Reductive debromination by sponge-associated anaerobic bacteria coupled to carbon isotope fractionation

https://doi.org/10.1016/j.ibiod.2020.105093Get rights and content

Highlights

  • Compound specific isotope analysis (CSIA) was evaluated for monitoring reductive dehalogenation.

  • Reductive debromination of 2,6-dibromophenol to phenol results in carbon isotope fractionation.

  • CSIA can assess reductive debromination and estimate in vivo organohalide flux in a sponge animal.

Abstract

Marine sponges contain diverse brominated compounds as secondary metabolites and the sponge habitat appears to enrich for a population of anaerobic dehalogenating bacteria. Hence, there is interest in understanding how these natural and anthropogenic compounds are degraded in the marine environment. Compound specific isotope analysis (CSIA) is a useful tool to monitor and to quantify the degradation and fate of aquatic pollutants. The objective of this study was to evaluate whether reductive dehalogenation of brominated phenols by sponge-associated bacteria, including Desulfoluna spongiiphila, can be monitored by CSIA. Debromination of 2,6-dibromophenol to phenol by sponge-associated cultures resulted in measurable stable carbon isotope fractionation. All sponge-associated cultures showed similar isotopic enrichment factors (ε). The ε values for two independent sponge-derived dehalogenating cultures were −3.1 ± 1.5‰, and −3.0 ± 0.3‰, and that of sponge associated sediment cultures −2.0 ± 0.3‰. Thus, we demonstrate that reductive debromination of 2,6-dibromophenol resulted in measurable carbon isotope fractionation and that CSIA can be used to assess reductive debromination and to monitor and estimate in vivo dehalogenation in a sponge animal.

Introduction

Organobromine compounds are ubiquitous in marine sedimentary environments (Leri et al., 2010) and organic and inorganic bromine cycling is intertwined with the cycling of organic carbon. Marine organisms produce and transform a myriad of natural organohalides (Gribble, 2003, 2010), while the marine environment is also a sink for many organohalide pollutants, including brominated flame retardants, widely used to avoid fire accidents in electronics, indoor house-hold fabrics and even clothing (de Wit, 2002; Law et al., 2006). Brominated aromatics, such as tetrabromobisphenol A (TBBPA) and polybrominated diphenyl ethers (PBDEs), are among the most widely used brominated flame retardants (Birnbaum and Staskal, 2004). Because of their physicochemical properties, brominated compounds can accumulate in anoxic sediments, have ecotoxicological effects, or may biomagnify in the food chain (de Wit et al., 2010; Fernie and Letcher, 2010; Bartrons et al., 2012). For example, several brominated flame retardants are thyroid and estrogen disruptors (Hamers et al., 2006, 2008). The wide use and increasing prevalence of these brominated compounds has prompted the study of their fate and transport in the environment.

Interestingly, the marine environment is also a particularly rich source of biogenic halogenated compounds produced by diverse marine organisms, including sponges (Gribble, 2003, 2010). The organohalides present in marine sponges are usually bioactive, with antifouling or/and antibiotic characteristics. Some of these organohalides resemble anthropogenic pollutants, such as halogenated dibenzo-p-dioxins (Bowden et al., 2000) and flame retardants (Utkina et al., 2001). This long-term exposure to biogenic organohalides is believed to have supported the evolution of reductive dehalogenating bacteria (Krzmarzick et al., 2012). Marine sponges are some of the most prolific organohalide producers. Sponges are filter feeders, and although microorganisms are a major component of their diet, complex sponge-specific microbial communities thrive within the mesohyl of the sponge (Hentschel et al., 2006, 2012; Taylor et al., 2007a,b, 2007b; Thacker and Freeman, 2012; Webster and Taylor, 2012). The combined abundance of organohalides and the high density of their associated microbes, for example in the Aplysina aerophoba sponge, prompted Ahn et al. (2003) to determine whether sponge-associated microbes were capable of dehalogenation. Subsequently, a novel sponge-associated bacterial species, Desulfoluna spongiiphila, capable of organohalide respiration via reductive debromination of tri-, di-, and mono-bromophenols was isolated (Ahn et al., 2009). A novel reductive dehalogenase of D. spongiiphila was upregulated during respiration of bromophenols (Liu et al., 2017, 2020; Peng et al., 2020). Sponge-produced organohalides, thus, appear to select for microorganisms that can utilize these compounds as a source of energy. Desulfoluna spp. are a peculiar group of dehalogenating microorganisms active in metazoans and represent a unique contrasting group from the other known dehalogenating bacteria. Reductive dehalogenation of brominated phenolics has also been observed in other habitats, including estuarine sediments (e.g., Monserrate and Häggblom, 1997; Boyle et al., 1999; Watson et al., 2000; Fennell et al., 2004; Arbeli et al., 2006), and reductive dehalogenase genes appear to be widely distributed in marine Desulfobacterota (Liu and Häggblom, 2018). There is still much to understand about the in situ activity of dehalogenating microbes in marine and estuarine environments (Zanaroli et al., 2015) and elucidation of the fate and transport of brominated pollutants.

