Studies investigating the fate of diazotrophs through the microbial food web
are lacking, although N
In the south-west Pacific ocean, the natural occurrence of abundant and
diverse plankton taxa capable of dinitrogen (N
Through the VAHINE (VAriability of vertical and tropHIc transfer of diazotroph derived N in the south wEst Pacific) programme
(
Position of mesocosms implemented in the south-west lagoon of New Caledonia.
Three large mesocosms (
Example (day 23 M3 1 m) of flow-cytometry cytogram dot plot of
Flow-cytometry analyses were carried out at the flow-cytometry
platform of the laboratory (
Heterotrophic bacterial production (BP) was estimated daily using the
The availability of phosphorus (P), nitrogen (N) and organic carbon (C) for
heterotrophic bacteria was investigated by measuring changes in bacterial
production following additions of DIP (0.25
Total alkaline phosphatase activity (APA) was measured at the three depths
in M1, M2 and M3 and in Nouméa lagoon waters using the analog substrate
methylumbelliferone phosphate (MUF-P, 1
Non-parametric Mann–Whitney and Kruskal–Wallis tests were used to compare differences of each parameter studied between mesocosms, periods of time or effects of various amendments on BP in the nutrient addition experiments. Model I linear regressions and Pearson correlation coefficient were used to study log–log relationships between BP and Chl or PP; and evolution of DOC and POC with time.
Salinity and temperature measurements show that the water column was not stratified over the course of the experiment, except in the first two days, which were characterized by a slight stratification both inside and outside of the mesocosms (Bonnet et al., 2016b). No vertical stratification was observed in the mesocosms for bacterial production or alkaline phosphatase activity (APA; see exemplary data for M1 in Supplement Fig. S1) as for most of the parameters (Bonnet et al., 2016b; Turk-Kubo et al., 2015; Berthelot et al., 2015). For all descriptions of biogeochemical stocks and fluxes, we thus used the average of the three depths to plot the temporal evolution within each mesocosm.
Evolution of
Based on the Chl and PP dynamics, two periods P1 (days 5–14) and P2 (days
15–23) were identified after DIP fertilization, which were also identified
by Berthelot et al. (2015) based on biogeochemical
characteristics and by Turk-Kubo et al. (2015) based on
changes in abundances of targeted diazotrophs. Diatom heterocyst-forming
symbionts associated with diatoms were abundant during P1, while a bloom of
the unicellular N
Log–log relationships between heterotrophic bacterial production
(BP) and
Response of heterotrophic bacterial production to the enrichment
experiments conducted on days 2 and 20. Asterisks show significant responses
in comparison to the unamended control (Co) after the Mann–Whitney test (
Carbon budget of the mesocosms with time (
Averages
Mann–Whitney tests were performed to test
significant differences between P1 and P2:
Abundances of heterotrophic bacterioplankton (HBA) varied 10-fold, from 1.7
(day 9, M1, 1 m) to 12.8
Log–log relationships between BP
APA was homogeneous between the three depths sampled from the mesocosms
(example for M1 on Fig. S1), but this was not the case in the Nouméa
lagoon, where activity was often higher at 1 m depth compared to the two
other depths (data not shown). A slight but very reproducible decrease of
APA occurred on days 5 and 6 in all three mesocosms where DIP fertilization
took place and in lagoon waters only on day 5 (Fig. 3). DIP was consumed
more rapidly in M1, mirrored by higher APA and lower TDIP between day 9 and
18 in this mesocosm (Fig. 3). APA then increased very rapidly in M1 and M2
after day 17, but only after day 21 in M3. Such delays were in agreement
with the evolution of DIP, which was less rapidly consumed in M3 compared to
M1 and M2 (Berthelot et al., 2015). Consequently, although mean APA
increased significantly in all three mesocosms between P1 and P2 (Table 1),
it was lower in M3 compared to M1 and M2 during P2 (3.1 vs. 7.5–7.9 nmole MUF-P hydrolyzed L
In the two 48 h nutrient enrichment experiments performed on day 4 and on
day 20, BP increased 3-fold after nitrogen addition (NH
Among the different groups sorted by flow cytometry, significant
cell-specific leucine incorporation rates into macromolecules were obtained
for heterotrophic bacterioplankton. LNA, HNA and hi-HNA cells had specific
activities ranging from 20 to
554
Specific leucine activities of main groups sorted: PRO
(
We used the advantage of a day-to-day sampling in an enclosed system to
compute a carbon budget that will allow us to estimate the fate of
phytoplankton-derived organic carbon and the metabolic balance. This carbon
budget was calculated using time-integrated data and thus took into account the
whole data set. First, each time point was averaged for the three sampling
depths, and then time integration was calculated separately for each
mesocosm assuming a linear trend between two successive days. A mesocosm
average was calculated based on the time-integrated data obtained in each of
the three mesocosms, with error bars representing the standard deviation
(SD) among the three mesocosms (Fig. 6a). Gross primary production (GPP) is
derived from PP assuming GPP
Linear regression fits on temporal trends of POC and DOC in M1, M2
and M3 from days 5 to 23. DOC has been sampled only at 6 m depth in the
three mesocosms. df: degree of freedom,
Overall, M3 exhibited maximum peaks of chlorophyll biomass as well as PP and BP rates, and these different responses were particularly seen during P2. A time lag of a few days in the succession of the different planktonic populations was noticed, particularly for nitrogen fixers (Turk-Kubo et al., 2016), and DIP was consumed more rapidly in M1. However, slight divergence in biological and chemical evolution among different replicated mesocosms is not uncommon, particularly after the first week of enclosure (Martínez-Martínez et al., 2006; Pulido-Villena et al., 2014). Here, the divergence probably resulted from a combination of bottom-up (availability of DIP and nitrogen) and top-down controls (grazing pressure and viral lysis). The initial conditions prevailing before the DIP enrichment could occur also at the origin of the divergence. Indeed mesocosms were closed three days before the DIP addition, and many species of diazotrophs exhibit a patchy distribution (Bombar et al., 2015). In addition, Hunt et al. (2016) noticed larger amount of zooplankton individuals in M3 at the beginning of the experiment, some of which, stressed by the mesocosms, might have died (some larger amounts of “swimmers” were recovered in the traps in M3), contributing to supplementary sources of N in M3. Nevertheless, overall the replicability among mesocosms was considered sufficiently correct for most of the biogeochemical stocks, fluxes and abundances of phytoplankton groups (Bonnet et al., 2016b) and thus our results are discussed based on averages.
