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\section{Discussion}
\subsection{Biomass Pool Size}
The goal of this study was to evaluate how changes in resource stoichiometry
affected the biomass pool size, membership and structure of planktonic and
biofilm communities. Our results suggest that
carbon C subsidies increased
bacterial biomass in both plankton and biofilm communities as predicted. Carbon
subsidies also resulted in decreased algal biomass in the plankton community,
but there was no signifcant change in algal biomass of the biofilm communities
among resource treatments. The changes in the biomass pool size that did occurr
were consistent with changing relationships (commensal to competitive) between
the autotrophic and heterotrophic components of the plankton communities but
not neccessarily of the biofilm communities.
\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:P affected a) the membership and structure of each
community, and b) the recruitment of plankton during biofilm community
assembly. Here we highlight three key results that we find important for
understanding the assembly of aquatic biofilms. First, biofilm community
richness was consistently higher than planktonic community
richness. richness
(Figure~\ref{fig:rarefaction}). 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 vs. biofilm) than within a resource treatment.
However, for the bacteria in the highest C:P 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 5). (Figure~\ref{fig:pcoa}). Third,
carbon C subsides acted differently on the algal
and bacteria communities. Specifically while the highest level of
carbon 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 algal biofilm and plankton communities
which had distinct membership in all treatments.
We propose two potential mechanisms that could result in the increased
diversity of the biofilm communities relative to the planktonic communities.
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. 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 effects) but also select and enrich
(i.e. species sorting)
the
least lease abundant members of
the planktonic community resulting in a higher level
of detectable
alpha-diversity. alpha diversity.
The second mechanism would result if the biofilm enivronment represented a more
diverse habitat including sharply delineated oxygen, 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 indentical
between the time points
(Figure 5), (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
carbon 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 selecting for a unique
community that was composed of distinct populations when compared to the most
abundant members of the plankton community. As noted above, in the highest
carbon C treatment (C:P = 500) the bacterial biofilm and plankton community
membership had significant
overlapp overlap at the final timepoint
(Figure 5). (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. The first is the
increasing similarity between the plankton and the biofilm communities over
time in the highest resource C treatment. This suggests that selection pressure
of the \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 6) (Figure~\ref{fig:rank_abund}) shows
that the least abundant members of the plankton community were rountinely
highly abundant within the biofilm community. This was true for both algal 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 algal and
bacterial planktonic bacterioplanktonic community that were in very low
abundance in the planktonic habitat but readily became major constituents of
the biofilm community.
Very few studies have previously 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
\cite{22237539}. \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.
Besemer et al. (2012) \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
suprising. While biofilm communities were establishsed 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 very 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
200m from the shore during July when communities are more stable over the 17
day perios of the incubation. A separate study conducted in alpine and
sub-alpine streams of the Rocky Mountains clearly showed that stream plankton
communities reflected localized precipitation events and could be traced
largely to sources of soil communities of drainages within the watershed
\cite{22626459}. \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
\cite{22378536}. \citep{22378536}.
In addition to the diverse source communities the
\cite{22237539} \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
(algae and bacteria) against sample-wise biofilm libraries, we find more total
bacterial and algal planktonic OTUs than in any given single biofilm sample. It
appears, however, that sample-wise bacterial 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 itegrated planktonic
library. library (Figure~\ref{fig:integ_rarefaction}). This
result is consistent with our conclusion that temporal heterogenetity 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 Besemer
et al. (2012) compared to this study.
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 over time
although with only two time points it was unclear how pronounced this effect
was
(Figure 4). (Figure~\ref{fig:rarefaction}). Since biomass of the plankton and the
biofilm increased with increasing
carbon 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 with this result our experimental
design did not allow us to tell whether resources drove productivity that drove
changes in diversity or whether resoruces drove diversity which altered
productivity. Rather we note that, as diversity decreased in the highest C
treatment
bacterial plankton bacterioplankton and biofilm membership became increasingly
similar. This suggests that enviornments that contain high amounts of labile
carbon C selected for fewer dominant taxa that came to dominate the biofilm
community, overwhelming the 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
carbon C treatment (C:P = 500) were
surrounded by what appeared to be EPS. Because biofilm EPS appeared also to
increase moving from the low to high
carbon C treatments
(Figure 3) (Figure~\ref{fig:microscope}) it is possible that more 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 floculating DOC which has been shown to dominate high DOC
environments in nature, we did measure a substanial 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 bacterial membership in the
highest C treatment.
