Improved enrichment culture technique for methane-oxidizing bacteria from marine ecosystems: the effect of adhesion material and gas composition.

Cultivation of microbial representatives of specific functional guilds from environmental samples depends largely on the suitability of the applied growth conditions. Especially the cultivation of marine methanotrophs has received little attention, resulting in only a limited number of ex situ cultures available. In this study we investigated the effect of adhesion material and headspace composition on the methane oxidation activity in methanotrophic enrichments obtained from marine sediment. Addition of sterilized natural sediment or alternatively the addition of acid-washed silicon dioxide significantly increased methane oxidation. This positive effect was attributed to bacterial adhesion on the particles via extracellular compounds, with a minimum amount of particles required for effect. As a result, the particles were immobilized, thus creating a stratified environment in which a limited diffusive gas gradients could build up and various microniches were formed. Such diffusive gas gradient might necessitate high headspace concentrations of CH4 and CO2 for sufficient concentrations to reach the methane-oxidizing bacteria in the enrichment culture technique. Therefore, high concentrations of methane and carbon dioxide, in addition to the addition of adhesion material, were tested and indeed further stimulated methane oxidation. Use of adhesion material in combination with high concentrations of methane and carbon dioxide might thus facilitate the cultivation and subsequent enrichment of environmentally important members of this functional guild. The exact mechanism of the observed positive effects on methane oxidation and the differential effect on methanotrophic diversity still needs to be explored.

New Methyloceanibacter diversity from North Sea sediments includes methanotroph containing solely the soluble methane monooxygenase.

Marine methylotrophs play a key role in the global carbon cycle by metabolizing reduced one-carbon compounds that are found in high concentrations in marine environments. Genome, physiology and diversity studies have been greatly facilitated by the numerous model organisms brought into culture. However, the availability of marine representatives remains poor. Here, we report the isolation of four novel species from North Sea sediment enrichments closely related to the Alphaproteobacterium Methyloceanibacter caenitepidi. Each of the newly isolated Methyloceanibacter species exhibited a clear genome sequence divergence which was reflected in physiological differences. Notably one strain R-67174 was capable of oxidizing methane as sole source of carbon and energy using solely a soluble methane monooxygenase and represents the first marine Alphaproteobacterial methanotroph brought into culture. Differences in maximum cell density of >1.5 orders of magnitude were observed. Furthermore, three strains were capable of producing nitrous oxide from nitrate. Together, these findings highlight the metabolic and physiologic variability within closely related Methyloceanibacter species and provide a new understanding of the physiological basis of marine methylotrophy.

Draft Genome Sequences of Eight Obligate Methane Oxidizers Occupying Distinct Niches Based on Their Nitrogen Metabolism.

The genome sequences of Methylomonas methanica (NCIMB 11130(T), R-45363, and R-45371), Methylomonas koyamae (R-45378, R-45383, and R-49807), Methylomonas lenta (R-45370), and Methylosinus sp. (R-45379) were obtained. These aerobic methanotrophs were isolated from terrestrial ecosystems, and their distinct phenotypes related to nitrogen assimilation and dissimilation were previously reported.

Genome Characteristics of Two Novel Type I Methanotrophs Enriched from North Sea Sediments Containing Exclusively a Lanthanide-Dependent XoxF5-Type Methanol Dehydrogenase.

Microbial methane oxidizers play a crucial role in the oxidation of methane in marine ecosystems, as such preventing the escape of excessive methane to the atmosphere. Despite the important role of methanotrophs in marine ecosystems, only a limited number of isolates are described, with only four genomes available. Here, we report on two genomes of gammaproteobacterial methanotroph cultures, affiliated with the deep-sea cluster 2, obtained from North Sea sediment. Initial enrichments using methane as sole source of carbon and energy and mimicking the in situ conditions followed by serial subcultivations and multiple extinction culturing events over a period of 3 years resulted in a highly enriched culture. The draft genomes of the methane oxidizer in both cultures showed the presence of genes typically found in type I methanotrophs, including genes encoding particulate methane monooxygenase (pmoCAB), genes for tetrahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, and genes of the ribulose monophosphate (RuMP) pathway. The most distinctive feature, when compared to other available gammaproteobacterial genomes, is the absence of a calcium-dependent methanol dehydrogenase. Both genomes reported here only have a xoxF gene encoding a lanthanide-dependent XoxF5-type methanol dehydrogenase. Thus, these genomes offer novel insight in the genomic landscape of uncultured diversity of marine methanotrophs.

Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis.

BACKGROUND: Firmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. The recent renewed interest in their nitrogen metabolism has revealed many interesting features, the most striking being their wide variety of dissimilatory nitrate reduction pathways. In the present study, nitrous oxide production from Bacillus licheniformis, a ubiquitous Gram-positive, spore-forming species with many industrial applications, is investigated. RESULTS: B. licheniformis has long been considered a denitrifier but physiological experiments on three different strains demonstrated that nitrous oxide is not produced from nitrate in stoichiometric amounts, rather ammonium is the most important end-product, produced during fermentation. Significant strain dependency in end-product ratios, attributed to nitrite and ammonium, and medium dependency in nitrous oxide production were also observed. Genome analyses confirmed the lack of a nitrite reductase to nitric oxide, the key enzyme of denitrification. Based on the gene inventory and building on knowledge from other non-denitrifying nitrous oxide emitters, hypothetical pathways for nitrous oxide production, involving NarG, NirB, qNor and Hmp, are proposed. In addition, all publically available genomes of B. licheniformis demonstrated similar gene inventories, with specific duplications of the nar operon, narK and hmp genes as well as NarG phylogeny supporting the evolutionary separation of previously described distinct BALI1 and BALI2 lineages. CONCLUSIONS: Using physiological and genomic data we have demonstrated that the common soil bacterium B. licheniformis does not denitrify but is capable of fermentative dissimilatory nitrate/nitrite reduction to ammonium (DNRA) with concomitant production of N2O. Considering its ubiquitous nature and non-fastidious growth in the lab, B. licheniformis is a suitable candidate for further exploration of the actual mechanism of N2O production in DNRA bacteria and its relevance in situ.

Regulation of nitrogen metabolism in the nitrate-ammonifying soil bacterium Bacillus vireti and evidence for its ability to grow using N2 O as electron acceptor.

Bacillus vireti is a nitrate-ammonifying bacterium and a partial denitrifier, reducing NO3 (-) , NO2 (-) , NO and N2 O with NarG, NrfA, CbaA and NosZ respectively. Growth is optimized through successive use of the electron acceptors O2 and NO3 (-) , followed by NO2 (-) , NO and N2 O. Fermentation takes place simultaneously with anaerobic respiration. When grown in batch culture with 5 mM initial NO3 (-) , transcription of nrfA was high and most NO3 (-) was reduced to NH4 (+) . With 20 mM initial NO3 (-) , nrfA transcription was lower and more than 50% of the nitrate was recovered as NO, N2 O and N2 . Analysis of gene transcription patterns and corresponding gas kinetics indicated that O2 and NO2 (-) or NO are main controllers of nrfA, nirB, cbaA and nosZ transcription. This was corroborated by analyses of putative binding regions for specific transcriptional regulators. Furthermore, we demonstrate that N2 O reduction in B. vireti supports growth. The high nosZ transcription but low N2 O production seen at 5 mM NO3 (-) implies that this organism can use N2 O reductase to scavenge N2 O from other organisms in the soil, thus possibly acting as a net sink for N2 O.

Dissimilatory nitrogen reduction in intertidal sediments of a temperate estuary: small scale heterogeneity and novel nitrate-to-ammonium reducers.

