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COI Funded Project: Dynamics and Microbiology of Coastal Marine Carbon Monoxide Bio-Oxidation

Project Duration: 7/18/01-7/17/02

Proposed Research

As colored dissolved organic matter in seawater absorbs UV solar radiation, a variety of simple chemical species are produced, including carbon monoxide. As the ocean surface water is saturated with respect to CO, it is a source of CO to the atmosphere. CO reacts with and removes free-radical compounds, and may itself contribute to the 'greenhouse' gas content of the atmosphere. An important sink for this chemical species in seawater is the biological oxidation of CO to CO2 by marine microorganisms. Past measurements of the rate of CO oxidation have been limited to dark incubations where CO is not photolytically produced, and have been considered representative of oxidation rates throughout the photoperiod. It is likely that high light intensity in the upper water column is inhibitory to the microorganisms responsible for CO oxidation and results in depressed rates of CO oxidation during daylight hours. We have developed a new radioisotopic tracer technique of measuring the CO oxidation rate that allows the measurement to be made in the light and in the presence of CO production. Determining the extent of daytime CO oxidation rate depression will allow a more accurate assessment of the removal of CO from the water column and of the contribution of CO to the atmosphere by coastal waters.

Final Report

Abstract
As colored dissolved organic matter in seawater absorbs UV solar radiation, a variety of simple chemical species are produced including carbon monoxide. As the ocean surface water is saturated with respect to CO, it is a source of CO to the atmosphere where it reacts with and removes free-radical compounds and may itself contribute to the 'greenhouse' gas content of the atmosphere. An important sink for this chemical species in seawater is the biological oxidation of CO to CO2 by marine microorganisms.

The objectives of this study are to identify component members of the microbial community responsible for the oxidation of CO in coastal marine environments through a combination of recent microbiological and molecular approaches, and to estimate their contributions to total in situ CO bio-oxidation. We utilize a simple direct plating method for screening and isolating microorganisms with a particular phenotype.

The method involves cultivation of bacteria on membrane filters placed atop an oligotrophic liquid mineral medium, subsequent incubation with radiolabeled CO, and the use of autoradiography to screen colonies with the desired phenotype amidst a potential background of other colonies. Since this method spatially separates otherwise competing populations, simultaneous recovery of organisms with different growth rates and nutritional requirements is possible, and large numbers of bacteria can be screened rapidly at one time. Cell-specific CO-oxidation activity is determined for selected strains with a time-series 14CO-oxidation assay. Molecular phylogeny based on 16S-rRNA sequence information determines the phylogenetic relatedness and identity of marine CO-oxidizing bacteria that result from our cultivation program.

Preliminary results indicate that 20 – 33% of in situ CO oxidation can be attributed to marine microorganisms belonging to the Roseobacter clade of the alpha subclass of Proteobacteria. We have developed a protocol using a combination of simple assays for directly assessing the activity and relative cell density of a sub-population of the natural microbial assemblage, which allows us to determine early on which strains are important in performing a specific metabolism and are thus candidates for further investigation.

Introduction
Carbon monoxide (CO) is the third most abundant carbon species in the atmosphere. It is one of the most important reactive gasses in the atmosphere because of its reaction with the hydroxyl radical (OH), which forms a major sink for both atmospheric CO and hydroxyl radicals (Logan et al., 1981; Johnson & Bates, 1996; Pos et al., 1998; Zuo et al., 1998. Although the dominant sources of atmospheric CO are anthropogenic (Müller, 1992; Conrad & Seiler, 1982a), it is well documented that sunlight-initiated photodegradation of colored dissolved organic matter (CDOM) is primarily responsible for producing CO in sunlit waters, thus the surface waters of the world's oceans are saturated with CO with respect to the atmosphere and are a source of atmospheric CO (Swinnerton et al., 1970; Conrad et al., 1982).

It is likely that most of the CO produced in situ is consumed by microbial activities (Conrad et al., 1982). A global "blue-water" photochemical source of CO, 50 ? 10 Tg carbon from CO per year (CO-C a-1) and its microbial sink, 32 ? 18 Tg CO-C a-1 (Zafiriou et al., 2002) has been estimated, suggesting that CO processes are a non-trivial component of the oceanic carbon budget, and most CO is cycled internally by microbial processes. The global CO gas exchange flux has been estimated by Bates et al., (1995) as ~5.5 Tg CO-C a-1; the flux of CO from the ocean into the atmosphere would therefore represent a minor fraction of the total CO produced in the ocean (Conrad and Seiler, 1980; Conrad et al., 1982). Whether the CO is transferred into the atmosphere or oxidized to CO2 it represents a direct loss of otherwise refractory DOM carbon in natural waters (Zuo & Jones, 1997).

