Growth Rate‐Dependent Synthesis of Halomethanes in Marine

Environmental Microbiology Reports (2021) 13(2), 77–85 doi:10.1111/1758-2229.12905

Brief report

Growth rate-dependent synthesis of halomethanes in marine heterotrophic bacteria and its implications for the ozone layer recovery

Laura Gómez-Consarnau, 1,2* Nick J. Klein,1 and that their contribution to the atmospheric halo- Lynda S. Cutter1 and Sergio A. Sañudo-Wilhelmy1 gen budget could increase in the future, impacting 1Department of Biological Sciences, University of the ozone layer recovery. Southern California, Los Angeles, CA, 90089. 2 Departamento de Oceanografía Biológica, Centro de Introduction Investigación Cientíﬁca y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California, 22860, A major environmental achievement of the 20th century Mexico. was the implementation of the Montreal protocol and its subsequent amendments that phased out the production and consumption of human-made ozone-depleting sub- Summary stances (ODSs) (e.g., anthropogenic chloroﬂuorocar-

Halomethanes (e.g., CH3Cl, CH3Br, CH3I and CHBr3) bons; CFC) (Carpenter and Riemann, 2014). Since the are ozone-depleting compounds that, in contrast to protocol’s implementation, global emissions and atmo- the human-made chlorofluorocarbons, marine organ- spheric concentrations of anthropogenic ODSs have isms synthesize naturally. Therefore, their production declined by more than 90% (Hegglin et al., 2015; Prinn cannot be totally controlled by human action. How- et al., 2018; Engel and Rigby, 2018). Consistent with ever, identifying all their natural sources and under- these reductions, the recovery of the ozone layer is standing their synthesis regulation can help to already detectable in some areas of the world as well as predict their production rates and their impact on the in the upper stratosphere (Shepherd et al., 2014; Strahan future recovery of the Earth’s ozone layer. Here we et al., 2018; Kuttippurath et al., 2018). However, strato- show that the synthesis of mono-halogenated halo- spheric ozone layer recovery still lags the reductions of fi carbons CH3Cl, CH3Br, and CH3I is a generalized pro- reactive halogen gas loading (Chipper eld et al., 2017; cess in representatives of the major marine Liang et al., 2017). Full recovery (or return to historical heterotrophic bacteria groups. Furthermore, 1980 levels) is not expected to occur until the end of the halomethane production was growth rate dependent 21st century (Eyring et al., 2010; Ball et al., 2018), in all the strains we studied, implying uniform synthe- assuming that emissions of ODSs continue to decline. sis regulation patterns among bacterioplankton. Today, the 1987 Montreal Protocol still regulates the Using these experimental observations and in situ production and consumption of anthropogenic ODSs. halomethane concentrations, we further evaluated Nonetheless, unregulated and natural sources of haloge- the potential production rates associated with higher nated hydrocarbons (e.g., halomethanes) continue to cat- bacterial growth rates in response to global warming alytically destroy atmospheric ozone (Liang et al., 2017; in a coastal environment within the Southern Califor- Engel and Rigby, 2018). In contrast to CFC, the emis- nia Bight. Our estimates show that a 3 C temperature sions of some biogenic halomethanes (e.g., CH3Br, – rise would translate into a 35% 84% increase in CH3I) are overwhelmingly dominated by natural sources halomethane production rate by 2100. Overall, these (Redeker et al., 2000; Bell et al., 2002; Youn data suggest that marine heterotrophic bacteria are et al., 2010), and their atmospheric concentrations are significant producers of these climate-relevant gases currently increasing (Engel and Rigby, 2018; Fang et al., 2019). As opposed to their human-made counter- parts, their sources and transport to the atmosphere are Received 14 July, 2020; accepted 9 November, 2020. *For corre- spondence.E-mail [email protected]; Tel. (+1) 213 740 8779; Fax not yet well constrained (Engel and Rigby, 2018; Youn (213) 740-8123 et al., 2010; Ordóñez et al., 2011; Montzka et al., 2011).

