Joanna L. Dixon

Microbial methanol uptake in northeast Atlantic waters

Methanol is the predominant oxygenated volatile organic compound in the troposphere, where it can significantly influence the oxidising capacity of the atmosphere. However, we do not understand which processes control oceanic concentrations, and hence, whether the oceans are a source or a sink to the atmosphere. We report the first methanol loss rates in seawater by demonstrating that 14C-labelled methanol can be used to determine microbial uptake into particulate biomass, and oxidation to 14CO2. We have found that methanol is used predominantly as a microbial energy source, but also demonstrated its use as a carbon source. We report biological methanol oxidation rates between 2.1 and 8.4 nmol l−1 day−1 in surface seawater of the northeast Atlantic. Kinetic experiments predict a Vmax of up to 29 nmol l−1 day−1, with a high affinity Km constant of 9.3 nM in more productive coastal waters. We report surface concentrations of methanol in the western English channel of 97±8 nM (n=4) between May and June 2010, and for the wider temperate North Atlantic waters of 70±13 nM (n=6). The biological turnover time of methanol has been estimated between 7 and 33 days, although kinetic experiments suggest a 7-day turnover in more productive shelf waters. Methanol uptake rates into microbial particles significantly correlated with bacterial and phytoplankton parameters, suggesting that it could be used as a carbon source by some bacteria and possibly some mixotrophic eukaryotes. Our results provide the first methanol loss rates from seawater, which will improve the understanding of the global methanol budget.

XoxF encoding an alternative methanol dehydrogenase is widespread in coastal marine environments

The xoxF gene, encoding a pyrroloquinoline quinone-dependent methanol dehydrogenase, is found in all known proteobacterial methylotrophs. In several newly discovered methylotrophs, XoxF is the active methanol dehydrogenase, catalysing the oxidation of methanol to formaldehyde. Apart from that, its potential role in methylotrophy and carbon cycling is unknown. So far, the diversity of xoxF in the environment has received little attention. We designed PCR primer sets targeting clades of the xoxF gene, and used 454 pyrosequencing of PCR amplicons obtained from the DNA of four coastal marine environments for a unique assessment of the diversity of xoxF in these habitats. Phylogenetic analysis of the data obtained revealed a high diversity of xoxF genes from two of the investigated clades, and substantial differences in sequence composition between environments. Sequences were classified as being related to a wide range of both methylotrophs and non-methylotrophs from Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. The most prominent sequences detected were related to the family Rhodobacteraceae, the genus Methylotenera and the OM43 clade of Methylophilales, and are thus related to organisms that employ XoxF for methanol oxidation. Furthermore, our analyses revealed a high degree of so far undescribed sequences, suggesting a high number of unknown bacterial species in these habitats.

Quantification of oxygenated volatile organic compounds in seawater by membrane inlet-proton transfer reaction/mass spectrometry

The role of the ocean in the cycling of oxygenated volatile organic compounds (OVOCs) remains largely unanswered due to a paucity of datasets. We describe the method development of a membrane inlet-proton transfer reaction/mass spectrometer (MI-PTR/MS) as an efficient method of analysing methanol, acetaldehyde and acetone in seawater. Validation of the technique with water standards shows that the optimised responses are linear and reproducible. Limits of detection are 27 nM for methanol, 0.7 nM for acetaldehyde and 0.3 nM for acetone. Acetone and acetaldehyde concentrations generated by MI-PTR/MS are compared to a second, independent method based on purge and trap-gas chromatography/flame ionisation detection (P&T-GC/FID) and show excellent agreement. Chromatographic separation of isomeric species acetone and propanal permits correction to mass 59 signal generated by the PTR/MS and overcomes a known uncertainty in reporting acetone concentrations via mass spectrometry. A third bioassay technique using radiolabelled acetone further supported the result generated by this method. We present the development and optimisation of the MI-PTR/MS technique as a reliable and convenient tool for analysing seawater samples for these trace gases. We compare this method with other analytical techniques and discuss its potential use in improving the current understanding of the cycling of oceanic OVOCs.

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined deoxyribonucleic acid-stable isotope probing (DNA-SIP) with metagenomics and metaproteomics to characterize an uncultivated marine methylotroph that actively incorporated carbon from (13) C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which the culture-independent techniques of DNA-SIP and protein-SIP have been used to characterize the metabolism of a naturally occurring Methylophaga-like bacterium in the marine environment (i.e. Methylophaga thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment.

Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean

Methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. We report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d−1 (∼10 nmol l−1 d−1). On average, coastal upwelling waters were 11 times greater than open ocean northern temperate (NT) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (EU) waters; suggesting that all upwelling waters upon reaching the surface (⩽20 m), contain a microbial population that uses a relatively high amount of carbon (0.3–10 nmol l−1 d−1), derived from methanol, to support their growth. In open ocean Atlantic regions, microbial uptake of methanol into biomass was significantly lower, ranging between 0.04–0.68 nmol l−1 d−1. Microbes in the Mauritanian coastal upwelling used up to 57% of the total methanol for assimilation of the carbon into cells, compared with an average of 12% in the EU, and 1% in NT and gyre waters. Several methylotrophic bacterial species were identified from open ocean Atlantic waters using PCR amplification of mxaF encoding methanol dehydrogenase, the key enzyme in bacterial methanol oxidation. These included Methylophaga sp., Burkholderiales sp., Methylococcaceae sp., Ancylobacter aquaticus, Paracoccus denitrificans, Methylophilus methylotrophus, Methylobacterium oryzae, Hyphomicrobium sp. and Methylosulfonomonas methylovora. Statistically significant correlations for upwelling waters between methanol uptake into cells and both chlorophyll a concentrations and methanol oxidation rates suggest that remotely sensed chlorophyll a images, in these productive areas, could be used to derive total methanol biological loss rates, a useful tool for atmospheric and marine climatically active gas modellers, and air–sea exchange scientists.

The effect of water depth on bacterial numbers, thymidine incorporation rates and C:N ratios in northeast Atlantic surficial sediments

The effect of water depth on bacterial biomass and their ability to synthesise DNA, by measuring their rate of [3H]-thymidine incorporation, was investigated in the northeast Atlantic at three sites of varying water depth (1100–3580 m) and sediment characteristics. Thymidine incorporation rates (y) in surficial sediments varied between 0.028 and 1.44 pmol h−1 g−1 and showed an exponential relationship with depth (x) according to the equation y= 2.05e−0.0011x (r=0.9830 for n=7, P<0.001). However, this relationship failed when a layer of phytodetritus was found overlying the surface sediment and [3H]-thymidine incorporation rates increased by 80–339%. In contrast, bacterial numbers varied between 1.09 and 11.96 × 108 cells g−1 (dry weight) and showed no significant relationships with water depth or sediment POC/TN content. Significant exponential relationships were also found between water depth (x) and the POC (y1) and total nitrogen (TN, y2) content of surficial sediments according to the following equations: where y1 = 719e−0.0003x (r=0.8700 for n=9, P<0.01) and y2 = 76e−0.0002x(r=0.7582 for n=9 P<0.02). These relationships were irrespective of the presence or absence of an overlying layer of phytodetritus. This suggests that the POC and TN content of these surficial deep sea sediments is directly related to the flux of material through the water column, which significantly impacts bacterial production.

Macro and micro nutrient limitation of microbial productivity in oligotrophic subtropical Atlantic waters

Environmental context. The subtropical oceans comprise ~70% of the world’s ocean surface and profoundly affect global biogeochemistry and climate. They are characteristically low-nutrient regions, but, owing to their large extent and often rapid nutrient turnover, may contribute to greater than 30% of the total marine primary production. However, there remains long-standing uncertainty as to what individual or combination of resources, e.g. macro (N, P) and micro (trace metals) nutrients, limit or co-limit marine productivity and thus total carbon fixation in these spatially dominant gyre systems. Abstract. The subtropical oceans are characteristically low-nutrient low-chlorophyll regions, but owing to their geographical dominance and rapid nutrient cycling may contribute >30% of the total marine primary production. The present study investigates the addition of P, Fe, Co and Zn on rates of primary production and heterotrophic bacterial production, through a combination of mesoscale in situ (P, and P + Fe) and in vitro (Co or Zn) bioassay incubation experiments. Results from the bioassay incubation experiments suggest that primary production and chlorophyll a biomass are limited by N and P in this oligotrophic region. However, both were increased further after addition of trace metal micronutrients in the order Fe + Zn ≥ Fe + Co > Fe ≈ Co. In contrast, rates of heterotrophic bacterial production did not appear to be P, or significantly, P + Fe limited, although in situ rates did increase during the first 12 h of mesoscale P fertilisation (which were not mirrored in the mesoscale P + Fe addition). The addition of Co to unfertilised waters increased heterotrophic bacterial production and the numbers of heterotrophic bacteria, Prochlorococcus spp. and Synechococcus spp., suggesting Co limitation. Prochlorococcus spp. were the most abundant autotrophs. The highest increases in both heterotrophic and autotrophic carbon assimilation were shown after in vitro addition of either Co or Zn to mesoscale enriched P + Fe waters, suggesting multiple limitation of microbial growth rates in the subtropical oligotrophic north-east Atlantic.

