Microbial consumption of phytoplankton-derived organic
carbon in the pelagic food web is an important component of the global C
cycle. We studied C cycling in two phytoplankton–bacteria systems
(non-axenic cultures of a dinoflagellate Apocalathium malmogiense and a cryptophyte Rhodomonas marina) in two
complementary experiments. In the first experiment we grew phytoplankton and
bacteria in nutrient-replete conditions and followed C processing at early
exponential growth phase and twice later when the community had grown
denser. Cell-specific primary production and total community respiration
were up to 4 and 7 times higher, respectively, in the A. malmogiense treatments. Based on
the optical signals, accumulating dissolved organic C (DOC) was degraded
more in the R. marina treatments, and the rate of bacterial production to primary
production was higher. Thus, the flow of C from phytoplankton to bacteria
was relatively higher in R. marina treatments than in A. malmogiense treatments, which was further
supported by faster 14C transfer from phytoplankton to bacterial
biomass. In the second experiment we investigated consumption of the
phytoplankton-derived DOC by bacteria. DOC consumption and transformation,
bacterial production, and bacterial respiration were all higher in R. marina
treatments. In both experiments A. malmogiense supported a bacterial community
predominated by bacteria specialized in the utilization of less labile DOC
(class Bacteroidia), whereas R. marina supported a community predominated by
copiotrophic Alpha- and Gammaproteobacteria. Our findings suggest that large
dinoflagellates cycle relatively more C between phytoplankton biomass and
the inorganic C pool, whereas small cryptophytes direct relatively more C to
the microbial loop.
Introduction
Dissolved organic carbon (DOC) forms the largest aquatic organic C pool
(∼ 660 Pg C; Hansell et al., 2009),
comparable in magnitude to atmospheric CO2 (∼ 780 Pg C;
Emerson and Hedges, 2008). Phytoplankton are the most important
source of autochthonous DOC in marine systems
(Thornton, 2014). DOC is the main
energy source for pelagic heterotrophic bacteria (Ducklow and
Carlson, 1992), which quickly consume the most bioavailable organic
molecules. As a result, the bulk of the marine DOC pool consists of refractory
DOC (Jiao et al., 2010). Depending on the
composition of DOC and surrounding conditions, DOC may accumulate in the
water column (Hedges, 1992; Jiao et
al., 2010; Mari et al., 2017), aggregate and sink (Engel
et al., 2004), or be consumed (Azam et al.,
1983; Kujawinski, 2011). The rates of these processes determine the
prevalent fate of DOC and thus greatly determine total C cycling pathways.
The proportion of a phytoplankton species in a mixed community may affect
the release of dissolved organic matter (DOM) within that community. The
composition of phytoplankton-derived DOM is generally affected by growth
phase (Urbani et al.,
2005), environmental conditions (e.g., increased C : nutrient ratios of
released DOM under nutrient limitation;
Saad et al., 2016), and
physiological state of the phytoplankton community (e.g., release of specific
compounds as a result of cell death; Orellana et al.,
2013). Different phytoplankton species produce different kinds of DOM
(Becker
et al., 2014; Mühlenbruch et al., 2018; Romera-Castillo et al., 2010;
Sarmento et al., 2013), which shape the composition of the bacterial community
depending on the composition of the released DOM
(Romera-Castillo
et al., 2011; Sarmento et al., 2013; Sarmento and Gasol, 2012; Teeling et
al., 2012). Bacteria remineralize and transform organic matter and as a
result produce different types of DOC
(Kawasaki and Benner, 2006), nutrients
(Amin et al., 2009; Christie-Oleza et al.,
2017), and other substances (Croft et al.,
2005) that become available to phytoplankton. Interactions among
phytoplankton and bacteria may affect the composition of DOM released by
phytoplankton (reviewed by Mühlenbruch et al.,
2018).
Metabolic capability to utilize rapid pulses of phytoplankton-derived DOM
varies among bacteria, and thus phytoplankton blooms are followed by distinct
succession patterns of various bacterial genera commonly from the classes
Gammaproteobacteria, Alphaproteobacteria, and Bacteroidia
(Mühlenbruch et al., 2018;
Teeling et al., 2012). Marine bacteria are often functionally divided in
copiotrophs and oligotrophs based on their C uptake strategies. Oligotrophs
are specialized in low nutrient concentrations, whereas copiotrophs thrive
in high nutrient and DOM concentrations. Labile DOM attracts copiotrophic
bacteria capable of quickly draining the DOM pool of its most bioavailable
labile components (Pedler et al.,
2014). Copiotrophic bacteria can become abundant, e.g., after phytoplankton
blooms, but because oceans are mostly a low-nutrient environment
copiotrophic bacteria are not as ubiquitous as oligotrophic bacteria, e.g.,
alphaproteobacterial SAR11 clade (Morris et al., 2002).
Optical properties of colored and fluorescent DOM (CDOM and FDOM,
respectively) can be used as proxies of DOM bioavailability and source
(Coble, 1996). Proxies for properties such as molecule size and
amino acid content can be used to make predictions of the ecological
function of the DOM pool by, for example, identifying DOM produced by phytoplankton
blooms (Suksomjit et al., 2009), DOM degraded
by bacteria (Kinsey et al., 2018), or DOM of freshwater
origin (Coble, 1996). CDOM produced by phytoplankton differ in
composition depending on phytoplankton species
(Fukuzaki et al., 2014; Romera-Castillo et
al., 2010). The composition is further altered by bacterial DOM utilization
(Guillemette and del Giorgio, 2012;
Romera-Castillo et al., 2011).
Mixed species communities mask C cycling differences that stem from the
traits of individual phytoplankton species. Even during single species
blooms previous environmental conditions may affect C cycling and DOM
processing. Knowledge on the full cascade of C cycling through manipulated
phytoplankton–bacteria communities aids to understand the contribution of
individual phytoplankton species to C cycling in mixed communities. This is
especially important because the composition of natural mixed phytoplankton
communities seems to have little effect on the chemical composition of the
accumulated autochthonous DOM, apparently due to rapid bacterial DOM
processing (Haraguchi et al., 2019). In mixed phytoplankton
communities it is also difficult to detect how the age and physiological
state of individual phytoplankton species affects bacterial community
composition (Grossart et al., 2005).
Environmental change has affected the composition of phytoplankton
communities (Li et al., 2009). In the Baltic Sea spring
blooms have shifted towards dinoflagellate predominance
(Klais et al., 2011),
and the ecological consequences of this shift are currently being
investigated (Spilling et al., 2018). In this study we
investigated how ecophysiology of two phytoplankton species affects
microbial C cycling. We compared two common coastal phytoplankton species: a
larger dinoflagellate Apocalathium malmogiense and a smaller, fast-growing cryptophyte Rhodomonas marina. In a broad
sense they could be considered a K-strategist and an R-strategist,
respectively (A. malmogiense can produce cysts (Kremp and
Heiskanen, 1999), can use allelopathy to inhibit growth of competitors
(Suikkanen et al., 2011), and has slower growth
rate than R. marina). A. malmogiense is a common, bloom-forming species in the Baltic Sea during
spring, and R. marina was chosen as a general model organism representing a smaller,
faster-growing phytoplankton. We hypothesized that these two
phylogenetically and physiologically different phytoplankton species show
differences in C cycling. The focus of the experiment was to investigate
whether these differences can be detected consistently on all levels of C
cycling, from dissolved inorganic C (DIC) fixation via DOM release to
bacterial DOM uptake and processing. Using phytoplankton cultures inoculated
with natural bacterial community from the Baltic Sea, we experimentally
investigated how species-specific differences in primary production (PP) and
DOM production affect C flow from phytoplankton to bacteria as well as bacterial
DOM consumption, production, and community composition. Our results on the
effects of individual species on C cycling will increase understanding on
how community shifts driven by environmental change will affect C cycling in
aquatic environments.
MethodsExperimental setup
The ecophysiology of two different phytoplankton species and its effect on
microbial C cycling from DIC uptake to bacterial DOC processing were
investigated in an experimental study design. A larger (cell volume
3391–12764 µm3; Olenina et al., 2006)
dinoflagellate Apocalathium malmogiense (Lars Gunnar Sjöstedt) Craveiro, Daugbjerg, Moestrup and Calado 2016 was compared to a smaller (mean cell volume 217 µm3;
Olenina et al., 2006), fast-growing cryptophyte Rhodomonas marina
(Pierre Augustin Dangeard) Lemmermann 1899. Both species are common in the Baltic Sea
during spring. Phytoplankton cultures were acquired from the FINMARI
culture collection/SYKE Marine Research Centre (A. malmogiense (syn. Scrippsiella hangoei), culture ID:
SHTV-2, isolated in Storfjärden, Tvärminne, by Anke Kremp in 2002;
R. marina, culture ID: Crypto08-A2, isolated in Storfjärden, Tvärminne by
Anke Kremp in 2008). These non-axenic unialgal phytoplankton cultures were
grown in artificial seawater to minimize the effect of growth medium on
optical DOM properties. Cultures were inoculated with the natural bacterial
community from the Baltic Sea (hereafter called A. malmogiense treatment and R. marina treatment)
and then investigated experimentally for the effect of species-specific
differences in PP and DOM production on C flow from phytoplankton to
bacteria as well as bacterial DOM consumption, production, and community
composition.
