charged species at physiological pH and has more complex removal mechanism. Thus, although total acrylate concentrations were low (1.4-3.4 nmol L-1) and only a few samples were analyzed, a significant fraction of total acrylate (50-61%) was present in the particulate phase, consistent with algal culture studies (Tyssebotn 2015; Kinsey et al. 2016). By contrast, DMS should mostly (~100%) be in the dissolved phase (Spiese et al. 2016).
Another important difference between acrylated and DMS was that DMS removal rates from the dissolved phase were faster than for acrylated, for both photolysis (J. D. Kinsey unpubl.) and biological uptake (Motard-Côté et al. 2016). The first-order DMS photolysis rate constant during our cruise (0.04-0.20 h-1, J. D. Kinsey unpubl.) was more than an order of magnitude greater than the rate constant for acrylated photolysis (0.6-1.5 × 10-3 h-1, Tyssebotn 2015). With a lower rate constant and similar DMS and acrylated concentrations, corresponding photolysis rates will be slower for acrylated (Tables 1 and 4).
Biological uptake rates of acrylated at ambient concentrations were also generally slower than for DMS, but mostly at the oceanic stations. For the coastal stations, VSn for DMS,
determined from dark incubations of 35S-DMS-amended unfiltered seawater (Motard-Côté et al.
2016), were comparable to those for acrylated (Tables 2 and 4, Stns. 2, 9, 17). In oceanic waters (Stns. 4, 6, 8, 14, 19) DMS VSn were substantially greater than the corresponding acrylate VSn by a factor of 4.6-12 (Tables 2 and 4). These large differences were not due to differences in Sn
which by comparison were small (median 1.5 and 2.7 nmol L-1, respectively, for acrylated and DMS) at all sampling stations. Differences in VSn for DMS and acrylated were also independent of changes in bacterial production and Chl a concentrations (Tables 1 and 2, Fig. 3; Motard-Côté et al. 2016), implying that there was a tight coupling between production and removal rates for these compounds.
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Overall, similar acrylated and DMS concentrations (Fig. 3, Tables 1 and 4), but lower acrylate VSn (at the open-ocean sites) and photolysis rates (0.6-1.5 × 10-3 h-1, Tyssebotn 2015), suggest that rates of acrylate input to the dissolved phase were less than corresponding DMS inputs into northern Gulf of Mexico waters. This might be expected because bacteria that take up DMSPd and cleave it intracellularly, likely metabolize acrylate further while losing DMS to diffusion out of the cell (Ansede et al. 2001; Kiene et al. 2000; Spiese et al. 2016). Similarly, smaller fluxes of acrylate into the dissolved phase from phytoplankton cells might be expected because some acrylate is retained in phytoplankton cells (Tyssebotn 2015; Kinsey et al. 2016), and marine particulate matter (this study), while DMS will rapid diffuse out of phytoplankton cells into the dissolved phase (Spiese et al. 2016). Thus extracellular releases of acrylate will mostly depend on processes such as viral lysis of or grazing on phytoplankton cells, which one would expect to be relatively low under non-bloom conditions. In addition, DMS has other sources that do not result in acrylate production. Three potential sources of DMS that would not lead to acrylate production are DMSO reduction (Fuse et al. 1995; Spiese et al. 2009),
methylation of methanethiol (Kiene and Taylor 1989; Carrión et al. 2015), and the DddD gene-encoded cleavage of DMSP to form DMS and β-hydroxypropionate (Todd et al. 2007).
Since acrylated concentrations were uniformly low during the entire study in the northern Gulf of Mexico (0.8-2.1 nmol L-1, median 1.5 nmol L-1), then it can be assumed that the system was approximately in steady state, with the production of acrylated (i.e., input into the dissolved phase, both from the particulate phase and DMSPd metabolism) approximately balanced by its losses from the dissolved phase through photolysis and biological uptake. With this assumption, the net production rate of acrylated from all sources can be estimated. On average the biological uptake of acrylated in the study area was 0.53 nmol L-1 d-1. Since the biological uptake was
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significantly greater than the rate of photolysis (ca. 0.01 nmol L-1 d-1), we infer that the net production term must be low, ca. 0.53 nmol L-1 d-1, and therefore conclude, that inputs of acrylated from DMSP cycling and particulate sources must be slow. By comparison, the
biological production of DMS from the biological consumption of DMSPd in the same study area ranged from 1.2 to 5.0 nmol L-1 d-1 (Motard-Côté et al. 2016).
