In open-ocean regions, as is the Eastern Tropical North Atlantic (ETNA),
pelagic production is the main source of dissolved organic matter (DOM) and
is affected by dissolved inorganic nitrogen (DIN) and phosphorus (DIP)
concentrations. Changes in pelagic production under nutrient amendments were
shown to also modify DOM quantity and quality. However, little information
is available about the effects of nutrient variability on chromophoric
(CDOM) and fluorescent (FDOM) DOM dynamics. Here we present results from two
mesocosm experiments (“Varied P” and “Varied N”) conducted with a
natural plankton community from the ETNA, where the effects of DIP and DIN
supply on DOM optical properties were studied. CDOM accumulated
proportionally to phytoplankton biomass during the experiments. Spectral
slope (
Dissolved organic matter (DOM) is the largest dynamic pool of organic carbon in the ocean. Its global inventory comprises of approximately 662 pentagrams of carbon (PgC; Hansell et al., 2009). Labile and semi-labile high molecular weight (HMW) DOM is released primarily by phytoplankton (Carlson and Hansell, 2015). It is used as substrate by heterotrophic communities, which, in turn, release less bioavailable semi-refractory or even refractory DOM, thereby modifying the quantity and quality of the DOM pool (Azam et al., 1983; Ogawa et al., 2001; Jiao et al., 2010). Therefore, natural DOM is a complex mixture of organic compounds with different characteristics, such as molecular structure and molecular weight, resulting in different optical properties (Stedmon and Nelson, 2015).
For instance, the presence of conjugated double bonds (polyenes) results in
the absorption of light in the UV and visible wavelengths (Stedmon and
Álvarez-Salgado, 2011). The light absorbing DOM fraction is referred to
as “chromophoric” or “colored” DOM (CDOM; Coble, 2007). Due to its
abilities to absorb in a wide wavelength range, CDOM may protect primary
producers from harmful UV irradiation in the water column, but may also
reduce photosynthetically active radiation, as it absorbs at a similar
wavelength to the first chlorophyll absorption maximum (
CDOM absorption has often been used as an indicator for dissolved organic carbon (DOC) concentrations in the Ocean (Fichot and Benner, 2011, 2012; Rochelle-Newall et al., 2014). For example, DOC concentration in estuarine surface waters can be derived from CDOM absorption by remote-sensing techniques, assuming a direct relationship between CDOM absorption and DOC concentrations (Del Castillo, 2007). In the open ocean, however, this relationship varies throughout the water column (Nelson and Siegel, 2013), and factors affecting it are poorly understood.
A better knowledge on factors influencing the CDOM/DOC relationship could improve our understanding of DOM cycling, as well as of the regulation of light attenuation in the ocean. Furthermore, the knowledge of the factors influencing the open-ocean CDOM/DOC relationship would be useful for the estimation of DOC concentrations from CDOM absorption measurements by remote-sensing techniques.
As CDOM embodies a complex mixture of organic compounds that have overlapping absorption spectra with, generally, no single compound dominating (Del Vecchio and Blough, 2004), CDOM absorbance spectra decrease exponentially toward a longer wavelength with no discernible peaks. Therefore, the CDOM concentration is commonly expressed as absorption coefficient at a chosen wavelength (e.g., 325, 355, 375 nm; Stedmon and Markager, 2001; Fichot and Benner, 2012; Nelson and Siegel, 2013).
To derive information on CDOM quality, such as molecular weight and
modification processes, spectral slopes (
The ratio of
The presence of aromatic rings in CDOM often also results in fluorescence (Stedmon and Álvarez-Salgado, 2011). Fluorescent DOM (FDOM) excitation/emission (Ex/Em) spectra allow discriminating between different pools of CDOM (Coble, 2007; Stedmon and Bro, 2008; Mopper et al., 2007; Yamashita et al., 2010). The substances that are excited and emit in the UV spectral range commonly correspond to labile proteinaceous DOM, and therefore are referred to as amino acid-like (tyrosine- and tryptophan-like) FDOM (e.g., Coble, 1996). The substances that are excited in the UV spectral range, but emit in the visible spectral range were identified as fulvic- and humic-like FDOM (Gueguen and Kowalczuk, 2013). Tyrosine- and tryptophan-like substances have been used for the assessment of in situ primary productivity, while humic-like substances are used for the indication of allochthonous (e.g., riverine) DOM or microbial DOM transformation (Coble, 1996).