The experiment was conducted at Tvärminne Zoological Station (59.844966,
23.249642) during winter 2017–2018 in two parts: the DOM release experiment and the DOM consumption experiment (Fig. 1).
The purpose of the DOM release experiment was to study the long-term net
accumulation and alterations of DOM in conditions where phytoplankton
produce new DOM and bacteria consume it. The purpose of the DOM consumption
experiment was to study the effect of bacteria on DOM processing when the
phytoplankton are removed. The experiment was timed to winter and spring
months because the phytoplankton used were spring bloom species and we
wanted the natural bacterial inoculum to represent winter and spring bloom
bacteria. Phytoplankton batch cultures were grown in two triplicate series,
one for each part of the experiment with identical growth conditions.
Cultures were grown in F/2 growth medium in 5 L Erlenmeyer flasks in 4 ∘C (local seawater winter temperature) in approximately 60 µmol photons s-1 m-2 under a light–dark regime of 14 and 10 h. The growth medium was prepared in artificial seawater (autoclaved MQ
water adjusted to salinity 6 using Tropic Marin Classic Sea Salt). Vials
were stirred manually every 1–2 d and prior to any sampling.
A schematic description of the DOM release experiment (a) and the
DOM consumption experiment (b). Both experiments were conducted separately
for A. malmogiense and R. marina. The black timeline arrow at the far left starts from the inoculation
of phytoplankton into 5 L growth vials. During the timeline phytoplankton
abundance and optical properties of DOM were measured one to three times a week. Grey
arrows depict the flow of water through different filtration, mixing, and
measurement steps. (a, bracketed area) Procedure for an individual key point
incubation, which were conducted thrice for each species. On day 1 the bacterial
inoculum was prepared, on day 2 the incubation was initiated (i.e.,
phytoplankton treatments were established) and measurements (production
line: primary production, bacterial production, 14C flow; DOM line:
DOC, CDOM, bacterial abundance) were taken at 4 h intervals, and on day 3 the
final primary production measurement was taken (for net primary production).
(b, bracketed area) Incubation of the DOM consumption experiment, which was
conducted once for each species. On preparation day 1 bacterial inoculum was
prepared, on preparation day 2 the cultures were filtered, on day 1 the
incubation was initiated (i.e., phytoplankton treatments were established),
and measurements (DOC, CDOM, bacterial abundance, production, respiration,
community composition, and growth efficiency) were taken daily until day 7.
The variables measured at each phase of the two experiments are listed in
Table 1. KPI: key point incubation (see text); BR: optical O2
consumption (bacterial respiration) measurement; F/2: F/2 growth medium
(control); TFF: concentration of bacteria by tangential flow filtration.
In the first part of the experiment, the DOM release experiment (Fig. 1a),
the phytoplankton and bacteria present in the cultures were grown together
for over 4 months, and phytoplankton abundance and optical properties of DOM
were monitored one to three times a week. At the beginning of the exponential growth
phase and at two later stages the C flow from phytoplankton to bacteria, DOM
alterations, and bacterial activity were measured using day-long incubations.
During these three measurement occasions (hereafter referred to as first,
second, and third KPIs after key point incubation), subsamples of
phytoplankton cultures were incubated with an inoculum of natural bacterial
community for 24 h and sampled at 0, 4, 8, and 12 h (+ extra sampling at
24 h for net PP). This experiment addressed the C flow in a combined
phytoplankton–bacteria community system.
In the DOM release experiment concentrations of chlorophyll a (chl a) and
particulate organic C and N (POC and PON, respectively) were measured, and
bacterial community composition was determined, before each KPI. Two
separate sample sets were incubated at each KPI.
In the first set, hereafter referred to as production line (Fig. 1a), phytoplankton
cultures and bacteria (90 % vol phytoplankton culture +10 % Vol mL
bacterial inoculum in 10 mL aliquots) were incubated in light and PP, and
bacterial production (BP) and 14C transfer from 14C-NaHCO3
via phytoplankton to the DOC pool and bacterial biomass were measured. Transfer
of 14C to DOC was investigated by filtering PP samples through 0.45 µm GD/X (Whatman) syringe filters and by measuring the radioactivity
in the filtrate. Transfer of 14C from DOC to bacterial biomass was
investigated by incubating the previously mentioned filtrate for 4 h in dark,
after which the incubation was stopped by addition of 50 % trichloroacetic
acid, and the particulate biomass in the samples was centrifuged for analysis
of radioactivity. The protocol for the production line is depicted in detail
in Fig. 2.
Schematic description of the production line of the DOM release
experiment. The schematic starts at the top from the “Production line mix”,
which consisted of phytoplankton culture (90 % Vol.) and inoculum of seawater bacteria (explained in Fig. 1a). This was divided into five 10 mL
aliquots in 20 mL scintillation vials, which were spiked with 125 µL
of 23.43 µCi mL-114C-NaHCO3 and incubated in light for 0
to 24 h. At 0, 4, 8, and 12 h one set of incubation vials was divided in two.
One half were terminated with 0.1 % formaldehyde for
14C-NaHCO3-incorporation measurement (i.e., primary production). To remove the remaining 14C-NaHCO3
prior to scintillation counting, 24 h acidification with HCl was used. The other half was filtered to remove the
majority of 14C-labeled phytoplankton and bacterial cells and mixed
(50:50 Vol.) with non-spiked production line mix (termed “mix” in the
lower left part of the schematic). This new mixture was then spiked with
either only 3H-thymidine or both 3H-thymidine and 14C-leucine
and incubated for 4 h. The mixture spiked with 3H-thymidine was used to
measure incorporation of 14C-labeled DOC originating from phytoplankton
into bacterial biomass (3H-thymidine incorporation was measured as a
control for bacterial activity). The mixture spiked with 3H-thymidine
and 14C-leucine was used to measure incorporation rate of both
radioisotope tracers and, subsequently, bacterial production based on both
tracers. These mixtures were terminated with trichloroacetic acid (TCA,
final concentration: 5 %) and measured according to the centrifugation method
(Smith and Azam, 1992). Instagel Plus
(PerkinElmer) was used as the scintillation cocktail, and liquid
scintillation counting was done with Wallac 1414 LSC.
In the second set, hereafter referred to as DOM line (Fig. 1a), phytoplankton were
removed by 0.8 µm filtration, and bacteria (225 mL filtered
phytoplankton culture +25 mL bacterial inoculum) were incubated in dark
and DOC concentration, optical properties of DOM, and bacterial abundance
were measured. This incubation was conducted in the dark to see if the
modified bacterial community influences the DOM pool already during the 12 h
KPI when photosynthesis is stopped. A separate incubation mix was needed
because, for technical reasons, we could not measure these variables from
the mix containing radioisotopes. Inorganic nutrients (NO3-, including
NO2-, and PO43-) were measured in untreated culture and DOM
line samples at 0 and 12 h.
Except for PP measurements, there were no trends in any other production
line or DOM line variables from 0 to 12 h measurements. Therefore the
measurements at all time points within a KPI were pooled for statistical
analysis and presentation. The variables measured at each phase of the DOM
release experiment are listed in Table 1.
Measured variables at each stage of both parts of the experiment
(DOM release experiment and DOM line experiment). The stages of the
experiments are explained in the methods and in Fig. 1. In short, in the
DOM release experiment monitoring of culture growth refers to the total time
the cultures were grown. During this time three shorter key point
incubations (KPIs) were conducted. In the DOM consumption experiment,
monitoring of culture growth refers to the period before the experimental
incubation.
DOM release experiment DOM consumption experiment MeasurementMonitoring ofculture growthKPI: phytoplanktonculture onlyKPI:production line mixKPI: DOM line mixMonitoring ofculture growthExperimentalincubation (7 d)Phytoplankton abundanceOne to three times per weekOne to three times per weekBacterial abundance0, 4, 8, and 12 hDailyBacterial communitycompositionAt the start of each KPI (also from seawater)At the start of each KPIDaily (also from seawater at the start of theincubation)14C incorporation0, 4, 8, 12, and 24 h3H and 14C incorporation0, 4, 8, and 12 hDailyBacterial respirationDailyNutrients (NO3-(including NO2-) and PO43-)At the start and end ofeach KPI (same time asfrom DOM linesamples)0 and 12 hParticulate organic C andN and chl aAt the start of each KPIDissolved organic C and total dissolved N0, 4, 8, and 12 hDailyOptical properties of dissolvedorganic matterOne to three times per week0, 4, 8, and 12 hDaily
In the second part of the experiment, the DOM consumption experiment (Fig. 1b), the phytoplankton were grown to high density (A. malmogiense:
∼ 1 × 104 cells mL-1, R. marina∼ 9 × 104 cells mL-1), after which the
phytoplankton and most of the bacteria were removed by filtering (GF/F
filters pre-combusted 450 ∘C for 4 h; Whatman). The filtrate,
inoculated with a natural bacterial community (1480 mL filtered phytoplankton
culture + 120 mL bacterial inoculum), was incubated for 7 d to study
the DOM processing and C flow in the bacterial compartment without
phytoplankton present. To ensure nutrient-replete conditions, 18 µmol
NH4Cl and 11 µmol NaH2PO4 were added in the
experimental mixtures at the start of the incubation. Temperature during the
incubation was increased to 10 ∘C to enhance the bacterial
processes for easier detection. A control treatment containing only F/2
medium and a natural bacterial community inoculum was used to investigate how
the natural bacterial community develops and how their DOC processing
differs in the growth medium in the absence of DOM derived from the cultured
phytoplankton and competition from cultured bacteria. During the 7 d
incubation of the DOM consumption experiment DOC concentration, optical
properties of DOM and bacterial abundance, production, respiration,
community composition, and growth efficiency were measured daily. The
variables measured at each phase of the DOM consumption experiment are
listed in Table 1.