CO distribution and diel variability
The major source of CO in the surface waters of the ocean is the abiotic photo-oxidation of chromophoric dissolved organic material (CDOM) initiated by UV or near-UV light (Conrad et al., 1982 and many others). The dependence of CO production of the presence of light and oxygen and its independence of the presence of plankton or bacteria indicates that CO production is a photochemical rather than photometabolic process. At night photoproduction of CO ceases; thus the nighttime decrease in CO concentration results from two primary mechanisms, sea-to-air exchange and oxidation by microbes (Conrad & Seiler, 1980; Jones, 1991; Johnson & Bates, 1996).

Several biogeochemical studies have estimated the diel, annual, and global rates of CO oxidation but few have attempted to investigate beyond the black-box? level of the phenomena occurring in marine environments. There have been no definitive investigations concerning the identity of the microbes responsible for CO bio-oxidation in the open-water marine environment; only circumstantial evidence for CO metabolizing marine organisms based on ancillary ability of certain bacteria to also metabolize CO. The question of which organisms are performing CO bio-oxidation in coastal or oceanic surface waters has not been satisfactorily resolved. As the marine microflora are thought to be actively oxidizing CO, the main problem is to find out if there exists a specialized group of microorganisms that is capable of utilizing CO at nM concentrations, or if CO at nM concentrations is oxidized by the ordinary microflora.

This work provides the first definitive identification of the CO metabolizing microorganisms that exist in a coastal marine environment, infers the phylogeny of important members of the microbial community responsible for oxidation of CO, and determines the relative contributions of these organisms to total CO oxidation observed in natural waters.

Methods/Materials
Sampling location and procedure
The primary coastal sampling site for this study is at the WHOI Shore Lab pier located 1 km east of Nobska Light, Woods Hole, on a south-facing beach on Vineyard Sound, Cape Cod, in Massachusetts (Figure 1). This location was chosen for its proximity to the laboratory, ease of sampling, and distance from coastal pond- and freshwater discharge. Syringe or bottle samples are collected 0.5 m below the surface to avoid atmospheric CO contamination, in water depth of approximately 2 m. Reduced gas analysis (Trace Analytical RGA) of in situ CO concentrations, rates of CO consumption, and turnover times of CO at this site are consistent with other coastal CO oxidation measurements in similar environments (Jones & Amador, 1993; Jones, 1991).

Isolation procedure
To isolate CO metabolizing microorganisms, we utilize a simple direct plating method for screening and isolating microorganisms with particular phenotypes. The method involves cultivation of bacteria on membrane filters atop a combusted glass-fiber filter saturated with an oligotrophic liquid mineral medium, subsequent incubation with radiolabelled CO, and the use of autoradiography to identify colonies with desired phenotypes amidst a potential background of other colonies. Since this method spatially separates otherwise competing populations, simultaneous recovery of organisms with different growth rates and nutritional requirements is possible, and large numbers of bacteria can be screened rapidly at one time. The defined mineral media to be provided supports growth of carboxydotrophic bacteria and all known phenotypes able to metabolize CO, and selects against diverse heterotrophs.

CO oxidation rate determination
Water was drawn without contact with the atmosphere in 100 ml ground-glass syringes. Samples were subdivided (25 ml) into 75 ml glass serum vials and septum sealed. 1-ml aliquots of 14CO stock were withdrawn with a gas-tight syringe and injected into each of duplicate sample-incubation vials. This provided a relatively large (>100 ml) headspace CO reservoir, and a potential doubling of aqueous [CO] due to photoproduction changed 14CO specific activity (hence, measured rates) by <5%. Ambient CO concentration in the room air at the time of sealing was determined with a reduced gas analyzer (Trace Analytical). Replicate samples were incubated in the dark at within 2°C of the in situ temperature. Biological activity was stopped at discrete time points during four point time series incubations by injecting NaOH sufficient to raise the pH of the sample to >9, and shaken to sequester any CO2 remaining in the headspace. Zero-time bottles were fixed by NaOH injection within 5 minutes of 14C-CO injection, and time-point duplicates were fixed at three-hour intervals. The NaOH-stabilized sample-vials were then stored until 14C-activity could be assessed. Sample incubation vials were opened to allow excess 14CO to exchange overnight. The vials were resealed with a CO2-trapping wick (fluted filter paper treated with 0.2 ml hyamine hydroxide) suspended in the headspace above the liquid, acidified by injection of 1.0 ml 3.6 N H2SO4 sufficient to lower the pH below 2, and left overnight on a shaker-table at 100 rpm. The CO2-traps containing the product of microbial bio-oxidation of 14CO were then measured for activity by liquid scentillation spectroscopy.

