Discussion and Conclusions

1. k600-N2O k600-N2O k600-CH4 k600-CH4 k600-SF6 k600-SF6 10 Invasive 8 Evasive 6 Invasive - N O k 00 2 6 Invasive - CH k 600 4 Evasive 4 - SF k Evasive 600 6 ) -1 2 (cm hr 600 0 k Surfactant Environmental Deactivated Fig.3. Apparent k600 for gas invasion and evasion for The bacteria-specific controls. YO-Inv KO-Inv Killed-Inv KO-Ev OB3B-Inv Killed-Ev 9232-Inv OB3B-Ev O4-Ev 9232-Ev Surfactant Ev Surfactant Inv Community-Inv Environmental-Ev Community-Ev Environmental-Inv Fig 2. k600 for invasion and evasion in the laboratory gas exchange tank estimated from changes in water phase gas partial pressures with time. Bacterial monocultures (9232, O4, YO, KO) were added separately and as a mixed community. Due to baffle motor breakdown the two “community” experiments ran at a lower degree of turbulence than the others. k600 is the equivalent gas transfer velocity for a Schmidt number of 600. 8 7 7 6 6 5 5 k600 (cm hr-1) 4 4 3 3 2 2 1 1 0 0 9232-Inv KO-Inv O4-Inv YO-Inv Community-Inv 9232-Ev KO-Ev O4-Ev Community-Ev Invasive Experiments Evasive Experiments Bacterioneuston control of air-water CH4 exchange determined with a laboratory gas exchange tank. Rob Upstill-Goddard, Tom Frost, & Gordon Henry: School of Marine Science & Technology, Univ. of Newcastle NE1 7RU, UK; Colin Murrell & Mark Franklin: School ofBiological Sciences, Univ. of Warwick, Coventry CV4 7AL UK; Nick Owens: Plymouth Marine Laboratory, Plymouth PL1 3DH, UK University of Newcastle Introduction The rate-limiting step in gas exchange is slow molecular transport across the “surface microlayer”, a region only tens of microns thick yet chemically and microbiologically distinct from the underlying water [1]. Bacterial enrichment of the microlayer (the “Bacterioneuston”) is well-known [2,3] and evidence implies associated modifications to sea-air exchange [2,4,5]. Even so, the microlayer remains under-represented in global change science. We measured invasive and evasive exchange of CH4, N2O and SF6 in a 1.0 x 0.5 x 0.5 m closed laboratory exchange tankat varying turbulence levels and calculated “apparent gas transfer velocities” for both exchange directions. We focused on potential bacterioneuston control of CH4 exchange using live or deactivated methanotrophs or bacterial cultures devoid of methane mono-oxygenase (MMO) activity, and bacterially-associated surfactant. N2O was used as a reactive gas unaffected by methanotrophy and SF6 acted as an inert control [6]. The Bacterioneuston - Fact or Fiction ? In accompanying work [7] we collected North Sea microlayer (top 30-40 mm) samples on polycarbonate membranes and subsurface (1m) samples in carbuoys. Samples were DNA extracted and used to construct clone libraries using bacterioneuston and pelagic DNA as templates with polymerase chain reaction (PCR) primers for bacterial 16S rRNA genes and pmoA (specific for the active site of particulate CH4 monooxygenase in all methanotrophs). Samples were also challenged with PCR primers specific for mmoX (soluble CH4 monooxygenase active site) and mxaF (encoding methanol dehydrogenase, a key enzyme in CH4 oxidation). We cloned the PCR products, compiled clone libraries and sorted data into operational taxonomic units (OTU’s). Representatives of each group were DNA sequenced. 16S rRNA gene sequences were very different for the two samples (Fig. 1). For subsurface water, of the 120 clones analysed OTU groups each containing ~ 4-6% of the library were observed for Roseobacter, Rhodobacter, Cytophaga, Oceanospirillum, Colwellia and Polaribacterium. Genera such as Bacteroides, Flexibacter, Shewanella, Leucothrix, Pseudomonas, Janthinobacter, and Cryptomonas each accounted for ~ 1-3% of the library (Fig. 1). In contrast, 74% of 16S rRNA clonesin the bacterioneuston library were from Vibrio spp, with 22% from Pseudoalteromonas spp. The rest comprised 7 OTU groups, each one Most importantly, in contrast to the pure methanotroph strains none of the controls showed any enhancement of apparent k600-CH4 relative to K600-N2O or K600-SF6, hence Figs. 2 and 3 are consistent with the notion of active control of k600-CH4 by added methanotrophs. Fig. 4. quantifies the amount by which apparent k600-CH4 was