The natural bacterial community inoculum was prepared the same way for both
parts. Seawater was collected at the pier of the station, and bacterial
abundance and community composition were measured. Seawater was vacuum
filtered using 10 mm Hg pressure through a 0.8 µm pore size
polycarbonate membrane filter (∅ 47 mm; Whatman) to remove grazers
including heterotrophic nanoflagellates, and 3 L of the filtrate was
concentrated to about 30 mL using tangential filtration (Pall Minimate 100 kDa TFF capsule), and then diluted to 300 mL with artificial seawater, after
which bacterial abundance was measured again. All handling of seawater and
bacterial concentrate was done in 4 ∘C. The purpose of this treatment
was to concentrate the seawater bacterial concentration 10-fold and to
remove most of the marine DOM. Phytoplankton culture and bacterial
concentrate were mixed in a volume ratio of 90:10 % to recreate the natural
concentration of seawater bacteria. Time limitations of filtrations forced
the use of only 92.5:7.5 % in the DOM consumption experiment. However, the
concentration of seawater bacteria proved to be inefficient and the final
ratio of seawater bacteria to bacteria present in the culture was small
(DOM release experiment: A. malmogiense: 7.53 %, 0.02 %, and 0.03 %, R. marina: 18.11 %,
0.03 %, and 0.02 %, at first, second, and third KPIs, respectively;
DOM consumption experiment: A. malmogiense: 3.38 %, R. marina: 1.70 %).
Laboratory analysesCell abundance
Phytoplankton and bacterial abundance were analyzed using flow cytometry (BD
Accuri C6 Plus). Phytoplankton abundance was analyzed in untreated samples
by plotting red fluorescence (670 nm long-pass filter, 488 nm excitation)
against forward scatter. Samples for bacterial abundance were fixed with
paraformaldehyde (final concentration: 0.9 %) and glutaraldehyde (final
concentration: 0.045 %), incubated in the dark at room temperature for 30 min,
frozen in liquid N, and stored in -80 ∘C until analysis. After
thawing, samples were diluted 10–100-fold with pH 8 TE buffer, stained with
Sybr Green I nucleic acid stain (final concentration 1:10000 Vol.), and
incubated in the dark at room temperature for 10 min. Heterotrophic bacteria
were detected and counted by plotting green (530/30 bandpass filter, 488 nm
excitation) fluorescence against red (670 nm long-pass filter, 488 nm
excitation) fluorescence so that they could be differentiated from cells
containing chl a. Cytometer data were analyzed with FCS Express 5 software
(De Novo software).
Primary production and 14C transfer
DIC was analyzed with an Elektro-Dynamo URAS-3E C analyzer against NaHCO3
standards. PP was measured from the mean of light sample 14C activity
corrected with dark sample 14C activity according to Gargas (1975) with the modifications described in Fig. 2. PP measured at each KPI
represents cumulative gross PP divided by time (GPP). PP at 24 h is the net
PP including the dark period (NPP). PP was used to calculate community
respiration according to Spilling et al. (2019) using
Eq. (1). Actual respiration measurements were not available for either
phytoplankton species so uniform respiration rates for light and dark
periods were assumed.
Respiration=(GPP×14)-(NPP×24)
GPP in A. malmogiense treatments decreased from 4 to 12 h at the second and third
KPI. This might have been caused by high respiration (see results) but also
by insufficient addition of 14C-NaHCO3, which might have resulted
in underestimation of GPP, which we did not want to carry over to other
results. Therefore GPP at 4 h was used in calculation of community
respiration and 14C flow percentages.
Concentration of 14C originating from 14C-NaHCO3 in DOC and
bacterial biomass was measured after the PP incubations (Fig. 2). The
transfer of 14C originating from 14C-NaHCO3 measured in
percent from phytoplankton via DOC to bacteria was quantified by dividing
the accumulation rate of 14C in each compartment with the accumulation
rate in the previous compartment (i.e., GPP : DIC, DOC : GPP, and bacterial
biomass : DOC). Accumulation of 14C activity in DOC and in bacterial
biomass was calculated by dividing the time-normalized activity in samples
after 12 h incubation with specific activity of 14C-NaHCO3. Before
calculations, 14C accumulation in the DOC pool and in bacterial biomass
was corrected for the ratio of 14C-DIC to ambient DIC concentration.
This was done by multiplying them with the ratio of PP to bulk 14C
accumulation rate in phytoplankton biomass.
Bacterial production and respiration
Thymidine and leucine-based BP were measured with the centrifugation method
(Smith and Azam, 1992) with modifications for
the DOM release experiment described in Fig. 2. 3H-thymidine
incorporation was converted to bacterial biomass increase by using
conversion factors of 1.1 × 1018 cells mol-1
(Riemann et al., 1987) and 0.12 pg C × (µm3 cell-1)0.7 (Norland, 1993) using a theoretical
bacterial cell volume of 0.063 µm3 cell-1
(Kuparinen, 1988). 14C-leucine incorporation was converted to
bacterial biomass increase with a conversion factor of 1.55 kg C mol-1.
The incorporation ratio of 3H-thymidine and 14C-leucine was calculated
to investigate departures from balanced growth of bacteria
(Chin-Leo and Kirchman, 1990).
From each experimental treatment in the DOM release experiment 100 mL was
enclosed in airtight septum-sealed Duran bottles at the start of the
incubation for bacterial respiration (BR) measurements (Fig. 1b). A needle-sheathed oxygen optode (PreSens NTH-PSt1-L5-TF-NS120/0.8-YOP) was pierced
through the septum to monitor bacterial oxygen consumption with an OXY-4
micro oxygen meter (PreSens). Prior to measurements oxygen optodes were
calibrated to 0 % and 100 % air saturation by exposing the optode to
Na2SO3 solution and water-vapor-saturated air, respectively.
Relative oxygen concentration was recorded every 10 min through the
incubation. BR for each day was calculated by dividing the difference in
relative O2 saturation between the start of the experiment and each day
with time. Oxygen solubility of 678.8 mmol L-1 (at 10 ∘C, 1 atm, and salinity 6) was used to convert relative O2 saturation to molar
concentration, which was then converted to units of mol C L-1 h-1.
Bacterial growth efficiency (BGE) each day during the DOM consumption
experiment was calculated separately for thymidine and leucine-based BP
using Eq. (2):
BGE=BP/(BP+BR),
where BP is bacterial production and BR is bacterial respiration calculated
as the change in C during the previous day and converted to h-1.
Therefore BGE is reported starting from day 2.
Bacterial community composition
A total of 500 mL (seawater) or 100 mL (DOM release experiment water (DOM line mix)
and DOM consumption experiment water) was vacuum filtered onto sterile 0.22 µm pore size membrane filters (∅ 47 mm; Whatman), frozen in liquid
N, and stored at -80 ∘C until analysis. DNA was extracted from
filters with DNeasy Power Soil kit (Qiagen) 6 months after the experiments
and stored at -80 ∘C for further processing. In addition, negative
controls without a sample were extracted. Only one replicate from seawater
(both experiments) and cultures (DOM release experiment) was sequenced (Fig. 1). For sequencing, 16S ribosomal RNA gene region V4 was amplified with a
polymerase chain reaction, using the universal bacterial primers 341F and
785R (Klindworth et al., 2013). A two-step
polymerase chain reaction and Illumina MiSeq (Illumina Inc, San Diego, CA,
USA) paired-end multiplex sequencing were performed at the Institute of
Biotechnology, University of Helsinki, Finland. In total 16 × 106 paired raw reads were obtained with the Illumina MiSeq platform.
Primer removal was done with Cutadapt (settings – m 1 – O 15 – e 0.2, V 2.1
with Python 3.5.3; Martin, 2011). Reads were merged and
processed according to the DADA2 pipeline (DADA2 V 2.1.10 Rcpp V 1.0.0;
Callahan et al., 2016) with filterAndTrim maxEE = 3.
After filtering and trimming, a total of 11.2 × 106 sequences
remained from which 10.7 × 106 were merged and 9.1 × 106 were non-chimeric and used for further analyses. Taxonomic
classification of the amplicon sequence variants (ASVs) was done with DADA2
default parameters (minBoot = 50) using Silva for DADA2 (v. 132,
Quast et al., 2013,
https://zenodo.org/record/1172783#.Xila11 MzZgg, last access: 16 October 2019). Before the statistical
analyses, chloroplasts and mitochondria were removed, ending up with 4545 ASVs. Raw reads are deposited in the Sequence Read Archive of the National
Center for Biotechnology Information under BioProject accession number
PRJNA647035.