The activity measured for each vial was corrected for the t-naught background, and nanomoles of CO2 produced calculated by dividing by the 14CO specific activity inside the vial. This was converted to nanomolar (in 25 ml sample), and plotted vs. incubation time. The rate of oxidation over the plotted time series incubation period was determined as the best-fit slope of the line plotted through the four data points (Griffiths et al., 1982; Jones et al., 1984). Rate constants were calculated by normalizing the time-series rate (nM/hr) by the total equilibrated [CO] within each incubation vessel (moles CO in situ + moles CO headspace + moles CO injected) to give a rate constant in hr-1. The total equilibrated [CO] also required the Bunsen solubility coefficient for CO (Weisenburg & Guinasso, 1979), which depends on pressure, temperature, and salinity during the incubation.

Molecular phylogeny
The 16S rRNA gene of isolated pure clones of CO metabolizing microorganisms was amplified by PCR by a variety of treatments. In general, the bacterial universal primers 8f and 1492r were used resulting in a 1484 bp segment. Product of the correct size was confirmed by electrophoresis in 2% agarose gel at 100 volts for 25-30 minutes, with either a Low DNA MASS Ladder or Amplisize Molecular Ruler 50-2,000 bp ladder. The sequencing reactions were performed with the Big Dye Terminator (BDT) sequencing kit (Applied Biosystems), and separately using 3 different bacterial primers. For each reaction, 1 ?l BDT, 1 ?l primer (8f, 1492r, or 519f), 50 ng DNA, and H20 were combined to a total volume of 6 ?l. 32 isolates and 3 separate primer reactions filled a 96-well microplate. The thermocycler was set for 25 cycles: 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes.

Sequences from 30 isolates were edited using Sequencher 4.0.5 (Gene Codes Corporation, Ann Arbor, MI). Sequences from primers 8f, 519f, and 1492r were cleaned and edited separately. For the remaining isolates, the three fragment sequences were assembled into a contiguous sequence. The sequence fragment with multiple-strand redundancy was checked against the NCBI Entrez (BLAST) for initial identification. Sequences were aligned in the ARB software package (http://www.arb-home.de/) using the Ribosomal Database II (http://rdp.cme.msu.edu/html/) content of aligned sequences. A preliminary phylogenetic tree was constructed using the neighbor joining method. Bootstrap values are absent, pending further refinement of the alignments.

Preliminary Results
Thirty organisms were isolated in pure culture from the autoradiography / 14CO incubation of the natural microbial assemblage. These organisms were each tested for their CO oxidation activity in pure culture, using the time-series 14CO-oxidation assay described above. These determinations are still in progress at this writing, but it appears evident that there are two strains (JT-01 and JT-08) that oxidize CO at a fast rate on a per-cell basis, and these are considered "fast-oxidizers" in this study. Six other strains oxidize CO but at a slower rate; these organisms (JT-6b, JT-21, JT-22, JT-23, JT-27, JT-29) are designated as "meso-oxidizers" (Figure 3). A calculation to determine whether these organisms can account for the observed CO oxidation in the coastal environment shows that 20 ? 33 % of in situ CO oxidized can be attributed to these 8 organisms. The assumption of in situ cell density in this calculation was based on the volumetric density of positive signals that appeared on the autoradiographic plates. A true enumeration of these strains is pending, and will be made using strain-specific oligonucleotide probes and MPN-PCR.
Preliminary identification of these strains was performed by comparison of the 16S-rRNA gene sequence with sequences contained within the NCBI databank, however, there were several 'hits' for each strain with high degrees of sequence identity (95 ? 100%), and it is not possible to make a species determination in this manner. The 16S rRNA gene sequences of the two most active "fast" CO oxidizers (JT-01 and JT-08) were aligned to the 'universal alignment' and a rough phylogenetic tree indicates that these organisms both belong to the Roseobacter clade within the alpha subclass of Proteobacteria. This result is interesting in that Roseobacter has been recently determined to be a numerically important group within the total microbial assemblage (Gonzalez and Moran, 1997), and have been implicated in DMSP- and other sulfur metabolism in surface waters (Ledyard et al, 1993).

On-going research
This study has given us a good indication of the organisms most responsible for the observed oxidation of CO in the coastal marine environments, but before we can attribute all or most of in situ CO oxidation to these strains, it is necessary to enumerate the CO-oxidizers in natural samples. In this pursuit, we must determine how widespread the CO-oxidation behaviour is found within members of the Roseobacter and related clades by performing the 14CO-oxidation assay on defined bacterial cultures obtained from culture collections around the world (DSMZ, ATCC, Moran, Teske, Jannash, Ledyard). Once we determine whether this activity is performed by closely related organisms, a group-specific molecular probe will be designed based on 16S-rRNA sequence information to enumerate members of the group by Most-Probable-Number PCR, a dilution method combined with molecular amplification.

We have developed a protocol using a combination of simple assays for directly assessing the activity and relative cell density of a sub-population of the natural microbial assemblage, which allows us to determine, early on, which strains are important in performing a specific metabolism and are thus candidates for further investigation.

References cited:
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Originally published: January 1, 2001