Although the CDOM and FDOM distribution and cycling has been described for many open-ocean sites (Jørgensen et al., 2011; Kowalczuk et al., 2013; Nelson and Siegel, 2013), specific sources and factors influencing their composition and transformations are not yet well understood.
For example, CDOM accumulation is often related to nutrient remineralization (Swan et al., 2009; Nelson and Siegel, 2013). However, the effects of nutrient variability on CDOM concentration and on the relationship between CDOM and DOC are largely understudied.
Stedmon and Markager (2005) have reported that nutrients affect freshly produced marine FDOM pools in temperate climate conditions (Raunefjord, Norway). In their study, the amino acid-like fluorescence was enhanced under phosphate (P) and silica limitation, but was independent from phytoplankton composition. Bacterially produced humic-like FDOM components were reported to accumulate under P and silica limitation as well. Later, by addition of different synthetic dissolved organic and inorganic nitrogen (N) substrates to microbial incubations, Biers et al. (2007) emphasized the role of N in CDOM accumulation. They showed that CDOM and FDOM production by bacteria, cultured in natural seawater medium, can be affected to a different degree by the chemical composition and steric effects of the organic N source, while inorganic N sources do not contribute significantly to CDOM or FDOM accumulation. On the other hand, Kramer and Herndl (2004) demonstrated that bacteria may be able to transform about 30 % of taken up inorganic N into semi-labile to refractory humic DOM.
Stedmon and Markager (2005), however, revealed some doubts about a setup of P limitation. Besides, Kramer and Herndl (2004) and Biers et al. (2007) were based on single bacterial cultures, and phytoplankton and net-effects, associated with natural aquatic bacterial community, were excluded. Therefore, the influence of inorganic nutrients on CDOM concentration and FDOM components in natural waters remains to be resolved.
In the open-ocean regions, as is the Eastern Tropical North Atlantic (ETNA), pelagic production of DOM is, supposedly, of greater importance than terrestrial DOM input (e.g., Coble et al., 2007).
In classical view, the ETNA is considered an “excess N” region compared to
the “Redfield N : P ratio” of 16 (see Redfield, 1958 and Gruber and Sarmento,
1997) reflecting high rates of biological N-fixation due to Saharan dust
deposition, with N : P ratios of
16–25 at depth (see Fanning, 1992). It features
a shallow oxygen minimum zone (OMZ) at about 100 m depth with oxygen
concentrations of
about 60
Here we investigated the effects of different dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) concentrations and of their supply ratio (DIN : DIP) on DOM quantity and quality by using spectroscopic methods of DOM analysis (e.g., accumulation and properties of CDOM and FDOM) during a mesocosm study with natural pelagic community off the Cape Verdean Archipelago, an area affected by low oxygen-core eddies.
During these mesocosm experiments, we tested whether (1) pelagic production is a source of CDOM and FDOM, (2) CDOM and FDOM accumulation and composition are affected by changes in nutrient stoichiometry, and whether (3) the relationship between CDOM absorption and DOC concentrations is stable under variable nutrient concentrations.
To do so, DOC concentrations, CDOM absorption and CDOM spectral properties
(
Two 8-day mesocosm experiments were conducted consecutively in October 2012
at the Instituto Nacional de Desenvolvimento das Pescas (INDP), Mindelo,
Cape Verde. Seawater from 5 m depth was collected into four 600 L tanks on
the night of the 1/2 and 11/12 of
October for the first and second
experiment, respectively. The sampling was done with the RV
Nutrients were manipulated by adding different amounts of phosphate (DIP) and nitrate (DIN). In the first experiment, the DIP supply was varied (“Varied P”) at relatively constant DIN concentrations in 12 of the 16 mesocosms, while in the second experiment initial DIN concentrations were varied (“Varied N”) at the constant DIP supply in twelve of the sixteen mesocosms.