Dissolved DMSO was the dominant dissolved organosulfur compound measured in the Gulf of Mexico, a finding that is consistent with several studies in oceanic waters including the Arabian Sea (Hatton et al. 1996), Pacific Ocean (Bates et al. 1994), Mediterranean Sea (Simó et al. 1995), coastal Atlantic Ocean (Ridgeway et al. 1992), the Ross Sea (del Valle et al. 2009), and Antarctic coastal waters (Gibson et al. 1990). Dissolved DMSO concentrations showed no significant differences between stations or correlations with acrylated, DMSPd, a300, BCP, or Chl a. However, DMSOd concentrations showed a strong linear correlation to DMS in both surface waters (rp = 0.776, p = 0.024, n = 8) and when depth profile data were included (rp = 0.759, p < 0.001, n = 26). This relationship was expected as DMS is the main source of DMSOd
in the surface mixed layer through its photochemical and biological oxidation (Kieber et al.
1996; del Valle et al. 2009), and DMSOd losses are slow. Previous studies have also reported strong correlations between DMS and DMSO concentrations during the spring and summer in a variety of oceanic surface waters. Kiene et al. (2007) collected DMS and DMSOd concentration data from a Southern Ocean transect between New Zealand and the Ross Sea in three separate years, once in the austral summer (2004) and twice in the austral spring (2003 and 2005); they observed a relatively high correlation between DMS and DMSOd (rs = 0.630, p < 0.001, n = 108) for all data during this three-year period, although the correlation was not significant during the austral summer in 2004. When Hatton et al. (2004) combined several published data sets, a
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strong correlation was observed between DMS and DMSOd (r2 = 0.801, p < 0.001) irrespective of geographical location.
Depth profiles of DMSOd concentrations in the Gulf of Mexico (Fig. 3) are consistent with previously reported depth profiles in several oceanic water types ranging from tropical to polar regions (Andreae 1980; Ridgeway et al. 1992; Kiene and Gerard 1994; Hatton et al. 1996;
Simó et al. 1997; del Valle et al. 2009). In these studies, the highest DMSOd concentrations were nearly always detected in the surface mixed layer, followed by a rapid decrease in DMSOd with depth. This trend was observed in the present study (Fig. 3) and was likely controlled by photochemical and biological oxidation of DMS to DMSO (Kieber et al. 1996; del Valle et al.
2007a,b; del Valle et al. 2009) weakly offset by the slow biological uptake of DMSOd (vide infra) and weak vertical mixing due to the very calm conditions throughout the cruise (median wind speed 2.4 m s-1).
Biological uptake and transformations of acrylated and DMSOd
The low nanomolar Ks values reported here for the biological uptake of acrylated and DMSOd are consistent with the low nanomolar concentrations observed for these compounds in the northern Gulf of Mexico. For acrylated, these low values suggest that it was taken up
efficiently by the heterotrophic community through high-affinity uptake systems. For DMSOd, it will rapidly diffuse into the cell, reaching equilibrium within seconds (Spiese et al. 2009), and therefore specific cell-membrane transporters are not needed for its uptake. Nonetheless, we observed saturation kinetics for DMSO uptake that likely reflected the heterotrophic
community’s average Ks and Vmax for the enzymes responsible for the assimilation and respiration of cellular DMSO.
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Values of Ks and Vmax obtained for the uptake of acrylated and DMSOd are comparable to values of these kinetic parameters observed in marine waters for the biological uptake of other low molecular weight organic substrates including dissolved amino acids (Ferguson and Sunda 1984; Suttle et al. 1991), monosaccharides (Skoog et al. 1999), α-keto acids (Kieber et al. 1989), glycolate (Edenborn and Litchfield 1987), glycine betaine (Kiene and Hoffman Williams 1998), methanol (Dixon et al. 2011), and CO (Xie et al. 2005), all of which have Ks in the low
nanomolar range and Vmax between 10-10 and 10-9 mol L-1 h-1. Despite the similarity in the uptake kinetic constants, turnover times were considerably slower for acrylate and DMSO compared to several other biological substrates including amino acids, as discussed in the ensuing section.