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd 78 L. Gómez-Consarnau, N. J. Klein, L. S. Cutter and S. A. Sañudo-Wilhelmy As a result, the atmospheric halomethane budget Manley, 2002; Bayer et al., 2009; Butler et al., 2009; remains open, with sinks still outweighing all known Gutleben et al., 2019). Indeed, a positive correlation sources (Montzka et al., 2011; Engel and Rigby, 2018). between halomethane concentrations and phytoplankton While we would generally expect a delay in ozone layer distributions has rarely been observed (Smythe-Wright recovery with any increase in halomethane emissions et al., 2006). Instead, halomethane production correlates

(Fang et al., 2019), some iodine compounds – CH3Iin best with chlorophyll-a levels under post-bloom condi- particular – could have a dual impact on climate. On the tions (Arnold et al., 2010; Lai et al., 2011; Hu et al., 2016) one hand, when reaching the lower stratosphere, the when heterotrophic bacteria thrive. Thus, predicting any iodade ion of CH3I would trigger ozone layer destruction future impacts of climate change on natural emissions of as other halogens do. Still, given the right circumstances, halomethanes requires a more thorough characterization I− may also promote the formation of cloud condensation of the different marine organisms responsible for their nuclei, which would help mitigate global warming by synthesis. Here, we present evidence of widespread increasing cloud formation and solar reflectivity halomethane biosynthesis in a selection of cultured (O’Dowd, 1998; O’Dowd et al., 2002; Küpper marine heterotrophs representative of the main environ- et al., 2008). Therefore, understanding what regulates mentally relevant marine bacterial phyla. Our results the biological production of different halomethane com- show that halomethane synthesis rates are directly pro- pounds will be crucial to unravel the intricacies around portional to bacterial growth rate. Using these data, we solving the ozone layer depletion. further explore the halomethane production rates in the A variety of marine organisms (e.g., macroalgae, auto- natural marine environment and project their future devel- trophic phytoplankton, and heterotrophic bacteria) can opment under a global warming scenario. mediate the methylation of halides in marine environments (Scarratt and Moore, 1996, 1998; Manley and de la Cuesta, 1997; Schall et al., 1997; Manley, 2002; Materials and methods Smythe-Wright et al., 2006; Küpper et al., 2008; Brownell Experimental design to determine halocarbon et al., 2010; Hughes et al., 2011; Fujimori et al., 2012; production during bacterial growth Leedham et al., 2013; Hirata et al., 2017). However, identifying the primary halomethane producers and esta- Halogenating enzymes are ubiquitous in marine hetero- blishing predictive models for their synthesis has been trophic bacteria (Amachi et al., 2001; Manley, 2002; difficult, as this seems to be not only species or even Bayer et al., 2009; Butler et al., 2009; Atashgahi strain-specific (Johnson et al., 2015) but also dependent et al., 2018; Gutleben et al., 2019), and therefore we on different environmental conditions such as water tem- hypothesized that halomethane production is a wide- perature (Hughes et al., 2011; Liu et al., 2013). The spread process among these organisms. To date, impact of temperature on halomethane production is par- halomethane synthesis has only been confirmed in ticularly worrisome as global warming may increase the aquatic heterotrophic bacteria from the genera Pseudo- rate of the enzymatic reactions responsible for their syn- monas and Erythrobacter (Fujimori et al. 2012; Hirata thesis (Gillooly et al., 2001; Hirata et al., 2017). Several et al. 2017). Here we selected six taxonomically diverse studies have already shown higher halomethane produc- strains of marine bacteria representing the three main tion during summer seasons (Archer et al., 2007; heterotrophic oceanic groups: Alphaproteobacteria Yokouchi et al., 2014; Rhew et al., 2014) consistent with (DFL12, Biebl et al., 2005; MED193, Lekunberri a link between their synthesis and surface water temper- et al., 2014), Gammaproteobacteria (AND4, Gómez- ature (Hirata et al., 2017; Abe et al., 2017). Consarnau et al., 2010) and Bacteroidetes (MED134, Although marine heterotrophic bacteria are the most Gómez-Consarnau et al., 2007; MED152, Gonzalez abundant and widely distributed organisms in the ocean et al., 2008; MED217, Pinhassi et al., 2006; Fig. 1), to (with about three-orders of magnitude higher abundance test this hypothesis under laboratory conditions. An addi- than phytoplankton; Hobbie et al., 1977), they have rarely tional advantage of using these particular strains is that been assayed for halomethane production. Only a few some of their physiological characteristics have already studies have reported on halomethane production in been characterized, and they could therefore be consid- strains isolated from brackish water (Fujimori et al., 2012; ered model organisms within their phylogenetic groups. Hirata et al., 2017) or in taxa that are not widely distrib- Each bacterial strain was grown in axenic cultures using uted in the ocean (Amachi et al., 2001). Nevertheless, 120 ml of ZoBell medium in 250 ml gas-tight serum glass the limited literature available suggests that bacterial vials with silicone-septa caps (ca. 120 ml headspace) at halomethane production could be more significant than 22C on a shaker at 10 rpm under continuous light, using previously thought, given that halogenating enzymes are an artificial light source maintained at 125 μmol photons − − ubiquitous in these organisms (Amachi et al., 2001; m 2 s 1 until reaching stationary phase, which ranged