Dissolved C and N and optical properties of DOM
DOC and CDOM samples were prepared by filtering 20 mL of water through acid-washed and pre-combusted GF/F filters (450 ∘C, 4 h) into acid-washed and pre-combusted glass vials, which were then sealed with a septum
cap. DOC samples were acidified to pH 2 with 2 M HCl and stored at -20 ∘C until analysis of DOC with a Shimadzu TOC-V CPH total organic
carbon analyzer. Filtered CDOM samples were analyzed within 24 h. CDOM
absorption was measured using a Shimadzu 2401PC spectrophotometer with a 4 cm quartz cuvette over the spectral range from 200 to 800 nm with 1 nm
resolution. Ultrapure water (MQ) was used as the blank for all samples.
Excitation–emission matrices (EEMs) of FDOM were measured with a Varian Cary
Eclipse fluorometer (Agilent). Processing of the EEMs was done using the
eemR package for R software (Massicotte, 2016). A blank
sample was subtracted from the EEMs, and the Rayleigh and Raman scattering
bands were removed from the spectra after calibration. EEMs were calibrated
by normalizing to the area under the Raman water scatter peak 11 (excitation
wavelength of 350 nm) of an MQ water sample run on the same session as the
samples and were corrected for inner filter effects with absorbance spectra
(Murphy et al., 2010). For assessing the
characteristics and the quality of the DOM pool, fluorescence peaks
(Coble, 1996) were extracted from the EEMs. In this study the
following optical variables were used as proxies for DOM characteristics:
absorbance coefficient at 254 nm (aCDOM(254)) as a general indicator of
optically active molecules and light attenuation, absorption spectral slope
between 275 and 295 nm (S275-295) as a proxy of molecular size
(Helms et al., 2008), fluorescence peaks in T and C
(Coble, 1996) as proxies of protein-like and humic-like DOM,
respectively, and humification index (HIX; Zsolnay et
al., 1999) as an indicator of relative humification of DOM. Additional
optical variables were collected, but these were not included in the
detailed analysis and are only presented in Appendix A (Figs. A1, A2 and
Table A1).
Particulate organic C and N, chl a, and nutrients
For POC/N and chl a measurements, 20 mL of sample water was filtered through
GF/F filters (for POC/N they were pre-combusted in 450 ∘C for 4 h). The POC/N filters were wrapped in a foil and stored at -20 ∘C
until analysis with a Europa Scientific ANCA-MS 20-20 15N /13C mass
spectrometer. Chl a filters were placed in EtOH for extraction in the dark at
room temperature overnight, after which the extracts were stored at -20 ∘C until fluorometric analysis with a Varian Cary Eclipse
spectrofluorometer. A total of 300 µL of sample was added into a well plate, and
the fluorescence was measured (excitation/emission: 430/670 nm).
Fluorescence intensity was converted to chl a concentration using chl a
standards (Sigma).
Nutrient samples were frozen immediately after sampling at -20 ∘C
and stored frozen until measurement according to Grasshoff
et al. (1999) using a Thermo Scientific Aquakem 250 photometric analyzer.
Statistical analyses
All statistical analyses were done using R version 3.6.1 (R Core Team, 2019) and figures using package ggplot2 (Wickham,
2016). Differences in variables between treatments (species) and among KPIs
were analyzed using Welch's ANOVA, which allows for some difference in
variance among treatments. If there was no trend in measurements of all the
time points within a KPI, all the measurements were pooled for the
statistical analyses. For cumulative variables (14C accumulation rate
in DOC and in bacterial biomass), measurements at 12 h were used in the
statistical analyses. For GPP, measurements at 4 h were chosen for
statistical analyses (justified in results). Significant differences among
KPIs were investigated using Games–Howell post hoc test
(Peters, 2018). Differences in ANOVA were considered
significant at a p<0.05. Results of all Welch ANOVA tests are given
in Appendix B.
All multivariate analyses for bacterial community analysis were performed on
the Bray–Curtis dissimilarity matrix derived from square-root transformed
values. The bacterial community dynamics in the experiments was visualized
with principal coordinate analysis (PCoA). To determine whether the
bacterial communities differed significantly between different phytoplankton
species, a PERMANOVA (permutational multivariate analysis of variance;
Anderson, 2001) was performed using the function adonis
(9999 permutations) in the R package vegan (Oksanen et
al., 2019). Due to the lack of replicates, differences between phytoplankton
treatments with seawater and control treatments could not be tested. The
homogeneity of dispersion was tested using the function betadisper (9999
permutations) in the R package vegan (Oksanen et al.,
2019). To determine the association between the environmental parameters and
bacterial community composition, distance-based redundancy analysis (dbRDA)
with 9999 permutations (capscale; Oksanen et al., 2019)
was done. Significance of the model and the explanatory variables were
tested with analysis of variance (ANOVA; Oksanen et al.,
2019), using 9999 permutations. Biologically most relevant factors with
a variance inflation factor less than 10 that were statistically significant
(p<0.05) were chosen for the analysis (vif.cca;
Oksanen et al., 2019). Bar plots and PCoA (Bray–Curtis
dissimilarity) were made with phyloseq v. 1.26.1 (McMurdie and
Holmes, 2013).
(a) Abundance of phytoplankton (mean of three replicate
treatments; error bars indicate 1 standard deviation) during the DOM
release experiment. Vertical lines from left to right in each panel mark the
times of the first, second, and third KPIs. (b) Cell-specific gross
primary production in A. malmogiense (grey, left) and R. marina (white, right) treatments at each
KPI. The number above the boxplots shows the number of measurements. Lower and
upper hinges of boxes mark the first and third quartiles, respectively,
while the whiskers extend to the lowest and the highest values within 1.5
times the interquartile range.
ResultsPhytoplankton growth and primary production
In the DOM release experiment R. marina grew faster to maximum density and ended the
growth phase sooner than A. malmogiense (Fig. 3a). The average cell size of both
phytoplankton species remained unchanged throughout the experiment, as
indicated by the forward scatter results from flow cytometry (data not
shown). There was no indication of N limitation during the experiment as
nitrate concentrations remained high (Table A1). Phosphate was depleted in
filtered A. malmogiense treatments but not in unfiltered treatments (Table A1), suggesting
intracellular phosphate storing. We assumed this stored phosphate to be
available to phytoplankton and bacteria and, consequently, their growth not
being P limited. PP (gross, net, and cell specific) was higher in A. malmogiense
treatments (Fig. 3b, Table B1).
The timing of KPIs aimed to capture comparable growth phases for these short
incubations, but A. malmogiense cultures were still growing when spring bloom was closing
in, so we had to initiate the measurements at earlier stages while the
natural bacterial communities still resembled winter and spring communities
(Fig. 3a).
In A. malmogiense treatments a second population distinguished by flow cytometry
based on lower red fluorescence (chl a fluorescence) also started to grow at the
same time as the main population (Table A1). This population grew linearly
to about 15 % of maximum density of the main population. These were
considered to possibly be cysts and were not included in A. malmogiense abundance in
further analyses, although A. malmogiense does not usually produce cysts in the low
temperature used in this study
(Kremp et al., 2009). This decision
brings a certain bias to the interpretation of the results, but we did
relevant calculations with and without these cells and the results did not
change significantly. We chose to exclude the group because we could not be
certain of what the group consists of, and we wanted to avoid including, for example,
cysts in the population of active A. malmogiense cells. We considered that doing the
opposite would have introduced unknown distortion to our interpretation.
Bacterial abundance (a, d), cell-specific bacterial thymidine (b, e), and leucine (c, f) incorporation at each KPI (0–12 h measurements pooled)
in the DOM release experiment (boxplots) and in the DOM consumption
experiment (line graphs). Lines A, B, and C mark different replicates
(culture filtrate + seawater bacteria), and ctrl marks the control (F/2 + seawater bacteria). The number above the boxplots shows the number of
measurements. Lower and upper hinges of boxes mark the first and third
quartiles, respectively, while the whiskers extend to the lowest and the
highest values within 1.5 times the interquartile range. Grey boxes (left) show A. malmogiense, and white boxes (right) show R. marina. Due to a measurement error only 12 h
measurements of bacterial abundance are available in A. malmogiense treatments at the third
KPI.
Bacterial production and 14C transport
In the DOM release experiment differences in bacterial abundance between the
phytoplankton were modest at each KPI (Fig. 4a, Table B1). The ratio of bacteria
to phytoplankton was between 1×104 and 3×104 most of the time
except at the first KPI for R. marina, when it was much lower (Tables A1, B1).
Thymidine incorporation was slightly higher in A. malmogiense treatments at each KPI (Fig. 4b, Table B1; see Table A1 for BP), while leucine incorporation was of equal
magnitude between the species, except at the first KPI (Fig. 4c, Table B1). As a result, the ratio of leucine to thymidine incorporation was higher
in R. marina treatments at the second and third KPIs (Table A1, B1). In the DOM
consumption experiment, bacterial abundance increased considerably faster,
and thymidine and leucine incorporation was higher in R. marina treatments than in
A. malmogiense treatments (Fig. 4d–f; see Fig. A2 for BP). These observations from both
experiments suggest that the R. marina community can support a more productive bacterial
community in proportion to PP.