Although heterotrophic bacteria have been shown to take up DMSOd, its cellular transformations are not well understood. The only cellular transformation that has been
identified for DMSO in marine systems is the enzymatic reduction of DMSO to DMS (Fuse et al.
1995; Spiese et al. 2009), but this was not determined in the present study since any DMS formed from 14C-DMSO through this process would quickly partition to the dissolved phase (Spiese et al. 2016) and not be captured on a filter. Dimethylsulfide produced from DMSO in this way could be further metabolized as well, either back to DMSO or to other mineralized end products (Wolfe and Kiene 1993; del Valle et al. 2007b). Enzymes capable of reducing DMSO to DMS appear to be ubiquitous in marine bacteria (Fuse et al. 1995) and marine algae (Spiese et al. 2009). It is unknown whether enzymatic reduction of DMSO to DMS occurred at an
appreciable rate in the Gulf of Mexico water column, but it has been shown to be important in Antarctic sea-ice brine communities wherein DMSO reduction, as opposed to DMSP cleavage, was the main source of DMS (Asher et al. 2011).
Despite the lack of understanding of cellular transformations of DMSO in marine waters,
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our results indicate that DMSO is mostly oxidized for energy with a small percentage assimilated for growth. The majority of DMSOd taken up by the heterotrophic community was respired (62-75%, Table 3). This degree of respiration is generally greater than seen for other biological substrates that have been studied in seawater including glucose (e.g., 24-41%, Arctic, Griffiths et al. 1984; 30-60%, equatorial Pacific, Rich et al. 1996), glycine betaine (40-60%, Gulf of Mexico, Kiene and Hoffman Williams 1998), leucine (e.g., < 10%, Sargasso Sea, Suttle et al. 1991; 30-70% Mediterranean Sea, del Giorgio et al. 2011), or dissolved free amino acids (e.g., 30-65%, Kysing Fjord, Jørgensen 1982; 42-68%, Arctic, Griffiths et al. 1984). A few substrates have been found to have similarly high percentages of respiration to those of DMSO, including mannose, which was 60-90% respired in the equatorial Pacific (Rich et al. 1996), and glutamate, which was 60-65% respired in the Kysing Fjord (Jørgensen 1982) and 60-80% respired in the Sargasso Sea (Suttle et al. 1991), or even a higher percentage as observed for CO which is essentially all respired (~99%, Tolli and Taylor 2005). It is interesting to note that for DMSOd, unlike acrylated, there was no significant difference in the percent respiration between the low and high bacterial productivity stations in the Gulf of Mexico; a high percentage of respiration was observed at all stations. This implies that the DMSO carbon moieties were mostly
metabolized for energy, irrespective of the amount or lability of dissolved carbon or nutrient status of the seawater.
Unlike DMS, DMSP, or DMSO, no field studies have previously been conducted to determine biological uptake rates of acrylated in seawater. As previously discussed, a variety of microbes have been isolated from diverse marine environments that use acrylate as a carbon source in culture (e.g., Diaz et al. 1992; Ledyard et al. 1993; Ansede et al. 1999, 2001; Gonzalez et al. 1999; Noordkamp et al. 2000; Raina et al. 2009; Todd et al. 2010; Garren et al. 2014). It is
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therefore not surprising that there was biological uptake of acrylate in northern Gulf of Mexico waters. However, acrylated exhibited a very different uptake pattern compared to DMSOd. First, acrylate was assimilated into macromolecules at percentages comparable to or greater than its respiration (Table 3). Second, as %R increased, %A decreased for acrylate uptake when going from the high CDOM and bacterial productivity coastal stations to the low CDOM and bacterial productivity oceanic stations (Fig. 7). This indicated that acrylate was metabolized more for energy than assimilated for growth in offshore waters compared to coastal waters, similar to what is generally expected for the BGE when comparing productive nearshore waters to oligotrophic waters (del Giorgio and Cole 1998). The third difference in biological uptake between the two compounds was that a significant percentage (ca. 33% on average) of the total acrylate uptake was not accounted for by either assimilation into macromolecules or respiration, unlike DMSO whose respiration and assimilation accounted for nearly 100% of its total uptake.