In addition to this, four “corner points”, where both DIN and DIP were varied, were chosen to be repeated during both experiments (see target DIN and DIP values in Table 1). However, during the first experiment, setting the nutrient levels in one of the “corner point” mesocosms (mesocosm 10) was not successful and it was decided to adjust the DIN and DIP concentrations in this mesocosm to “Redfield N : P ratio” of 16 (Redfield, 1958) and therefore add another replicate to the treatment 12.00N/0.75P. Another “corner point” mesocosm (mesocosm 5) during the first experiment was excluded from further analyses as no algal bloom had developed.
Initial sampling for biogeochemical parameters was accomplished immediately after the mesocosms filling (day 1). Nutrients were added after the initial sampling. Daily water sampling was conducted between 09:00 and 10:30 a.m. on days 2 to 8.
The target and actual nutrient concentrations are shown in Table 1 and the corresponding treatment indications will be used in the following sections.
Varied P and Varied N: target and measured concentrations of DIN and DIP and treatment identifications according to target values.
Samples of 500 mL for chl
For bacterial cell counts, samples (5 mL) were fixed with 2 %
formaldehyde, frozen at
DOC duplicate samples (20 mL) were filtered
through combusted GF/F filters and collected in combusted glass ampoules.
Samples were acidified with 80
DOC samples were analyzed by applying the high-temperature catalytic
oxidation method (TOC -VCSH, Shimadzu) adapted from Sugimura and Suzuki (1988). The instrument was calibrated every 8–10 days by measuring
six standard solutions of 0, 500, 1000, 1500, 2500 and 5000
For CDOM and FDOM, duplicate samples of 35 mL for each parameter were
collected daily into combusted (450
Absorption of CDOM was detected using a 100 cm path length liquid waveguide cell (LWCC-2100, World Precision Instruments, Sarasota, Florida) and a UV-VIS spectrophotometer (Ocean Optics USB 4000) in conjunction with the Ocean Optics DT-MINI-CS light source. The absorbance was measured against ultrapure water (MilliQ) by injection to the cell with a peristaltic pump. The measurements were done over a spectral range of 178.23 to 885.21 nm at 0.22 nm interval.
For the determination of FDOM, 3-D
fluorescence spectroscopy – Excitation-Emission Matrix Spectroscopy (EEM) –
was performed using a Cary Eclipse Fluorescence Spectrophotometer (Agilent
Technologies) equipped with a xenon flash lamp. The fluorescence spectra for
samples were measured in a 4 optical window 1 cm Quartz
SUPRASIL® precision cell (Hellma® Analytics).
The blank 3-D fluorescence spectra and Water Raman scans were performed daily
using an Ultra-Pure Water Standard sealed cuvette (3/Q/10/WATER; Starna
Scientific Ltd). The experimental wavelength range for sample and ultra-pure
water scans was 230 to 455 nm in 5 nm intervals on excitation and 290 to 700 nm in 2 nm intervals on emission. Water Raman scans were recorded from 285
to 450 nm at 1 nm intervals for emission at the 275 nm excitation wavelength
(Murphy et al., 2013). All fluorescence measurements were managed at
19
The measured CDOM absorbance spectra were corrected to the refractive index
of remaining particulate matter and colloids after Zhang et al. (2009) and
for salinity after Nelson et al. (2007), and converted to absorption
coefficients according to Bricaud et al. (1981):
In rivers and the coastal waters, absorption coefficients at 355
(
Spectral slopes for the intervals 275–295 nm (
The CDOM alteration indicator,
To describe changes in CDOM spectral properties along with changes in CDOM
concentration, the following equation was used:
The variability of the relationship
The 3-D fluorescence spectra were corrected for spectral bias, background signals and inner filter effects. Each EEM was normalized to the area of the ultra-pure water Raman peaks, measured in the same day. EEMs were combined into three-dimensional data array, analyzed by PARAFAC (Stedmon and Bro, 2008) and validated by split-half analysis using “drEEM toolbox for MATLAB” after Murphy et al. (2013).
Only up to three components could be validated. For models with more than three components the results varied during split-half analysis (see Murphy et al., 2013), indicating the possibility of identifying the instrument noise as a signal (e.g., Stedmon and Markager, 2005). The fluorescence of each component is stated as fluorescence at excitation and emission maximums in Raman units (RU).
High variability of CDOM components (Fig. S1 in the Supplement) was observed on day 1 and day 2
of Varied P and day 1 of Varied N. This variability was likely associated to
the filling and manipulation of the mesocosm bags and vanished afterwards.
