, heterotrophic plankton and biofilm
membership became increasingly similar. This suggests that environments that
contained high amounts of labile C selected for fewer dominant taxa,
overwhelming the lifestyle species sorting mechanisms that appeared to dominate
biofilm community assembly in all other treatments. Similarly, while we did not
measure extracellular polymeric substances (EPS), direct microscopy showed that
planktonic cells in the highest C treatment (C:P $=$ 500) were surrounded by
what appeared to be EPS. Because biofilm EPS also appeared to increase
from the low to high C treatments it is possible that abundant
planktonic cells were more readily incorporated into biofilms due both to
increased "stickiness" of the planktonic cells as well as the biofilm itself.
While we did not observe flocculating DOC which has been shown to dominate high
DOC environments in nature, we did measure a substantial increase in DOC in the
C:P $=$ 500 treatment which was more than 2-fold higher than any of the other
treatments. Thus additional adhesion of the plankton and the biofilm may also
explain the merging of the planktonic and biofilm heterotroph membership in the
highest C treatment.

\subsection{Lifestyle (biofilm or planktonic)  Enriched OTUs} There are only a
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, with 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
photoautotroph and heterotroph biofilm communities are comprised of phylogenetically
distinct organisms that exist in low abundance in the surrounding habitat (i.e.
the plankton) but are readily enriched in the biofilm lifestyle. Most of the
biofilm enriched photoautotroph OTUs were \textit{Bacillariophyta} although
there were also many \textit{Bacillariophya} OTUs enriched in the planktonic
libraries. We also found more \textit{Cryptophyta} and \textit{Viridiplantae}
were enriched in the planktonic photoautotroph libraries. It appears that
these broad taxonomic groups were selected against in biofilms under our
experimental conditions. Heterotroph OTUs enriched in planktonic samples
displayed more dramatic differential abundance patterns than heterotroph OTUs
enriched in biofilm samples, but, biofilm enriched heterotroph OTUs were spread
across a greater phylogenetic breadth (Figure~\ref{fig:l2fc}). This is also
consistent with the idea of greater niche diversity in the biofilm environment
as opposed to the plankton. Greater niche diversity should select for a more
diverse set of taxa but individual taxa would not be as numerically dominant in
a more uniform environment like the planktonic environment. At the Order level,
enriched heterotroph OTUs tended to have members that were enriched in both the
plankton and the biofilm suggesting the phylogenetic coherence of lifestyle is
not captured at the level of Order. It should be noted, however, that taxonomic
annotations in reference databases and therefore environmental sequence
collections show little equivalency in phylogenetic breadth between groups at
the same taxonomic rank \citep{Schloss_2011}. Unfortunately, at higher
taxonomic resolution (e.g. Genus-level), groups did not possess a sufficient
numbers of OTUs to evaluate coherence between taxonomic annotation and
lifestyle. Carbon amendments did not affect photoautotroph library membership
and structure to the same degree as it affected heterotroph library
composition. As expected, heterotroph OTUs enriched in the high C amended
mesocosm (C:P $=$ 500) include OTUs in classic copiotroph families such as
\textit{Altermonodales} and \textit{Pseudomonadaceae}. Interestingly, the most
depleted OTU in the high C treatments is annotated as being in the HTCC2188
order of the \textit{Gammaproteobacteria} and shares 99\% sequence identity
with another "HTCC" strain (accession AY386332). HTCC stands for 'high
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 heterotroph 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 heterotroph 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 bacterial heterotrophs
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 that are found in low abundance in the neighboring
communities. This membership was affected by C amendments for heterotrophic but
not photoautotrophic 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.