A. malmogiense cells were more efficient in incorporating DIC (i.e., higher PP, Fig. 3b),
but they also respired more than R. marina cells, as shown by higher cell-specific
community respiration in the DOM release experiment (Fig. 5a, Table B1). In
the DOM consumption experiment, cell-specific BR was higher in R. marina treatments
(Fig. 5b). Although BP was higher in R. marina treatments, the lower BR in A. malmogiense
treatments led to comparable BGE in both treatments (Fig. 5c). Since BR was
consistently higher in R. marina treatments (DOM consumption experiment) and the
ratio of bacteria to phytoplankton cells was higher in the A. malmogiense treatments only
at the first KPI (DOM release experiment, Table A1), the higher
cell-specific community respiration in A. malmogiense treatments is likely mainly caused
by respiration of phytoplankton.
14C-DOC, originating from 14C-NaHCO3, was produced by both
species at each KPI (Fig. A3). 14C originating from
14C-NaHCO3 was incorporated in bacterial biomass at the second and
third KPIs. There were considerable uncertainties with this measurement:
14C-DOC was also produced in dark controls and surprisingly much at the
first KPI compared to the latter KPIs when phytoplankton biomass was much
higher (Fig. A3). Regardless, these uncertainties concern both phytoplankton
species, so we consider the analysis to be suitable for comparing the
species, despite uncertainties in the absolute quantities of 14C in
different compartments. Higher PP of A. malmogiense led to a higher fraction of
14C-NaHCO3 pool being incorporated into phytoplankton biomass
compared to R. marina (Table 2). However, a larger fraction of PP ended in filtrate
in R. marina treatments (Table 2). In R. marina treatments, a larger fraction of
14C-organic matter was also incorporated into bacterial biomass at the second
and third KPIs (Table 2), although the difference was very small at
the third KPI (at the first KPI no activity was detected in R. marina treatments so
comparisons could not be made). Of all the 14C that was fixed by PP,
about 5 and 4 times more ended up in bacterial biomass in R. marina treatments at the
second and third KPIs, respectively (Table 2).
(a) Community respiration divided by phytoplankton abundance in
A. malmogiense treatments (grey, left) and R. marina treatments (white, right) at each KPI in the
DOM release experiment. Cell-specific BR (b) and BGE calculated from leucine
incorporation-based BP (c) in the DOM consumption experiment. Lines A, B, and
C mark different replicates (culture filtrate + seawater bacteria), and
ctrl marks the control (F/2 + seawater bacteria). The number above the
boxplots shows the number of measurements. Lower and upper hinges of boxes
mark the first and third quartiles, respectively, while the whiskers extend
to the lowest and the highest values within 1.5 times the interquartile
range.
Flow of 14C originating from 14C-NaHCO3 between
different phases of the C cycle. Numbers are percentages of 14C
accumulation rates between the phases indicated in the left column (PP : DIC
is an exception as PP is a rate but DIC is a concentration). Stars indicate
the significance (p values) at the side of the significantly higher
percentage between species at the corresponding KPI, compared with
Welch's ANOVA (***<0.001, **<0.01, *<0.05). Welch ANOVA results are presented in Table B2.
In both experiments, bacterial communities resembled those in the cultures
and were distinct from the seawater community (bacterial inoculum),
suggesting that the addition of seawater bacteria had a negligible
contribution to the composition of the total bacterial community (Fig. 6).
In the DOM release experiment classes, Alphaproteobacteria,
Gammaproteobacteria, and Bacteroidia predominated the bacterial communities
(Fig. 6). Alphaproteobacteria increased and Gammaproteobacteria decreased
from the first KPI to the second KPI, whereas Bacteroidia had its peak
at the second KPI. The relative share of different classes differed
between the treatments: Bacteroidia (average: 45.6 %, genera
Algoriphagus and Polaribacter) and Alphaproteobacteria (average: 44.7 %, genera
Pseudorhodobacter and Sphingorhabdus) were the most abundant classes in A. malmogiense treatments while Alphaproteobacteria (average:
49.9 %, genus Pseudorhodobacter) and Gammaproteobacteria (average: 38.8 %, genus
Rheinheimera and RS62 marine group) predominated in the R. marina treatments. Interestingly, the
class Actinobacteria (average: 2 %, genus Candidatus Aquiluna) appeared in A. malmogiense treatments and
slightly increased along the experiment. Bacterial communities in both
phytoplankton treatments changed between the KPIs (PCoA, Fig. 7a, c). There
were also differences in bacterial communities between the phytoplankton
species in relation to selected environmental variables: thymidine-based BP
correlated with A. malmogiense at the second and third KPIs whereas aCDOM(254) and
S275-295 correlated with R. marina at the second and third KPIs (Fig. 7c).
In total, dbRDA axes 1 and 2 explained 51.33 % of the variation in
bacterial community analysis.
Class-level (upper panels) and genus-level (lower panels)
bacterial diversity of 16S ribosomal RNA (rRNA) gene sequences representing
>1 % of all amplicon sequence variants (ASVs) in the DOM release
experiment (a, c) and in the DOM consumption experiment (b, d). (a, c) The bars
labeled control refer to cultures before the addition of bacterial inoculum. (b, d) The bars labeled control refer to the control treatments (F/2 + seawater bacteria).
In the DOM consumption experiment class Bacteroidia (average: 70 %, genera
Algoriphagus and Polaribacter) and Alphaproteobacteria (average: 19.9 %,
genera Pseudorhodobacter, Sphingorhabdus, and Seohaeicola) predominated bacterial communities in A. malmogiense treatments (Fig. 6).
Congruently with the DOM release experiment, the class Actinobacteria (average:
5.7 %, genus Candidatus Aquiluna) was present throughout the experiment. In the small share
of the class Gammaproteobacteria (average: 1.7 %), the most abundant were the
order Betaproteobacteriales (genera Hydrogenophaga, Kerstersia, Limnobacter, Methylotenera). In R. marina Alphaproteobacteria (day
1–3 average 82 %, genus Pseudorhodobacter) predominated the bacterial communities until
day 3, after which they began to decrease (day 4–7 average: 36.1 %) and
Bacteroidia (day 4–7 average: 49.9 %; genus Flavobacterium) increased. Also,
Gammaproteobacteria increased slightly towards the end of the experiment
(day 4–7 average: 7.7 %, genera Shewanella, Marinomonas, and Polynucleobacter). In R. marina, bacterial community
composition changed with time (PCoA, Fig. 7b). Bacterial communities
differed significantly between the different phytoplankton treatments
(adonis: R2=0.67, p<0.001), but due to the tight grouping
in A. malmogiense, the homogeneity of variance was violated (betadisp: p>0.05, Fig. 7d). However, because groups were not overlapping, it can be
assumed that the observed differences are true. The shift in bacterial
community was also observed in relation to selected environmental
parameters: bacterial communities on days 1–3 correlated with peak T and
aCDOM(254), whereas on days 4–7 they correlated with thymidine-based BP
(Fig. 7d). In total, dbRDA axes 1 and 2 explained 68.89 % of the variation
in bacterial community analysis.
(a, b) Principal coordinate analysis plots showing bacterial
community dynamics in different experiments and (c, d) dbRDA biplots showing the
relationship between bacterial communities and selected significant
environmental variables (ANOVA, p<0.05) in the DOM release experiment
(a, c) and DOM consumption experiment (b, d). THY: thymidine based
bacterial production; a254: aCDOM(254); S275-295: S275-295.
In the control treatments of the DOM consumption experiment (seawater
inoculum + growth media) bacterial communities were comparable with the
seawater community in the beginning of the experiment in both experimental
treatments but later developed into cultures which were different from
communities in both seawater and experimental treatments (Fig. 6).
Interestingly, the class Campylobacteria (genus Argobacter), which was not abundant in
either of the phytoplankton treatments, began to increase in both control
treatments on day 4 (Fig. 6b, d).
DOM transformations
During the DOM release experiment DOC concentrations increased in both
phytoplankton treatments; however, only a very small increase from the second
to third KPIs was observed in R. marina treatments (Fig. 8a). DOM absorbance and
fluorescence generally started to increase when the phytoplankton started to
grow (Fig. 8, Fig. A1). The general trend was the accumulation of lower-molecular-weight molecules and potentially more refractory molecules, as seen by the
increase in aCDOM(254), S275-295, humic-like DOM peak C, and HIX.
S275-295, peak C, and HIX increased faster in R. marina treatments (Table B1).
While aCDOM(254) increased in both species during the experiment,
DOC-normalized absorbance at 254 nm (Weishaar
et al., 2003) increased slightly in R. marina treatments, whereas it decreased in A. malmogiense
treatments (Table A1). This suggests that the increase in aCDOM(254) in
R. marina treatments was caused mainly by the increase in DOC absorbing in UV region
with relatively higher intensity than the bulk material, whereas in A. malmogiense
treatments the increase in aCDOM(254) was caused by increased absorbance
due to higher bulk DOC concentration. This was also supported by the 3
times higher aCDOM(254) production by R. marina when the aCDOM(254)
consumption by bacteria during the DOM consumption experiment is subtracted
from the net accumulation of aCDOM(254) in the DOM release experiment.
