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 V_max of up to 29 nmol l⁻¹ day⁻¹, with a high affinity K_m constant of 9.3 n in more productive coastal waters. We report surface concentrations of methanol in the western English channel of 97±8 n (n=4) between May and June 2010, and for the wider temperate North Atlantic waters of 70±13 n (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.     

Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere

Anaerobic oxidation of ammonium (anammox) is recognized as an important process for nitrogen (N) cycling, yet its role in agricultural ecosystems, which are intensively fertilized, remains unclear. In this study, we investigated the presence, activity, functional gene abundance and role of anammox bacteria in rhizosphere and non-rhizosphere paddy soils using catalyzed reporter deposition–fluorescence in situ hybridization, isotope-tracing technique, quantitative PCR assay and 16S rRNA gene clone libraries. Results showed that rhizosphere anammox contributed to 31–41% N₂ production with activities of 0.33–0.64 nmol N₂ g⁻¹ soil h⁻¹, whereas the non-rhizosphere anammox bacteria contributed to only 2–3% N₂ production with lower activities of 0.08–0.26 nmol N₂ g⁻¹ soil h⁻¹. Higher anammox bacterial cells were observed (0.75–1.4 × 10⁷ copies g⁻¹ soil) in the rhizosphere, which were twofold higher compared with the non-rhizosphere soil (3.7–5.9 × 10⁶ copies g⁻¹ soil). Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated that two genera of ‘Candidatus Kuenenia'' and ‘Candidatus Brocadia'' and the family of Planctomycetaceae were identified. We suggest the rhizosphere provides a favorable niche for anammox bacteria, which are important to N cycling, but were previously largely overlooked.     

Production of lactic acid using a new homofermentative Enterococcus faecalis isolate

Lactic acid is an intermediate-volume specialty chemical for a wide range of food and industrial applications such as pharmaceuticals, cosmetics and chemical syntheses. Although lactic acid production has been well documented, improved production parameters that lead to reduced production costs are always of interest in industrial developments. In this study, we describe the production of lactic acid at high concentration, yield and volumetric productivity utilizing a novel homofermentative, facultative anaerobe Enterococcus faecalis CBRD01. The highest concentration of 182 g lactic acid l⁻¹ was achieved after 38 h of fed-batch fermentation on glucose. The bacterial isolate utilized only 2–13% of carbon for its growth and energy metabolism, while 87–98% of carbon was converted to lactic acid at an overall volumetric productivity of 5 g l⁻¹ h⁻¹. At 13 h of fermentation, the volumetric productivity of lactate production reached 10.3 g l⁻¹ h⁻¹, which is the highest ever reported for microbial production of lactic acid. The lactic acid produced was of high purity as formation of other metabolites was less than 0.1%. The present investigation demonstrates a new opportunity for enhanced production of lactic acid with potential for reduced purification costs.     

Depleted dissolved organic carbon and distinct bacterial communities in the water column of a rapid-flushing coral reef ecosystem

Coral reefs are highly productive ecosystems bathed in unproductive, low-nutrient oceanic waters, where microbially dominated food webs are supported largely by bacterioplankton recycling of dissolved compounds. Despite evidence that benthic reef organisms efficiently scavenge particulate organic matter and inorganic nutrients from advected oceanic waters, our understanding of the role of bacterioplankton and dissolved organic matter (DOM) in the interaction between reefs and the surrounding ocean remains limited. In this study, we present the results of a 4-year study conducted in a well-characterized coral reef ecosystem (Paopao Bay, Moorea, French Polynesia) where changes in bacterioplankton abundance and dissolved organic carbon (DOC) concentrations were quantified and bacterial community structure variation was examined along spatial gradients of the reef:ocean interface. Our results illustrate that the reef is consistently depleted in concentrations of both DOC and bacterioplankton relative to offshore waters (averaging 79 μmol l⁻¹ DOC and 5.5 × 10⁸ cells l⁻¹ offshore and 68 μmol l⁻¹ DOC and 3.1 × 10⁸ cells l⁻¹ over the reef, respectively) across a 4-year time period. In addition, using a suite of culture-independent measures of bacterial community structure, we found consistent differentiation of reef bacterioplankton communities from those offshore or in a nearby embayment across all taxonomic levels. Reef habitats were enriched in Gamma-, Delta-, and Betaproteobacteria, Bacteriodetes, Actinobacteria and Firmicutes. Specific bacterial phylotypes, including members of the SAR11, SAR116, Flavobacteria, and Synechococcus clades, exhibited clear gradients in relative abundance among nearshore habitats. Our observations indicate that this reef system removes oceanic DOC and exerts selective pressures on bacterioplankton community structure on timescales approximating reef water residence times, observations which are notable both because fringing reefs do not exhibit long residence times (unlike those characteristic of atoll lagoons) and because oceanic DOC is generally recalcitrant to degradation by ambient microbial assemblages. Our findings thus have interesting implications for the role of oceanic DOM and bacterioplankton in the ecology and metabolism of reef ecosystems.     