Fig 1. Phylogenetic tree of 16S rRNA partial sequences of marine bacteria representative of the major marine groups: Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes (Fuhrman and Hagstrom, 2008), using the neighbour-joining method in MEGA X (Kumar et al., 2018). The scale bar represents substitutions per site. All sequences in the tree belong of marine bacteria isolated from different marine systems and are available in culture collections. In parentheses are the GenBank accession numbers. Red circles denote the strains that were used in the present study. Additional information about these strains is available in the literature: DFL12 (Biebl, 2005), MED193 (Lekunberri et al., 2014), AND4 (Gómez-Consarnau et al., 2010), MED134 (Gómez-Consarnau et al., 2007), MED152 (Gonzalez et al., 2008), MED217 (Pinhassi et al., 2006). Blue circles denote published cyanobacterial cultures assayed for halomethane production; CC9311 reported by Johnson et al. (2015), MED4 by Brownell et al. (2010). from ca. 8 to 100 h. Each litre of ZoBell medium was pre- to account for potential abiotic synthesis were assayed at pared with 750 ml of filtered surface seawater collected the beginning and end of the experiment, and blank at the San Pedro Ocean Time Series Station (SPOT), values were never more than 10% of those measured 250 ml of MilliQ water, 1 g of yeast extract (DIFCO™) from the cultures and are subtracted from data pres- and 5 g of peptone (DIFCO™). Halocarbon samples were ented. For cell abundance, duplicate 2 ml samples were collected every 8–10 h for DFL12, MED193, MED134, fixed with 10% formalin (3.7% formaldehyde), stained MED152 and MED217 and every 1–2 h for the fast- with acridine orange (Hobbie et al., 1977), filtered onto growing Vibrio sp. AND4. For each time-point, two 0.2 μm pore-size black polycarbonate track-etched 250 ml glass vial replicates were collected and (PCTE) filters (25 mm, Fisher Scientific) and counted with ‘sacrificed’ for each measurement. Cultures reached epifluorescence microscopy. highest cell densities typically on the order of 107–108 − cells ml 1 during late exponential growth phase, and one Halocarbon quantification final ‘late’ time-point for halomethane quantification was taken 24 h after the last exponential time point to assess Purge-and-trap capillary column gas chromatography the production during stationary phase. For halocarbon with electron capture detection (GC-ECD) was employed quantification, duplicate culture vials were fixed by acidifi- for dissolved halocarbon analysis (Schall and cation to ca. pH 2.0 with HCl (Munch, 1995) (0.5 ml 3 M Heumann, 1993). About 25 ml samples were transferred solution for 125 ml culture volume), which was injected using gastight syringes with on/off valves from septum through the silicone septum using a syringe, and refriger- topped vials through septum injections ports on the purge ated at 4C in the dark (for a maximum of 14 days) apparatus and purged with ultra-high purity He for 45 min −1 (Munch 1995) until analysis. Media blanks fixed with HCl at a flow rate of 60 ml min through an in-line K2CO3