DOC concentration (a, d), absorbance coefficient at 254 nm (b, e),
spectral slope between 275 and 295 nm (c, f) fluorescence peaks T (g, j) and C (h, k), and humification index (i, l) at each KPI (0–12 h measurements
pooled) in the DOM release experiment (boxplots) and in the DOM consumption
experiment (line graphs). Lines A, B, and C mark different replicates
(culture filtrate + seawater bacteria), and ctrl marks the control (F/2 + seawater bacteria). The number above the boxplots shows the number of
measurements. Lower and upper hinges of boxes mark the first and third
quartiles, respectively, while the whiskers extend to the lowest and the
highest value within 1.5 times the interquartile range. Grey boxes (left) show A. malmogiense, and white boxes (right) show R. marina. Optical variables not included in the
detailed analysis are presented in Fig. A1 and Table A1 (DOM release
experiment) and Fig. A2 (DOM consumption experiment).
While PP (Fig. 3b) and DOC accumulation (Fig. 8a) are both higher in A. malmogiense
treatments during the DOM release experiment, the difference in PP between
the phytoplankton treatments is higher than the difference in DOC
accumulation at corresponding KPIs. Thus, it can be assumed that a relatively
larger share of fixed C is directed to respiration by A. malmogiense, as also suggested by
higher community respiration (Fig. 5a), and to DOC release by R. marina. This is also
supported by the higher 14C-DOC production relative to PP in R. marina
treatments (Table 2). These results also suggest that DOM released by R. marina is
more bioavailable to bacteria than DOM released by A. malmogiense and that the microbial
loop is favored more strongly when DOM originates from R. marina.
At the beginning of the DOM consumption experiment DOC concentrations were
comparable between the two phytoplankton treatments and higher than in the
control treatments (Fig. 8d), indicating that considerable DOC production by
phytoplankton had occurred in both treatments despite the difference in the
phytoplankton abundance before the start of the incubation (R. marina:
∼9×104 cells mL-1, A. malmogiense: ∼1×104 cells mL-1). During the incubation the DOC concentration did not change
much in A. malmogiense treatments but decreased in R. marina treatments, especially during the
first 4 d.
Contrary to the DOM release experiment, in the DOM consumption experiment DOM
absorbance decreased during the incubation, although often no clear change
could be detected in A. malmogiense treatments (Fig. 8, Fig. A2). Peak C and HIX increased
at first, as in the DOM release experiment, but started to decline at day 4
(Fig. 8k, l). Likely in the DOM release experiment the continuous production
of fresh DOM by phytoplankton supplied the bacteria with bioavailable DOM,
that was consumed and transformed to more refractory, UV-absorbing
material. In the DOM consumption experiment the phytoplankton were no longer
present as a fresh DOM source, so the bacteria started to use the more
refractory material. This would also explain the bell-shaped curves
(increase until day 4 and then decrease) of peak C and HIX in the DOM
consumption experiment. Until day 4 the bacteria still used more
bioavailable material which was left from the phytoplankton and converted it
to optically active molecules, but on day 4 this material ran out and the
bacteria switched to consuming more refractory, optically active material.
Discussion
Species-specific differences in carbon cycling were found in both
experiments: in the DOM release experiment, where simultaneous processing of
DOM by phytoplankton and bacteria were investigated, and in the DOM
consumption experiment, where bacterial DOM processing was investigated.
Differences were clear at every level of carbon cycling: PP, flow of 14C
from DIC to bacterial biomass, optical properties of DOM, and the response in
the bacterial community. A. malmogiense displayed higher PP and higher community respiration
and, based on the optical properties of DOM and the composition of the
bacterial community, produced relatively less labile DOM than R. marina. R. marina, instead,
displayed lower PP but produced more labile DOM, which supported a more
productive bacterial community with a higher proportion of copiotrophs.
DOC production, transformation, and consumption
In the DOM production experiment the trends in DOC concentration and optical
DOM characteristics were similar between the two phytoplankton treatments
through KPIs 1–3, suggesting that there was no qualitative shift from
production to consumption of any DOM fraction detected by the optical
methods. As this was true for both species even though the cultures were not
at the same growth phase at each KPI, the observed changes in the optical
DOM properties were likely more related to the age of the culture than to
growth phases. Of course, the optical method does not detect changes in the
concentrations of optically inert molecules, such as simple carbohydrates,
and there may have been growth-phase-dependent changes in their production
(Chen and Wangersky,
1996; Urbani et al., 2005). We recognize the bias caused by measuring the
two phytoplankton cultures at different growth phases; the DOM processing in
the two phytoplankton treatments might have been more similar if the KPI
incubations and the initiation of the DOM consumption experiment had been
initiated at the same growth phases. But with the previously presented
reasoning, we concluded that comparing the two phytoplankton treatments
would show real differences in DOM processing of these species.
The decline in the abundance of R. marina was not fast or linear, and occasionally
abundance increased again, suggesting that conditions were still quite
favorable for R. marina during all KPIs. The resumption of growth might have been due
to the cells turning to heterotrophy, as some Rhodomonas species are known to be
mixotrophs (Ballen-Segura et al., 2017). As
nutrient limitation was most likely not significant, C limitation could be
another possible cause for population decline and a switch to support growth
with heterotrophy. Total dissolved C was high in both R. marina and A. malmogiense treatments even
at the third KPI, but since pH was not measured the relative fractions of
different forms of inorganic C are not known. To our knowledge, the capacity
of A. malmogiense to use different forms of inorganic C is not known, but many
dinoflagellates are able to use bicarbonate (Nimer et al.,
1997), suggesting that A. malmogiense was likely not C limited. The potential for C
limitation of R. marina is not clear since the use of different forms of inorganic C
by R. marina is not known. Some Rhodomonas species use only free CO2
(Elzenga et al., 2000), while some also seem to use
bicarbonate (Camiro-vargas et al., 2005).
Optical characteristics of DOM revealed potential sources and consumption
patterns in the experiments. Usually fluorescence peak T is interpreted as a
proxy for bioavailable DOM
(Nieto-Cid et al.,
2006), but it increased in both treatments together with the signals for
potentially less labile DOM (e.g., S275-295, peak C and
HIX) throughout the DOM release experiment. An increase in protein-like DOM
fluorescence has been connected to phytoplankton growth during simulated
(Stedmon and Markager, 2005)
and natural (Suksomjit et al., 2009)
phytoplankton blooms, but bacterial processing can decrease protein-like
fluorescence while increasing humic-like fluorescence
(Romera-Castillo et al., 2011; Yamashita and
Tanoue, 2004b). Therefore, simultaneous increases in peaks T and C likely
occurred because of (1) excess production of protein-like DOM by
phytoplankton, (2) production of less labile protein-like DOM by
phytoplankton, (3) production of protein-like DOM by bacteria, or
combinations of these. Not all protein-like DOM fractions are equally
degradable (Yamashita and Tanoue,
2004a), and some protein-like FDOM can accumulate in the pelagic environment
(Asmala et al.,
2018; Yamashita et al., 2017). Production of peak T by bacteria might be due
to bacterial reworking of initially labile (non-colored) autochthonous DOM
into small, UV-absorbing molecules
(Asmala et
al., 2018; Berggren et al., 2009). In the case of R. marina, this could possibly
result from the bacterial consumption of monosaccharides, which R. marina can produce
in high amounts (Fernandes et al., 2017), as
several bacterial species have been shown to produce peak T when grown on
glucose (Fox et al.,
2017).
Just like the simultaneous increase in most optical DOM variables in the DOM
release experiment, the decrease in most of the FDOM variables towards the
end of the DOM consumption experiment is surprising, given that bacterial
processing of phytoplankton-derived DOM is usually connected to an increase in
FDOM (Romera-Castillo et al., 2011). The high abundance of
Pseudorhodobacter might explain part of this as Rhodobacteraceae have been connected to
reduced FDOM intensities when using dinoflagellate-derived DOM
(Tada et al., 2017). Bacteria
may also change from a net source of protein-like FDOM to a net sink as
bacterial activity increases (Guillemette and del
Giorgio, 2012). This is in agreement with the decreasing peak T during the
DOM consumption experiment, as the higher temperature used in the DOM
consumption experiment may have directly enhanced bacterial activity.
Guillemette and del Giorgio (2012) also showed that production of humic-like
FDOM increases with increasing BGE, which is in line with the increase in
humic-like peak C and HIX concurrent with BGE until day 4, although after
that the FDOM signals decreased while BGE did not. The change in DOM
processing patterns on day 4, which was suspected to have been caused by the
depletion of fresh labile DOM originating from phytoplankton, was
also interesting because the production of humic-like DOM should increase
when bacteria shift from processing labile DOM to semi-labile DOM
(Jørgensen et al., 2015).