Microbial acetone oxidation in coastal seawater

Acetone is an important oxygenated volatile organic compound (OVOC) in the troposphere where it influences the oxidizing capacity of the atmosphere. However, the air-sea flux is not well quantified, in part due to a lack of knowledge regarding which processes control oceanic concentrations, and, specifically whether microbial oxidation to CO2 represents a significant loss process. We demonstrate that 14C labeled acetone can be used to determine microbial oxidation to 14CO2. Linear microbial rates of acetone oxidation to CO2 were observed for between 0.75-3.5 h at a seasonally eutrophic coastal station located in the western English Channel (L4). A kinetic experiment in summer at station L4 gave a Vmax of 4.1 pmol L-1 h-1, with a Km constant of 54 pM. We then used this technique to obtain microbial acetone loss rates ranging between 1.2 and 42 pmol L-1 h-1.(monthly averages) over an annual cycle at L4, with maximum rates observed during winter months. The biological turnover time of acetone (in situ concentration divided by microbial oxidation rate) in surface waters varied from ~3 days in February 2011, when in situ concentrations were 3 ± 1 nM, to >240 days in June 2011, when concentrations were more than twofold higher at 7.5 ± 0.7 nM. These relatively low marine microbial acetone oxidation rates, when normalized to in situ concentrations, suggest that marine microbes preferentially utilize other OVOCs such as methanol and acetaldehyde.

Comparative Genomics and Mutational Analysis Reveals a Novel XoxF-Utilizing Methylotroph in the Roseobacter Group Isolated From the Marine Environment

The Roseobacter group comprises a significant group of marine bacteria which are involved in global carbon and sulfur cycles. Some members are methylotrophs, using one-carbon compounds as a carbon and energy source. It has recently been shown that methylotrophs generally require a rare earth element when using the methanol dehydrogenase enzyme XoxF for growth on methanol. Addition of lanthanum to methanol enrichments of coastal seawater facilitated the isolation of a novel methylotroph in the Roseobacter group: Marinibacterium anthonyi strain La 6. Mutation of xoxF5 revealed the essential nature of this gene during growth on methanol and ethanol. Physiological characterization demonstrated the metabolic versatility of this strain. Genome sequencing revealed that strain La 6 has the largest genome of all Roseobacter group members sequenced to date, at 7.18 Mbp. Multilocus sequence analysis (MLSA) showed that whilst it displays the highest core gene sequence similarity with subgroup 1 of the Roseobacter group, it shares very little of its pangenome, suggesting unique genetic adaptations. This research revealed that the addition of lanthanides to isolation procedures was key to cultivating novel XoxF-utilizing methylotrophs from the marine environment, whilst genome sequencing and MLSA provided insights into their potential genetic adaptations and relationship to the wider community.

Basin-scale variability of microbial methanol uptake in the Atlantic Ocean

Methanol is a climate-active gas and the most abundant oxygenated volatile organic compound (OVOC) in the atmosphere and seawater. Marine methylotrophs are aerobic bacteria that utilise methanol from seawater as a source of carbon (assimilation) and/or energy (dissimilation). A few spatially limited studies have previously reported methanol oxidation rates in seawater; however, the basin-wide ubiquity of marine microbial methanol utilisation remains unknown. This study uniquely combines seawater 14C labelled methanol tracer studies with 16S rRNA “pyrosequencing” to investigate variability in microbial methanol dissimilation and known methanol-utilising bacteria throughout a meridional transect of the Atlantic Ocean between 47 N to 39 S. Microbial methanol dissimilation varied between 0.05 and 1.68 nmol L−1 h−1 in the top 200 m of the Atlantic Ocean and showed significant variability between biogeochemical provinces. The highest rates of methanol dissimilation were found in the northern subtropical gyre (average 0.99± 0.41 nmol L−1 h−1), which were up to 8 times greater than other Atlantic regions. Microbial methanol dissimilation rates displayed a significant inverse correlation with heterotrophic bacterial production (determined using 3H-leucine). Despite significant depth stratification of bacterial communities, methanol dissimilation rates showed much greater variability between oceanic provinces compared to depth. There were no significant differences in rates between samples collected under light and dark environmental conditions. The variability in the numbers of SAR11 (16S rRNA gene sequences) were estimated to explain approximately 50 % of the changes in microbial methanol dissimilation rates. We estimate that SAR11 cells in the Atlantic Ocean account for between 0.3 % and 59 % of the rates of methanol dissimilation in Atlantic waters, compared to < 0.01 %–2.3 % for temperate coastal waters. These results make a substantial contribution to our current knowledge and understanding of the utilisation of methanol by marine microbial communities, but highlight the lack of understanding of in situ methanol production mechanisms.