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 13, 77–85 80 L. Gómez-Consarnau, N. J. Klein, L. S. Cutter and S. A. Sañudo-Wilhelmy drying tube and onto a liquid nitrogen trap. The purge Dickinson FACScalibur). Incorporation rates of the nucle- vessel was rinsed with methanol and the drying trap rep- otide [3H]thymidine were used to estimate of bacterial laced with 0.75 g fresh K2CO3 between individual ana- growth rates in seawater at SPOT (Fuhrman and lyses. Cryo-concentrated samples were introduced into Azam, 1982). an Agilent 8390 GC using a splitless injection with sweep pressure at 50 psi for 1.5 min returning to analytical col- Results and discussion umn pressure of 18 psi 2.5 min after injection. Inlet temperature was set to 60C to facilitate cryo-focusing on the The synthesis of halomethanes in heterotrophic bacteria column. Initial oven temperature was set at 40C for has been previously described only in a handful of bacte- − 10 min, increasing to 120Cby4C min 1 and held there ria isolated from marine and brackish waters (Amachi for another 2 min. Temperature was then ramped to a et al., 2001; Fujimori et al., 2012; Hirata et al., 2017). − ﬁnal 240C at a rate of 5C min 1 and held for 20 min, Thus, it remains unknown how widespread halomethane the last ca. 15 min serving as a bakeout and cleaning synthesis is in bacterial clades that are more widely dis- step in preparation for successive analytical runs. If early tributed in the ocean ecosystem. In this study, we eluting peaks were not sufﬁciently resolved, reducing ini- explored halomethane production in a selection of cul- tial column pressure below 18 psi and slowing tempera- tured marine heterotrophic representatives of the major ture ramps effectively separated the more volatile three environmentally relevant taxa (Alphaproteobacteria, components. Gammaproteobacteria and Bacteroidetes; Fig. 1). The

Concentrations were determined relative to CBrCl3 as production of methyl halides with single halogenations an internal standard—an anthropogenic halocarbon (CH3Br, CH3I, CH3Cl) was observed in all assayed bacte- never significantly used industrially—using PeakSimple rial strains (Fig. 2; Supporting Information Fig. S1), data system and software. To account for the headspace, suggesting that biologically mediated mono-halogenation Henry’s law constants were used to estimate equilibrium reactions are common in bacterioplankton. Bromoform gas-phase concentrations at storage temperature (refrig- (CHBr3) was only detected in the fast-growing Vibrio erated at 4C) and the results are reported as aqueous sp. AND4 culture (Table 1; Supporting Information plus estimated equilibrium gas phase. Detection limits as Figs. S1 and S2). This suggests that, while the produc- 3x standard deviation of the lowest standard were tion of mono-halomethanes seems to be a common trait −1 −1 −1 0.24 pmol l CH3Cl, 0.18 pmol l CH3Br, 0.27 pmol l in marine bacteria, polyhalomethane synthesis is more −1 for CH3I, and 0.38 pmol l for CHBr3. All analytes were species-dependent, as previously reported (Manley and undetectable in Milli-Q or sterile media blanks. de la Cuesta 1997; Hughes et al., 2011; Fujimori et al., 2012; Hirata et al., 2017; Johnson et al., 2015; Hughes et al., 2013). Halocarbon quantifications in natural seawater Halomethane concentrations measured over the Methyl halides were quantified in environmental samples course of the growth experiment fitted an exponential collected at the San Pedro Ocean Time Series Station response, similar to the exponential bacterial growth (SPOT) located within the Southern California Bight. curve (Fig. 2; Supporting Information Fig. S1). Hence, SPOT is located in the center of the San Pedro basin while the concentrations of halomethanes increased by (3333’N, 11824’W), where monthly sampling of the several orders of magnitude during bacterial growth water column for biologic and hydrographic parameters (Fig. 2A; Supporting Information Fig. S1), the standard- has been conducted since 1998. For this study samples ized per-cell concentrations were relatively constant dur- were collected at the surface (5 m depth) during the ing the exponential phase growth (Fig. 2B; Supporting spring upwelling season in April of 2017 and during oligo- Information Fig. S2). These data show that the production trophic summer conditions in June of 2017. The natural of halomethanes – and monohalomethanes in particular seawater measurements were used to validate and con- – is directly associated with growth rate and metabolic strain some of the culture-based results as the activity. laboratory-controlled experiments were carried out at Of all halomethanes measured, CH3I was the com- optimum growing conditions that may not always reflect pound produced at highest per-cell rate by all heterotro- − the natural environment. phic bacteria strains (from 1.5 10 8 to 3.7 − − − 10 7 pmol cell 1 day 1) up to an order of magnitude −10 higher than CH3Br (3.0 10 to 2.1 Bacterial cell abundance and growth rate −8 −1 −1 −9 10 pmol cell day ) and CH3Cl (1.0 10 to 3.8 measurements in situ − − − 10 8 pmol cell 1 day 1; Table 1). Although the patterns of Bacterial cell counts from seawater were determined by halomethane synthesis were related to growth rate for all flow cytometry (Gasol and del Giorgio, 2000) (Becton– heterotrophic bacteria tested in culture, each isolate