These days were excluded from further calculations, and day 3 and day 2 were
defined as “start” or “beginning” of Varied P and Varied N,
respectively. Day 8 was defined as the “end” of both experiments. To exclude
initial variability, changes of the different DOM parameters over time were
calculated as the difference between sampling day and start day:
For the presentation of the development over time, POM and DOM
The “corner points” are not presented in the DOM development plots, since both DIN and DIP in them were modified. Therefore, including these treatments could bias the interpretation of effects induced by single inorganic nutrients. However, in plots and analyses where DIP or DIN influence was investigated all treatments were included to avoid a single nutrient effect overestimation.
For an estimation of the drivers of changes in DOM optical properties, the
covariance of total accumulation of DOM compounds (
Mean normalized deviations (mean dev. %), calculated as
All statistical tests in this work were performed by the use of Sigma Plot
12.0 (Systat). The significance level was
After nutrient addition, a phytoplankton bloom development was observed in
all mesocosms during both experiments. Maximum chl
Bacterial abundance increased until day 6 (paired
Mean development of chl
The initial DOC concentration (day 3), did not differ significantly between
treatments in Varied P (one way ANOVA:
During both experiments, DOC accumulated significantly over time (paired
Initial average CDOM absorption at 325 nm (
Accumulation over time: of DOC (
CDOM accumulated over time during both experiments (paired
Spectral slopes, calculated within the 275–295 nm spectral range,
(
In the relationship between
Estimated linear change (per day) of spectral slope parameter
(d
Spectral slope
The
Three FDOM components with distinct spectral properties were identified during PARAFAC analysis of our data set. The first FDOM component (Comp.1) was excited at 235 nm and emitted at 440–460 nm, the second (Comp.2) and the third (Comp.3) FDOM components were excited at 275 and 265 nm and emitted at 340 and 294 nm respectively. Both also had secondary excitation peaks at wavelengths less than 230 nm (Table 3, Fig. 4).
The initial fluorescence of Comp.1 was 0.019
Subtracting the initial fluorescence of Comp.1 (
Spectral characteristics of excitation and emission maxima and range of intensities of the three fluorescent components identified by PARAFAC modeling in this study and their comparison with previously reported ones.
Spectral loadings (upper panels) and fingerprints (lower panels) of the FDOM components.
The fluorescence intensities of Comp.2 were almost identical at the start of
Varied P and Varied N, yielding 0.029
Comp.2 fluorescence increased in all mesocosms over time (paired
The Comp.3 fluorescence intensity was highly variable during both
experiments (Fig. 2m, n). Its starting values were not statistically
different between Varied P and Varied N (two way ANOVA:
In Varied P, Comp.3 fluorescence intensity increased from the start until day 5
(paired
To investigate a potential influence of phytoplankton or bacteria abundances
on DOC concentrations and CDOM and FDOM accumulation, cumulative sums of chl
Values of
CDOM accumulation (
Similar to
In contrast to
To assess the nutrient influence on DOM accumulation, mean normalized
deviations (mean dev. %) of
DOC accumulation was related to the initial inorganic nutrient supply in
both experiments. Higher
Mean normalized deviations of DOM accumulation against initial
nutrients supply. The
In contrast,
In contrast to both previous FDOM components,
No overall effect of DIN : DIP ratios on
Hence, accumulation of Comp.1 was dependent on the initial DIN concentrations, accumulation of Comp.2 increased with an increase of initial DIP concentrations and Comp.3 was unaffected by nutrient treatments.
Regression plots of
To investigate the relationship between CDOM absorption and DOC
concentrations during the course of the experiments, daily DOM accumulation
(
The estimated slopes of linear regressions, determined for each mesocosm for
Although the relationship between CDOM and DOC revealed a dependency
on initial DIN supply, the values of
All data of
Our results indicate that chl
Initial DOC concentrations, measured in both experiments (Fig. S1a, b), were in the range or slightly higher than those previously reported and modeled for the upper 30 m of the Tropical Atlantic Ocean watercolumn (Hansell et al., 2009).