enhanced by methanotrophy. For a situation where CH4 is inert k600-CH4 / k600-SF6 =1. Results for all evasive bacterial enrichments are significantly different from the control results, whereas for invasive experiments Table 1. Methanotroph species used in the gas exchange experiments. Strain Habitat Species Source / Reference 9232 Marine Methylococcus Donated by Dr K. Nanba, Univ. Tokyo O4 Marine Methylobacter Donated by Dr K. Nanba, Univ. Tokyo YO Marine Methylomicrobium Fuse et al. (1998) [8] KO Marine Methylomicrobium Fuse et al. (1998) [8] Communitya Marine marine methanotrophs OB3B Freshwater Methylosinus trichosporium Whittenbury et al. (1970) [9]. a Manufactured from an equal part mixture of 9232,O4, Y4 and KO Strain YO deactivated with HgCl2 was used to examine possible modification of gas exchange by inactive methanotrophs. The effect of live bacterial cells devoid of MMO was investigated with Vibrio enriched North Sea water (subsequently referred to as “environmental”). Bacterial additions were 12 hours prior to each experiment to allow for acclimatization. Tank water surface tension fell by 80-85% in the first 2-3 hours after bacterial additions, consistent with a migration of bacterial components to the tank microlayer. The role of bacterially associated surfactant was examined using N-acetyl-D-glucosamine, a naturally occurring bacterial surfactant, at natural microlayer C concentrations [10]. Experimental Results Fig. 2 shows “apparent k600”for experiments with ASW and added methanotrophs. There are several features. First, for individual strains in many cases, apparent k600 forinvasion exceeds apparent k600for evasion. Second, for both invasion and evasion in general, apparent k600 differs between individual strains. Third, for all individual invasive experiments and for “community” (see Table 1) evasion, apparent k600 is statistically identical for all three gases. Fourth, for all pure methanotroph strains during gas evasion, apparent k600 for N2O and SF6 was identical whereas apparent k600for CH4 was always significantly higher (Fig. 2). only the results for the KO experiment differed significantly from the controls. By contrast, k600-N2O / k600-SF6 (not shown) never differed significantly from unity. Discussion and Conclusions For active methanotrophy in the tank microlayer during bacterial enrichments the true CH4 concentration gradient at the air-water interface would exceed that derived from tank water samples collected below the microlayer, hence apparent k600-CH4 would be expected to exceed k600-SF6.Although the evasive bacterial enrichments are consistent with this reasoning the invasive results are not (Fig. 4), however the apparent paradox is a consequence of experimental measurement constraints. For apparent k600-CH4 estimated from tank water concentrations, the additional Evasion Invasion k-CH4 / k-SF6 1 Fig. 4. k600-CH4 / k600-SF6 for gas invasion and evasion. Bacterial enrichment experiments are shown in blue and bacterial control experiments in red. containing 2% or less of clones. Why such comparatively low diversity was observed for the bacterioneuston is unclear. Could the neuston genera be more UV resistant?, or do they form a polysaccharide or cellulose matrix to create a surface film binding them together? What does seem clear is that the bacterioneuston is indeed a real phenomenon. We constructed a data base of pmoA and 16rRNA sequences from Invasive flux due to microbial consumption would not be observable in the bulk water due to its utilisation in the microlayer. Our estimates of k600-CH4 / k600-SF6 are ~ 1.12  0.10 (range 1.04-1.27). From preliminary calculations the rates of CH4 oxidation required to support these estimates are environmentally realistic [6]. Recent studies incorporating data from estuaries and shallow CH4 seeps speculate that the marine source of atmospheric CH4 could be as high as 30-50 Tg yr-1 [13-15]. Based on these data and our work described here consumption by the bacterioneuston might account for 6 Tg CH4 yr-1 globally. Coastal regions have high productivity and high bacterial numbers; hence the associated bacterioneuston might be especially well developed. Combined with high ambient levels of dissolved trace gases, this could make the coastal zone particularly important for trace gas processing by the bacterioneuston. Acknowledgements: We thank NERC (grant GR3/11144) & the Newcastle Research Committee for funding. References [1] GESAMP. The sea surface microlayer and its role in global climate change, GESAMP, 1995. [2] Hardy, J.T. Limnol. Oceanogr. 