contrast, the production of CH3Cl was on a similar range for both the Synechococcus strains and heterotrophic − − − bacterial cultures (10 9 pmol cell 1 day 1) but an order of magnitude lower for Prochlorococcus sp. MED4 −10 −1 −1 (10 pmol cell day ). CHBr3 synthesis was detected only in heterotrophic bacterial strain AND4 and in Synechococcus cultures, but with high variability among strains; AND4 showing 25x higher levels than strain Synechococcus strain CC9311 and 600x higher than the bromoperoxidase knockout mutant strain VMUT2 (Table 1). Overall, while both prokaryotic groups produce halomethanes, heterotrophic bacteria in particu-

lar appear to dominate the production of CH3I. The rea- sons behind the signiﬁcant differences in CH3I production by heterotrophs and cyanobacteria is currently unknown. Amachi et al. (2001) reported that the iodide concentrations in the media can affect the production levels of

CH3I in heterotrophic bacteria. However, the concentrations tested by these authors were on a much higher range than those usually found in seawater (1–104 μMof iodide compared to a maximum of 0.4 μM in seawater, Chance et al., 2014). Therefore, within the concentration range of natural seawater – which was used in our growth media – it is unlikely that changes in the initial iodide concentration had triggered these differences. Given the consistent relationship between halomethane production and bacterial growth rate in cultures, we estimated the in situ bacterial halomethane production in surface seawater at the coastal SPOT station. To accomplish this, we used bacterial growth rates inferred from bacterial production measurements and Fig 2. Bacterial growth and halomethane (CH3I, CH3Cl and CH3Br) production characteristics of Leeuwenhoekiella blandensis MED217. halomethane concentrations, both measured under This figure serves as an example of the bacterial growth and upwelling (spring) and oligotrophic (summer) conditions fi halomethane synthesis characteristics, both following a rst order in 2017 (Table 1). In contrast to what was observed in equation. Similar patterns were identified in all cultures assayed, as shown in the Supporting Information Figs. S1 and S2. Bacterial cultures of heterotrophic bacteria, the in situ per-cell syn- growth and halomethane synthesis rate constants (k) were obtained thesis rates of CH3I were the lowest from linearizations of cell counts and halocarbon concentrations −10 −1 −1 kt (10 pmol cell day ) while CH3Br were the highest using exponential growth expression Ct =Ci *e . − − − in surface natural seawater (7Á10 9 pmol cell 1 day 1). One explanation for the lower in situ concentrations of produced halocarbons at different per-cell basis rates. CH3I in natural seawater could be that the chemical spe- Dinoroseobacter shibae DFL12 displayed the lowest pro- ciation of iodine in the coastal waters was different than −10 −1 −1 duction rates of CH3Br (3.0 10 pmol cell day ) and that found in our media after autoclaving (iodate −9 −1 −1 CH3Cl (1.0 10 pmol cell day ) while Vibrio AND4 vs. iodide; Wong et al., 2002). Yet, it is currently unknown produced all halomethanes at the highest per-cell rates to what extent the different chemical forms of iodine are −8 −7 compared to all other bacteria (1.1 10 to 3.7 10 pmol used as CH3I precursors by bacteria. Furthermore, while −1 −1 cell day ; Table 1). We further compared these data all bacteria in culture produced CH3Cl, this halomethane with the halomethane production rates reported in the lit- was undetectable in the environmental samples. These erature for cyanobacteria cultures of Synechococcus differences between the culture and the field measure- strains CC9311 and VMUT2 (Johnson et al., 2015) and ments are somewhat expected, given the high microbial Prochlorococcus marinus MED4 (Brownell et al., 2010). diversity found in situ and the fact that pure bacterial cul-

Notably, the per-cell production rates of CH3I were more tures were grown in rich media conditions that rarely than three orders of magnitude lower in cyanobacteria occur in nature. In fact, bacteria in the SAR11 clade usu- (10−11 to –10−12 pmol cell−1 day−1) compared with het- ally dominate natural surface waters at SPOT (Chow − − − − erotrophic bacteria (10 7 to 10 8 pmol cell 1 day 1). In et al., 2013) during most of the year, and cultures of this

Table 1. Halomethane synthesis rates obtained in cultures of heterotrophic bacteria during exponential growth (this study), cyanobacteria (Syn- echococcus sp. from Johnson et al. 2015, Prochlorococcus marinus from Brownell et al. (2010), and environmental samples collected at SPOT.