Compound specific isotope analysis (CSIA) is a useful tool to monitor and to quantify the degradation and fate of aquatic pollutants (Meckenstock et al., 1999; Sherwood-Lollar et al., 1999; Hunkeler et al., 2005). Compounds analyzed for effects of biodegradation on isotope composition include aromatic hydrocarbons (Richnow et al., 2003; Braeckevelt et al., 2007), methyl tert-butyl ether (Somsamak et al., 2005); organohalides such as tetrachloroethene (Nijen-huis et al., 2005; Fletcher et al., 2011), dichloromethane (Nikolausz et al., 2006) and polychlorinated dibenzo-p-dioxins (Ewald et al., 2007; Liu et al., 2010). More recently, carbon isotope fractionation for microbial debromination of brominated alkanes and aromatics were published (Bernstein et al., 2013; Sohn et al., 2018; Woods et al., 2018). CSIA is based on the different reaction kinetics of bond formed by light vs. heavy isotopes. Chemical bonds containing heavier isotopes need more energy for cleavage than light isotopes and in either abiotic or biological transformations. Therefore, normal isotope effects with a preferential transformation of the 12C substrates are observed. This results in an enrichment of 13C with a change in the C isotope ratio (13C/12C) in the remnant substrate over time. Biological transformations commonly lead to a normal isotope effect accompanied with an enrichment of heavier isotopes in the residual fraction of the substrate (for reviews see, Meckenstock et al., 2004; Schmidt et al., 2004; Bombach et al., 2010; Elsner, 2010; Renpenning and Nijen-huis, 2016). The isotope enrichment factors can potentially be linked to specific biochemical reactions because the extent of fractionation is linked to the degradation pathway of the target compound and type of bonds being affected during initial cleavage (Nijen-huis and Richnow, 2016). Accordingly, CSIA represents a powerful tool for assessing biodegradation of organic chemicals in the field.

In order to develop an approach for understanding the metabolic activities of the sponge endomesohyl microbiota in the cycling of organohalogens in the marine environment we applied CSIA to analyze the bromophenol substrates and dehalogenation products. We tested whether CSIA could be used for assessing the reductive dehalogenation of brominated phenols and for monitoring the activity of anaerobic sponge-associated bacteria, and to eventually estimate the organohalide flux in vivo. 2,6-Dibromophenol (2,6-DBP) was used as a model compound to assess whether temporal analysis of carbon isotopic composition during reductive debromination by sponge derived cultures can be used to determine the isotopic enrichment factor (ε) for this process. Here we report on CSIA of brominated phenols during microbial degradation in a marine system.

Section snippets

Chemicals

2,6-DBP used as substrate and other phenolic compounds (2-bromophenol, 2-BP; 4-chlorophenol, 4-CP; 4-methylphenol; 2,4-dichlorophenol; phenol) used as standards were obtained from Aldrich Chemical Co. (Milwaukee, Wis., USA) and had a minimum of 99% purity.

Origin and cultivation of reductively debrominating bacterial cultures

Anaerobic dehalogenating cultures of Desulfoluna spongiiphila strain AA1 (AA1), a sponge enrichment culture (PPS) of an unidentified sponge (presumed to be of the order Haplosclerida; collected Bucco Sur, Tumbes, Peru), and a sediment

CSIA of debrominating cultures D. spongiiphila AA1, sponge enrichment culture PPS and sediment culture TS7

Three sponge-derived anaerobic dehalogenating cultures were compared to determine carbon isotopic fractionation during debromination of 2,6-DBP. All cultures showed sequential debromination of 2,6-DBP to 2BP and phenol in less than 14 days (Fig. 1a and b). In cultures of D. spongiiphila AA1 the δ13C of 2,6-DBP became enriched from an initial value of −30 to −25.6‰ at 79% debromination. Similarly, the δ13C of 2,6-DBP in the Peruvian sponge enrichment culture (PPS) and the Tuckerton NJ sediment

Discussion

Three sponge-derived anaerobic dehalogenating cultures sets showed sequential debromination of 2,6-DBP to 2BP and phenol with measurable stable carbon isotope fractionation (Fig. 1, Fig. 2). All sponge-associated cultures showed similar isotopic enrichment factors (ε) (Table 1). The values observed for the dehalogenating cultures from Aplysina and Haplosclerida sponges indicates that transformation of 2,6-DBP is mediated by similar mechanisms, comprising similar steps. CSIA provides an approach

Author contribution statement

IH-G, IN and MMH conceived and designed the research. IH-G, NAL and YA conducted the experiments. HHR contributed analytical tools. IH-G, IN and MH analyzed the data. IH-G, IN and MMH wrote the manuscript. All authors read and approved the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Dr. Matthias Gehre, Dr. Stefanie S. Weber, Ursula Gunther and the crew of the isotope lab of the Department of Isotope Biogeochemistry, Helmholtz-Centre for Environmental Research – UFZ, Leipzig, Germany for their assistance with CSIA. The work was supported in part by the USDA National Institute of Food and Agriculture Hatch project accession numbers 205270 and 1012785 through the New Jersey Agricultural Experiment Station (Hatch projects NJ00138 and NJ01160) and by the European Union

References (68)

  • D.A. Merritt et al.