The overall differences between A. malmogiense and R. marina are similar to those in a previous
study with dinoflagellates Heterocapsa circularisquama and Alexandrium catenella and the cryptophyte Rhodomonas ovalis
(Fukuzaki et al., 2014). They observed higher
biomass production for the dinoflagellates and higher apparent percentage of
net photosynthetic extracellular release for R. ovalis. In addition to the inherent
species-specific physiological differences between A. malmogiense and R. marina, some fraction of
the different DOM release might be caused by more general traits, such as
the size difference between the species. Higher release of bioavailable DOM
from R. marina might simply be caused by the smaller size of R. marina cells and, therefore,
higher passive release of DOC (Bjørnsen, 1988).
Even though both of the phytoplankton species can be assumed to be
mixotrophic
(Ballen-Segura et al.,
2017; Rintala et al., 2007) and phytoplankton can take up DOM in mixed
communities (Bronk and Glibert, 1993; Moneta et al.,
2014), significant DOM consumption by phytoplankton during this experiment
was unlikely. Uptake of organic N or P would be energetically unlikely in
the presence of light and available inorganic N and P. Towards the end of
the experiment, if the decline of R. marina was caused by C limitation, DOM
consumption would have been more likely, and an unknown fraction of changes
in the properties of DOM could maybe be attributed to reuptake by
phytoplankton. However, because there was a shift from increase to decrease
in some optical DOM properties between the DOM release experiment and the
DOM consumption experiment, the principal role of R. marina was likely still the
production of DOM rather than its consumption throughout the DOM release
experiment.
Because the observed changes in optical DOM properties seem to be
independent of the ratio of bacterial to phytoplankton abundance, the
observed changes in DOM characteristics have to arise primarily from the
traits of individual phytoplankton species (rate and type of produced DOM;
Fukuzaki et al., 2014) or bacterial species
(rate and type of consumed and produced DOM
(Fox et al.,
2017; Romera-Castillo et al., 2011) instead of only from the ratio of
producers to consumers. A general conclusion from DOM quality indicators is
that R. marina produces comparatively more DOM, when normalized to PP, than A. malmogiense and that
this DOM seems to be more efficiently consumed and altered by bacteria.
However, DOC release and the rate of DOC production to PP do not necessarily
reflect natural conditions precisely for either phytoplankton species since
the fraction of PP released as DOC from phytoplankton is generally higher in
situ than in cultures (Thornton, 2014).
Response of bacteria to DOC
The higher leucine : thymidine incorporation ratio in R. marina treatments indicates
that bacteria struggled to get enough C and/or energy from DOM for balanced
growth. A likely explanation for this is that the bacterial community in R. marina
treatments efficiently depleted the readily available labile DOM pool, and
the stable DOM release from R. marina could not keep up with the demands of the
bacterial growth. This idea was supported by a much higher BP : PP ratio in R. marina
treatments during the second and third KPIs. In the DOM consumption
experiment bacteria growing on R. marina filtrate invested more in thymidine
incorporation (lower leucine : thymidine ratio) than in the DOM release
experiment. This was most likely caused by the relaxed resource competition
due to dilution of bacterial abundance during the filtration and further
suggests that the higher leucine : thymidine ratio in the DOM release experiment
was caused by intense competition for DOM among bacteria.
In general, Bacteroidia, Alphaproteobacteria, and Gammaproteobacteria
predominated bacterial communities in both DOM release and consumption
experiments, and the communities reflected those in the phytoplankton
cultures, indicating that the bacterial communities emerged from the
phytoplankton cultures. In the beginning of the DOM consumption experiment
in R. marina, the class Alphaproteobacteria (mostly genus Pseudorhodobacter) comprised 82 % of the
bacterial community, which was related to the high peak T and high BP. This
kind of “feast and famine” growth mode is typical for copiotrophic bacteria
(Lauro et al., 2009). Alphaproteobacteria benefit from
phytoplankton blooms when there is a high concentration of labile DOM
available (Allers et al., 2007), and they are efficient in
using amino acids (Cottrell and Kirchman, 2000; Gasol et
al., 2008). The predicted high production of monosaccharides by R. marina
(Fernandes et al., 2017) may explain the higher
proportions of Alpha- and Gammaproteobacteria in R. marina treatments.
Pseudorhodobacter has also been detected in a previous mesocosm study with Baltic Sea water
(Camarena-Gómez et al., 2018) as well as in Baltic Sea
bacterioplankton (Herlemann et al., 2011).
In general, the share of the class Bacteroidia (genera Algoriphagus and Polaribacter) was higher in A. malmogiense
treatments in both experiments, likely reflecting the less labile DOM pool
consisting of higher-molecular-weight molecules as suggested by the slightly
lower S275-295 in the DOM release experiment. In the R. marina treatments of the
DOM consumption experiment, the class Bacteroidia (genus Flavobacter) became abundant only
after day 3 congruently with the drop in peak T, implicating that the
ratio of labile to semi-labile DOM dropped on day 4 and caused a shift in
bacterial community composition towards more moderate copiotrophs. The phylum
Bacteroidetes is well known for its capability to degrade high-molecular-weight DOM (Cottrell and Kirchman, 2000; Romera-Castillo et
al., 2011) with their polysaccharide utilizing enzymes (Grondin
et al., 2017). Both Polaribacter and Flavobacterium are common moderate copiotrophs and detected from
phytoplankton blooms
(Mühlenbruch et al., 2018;
Teeling et al., 2012). In addition, Polaribacter and Algoriphagus have been detected in previous
Baltic Sea mesocosm studies (Camarena-Gómez et al.,
2018; Herlemann et al., 2017).
Actinobacteria, which were present in A. malmogiense treatments, are members of
autochthonous bacterioplankton in the Baltic Sea (Riemann et
al., 2008), occupying several different niches, and thus likely have various
different functions in the Baltic Sea food web
(Holmfeldt et al., 2009). They
have also occurred with dinoflagellates in a previous mesocosm experiment
with Baltic Sea water (Camarena-Gómez et al., 2018). In
the Baltic Sea, some Actinobacteria are linked to high DOC concentrations
and terrestrial DOM close to the land
(Holmfeldt et al., 2009), and
others are outcompeted by fast-growing copiotrophs when
phytoplankton-derived DOM is available
(Pérez and Sommaruga, 2006).
Possibly, in A. malmogiense treatments the presumably less labile DOM allowed them to
compete better with the copiotrophic Alpha- and Gammaproteobacteria. However,
it is also possible that the filtration in the DOM consumption experiment
caused a bias and favored them due to their small size
(Hahn et al., 2003).
In the DOM consumption experiment, a shift in the preferred substrate for
bacterial consumption and a concurrent shift in the bacterial community were
obvious in R. marina treatments even though DOC concentration was still high after
the incubation. This highlights the strong connection between phytoplankton
DOM release and bacterial processes. The existing DOM pool explains only
part of the mechanisms which structure the bacterial community. The fast
flow of 14C from the DIC pool through phytoplankton to the DOC pool and
bacterial biomass in DOM release experiment supports this statement. The
observed patterns in the bacterial community composition support the
interpretation from Sect. 4.1 that DOM was more labile in R. marina treatments than
in A. malmogiense.
In both phytoplankton treatments the final bacterial community at the end of
the DOM release experiment resembled the community at the end of the DOM
consumption experiment and was seemingly unaffected by the addition of
seawater bacteria. Most likely the low number of bacteria in the bacterial
inoculum could not compete with the high number of pre-existing bacteria.
This suggests that the phytoplankton–bacteria communities in the cultures
were somewhat stable and resistant to minor introductions of foreign
bacteria. This is in line with other studies which have shown stable and
predictable bacterial communities associated with certain phytoplankton
species (e.g.,
Schäfer
et al., 2002; Sapp et al., 2007; Goecke et al., 2013; Buchan et al., 2014;
Krohn-Molt et al., 2017; Mönnich et al., 2020). A variety of mutualistic
or algicidal interactions between bacteria and phytoplankton are known
(Seymour et al., 2017). Phytoplankton might
affect bacterial community composition by producing certain amino acids
(Tada et al., 2017), which may
in part explain why the development of the bacterial community in the DOM
consumption experiment was connected to peak T. The minor differences in the
thymidine and leucine incorporation between the species in the DOM release
experiment despite the major differences in PP and DOC processing, and the
comparable BGE between species in the DOM consumption experiment, suggest
that the bacterial communities, while different in composition, are
functionally optimized to grow using the DOC produced by the host
phytoplankton. The difference between control treatments and experimental
replicates of the DOM consumption experiment suggests that
phytoplankton-derived DOM, not the growth medium, is the main driver for
bacterial community and DOM processing dynamics.
Ecological implications of species-specific DOC dynamics
This experiment used phytoplankton batch cultures with higher concentrations
of phytoplankton and bacteria than in natural waters to better detect the
studied processes. The volume-related production rates in the experiment are
therefore assumed to be higher than in the natural experiments, but cell-specific production rates are likely closer to the natural environment. We
therefore did not compare the rates in the experiment with rates in the
natural environment. Instead we assumed that the differences in rates
between the two phytoplankton species would also be similar in the natural
environment. Likewise, even though most of the bacteria in the experiment
consisted of bacteria which had grown for long in culture conditions
together with the phytoplankton, their relationships with the investigated
DOM properties resembled relationships in natural communities. Thus, we
assume that these two phytoplankton species would also benefit the same bacterial
taxa in the natural environment.
