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 environ- ments (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, iden- tifying 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