18, 525-533, 1973. [3] Kjelleberg, S. & N. Hakansson. Mar. Biol. 39, 103-109, 1977. [4] Conrad, R. & W. Seiler. J. Atmos. Chem. 6, 83-94, 1988. [5] Garabetian, F. et al. Mar. Env. Res.35, 323-339, 1993. [6] Upstill-Goddard, R.C. et al. Glob Biogeochem. Cyc.17, 1108, doi:10.1029/2003GB002043, 2003. [7] Murrell, J.C. et al. Env. Microbiol. (in Press), 2004. [8] Fuse, H. et al. Biosci. Biotech., Biochem. 62, 1925-1931, 1998. [9] Whittenbury, R. et al. J. Gen. Microbiol. 61, 205-218, 1970. [10] Hunter, K.A & P.S. Liss. Organic sea surface films in Marine Organic Chemistry. Elsevier, 1981. [11] Frew, N.M. et al. J. Geophys. Res., 95, 3337-3351. [12] Wallace, D.W.R. & C.D. Wirrick. Nature 356, 694-696, 1992. [13] Bange, H.W. et al. Glob Biogeochem. Cyc.8, 465-480, 1994. [14] Upstill-Goddard, R.C. et al. Glob Biogeochem. Cyc.14, 1205-1217, 2000 [15] Kvenvolden, K.A. et al. Eos Trans. AGU, 82,457, 2001. Pseudoaltero- monas spp Vibrio spp. A B Fig. 1. The diversity of (A), the subsurface bacterial community and (B), the Bacterioneuston community. The bacterioneuston is dominated by Vibrio spp. and Pseudoalteromonas spp Observed contrasts in apparent k600 must reflect intrinsic bacterial properties because other experimental variables were constant (excepting turbulence in the “community” experiments, Fig. 2.). Modification of apparent k600 for N2O or SF6 by methanotroph uptake seems unlikely but k600 suppression by microbial surfactant release [11] might contribute to these differences. The most important observations however are the consistently elevated values of apparent k600-CH4 for pure methanotroph strains during gas evasion, which provides some evidence for gas transfer control by the bacterioneuston. Why are such effects not seen for the evasive “community” experiments or during gas invasion? For the “community” this could reflect microbial competition, but this must remain speculative without further investigation. Fig. 3 shows results from bacteria-specific controls. For the “environmental” experiments prior replacement of the baffle motor in part accounts for the higher apparent k600 observed. For each experiment individually, the three apparent k600 estimates are identical within experimental error and elevated k600-CH4 seen for gas evasion with added methanotrophs (Fig. 2) was not observed. However, for all three controls apparent k600 for invasion > apparent k600 for evasion. Similar results were seen for some methanotroph additions (Fig. 2). Invasive enhancements for all three gases imply some physical gas exchange control, perhaps due to periodic bubble formation [12], as observed during our experiments. marine methanotrophs to isolate strains for use in the exchange tank. We set up marine methanotroph enrichments and analysed extant marine methanotrophs from several culture collections. Molecular and PCR analyses showed the marine and culture collections to be dominated by Methylomicrobium pelagicum, and all pmoA sequences retrieved from bacterioneuston and pelagic samples were >98% identical to pmoA from extant Mb. pelagicum sequences. The marine enrichment cultures were used in the tank experiments. Gas Exchange Experiments Gas partial pressures in tank water (Milli-RO or artificial seawater, ASW) or headspace were modified to force invasive (air-to-water) or evasive (water-to-air) gas transfer. A variable speed baffle created turbulence. Rigorous cleaning protocols precluded residual biological contamination. Experiments were as follows: ASW and Milli-RO unamended; ASW and Milli-RO amended with active methanotrophs; ASW and Milli-RO amended with inactive methanotrophs; ASW and Milli-RO amended with livebacterial cells (Vibrio) devoid of methane mono-oxygenase (MMO); ASW and Milli-RO with added surfactant. We used one freshwater and four marine methanotroph strains (Table 1), and one strain of Vibrio.