This implies that, unlike DMSO, some 14C-acrylate uptake remained in the cytosol as acrylate or as a transformed low molecular weight 14C product. One potential transformation is the
conversion of acrylate to acrylyl-CoA, a toxic compound which may be further metabolized to β-hydroxypropionate potentially as a detoxification mechanism (Curson et al. 2014), followed by the accumulation of β-hydroxypropionate in the cytosol (Ansede et al. 1999).
Acrylated and DMSOd turnover times
Acrylated and DMSOd turnover times in the northern Gulf of Mexico during the late summer were on average 7.9- and 4.1-fold faster, respectively, at the coastal sites compared to oceanic stations, which was consistent with the approximately 7-fold higher bacterial carbon production rates in coastal waters (Table 2). No turnover-time estimates have been previously
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reported for acrylate, and despite extensive global measurements of DMSO concentrations, there are very few reported DMSO turnover times in oceanic waters.
The prevailing view in the literature is that DMSOd turnover times are slow in marine waters, and this is certainly true compared to reported turnover times for other simple organic molecules that are generally less than a day (vide infra). This has led to speculation by Hatton et al. (1996) and Ridgeway et al. (1992) that DMSO will serve as a net loss for DMS from the water column. This is likely correct over short time scales (days) because DMSOd turnover times in the Gulf of Mexico were slow (median 7.4 d). However, whether DMSO serves as a net loss for DMS or not, will depend on how much DMSO produced from DMS is enzymatically reduced back to DMS, as shown in culture (Fuse et al. 1995; Spiese et al. 2009), and therefore recycled, versus the amount of DMSO that is respired or assimilated.
Dissolved DMSO turnover times for coastal waters (median 3.2 d) were comparable to published DMSO turnover-time estimates, which are generally on the order of a few days in coastal waters (Kiene and Gerard 1994; Simó et al. 2000; Asher et al. 2011). In contrast, DMSOd turnover times in the Gulf of Mexico oceanic sites were 3 to 5 times longer (median 11.3 d) than previously reported in coastal waters, likely due to the correspondingly lower bacterial productivities (Table 2). These differences indicate that there may be considerable spatial and temporal variability in DMSOd turnover in oceanic waters, warranting a more detailed study to determine the factors controlling the biological consumption of DMSOd.
In comparison to other organosulfur substrates, DMSOd turnover times are slow, with a median (7.4 d) nearly five times longer than the median DMS turnover time (1.5 d, range 0.8-7.7 d) and 57 times longer than the corresponding median DMSPd turnover time (0.13 d, range 0.009-0.8 d) measured on the same cruise (Motard-Côté et al. 2016). Likewise, the median
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acrylated turnover time (4.8 d) is nearly 37 times longer than the median turnover time for DMSPd, and approximately 3 times longer than the median DMS turnover time. Turnover times for DMSOd and acrylated (Table 3, Fig. 8) are also generally much longer than dissolved free amino acids, which are generally less than 1 d (Ferguson and Sunda 1984; Fuhrman and Ferguson 1986; Kiel and Kirchman 1999; Suttle et al. 1991). Monosaccharide turnover times tend to be somewhat longer, on the order of 1-10 d (Suttle et al. 1991; Rich et al. 1996; Skoog et al. 1999), which are comparable to DMS and somewhat shorter than acrylated, but still
substantially shorter than DMSOd. Rich et al. (1996) observed considerable temporal variation in the uptake of glucose in the equatorial Pacific, with faster turnover (~2 d) in the summer months under normal sea surface temperature conditions compared to the following winter (~10 d) during an El Niño event. They also observed a large difference between mannose and glucose, with mannose exhibiting very little temporal variation in its turnover time, which was nearly 50 d on average. This is much slower than observed for acrylate or DMSO, but the reasons for these differences are unknown. Clearly, there will be interacting physical and
biogeochemical factors at play in contributing to these differences, not only between glucose and mannose but also among all the substrates discussed here. Temporal and spatial variations in temperature, light, the quality and quantity of organic matter and nutrients available to the microbial community, and in the makeup of microbial community itself will be important, as no one substrate will likely be taken up by all representative microbial groups. Additional studies are needed to relate substrate uptake to specific environmental conditions and the composition of the microbial community.