BP was significantly enhanced on a short-term scale (1–2 days) by
NO
A slight
BP was used in this study as a strict proxy of heterotrophic bacterial
production. As we incubated
Torréton et al. (2010) report mean Chl concentration around
0.3
Assuming negligible effect of a biofilm development on the mesocosms walls
(Knapp et al., 2015) on the plankton C budget, the main fate of
photosynthetically fixed organic carbon during the experiment was respiration
(71 % of GPP) then sedimentation (17 % of GPP). The different
responses between the triplicate mesocosms led to a great propagation of
errors and thus the variability of CR / GPP ratio was also high (70
In the lagoon, close to Grande Rade Bay, long residence times favoured
local degradation, refractorization of organic matter and not sedimentation
(Mari et al., 2007). However, as these authors discussed, modification of
phytoplankton community composition in Grande Rade Bay and the presence of
metals could influence sticking properties of polymers. The confinement of
the seawater inside the mesocosms probably favoured to some extent the
accumulation of UCYN-aggregates, as well as a possible reduction of grazing
pressure (by a factor of 1.6) in the mesocosms compared to those in the
lagoon waters (Turk-Kubo et al., 2015; Bonnet et al., 2016a; Hunt et al.,
2016). However, UCYN-C formed large aggregates (100–500
In order to calculate the fraction of GPP that directly or indirectly
channelled through the microbial food web, the bacterial carbon demand (BCD)
must be estimated through additional estimates of bacterial respiration (BR)
or bacterial growth efficiency (BGE). In an oligotrophic site inside the
Nouméa lagoon, BGE was estimated at 10 % using incubated samples
where oxygen changes were followed with time in the dark (Briand et al.,
2004). However, as suggested by Aranguren-Gassis et al. (2012),
using consistently low BGE, derived from size fractionation
experiments and long-duration incubations, leads to probable BGE
underestimation. In the lagoon, the use of a 10 % BGE would lead to BCD
values higher than GPP (Rochelle-Newall et al., 2008). If we assume such low
BGE in the mesocosms (10 %), cumulated BR from day 5 to 23 would rise to
93
The BGE values determined from C budget could be potentially related to a
beneficial effect of photoheterotrophy. Indeed, in a companion
metatranscriptomic study performed in M1 (Pfreundt et al., 2016a),
accumulation of proteorhodopsin transcripts was recurrently detected among
varying groups of bacteria notably Pelagibacteraceae and SAR86 These groups,
belonging to the alpha- and gammaproteobacteria, respectively, were also
abundant community members as observed through 16S sequencing (Pfreundt et
al., 2016b). Aerobic anoxygenic phototrophic (AAP) bacterial abundances are
reported to be particularly abundant in the South Pacific Ocean (Lami et al.
2007), but to date, AAP abundances are not available in the lagoon and they
were not counted in this experiment. Nevertheless, Pfreundt et al. (2016a)
detected expression of the
Assuming BGE values ranging from 27 to 43 %, the BCD
This study confirms that in the Nouméa lagoon, N
Sophie Bonnet was the chief scientist responsible for the VAHINE
programme. She designed and executed the experiment in mesocosms.
France Van Wambeke sampled for and analysed BP and APA, Thierry Moutin
sampled for and analysed
Funding for this research was provided by the Agence
Nationale de la Recherche (ANR starting grant VAHINE ANR-13-JS06-0002),
INSU-LEFE-CYBER programme, GOPS, IRD and M.I.O. The participation of UP and
WRH was supported by the German-Israeli Research Foundation (GIF), project
number 1133-13.8/2011 and the MiSeq-based microbial community analysis by
the EU project MaCuMBA (Marine Microorganisms: Cultivation Methods for
Improving their Biotechnological Applications; grant agreement no: 311975)
to WRH. The authors thank the captain and crew of the R/V