Macrofauna regulate heterotrophic bacterial carbon and nitrogen incorporation in low-oxygen sediments

Oxygen minimum zones (OMZs) currently impinge upon >1 million km² of sea floor and are predicted to expand with climate change. We investigated how changes in oxygen availability, macrofaunal biomass and retention of labile organic matter (OM) regulate heterotrophic bacterial C and N incorporation in the sediments of the OMZ-impacted Indian continental margin (540–1100 m; [O₂]=0.35–15 μmol l⁻¹). In situ pulse-chase experiments traced ¹³C:¹⁵N-labelled phytodetritus into bulk sediment OM and hydrolysable amino acids, including the bacterial biomarker 𝒟-alanine. Where oxygen availability was lowest ([O₂]=0.35 μmol l⁻¹), metazoan macrofauna were absent and bacteria assimilated 30–90% of the labelled phytodetritus within the sediment. At higher oxygen levels ([O₂]=2–15 μmol l⁻¹) the macrofaunal presence and lower phytodetritus retention with the sediment occur concomitantly, and bacterial phytodetrital incorporation was reduced and retarded. Bacterial C and N incorporation exhibited a significant negative relationship with macrofaunal biomass across the OMZ. We hypothesise that fauna–bacterial interactions significantly influence OM recycling in low-oxygen sediments and need to be considered when assessing the consequences of global change on biogeochemical cycles.     

Significance of archaeal nitrification in hypoxic waters of the Baltic Sea

Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread, and their abundance in many terrestrial and aquatic ecosystems suggests a prominent role in nitrification. AOA also occur in high numbers in oxygen-deficient marine environments, such as the pelagic redox gradients of the central Baltic Sea; however, data on archaeal nitrification rates are scarce and little is known about the factors, for example sulfide, that regulate nitrification in this system. In the present work, we assessed the contribution of AOA to ammonia oxidation rates in Baltic deep basins and elucidated the impact of sulfide on this process. Rate measurements with ¹⁵N-labeled ammonium, CO₂ dark fixation measurements and quantification of AOA by catalyzed reporter deposition–fluorescence in situ hybridization revealed that among the three investigated sites the highest potential nitrification rates (122–884 nmol l⁻¹per day) were measured within gradients of decreasing oxygen, where thaumarchaeotal abundance was maximal (2.5–6.9 × 10⁵ cells per ml) and CO₂ fixation elevated. In the presence of the archaeal-specific inhibitor GC₇, nitrification was reduced by 86–100%, confirming the assumed dominance of AOA in this process. In samples spiked with sulfide at concentrations similar to those of in situ conditions, nitrification activity was inhibited but persisted at reduced rates. This result together with the substantial nitrification potential detected in sulfidic waters suggests the tolerance of AOA to periodic mixing of anoxic and sulfidic waters. It begs the question of whether the globally distributed Thaumarchaeota respond similarly in other stratified water columns or whether the observed robustness against sulfide is a specific feature of the thaumarchaeotal subcluster present in the Baltic Deeps.     

Viral infections stimulate the metabolism and shape prokaryotic assemblages in submarine mud volcanoes

Mud volcanoes are geological structures in the oceans that have key roles in the functioning of the global ecosystem. Information on the dynamics of benthic viruses and their interactions with prokaryotes in mud volcano ecosystems is still completely lacking. We investigated the impact of viral infection on the mortality and assemblage structure of benthic prokaryotes of five mud volcanoes in the Mediterranean Sea. Mud volcano sediments promote high rates of viral production (1.65–7.89 × 10⁹ viruses g⁻¹ d⁻¹), viral-induced prokaryotic mortality (VIPM) (33% cells killed per day) and heterotrophic prokaryotic production (3.0–8.3 μgC g⁻¹ d⁻¹) when compared with sediments outside the mud volcano area. The viral shunt (that is, the microbial biomass converted into dissolved organic matter as a result of viral infection, and thus diverted away from higher trophic levels) provides 49 mgC m⁻² d⁻¹, thus fuelling the metabolism of uninfected prokaryotes and contributing to the total C budget. Bacteria are the dominant components of prokaryotic assemblages in surface sediments of mud volcanoes, whereas archaea dominate the subsurface sediment layers. Multivariate multiple regression analyses show that prokaryotic assemblage composition is not only dependant on the geochemical features and processes of mud volcano ecosystems but also on synergistic interactions between bottom-up (that is, trophic resources) and top-down (that is, VIPM) controlling factors. Overall, these findings highlight the significant role of the viral shunt in sustaining the metabolism of prokaryotes and shaping their assemblage structure in mud volcano sediments, and they provide new clues for our understanding of the functioning of cold-seep ecosystems.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Global abundance of planktonic heterotrophic protists in the deep ocean