In both experiments, DOC accumulated over time (Fig. 2a, b) and seemed to be
produced mainly through phytoplankton release. The highest DOC accumulation
was observed in the moment of rapid transition from nutrient-replete to
nutrient-depleted conditions (see also Engel et al., 2015). That is in line
with previous studies (Engel et al., 2002; Conan et al., 2007; Carlson and
Hansell, 2015) showing DOM accumulation after the onset of nutrient
limitation, while the chl
The effect of initial nutrient concentrations on DOC accumulation (Fig. 5a, b), observed in our study, was shown previously. In a mesocosm study with ETNA waters, Franz et al. (2012) observed that higher DOC concentrations developed when the initial inorganic nitrogen supply was high. As well, DOC concentrations in their study were even higher when high DIN concentrations were combined with high DIP supply. In their mesocosm experiment in Raunefjord, Conan et al. (2007) and Stedmon and Markager (2005) observed that at silicate-replete conditions, DOC concentrations under high initial DIN supply did not vary significantly from those under high initial DIP concentrations. In our study, silicate was also not limiting phytoplankton growth and higher DOC concentrations occurred at higher DIP as well as at higher DIN concentrations, supporting earlier findings.
Bacterial turnover may have influenced the composition of DOM (as it is seen by changes in spectral slope ratios and FDOM components) while DOC concentrations seemed to be not related to bacterial abundances. This observation may be explained by rapid bacterial consumption of labile DOM accompanied by the bacterial release of altered humic-like DOM (Azam et al., 1983; Ogawa et al., 2001), which are therefore not influencing measured DOC concentrations (e.g., Kirchman et al., 1991).
At the beginning of the experiment, CDOM absorption coefficients were in the
range of those previously reported for open waters of the Atlantic Ocean,
while the final CDOM absorptions were twice as high (Fig. S1c, d; Andrew et
al., 2013; Nelson and Siegel, 2013). Similar to our experiments, CDOM
absorption was previously shown to accumulate by a factor of 2 during mesocosm
studies, such as the study by Pavlov et al. (2014), where nutrient levels for
DIN were kept at 5
In our experiments, the accumulation of CDOM during the phytoplankton bloom
(Fig. 2c, d) as well as significant covariance to phytoplankton pigment –
chl
The decrease of CDOM spectral slopes over time (Fig. 2e, f) along with the increase in CDOM concentrations (Fig. 3) indicated that absorption in the visible wavelength range increased relatively to the UV wavelength range. As the absorption at longer wavelength is corresponding to larger molecules, we may assume that HMW-CDOM accumulated during both experiments. HMW-DOM was previously shown to be more labile for bacterial consumption than low molecular weight DOM (at molecular weight cutoff of 1 kDa; Benner and Amon, 2015), as bacterial activity was higher, when incubating with HMW-DOM (Amon and Benner, 1996). Furthermore, HMW-DOM typically accounts for 30 to 60 % of the total DOM released via phytoplankton (Biddanda and Benner, 1997; Engel et al., 2011). Therefore, we consider the spectral slope decrease over time as an indication of labile CDOM production via phytoplankton release.
In treatments with high initial DIN concentrations, bacterial abundance was significantly higher than in those with lower initial DIN concentrations. Furthermore, bacterial abundances in Varied N correlated significantly to CDOM concentrations. We therefore suggest that higher bacterial abundance may have been responsible for an additional production of CDOM in mesocosms, particularly in those with high initial DIN supply.
This suggestion is made also based on changes in optical properties during
our study. As Helms et al. (2008) and Zhang et al. (2009) showed before, the
spectral slope ratio –
However, due to its large uncertainties within treatments,
The CDOM to DOC ratio was also affected by variable initial DIN
concentrations. A significant positive correlation of CDOM accumulation over
time with DOC concentration was found in both experiments (Fig. 6a, b),
indicating that DOC and CDOM had been affected by the same processes.
Estimated slopes of
Factors influencing the ratio between CDOM absorption and DOC
concentrations are not well-understood so far. It is known that CDOM
absorption often co-varies with DOC concentration in river estuaries and
coastal seas, which are influenced to a high degree by conservative mixing
of riverine and marine waters (Nelson and Siegel, 2013; Rochelle-Newall et
al., 2014). However, in the open ocean, the relation is losing its
consistency (Nelson and Siegel, 2013). We suggest that under higher initial
DIN concentrations bacterial abundance is higher and such is the bacterial
reworking of DOM. Higher bacterial reworking, in turn, causes an
increase in the proportion of the colored fraction in DOM. Our results
suggest that an increase of initial DIN concentrations by 10
When CDOM properties, such as spectral slopes
Besides absorption, FDOM fractions were more sensitive to nutrient amendments. During our study, three different fluorescent components could be identified (Fig. 4).