Acrylated and DMSOd as carbon sources for bacteria
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Dissolved acrylate and DMSO contributed 0.013-0.13% and 0.04-0.14% of BCD, respectively (Table 2). Given the slow turnover times and low concentrations of acrylated and DMSOd, it is not surprising that they were not quantitatively important carbon substrates in northern Gulf of Mexico waters for the entire heterotrophic community during non-bloom conditions. Contributions of both compounds to BCD are approximately an order of magnitude lower than observed for DMSPd on the same cruise (1.1-7.0%) (Motard-Côté et al. 2016) or on a previous cruise in the Gulf of Mexico (0.4-6.5%) (Kiene and Linn 2000), and generally much lower than the 10 to ~100% contributions to BCD by dissolved free amino acids or glucose observed in numerous studies (for review see Nagata 2008). The low percent contribution of acrylated and DMSOd to the BCD determined here does not exclude the possibility that acrylated and DMSOd are important sources of carbon to specific bacterial groups. Our finding that acrylated and DMSOd biological uptake at ambient concentrations occurred through high-affinity (i.e., low nmol L-1 Ks) uptake systems indicates that at least some subset of the bacterial
community is poised to efficiently utilize these substrates in both productive coastal waters and under oligotrophic conditions.
During blooms of high DMSP-producing phytoplankton, acrylated and DMSOd may be much more important substrates to the microbial community than we observed in the Gulf of Mexico during non-bloom conditions. In a Phaeocystis antarctica bloom, DMSOd and acrylated
concentrations increased by an order of magnitude or more above background, non-bloom levels (e.g., Yang et al. 1994; Gibson et al. 1996; Hatton et al. 1998; del Valle et al. 2009). Their biological uptake rates are expected to increase dramatically as well if there is a corresponding increase in the number of acrylate-(or DMSO-) consuming bacteria, as was observed by Noordkamp et al. (2000). From this perspective, it will be essential in future work to test
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whether acrylate and DMSO make greater contributions to BCP in major DMSP- and DMS-producing blooms, and to assess the diversity of marine microbes that are capable of using acrylated and DMSOd as carbon sources for microbial energy and growth.
Conclusions
Our study significantly advances the field by filling in gaps about the fate of acrylate and DMSO in seawater. These two compounds are very often discussed in the context of DMSP and DMS cycling, but acrylate and DMSO have not previously been investigated at the detailed level of our study. We have shown that the microbial community takes up acrylated and DMSOd with high affinity systems, but biological turnover times are slow. The slow biological loss of
DMSOd, along with its photochemical and thermal stability, suggests that DMSOd serves as a significant short-term (days - weeks) dimethylsulfur storage species in the photic zone. Given the slow turnover rate of acrylate and DMSO, it is possible that these compounds are not
consumed by a wide array of microbes or by the dominant microbes present in seawater. This is in contrast to findings for DMSP, which is used by a wide spectrum of the microbial community (Malmstrom et al. 2004; Vila et al. 2004). Based on the relatively long biological turnover times of acrylated, we also conclude that supply rate of this compound to the dissolved pool is
relatively low in the northern Gulf of Mexico. This finding is consistent with the small fraction of DMSPd converted to DMS and acrylate by microbes (5-30% based on DMS yield; Motard-Côté et al. 2016) and the possible alternative fates for those compounds in photochemical (for DMS) and biological reactions in bacteria and phytoplankton. It also implies that very little of the DMSP-sulfur is cycled through DMSOd on a daily basis. Additional research is needed to
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understand the role these compounds play in cellular metabolism and in the microbial ecology of marine waters.
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Acknowledgments
The authors thank the officers and crew of the R/V Pelican for their technical support at sea, and
The authors thank the officers and crew of the R/V Pelican for their technical support at sea, and