The dark ocean is one of the largest biomes on Earth, with critical roles in organic matter remineralization and global carbon sequestration. Despite its recognized importance, little is known about some key microbial players, such as the community of heterotrophic protists (HP), which are likely the main consumers of prokaryotic biomass. To investigate this microbial component at a global scale, we determined their abundance and biomass in deepwater column samples from the Malaspina 2010 circumnavigation using a combination of epifluorescence microscopy and flow cytometry. HP were ubiquitously found at all depths investigated down to 4000 m. HP abundances decreased with depth, from an average of 72±19 cells ml⁻¹ in mesopelagic waters down to 11±1 cells ml⁻¹ in bathypelagic waters, whereas their total biomass decreased from 280±46 to 50±14 pg C ml⁻¹. The parameters that better explained the variance of HP abundance were depth and prokaryote abundance, and to lesser extent oxygen concentration. The generally good correlation with prokaryotic abundance suggested active grazing of HP on prokaryotes. On a finer scale, the prokaryote:HP abundance ratio varied at a regional scale, and sites with the highest ratios exhibited a larger contribution of fungi molecular signal. Our study is a step forward towards determining the relationship between HP and their environment, unveiling their importance as players in the dark ocean''s microbial food web.     

Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains

Methanol is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. In the present study, the methylotrophic and thermophilic bacterium Bacillus methanolicus was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg l⁻¹ during shake flask conditions. A volume-corrected concentration of 11.3 g l⁻¹ of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and Corynebacterium glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol.     

High Abundances of Aerobic Anoxygenic Photosynthetic Bacteria in the South Pacific Ocean

Little is known about the abundance, distribution, and ecology of aerobic anoxygenic phototrophic (AAP) bacteria, particularly in oligotrophic environments, which represent 60% of the ocean. We investigated the abundance of AAP bacteria across the South Pacific Ocean, including the center of the gyre, the most oligotrophic water body of the world ocean. AAP bacteria, Prochlorococcus, and total prokaryotic abundances, as well as bacteriochlorophyll a (BChl a) and divinyl-chlorophyll a concentrations, were measured at several depths in the photic zone along a gradient of oligotrophic conditions. The abundances of AAP bacteria and Prochlorococcus were high, together accounting for up to 58% of the total prokaryotic community. The abundance of AAP bacteria alone was up to 1.94 × 10⁵ cells ml⁻¹ and as high as 24% of the overall community. These measurements were consistent with the high BChl a concentrations (up to 3.32 × 10⁻³ μg liter⁻¹) found at all stations. However, the BChl a content per AAP bacterial cell was low, suggesting that AAP bacteria are mostly heterotrophic organisms. Interestingly, the biovolume and therefore biomass of AAP bacteria was on average twofold higher than that of other prokaryotic cells. This study demonstrates that AAP bacteria can be abundant in various oligotrophic conditions, including the most oligotrophic regime of the world ocean, and can account for a large part of the bacterioplanktonic carbon stock.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

High molecular weight dissolved organic matter enrichment selects for methylotrophs in dilution to extinction cultures

The role of bacterioplankton in the cycling of marine dissolved organic matter (DOM) is central to the carbon and energy balance in the ocean, yet there are few model organisms available to investigate the genes, metabolic pathways, and biochemical mechanisms involved in the degradation of this globally important carbon pool. To obtain microbial isolates capable of degrading semi-labile DOM for growth, we conducted dilution to extinction cultivation experiments using seawater enriched with high molecular weight (HMW) DOM. In total, 93 isolates were obtained. Amendments using HMW DOM to increase the dissolved organic carbon concentration 4x (280 μM) or 10x (700 μM) the ocean surface water concentrations yielded positive growth in 4–6% of replicate dilutions, whereas <1% scored positive for growth in non-DOM-amended controls. The majority (71%) of isolates displayed a distinct increase in cell yields when grown in increasing concentrations of HMW DOM. Whole-genome sequencing was used to screen the culture collection for purity and to determine the phylogenetic identity of the isolates. Eleven percent of the isolates belonged to the gammaproteobacteria including Alteromonadales (the SAR92 clade) and Vibrio. Surprisingly, 85% of isolates belonged to the methylotrophic OM43 clade of betaproteobacteria, bacteria thought to metabolically specialize in degrading C1 compounds. Growth of these isolates on methanol confirmed their methylotrophic phenotype. Our results indicate that dilution to extinction cultivation enriched with natural sources of organic substrates has a potential to reveal the previously unsuspected relationships between naturally occurring organic nutrients and the microorganisms that consume them.