The characteristics of the first component, Comp.1 (Table 3), were similar to those of the humic-like peak “A” described by Coble et al. (1996). The Comp.1 fluorescence was within the reported range of A-like peak fluorescence intensities for the open-ocean area (Jørgensen et al., 2011) or slightly higher towards the end of experiments depending on mesocosm treatment (Fig. S1i, j).
Marine humic substances were previously assigned to bacterially derived substances due to significant covariance of their concentrations to apparent oxygen utilization in deep open-ocean waters (Swan et al., 2009; Kowalczuk et al., 2013; Nelson and Siegel, 2013). As well, previous studies of Stedmon and Markager (2005), Kowalczuk et al. (2009) and Zhang et al. (2009) showed that humic-like components, similar in terms of spectral properties to Comp.1, were produced via microbial DOM reworking (Table 3).
In our study, in Varied N, Comp.1 was strongly correlated to initial DIN concentrations, as the final Comp.1 fluorescence intensity was almost three fold higher at the highest initial DIN supply than that in the treatments with lowest DIN supply. Thus, since bacterial abundance was DIN-dependent in this experiment and Comp.1 fluorescence intensities correlated significantly to bacterial abundances, the bacteria were likely responsible for Comp.1 occurrence during our experiments. The observed bacterial production of humic-like Comp.1 that is proportional to DIN is in agreement with Kramer and Herndl (2004) and Biers et al. (2007), where DIN and its organic derivatives were considered to be the primary drivers of humic-like DOM accumulation via bacterial reworking.
In Varied P, however, Comp.1 was not related to bacterial abundance. No significant differences between treatments were noticed for bacterial abundance and only few differences occurred for Comp.1 at similar initial DIN supply concentrations. Thus, under equal initial DIN concentrations bacterial reworking of DOM could occur at a similar degree, causing the absence of covariance of Comp.1 with bacterial abundance.
The higher concentrations of Comp.1 at the end of our experiments compared to concentrations measured in open ocean (Jørgensen et al., 2011) may be explained by slightly higher substrate availability in the mesocosms than that in the North Atlantic.
The fluorescence properties of the second FDOM component, Comp.2 (Table 3), were similar to that of the previously defined amino acid-like fluorescence (Mopper and Schulz, 1993; Coble et al., 1996; Stedmon and Markager, 2005): tryptophan-like peak “T” (Coble et al., 1996). The fluorescence intensities of this component were in the range of that previously reported for open-ocean area (Jørgensen et al., 2011) for the whole experimental period (Fig. S1k, l).
Similar in terms of spectral properties to Comp.2, amino acid-like compounds were previously hypothesized to represent the fluorescence of the bound-to-protein matrix amino acids tryptophan and tyrosine (Stedmon and Markager, 2005) and were assumed to be produced by phytoplankton (Mopper and Schulz, 1993; Coble et al., 1996). We, therefore, consider Comp.2 as an indicator of phytoplankton-produced proteinaceous DOM and as a possible precursor for humic-like FDOM.
In Varied P, Comp.2 accumulated proportionally to initial DIP concentrations
and its concentration did not correspond to chl
In Varied N, again no covariance of Comp.2 to chl
Previously, Stedmon and Markager (2005) showed an accumulation of an FDOM component, with spectral properties similar to Comp.2, during their mesocosm study treatments of high DIN and high DIP concentrations. This component was also shown to be consumed during dark and light incubations, when bacteria were added. Kirchman et al. (1991) showed that DOM uptake can be accompanied by a decrease in DIN concentrations, indicating the importance of DIN presence during bacterial reworking of labile DOM. Therefore, Comp.2 production might be dependent on initial DIP and DIN availability, similar to the increase of DOC concentrations. As well as at high initial DIN concentrations, Comp.2 may serve a substrate for developing bacteria, i.e., it can be consumed by bacteria that, in their turn, release humic-like Comp.1.