    Isotope-ratio-monitoring gas-chromatography mass-spectrometry methods for isotopic calibration

    Org. Geochem.

    (1994)
  • I. Nijen-huis et al.

    Recent advances in multi-element compound-specific stable isotope analysis of organohalides: achievements, challenges and prospects for assessing environmental sources and transformation

    Trends Environ. Anal. Chem.

    (2016)
  • I. Nijen-huis et al.

    Stable isotope fractionation concepts for characterizing biotransformation of organohalides

    Curr. Opin. Biotechnol.

    (2016)
  • H.H. Richnow et al.

    Microbial in situ degradation of aromatic hydrocarbons in a contaminated aquifer monitored by carbon isotope fractionation

    J. Contam. Hydrol.

    (2003)
  • B. Sherwood-Lollar et al.

    Contrasting carbon isotope fractionation during biodegradation of trichloroethylene and toluene: implications for intrinsic bioremediation

    Org. Geochem.

    (1999)
  • S.Y. Sohn et al.

    Evaluation of carbon isotope fractionation during dehalogenation of chlorinated and brominated benzenes

    Chemosphere

    (2018)
  • R.W. Thacker et al.

    Sponge-microbe symbioses: recent advances and new directions

    Adv. Mar. Biol.

    (2012)
  • A. Woods et al.

    Variable dual carbon-bromine stable isotope fractionation during enzyme-catalyzed reductive dehalogenation of brominated ethenes

    Chemosphere

    (2018)
  • G. Xu et al.

    Complete debromination of decabromodiphenyl ether using the integration of Dehalococcoides sp. strain CBDB1 and zero-valent iron

    Chemosphere

    (2014)
  • G. Zanaroli et al.

    Microbial dehalogenation of organohalides in marine and estuarine environments

    Curr. Opin. Biotechnol.

    (2015)
  • Y. Abe et al.

    Effect of molecule size on carbon isotope fractionation during biodegradation of chlorinated alkanes by Xanthobacter autotrophicus GJ10

    Isot. Environ. Health Stud.

    (2009)
  • Y.B. Ahn et al.

    Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba

    Appl. Environ. Microbiol.

    (2003)
  • Y.B. Ahn et al.

    Desulfoluna spongiiphila sp. nov., a dehalogenating bacterium in the Desulfobacteraceae from the marine sponge Aplysina aerophoba

    Int. J. Syst. Evol. Microbiol.

    (2009)
  • M. Bartrons et al.

    Pollutant dehalogenation capability may depend on the trophic evolutionary history of the organism: PBDEs in freshwater food webs

    PLoS One

    (2012)
  • A. Bernstein et al.

    Kinetic bromine isotope effect: example from the microbial debromination of brominated phenols

    Anal. Bioanal. Chem.

    (2013)
  • L.S. Birnbaum et al.

    Brominated flame retardants: cause for concern?

    Environ. Health Perspect.

    (2004)
  • P. Bombach et al.

    Current approaches for the assessment of in situ biodegradation

    Appl. Microbiol. Biotechnol.

    (2010)
  • B.F. Bowden et al.

    A new brominated diphenyl ether from the marine sponge Dysidea herbacea

    Aust. J. Chem.

    (2000)
  • A.W. Boyle et al.

    Isolation from estuarine sediments of a Desulfovibrio strain which can grow on lactate coupled to the reductive dehalogenation of 2,4,6-tribromophenol

    Appl. Environ. Microbiol.

    (1999)
  • D. Cichocka et al.

    Factors controlling the carbon isotope fractionation of tetra- and trichloroethene during reductive dechlorination by Sulfurospirillum ssp and Desulfitobacterium sp strain PCE-S

    FEMS Microbiol. Ecol.

    (2007)
  • T.B. Coplen

    Guidelines and recommended terms for expression of stable- isotope-ratio and gas-ratio measurement results

    Rapid Commun. Mass Spectrom.

    (2011)
  • M. Elsner

    Stable isotope fractionation to investigate natural transformation mechanisms of organic contaminants: principles, prospects and limitations

    J. Environ. Monit.

    (2010)
  • M. Elsner et al.

    A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants

    Environ. Sci. Technol.

    (2005)
  • E.M. Ewald et al.

    Microbial dehalogenation of trichlorinated dibenzo-p-dioxins by a Dehalococcoides-containing mixed culture is coupled to carbon isotope fractionation

    Environ. Sci. Technol.

    (2007)
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