\subsection{Sample Class \subsection{Lifestype (biofilm or planktonic) Enriched OTUs}
There are only a few studies that attempt to compare biofilm community
composition and the overlyng planktonic community
\cite{Besemer_2007, \citep{Besemer_2007,
22237539, Jackson_2001, Lyautey_2005}. Those studies illustrate community
composition among the two habitats are unique with very few taxa found in both.
This is consistent with our findings in this experimental system with a natural
marine planktonic source commmunity. In addition, our study also evaluated
algal community composition which showed a similar result suggesting that both
the algal and bacterial biofilm communities form from phylogenetically unique
organisms that exist in low abundance in surrounding habitat (i.e. the
plankton) but are readily enriched in the biofilm lifestyle. Most of the
biofilm enriched algal OTUs were \textit{Bacillariophyta} although there were
also many \textit{Bacillariophya} OTUs enriched in the planktonic libraries. We
also found \textit{Cryptophyta} and \textit{Viridiplantae} were more uniformly
enriched in the planktonic algal libraries. It appears that these broad
taxonomic groups were selected against in biofilms under our experimental
conditions. Bacterial OTUs enriched in planktonic samples displayed more
dramatic differential abundance patterns than bacterial OTUs enriched in
biofilm samples, but, biofilm enriched bacterial OTUs were spread across a
greater phylogenetic breadth
(Figure 6). (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 as in the more
uniform environment inhabited by the plankton. At the Order level, enriched
bacterial OTUs tended to have members in 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 databasees and therefore environmental sequence
collections show little equivalency in phylogenetic breadth between groups at
the same taxonomic rank
\cite{Schloss_2011}. \citep{Schloss_2011}. Unfortunatley, at higher
taxonomic resolution (e.g. Genus-level), groups did not possess a sufficient
number of OTUs to evaluate coherence between taxonomic annotation and
environment type preference. lifestyle. Similar to the richness results, we found the
shape of rank abundance distributions between biofilm and planktonic libraries
in our data
(Figure~\ref{fig:rank_abund_shape} to be in contrast to that
reported by \citet{22237539} although this may be an artifact of each study's
different source communities (as
discussed above). Carbon amendments did not affect algal library membership
and structure to the same degree as it affected bacterial library composition.
As expected, bacterial OTUs enriched in the high
carbon C amended mesocosm (C:P =
500) include
OTUS OTUs in classic copiotroph families such as \textit{Vibrionacaea}
and \textit{Pseudomonadaceae}. Interestingly, the one OTU depleted in the high
carbon C treatments is annotated as being in the HTCC2188 order of the
\textit{Gammaproteobacteria}. HTCC stands for 'high throughput culture
collection' and is a prefix for strains cultured under low nutrient conditions
\cite{Cho_2004, \citep{Cho_2004, Connon_2002}.
\subsection{Conclusion}
In summary this study shows mechanistic links between large scale community
level dynamics and the underlying constituent populations that compose them. We
found that autrotrophic pools and heterotrophic pools responded differently to
ammendments of labile
carbon C as hypothesized. Notably while
carbon C ammendments
altered both pool size and membership of the bacterial communities we did not
see similar dynamics within the algal communities. Planktonic
algal algae decreased
in response to
carbon C amendments presumably in response to increased
competition
for P from a larger bacterial community, however there was not a
simialr similar decrease in biofilm algal community. In addition membership of the
algal communities betweeen the plankton and biofilm lifestyles did not become
more similar in the algae as it did for the
bacterial 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
ammendements 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 biogeohemical processes comes from those taxa
primarily if not exclusively while they are part of a biofilm.
This suggests Our results point to lifestyle
is another (planktonic or biofilm) as an
important trait that explains a portion of the exceptional diversity found in
snapshots used to characterize environmental microbial
communities. communities in space and
time.