The spectral properties of the third fluorescent component – Comp.3 – were similar to that of amino acid-like fluorescence (Table 3; Mopper and Schulz, 1993; Coble et al., 1996; Stedmon and Markager, 2005): tyrosine-like peak “B” (Coble et al., 1996) and were in the range of those previously reported for open-ocean area (Jørgensen et al., 2011; Fig. S1m, n).
The development patterns as well as no clear response towards nutrient amendments of Comp.3 made it very difficult to interpret.
In Varied P, Comp.3 fluorescence intensities were highest at the day of chl
In Varied N, Comp.3 fluorescence intensities were generally low, but increased at the end of experiment (Fig. 2n). Therefore, the process of bacterial Comp.2 reworking could lead to Comp.3 release as a byproduct at the final stage of Varied N. On the other hand, Comp.3 accumulation towards the end of this experiment could be a result of extracellular release of higher amounts of amino acid-like substances, which accumulated under high DIN concentrations within phytoplankton tissues during its growth.
A fluorescent substance, similar by spectral properties to Comp.3, was previously hypothesized to represent the tryptophan and tyrosine in peptides by Stedmon and Markager (2005), as it had been previously found accumulating during the denaturation of proteins (Determann et al., 1998). In their study, Stedmon and Markager (2005) found no effect of microbial reworking on the abundance of this fluorescence substance in the dark and light incubations with bacteria. However, as this substance was removed during thier mesocosm experiment, they hypothesized spontaneous abiotic aggregation or photochemically induced flocculation as possible removal mechanisms.
We, therefore, conclude that Comp.3 potentially acted as an intermediate product during the formation or degradation of proteinaceous Comp.2 in our study. Still, the interpretation of the Comp.3 development remains speculative.
It was hypothesized previously that phosphorus limitation leads to an
accumulation of DOM that is more resistant to microbial degradation (Kragh and
Sondergaard, 2009), e.g., due to phytoplankton extracellular release of this
“poor quality” DOM or limitation of bacterial DOM consumption (Carlson and
Hansell, 2015). Based on changes in optical DOM properties (
Overall, the variances of CDOM and FDOM concentrations in the treatment with
DIN : DIP of 16 (12.00N/0.75P) for each experiment were higher than the
variance in this treatment between experiments. Therefore, the effects of
nutrients on CDOM and FDOM concentrations were considered much stronger
than possible effects caused by differences in initial sensitivity to
nutrient additions. However, due to the divergence in development pattern
for some of optically active parameters (
Another important aspect that could cause an additional CDOM and FDOM
variability, and, therefore, bias the interpretation of obtained results
during the mesocosm experiments, is the length of the sample storage. In our
study, CDOM and FDOM samples were filtered through 0.45
Our study shows that during phytoplankton blooms DOM is largely derived from phytoplankton, while its optical properties undergo considerable changes due to bacterial reworking. Thus, optically active proteinaceous substances are freshly produced by phytoplankton release. They are, however, consumed and reworked by bacteria, leading to an accumulation of less-bioavailable optically active humic substances.
Our experiments indicate that DIN is the major macronutrient regulating the accumulation of bacterially originated optically active humic substances, while the accumulation of labile proteinaceous substances via phytoplankton is rather regulated by DIN and DIP. An input of humic substances can increase the CDOM/DOC ratio and therewith affect predictions of DOC concentration based on CDOM absorption. Still, a relationship between CDOM spectral properties and CDOM and DOC concentrations can be derived, which is not influenced by differences in the nutrient supply.
This study was supported by the SFB754 project, “Climate-Biogeochemical Interactions in the Tropical Ocean” of the DFG. We thank all participants of our Cabo Verde 2012 research stay for joint work during water sampling and handling of mesocosms and also N. Vieira for help with initial water sampling. We also thank I. Monteiro, M. Schütt and P. Silva for help with logistics.
We are very grateful to P. Kowalczuk for valuable comments during our PARAFAC analysis, J. Roa for DOC analyses, U. Pankin for nutrient measurements, and S. Endres, L. Galgani, R. Flerus and C. Löscher for helpful discussions during the manuscript writing. We are very thankful also to P. Kowalczuk and an anonymous referee for reviewing and commenting on the manuscript, as well as to M. Mostofa for his short comment on the manuscript.
All data will be available at