A recent study connected a dinoflagellate community consisting of A. malmogiense and related
species to lower BP and a distinct bacterial community, compared to
communities with common spring bloom diatom species
(Camarena-Gómez et al., 2018). The results with A. malmogiense support
their view that DOC released from some dinoflagellate species may lead to
lower efficiency of the microbial loop.
When dinoflagellate blooms are not terminated in mass encystment, they are
expected to lyse in the water column and contribute to the pelagic DOC pool
(Spilling et al., 2018). Our results indicate that blooms
predominated by A. malmogiense indeed release high amounts of DOC, but this DOC may not be
readily bioavailable for bacteria coinciding with phytoplankton blooms and
may, therefore, stay in the pelagic system for longer. High biomass
production combined with release of less bioavailable DOC could lead to
direct grazing being favored over microbial loop. Thus, a probable long-term
effect of A. malmogiense predominance in natural communities on C cycling is the
accumulation of less bioavailable DOC at the expense of sedimentation and
microbial loop. Also, pronounced cycling of C between phytoplankton biomass,
and DIC pool can be expected, as community respiration was high and
dinoflagellates are generally considered to have high respiration rates
(Taylor and Pollingher, 1987).
Compared to A. malmogiense, R. marina produces less phytoplankton biomass and the DOC it releases
is more bioavailable. Thus, blooms predominated by R. marina may favor the microbial loop
and DOC processing over grazing. Since Alpha- and Gammaproteobacteria, which
were common in R. marina treatments, are heavily grazed by heterotrophic
nanoflagellates (Alonso-Sáez et al.,
2009), R. marina predomination may increase C transfer through the microbial loop. In
addition, the higher BR in the DOM release experiment may indicate that total C
fixation is lower during such blooms. Rhodomonas species have not traditionally been
connected to periods of high DOC release from phytoplankton
(Storch and Saunders, 1978), and, according to our
results, this might be the result of fast bacterial consumption of DOC
released by Rhodomonas species. The very fast consumption and transformation of DOC in
R. marina treatments in the DOM consumption experiment support the assumption of fast
DOC depletion in natural R. marina predominated blooms.
Strong extrapolations of these results to related phytoplankton species or
to phytoplankton of similar size should be made with caution, as even much
more closely related phytoplankton species may support differing bacterial
communities (Grossart et al., 2005) and, consequently,
different C cycling dynamics. Instead, we want to highlight the importance
of studying C cycling between individual phytoplankton species and related
bacterial communities in order to understand the mixed phytoplankton
communities of the natural environments. These community manipulation
experiments should also include protozoan grazers as their impact on DOM
composition and processing can be significant
(Kujawinski et al., 2016). Grazing may,
for example, alter bacterial community composition by removing groups which
are less resistant to grazing
(Alonso-Sáez et al., 2009), enhance DOM
production (Strom et al., 1997), and
affect the lability of the produced DOM
(Fouilland et al., 2014), all of
which can be assumed to affect C cycling. Better knowledge on C cycling at the
species level will help in predicting how the large-scale change in
phytoplankton community composition will affect C cycling at the ecosystem
level.
Conclusions
Two common phytoplankton species in the Baltic Sea, A. malmogiense and R. marina, produce DOM with
different bioavailability and support distinct bacterial communities
specialized in utilizing this specific DOM source. This results in different
C cycling patterns: A. malmogiense cells circulate more C between DIC and phytoplankton
biomass, while producing less labile DOC. R. marina releases more labile DOC, and
relatively more C is thus directed towards the bacterial community. DOC
released by R. marina is taken up, incorporated, and respired faster than DOC
released by A. malmogiense. Differences were clear at every level of C cycling: PP, flow
of 14C from DIC to bacterial biomass, optical properties of DOM, and the
response in the composition and activity of the bacterial community. This
experiment supports the view that phytoplankton and bacteria are intimately
connected through the rapid bacterial consumption of DOM released by
phytoplankton and that this connection explains bacterioplankton dynamics
better than the composition of the ambient DOM pool. An experimental
approach based on monocultures was necessary to quantify these differences
in C pathways. To better understand C cycling in a natural environment, it
may be beneficial to also see natural pelagic microbial communities as
collections of various linked phytoplankton–bacteria associations with
distinctive C cycling patterns.
Variables which were measured but not included in the detailed
analysis
CDOM and FDOM variables in phytoplankton cultures during the DOM
release experiment. (a–h) Absorption coefficients at different wavelengths.
(i, j, l) Spectral slopes between different wavelength ranges. (k) Ratio of
spectral slopes between 275–295 and 350–400 nm
(Helms et al., 2008). (n) Peak M, marine humic-like DOM
(Coble, 1996). (p) FI, fluorescence index
(Mcknight et al., 2001). (q) HIX, humification index
(Zsolnay et al., 1999). (r) BIX, biological index
(Huguet
et al., 2009). (s) DOC concentration at each KPI. Vertical lines from left
to right in each panel mark the times of the first, second, and third
KPIs. Error bars indicate 1 standard deviation. The number above the boxplots
shows the number of measurements. Lower and upper hinges of boxes mark the
first and third quartiles, respectively, while the whiskers extend to the
lowest and the highest values within 1.5 times the interquartile range. Grey
boxes (left) show A. malmogiense, and white boxes (right) show R. marina.
Those CDOM and FDOM variables (a–n) in the DOM consumption
experiment, which were measured but not included in the more detailed
analysis, and bacterial production measurements (o–p). (a–g) Absorption
coefficients at different wavelengths. (h, j) Spectral slopes between
different wavelength ranges. (i) Ratio of spectral slopes between 275–295 nm
and 350–400 nm (Helms et al., 2008). (k) Peak M, marine
humic-like DOM (Coble, 1996). (l) FI, fluorescence index
(Mcknight et al., 2001). (m) BIX, biological index
(Huguet
et al., 2009). (n) SUVA254, DOC-normalized absorbance at 254 nm
(Weishaar et al., 2003). Bacterial production based on thymidine (o) and
leucine (p) incorporation. Lines A, B, and C mark
different replicates (culture filtrate + seawater bacteria), and ctrl
marks the control (F/2 + seawater bacteria).
14C activity originating from 14C-NaHCO3 in the DOC
pool in the filtrates from light (a) and dark (b) primary production
incubation samples. 14C activity originating from 14C-NaHCO3
in bacterial biomass after 4 h of dark incubation of filtrate from light (c) and dark (d) primary production incubation samples amended with non-spiked
cultures. The number above the boxplots shows the number of measurements. Lower
and upper hinges of boxes mark the first and third quartiles, respectively,
while the whiskers extend to the lowest and the highest values within 1.5
times the interquartile range. Grey boxes (left) show A. malmogiense, and white boxes (right) show R. marina.
Mean±1 SD of additional variables at each KPI in the DOM
release experiment. S ratio: ratio of spectral slopes between 275–295
and 350–400 nm (Helms et al., 2008). Peak M: marine
humic-like DOM (Coble, 1996). FI: fluorescence index
(Mcknight et al., 2001). BIX: biological index
(Huguet
et al., 2009).
Welch ANOVA results of the DOM release experiment. Each line
presents the test of difference between the phytoplankton species for the
given variable at the given KPI.
Welch ANOVA results of 14C flow (Table 2). Each line presents
the test of difference between the phytoplankton species for the given ratio
at the given KPI.
Raw reads are deposited in the Sequence Read Archive of the National Center for
Biotechnology Information under BioProject accession number PRJNA647035.
Other data for this study were published open access at PANGAEA (Elovaara et
al., 2021).
Author contributions
SE, TT, and HK designed the experiment. SE conducted the experiment. EER
processed bacterial community composition data. EA processed DOM optics
data. SE processed remaining data. SE wrote the manuscript with
contributions from the other authors.
Competing interests
The contact author has declared that neither they nor their co-authors have any competing interests.
Disclaimer
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We thank Timo Tikka for help in establishing the experiments; Tvärminne
Zoological Station for producing facilities, practical aid, and analyses; and
Heidi Hällfors for aid and expertise related to phytoplankton cultures.
We thank the anonymous reviewers for considerably improving the manuscript. The
study utilized the Finnish Environment Institute Marine Research Centre
marine research infrastructure as a part of the national FINMARI RI
consortium.
Financial support
This research has been supported by the Walter ja Andrée de Nottbeckin Säätiö (grant “Phytoplankton mortality in marine ecosystems: Feedbacks to the pelagic detritus pool and biogeochemical cycling in a changing environment”), the Maa-ja vesitekniikan tuki ry (grant no. 13-8120-15), and the Societas pro Fauna et Flora Fennica (grant no. 5.12.2019). Eero Asmala was supported by the Academy of Finland (grant no. 309748). Tobias Tamelander was funded by the WANS and by the Swedish Cultural Heritage
Foundation. Open-access funding was provided by the Finnish Environment Institute.
Review statement
This paper was edited by Yuan Shen and reviewed by Youhei Yamashita and one anonymous referee.
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