Halomethane synthesis rate (pmol cell-1 day-1) (1010)

Heterotrophic bacteria cultures CH3Cl CH3ICH3Br CHBr3

AND4 376 ± 60 3704 ± 612 211 ± 40 122 ± 45 DFL12 10 ± 4 149 ± 43 3 ± 1 ND MED134 63 ± 37 393 ± 265 146 ± 58 ND MED152 55 ± 20 438 ± 112 31 ± 5 ND MED193 43 ± 24 394 ± 180 31 ± 15 ND MED217 47 ± 23 337 ± 177 22 ± 7 ND

Cyanobacteria cultures

Synechococcus sp. CC9311 12.0 ± 9.8 0.12 ± 0.13 7.4 ± 2.5 5.0 ± 1.8 Synechococcus sp. VMUT2 5.7 ± 15.8 0.1 ± 0.23 0.1 ± 1.0 0.2 ± 0.6 Prochlorococcus marinus MED4 0.90 ± 0.50 0.03 ± 0.00 0.13 ± 0.09 NA

Environmental samples SPOT

SPOT_Apr2017_5m ND 2.3 72.3 12.3 SPOT_June_2017_5m ND 0.3 70.3 17.2

ND, not detected. NA, not analyzed. group were not tested in our study. However, the temperature is a master variable that has a significant observed patterns of halomethane synthesis remained effect on bacterial metabolism (e.g. Ratkowsky remarkably consistent for the six marine bacterial strains et al., 1982; Zwietering et al., 1991; Zwietering tested, despite those belonging to three different taxo- et al., 1994). Our estimates suggest that, in coastal envi- nomical groups. Overall, these data suggest that a signifi- ronments such as SPOT, the temperature change could cant fraction of marine halomethanes could originate cause a 33%–84% increase in bacterial growth and from the activity of marine heterotrophic bacteria. In the halomethane synthesis rates (Table 2). This, together particular case of CHBr3, which production was only with a projected increase in bacterial abundance relative detected in the heterotroph AND4 and in cyanobacteria to phytoplankton in a warmer ocean (Sarmento cultures (Johnson et al., 2015), we can conclude that et al., 2008; Kvale et al., 2015) suggests that the relative phytoplankton and fast-growing heterotrophic bacteria contribution of heterotrophic bacteria to the atmospheric are probably its major synthesizers. budget of halomethanes could also rise in the future. Since bacterial metabolism and growth rate are pro- There are two contrasting outcomes of this projection. foundly impacted by changes in temperature (White On the one hand, an increase in the production of most et al., 1991; Gillooly et al., 2001), we projected halomethanes (e.g., CH3Br, CHBr3,CH3Cl) could accel- halomethane production rates under a global warming erate catalytic ozone destruction. However, similar to ‘business as usual’ scenario of a 3 C increase in sea sur- dimethyl sulfide, the nuclei condensation activity of CH3I face as predicted by the year 2100 (IPCC, 2014). For can trigger a negative climate feedback effect and help this, we used the model described by Ratkowsky mitigate global warming. Notably, CH3I can make up et al. (1982) on the effect of temperature on bacterial 20%–50% of all iodine forms present in the atmosphere growth (Table 2 and Supplementary Information). Our (Koenig et al., 2020). In this study, CH3I was the goal applying this simplified model was to try to under- halomethane produced at the highest per-cell rate by stand the magnitude of global warming on bacterial syn- marine heterotrophic bacteria, suggesting that its synthe- thesis of ozone-depleting halomethanes, as water sis could increase in future climate scenarios. Whether

Table 2. Current and predicted bacterial growth rates under a 3C temperature increase expected under the business as usual climate change scenario (IPCC, 2014).

Sample Current bacterial growth rate k (day−1) Projected bacterial growth rate k +3C (day−1) Rate percent increase

Apr 2017 5 m 0.38 0.51–0.70 33.15–82.76 Jun 2017 5 m 0.23 0.31–0.42 36.01–84.27

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