The estuarine nitrogen cycle can be substantially altered due to anthropogenic activities resulting in increased amounts of inorganic nitrogen (mainly nitrate). In the past, denitrification was considered to be the main ecosystem process removing reactive nitrogen from the estuarine ecosystem. However, recent reports on the contribution of dissimilatory nitrate reduction to ammonium (DNRA) to nitrogen removal in these systems indicated a similar or higher importance, although the ratio between both processes remains ambiguous. Compared to denitrification, DNRA has been underexplored for the last decades and the key organisms carrying out the process in marine environments are largely unknown. Hence, as a first step to better understand the interplay between denitrification, DNRA and reduction of nitrate to nitrite in estuarine sediments, nitrogen reduction potentials were determined in sediments of the Paulina polder mudflat (Westerschelde estuary). We observed high variability in dominant nitrogen removing processes over a short distance (1.6 m), with nitrous oxide, ammonium and nitrite production rates differing significantly between all sampling sites. Denitrification occurred at all sites, DNRA was either the dominant process (two out of five sites) or absent, while nitrate reduction to nitrite was observed in most sites but never dominant. In addition, novel nitrate-to-ammonium reducers assigned to Thalassospira, Celeribacter, and Halomonas, for which DNRA was thus far unreported, were isolated, with DNRA phenotype reconfirmed through nrfA gene amplification. This study demonstrates high small scale heterogeneity among dissimilatory nitrate reduction processes in estuarine sediments and provides novel marine DNRA organisms that represent valuable alternatives to the current model organisms.

A doubling of microphytobenthos biomass coincides with a tenfold increase in denitrifier and total bacterial abundances in intertidal sediments of a temperate estuary.

Surface sediments are important systems for the removal of anthropogenically derived inorganic nitrogen in estuaries. They are often characterized by the presence of a microphytobenthos (MPB) biofilm, which can impact bacterial communities in underlying sediments for example by secretion of extracellular polymeric substances (EPS) and competition for nutrients (including nitrogen). Pyrosequencing and qPCR was performed on two intertidal surface sediments of the Westerschelde estuary characterized by a two-fold difference in MPB biomass but no difference in MPB composition. Doubling of MPB biomass was accompanied by a disproportionately (ten-fold) increase in total bacterial abundances while, unexpectedly, no difference in general community structure was observed, despite significantly lower bacterial richness and distinct community membership, mostly for non-abundant taxa. Denitrifier abundances corresponded likewise while community structure, both for nirS and nirK denitrifiers, remained unchanged, suggesting that competition with diatoms for nitrate is negligible at concentrations in the investigated sediments (appr. 1 mg/l NO3-). This study indicates that MPB biomass increase has a general, significantly positive effect on total bacterial and denitrifier abundances, with stimulation or inhibition of specific bacterial groups that however do not result in a re-structured community.

Exploring methane-oxidizing communities for the co-metabolic degradation of organic micropollutants.

Methane-oxidizing cultures from five different inocula were enriched to be used for co-metabolic degradation of micropollutants. In a first screening, 18 different compounds were tested for degradation with the cultures as well as with four pure methane-oxidizing bacterial (MOB) strains. The tested compounds included pharmaceuticals, chemical additives, pesticides, and their degradation products. All enriched cultures were successful in the degradation of at least four different pollutants, but the compounds degraded most often were sulfamethoxazole (SMX) and benzotriazole (BTZ). Addition of acetylene, a specific methane monooxygenase (MMO) inhibitor, revealed that SMX and BTZ were mainly degraded co-metabolically by the present MOB. The pure MOB cultures exhibited less degradation potential, while SMX and BTZ were also degraded by three of the four tested pure strains. For MOB, copper (Cu(2+)) concentration is often an important factor, as several species have the ability to express a soluble MMO (sMMO) if the Cu(2+) concentration is low. In literature, this enzyme is often described to have a broader compound range for co-metabolic degradation of pollutants, in particular when it comes to aromatic structures. However, this study indicated that co-metabolic degradation of the aromatic compounds SMX and BTZ was possible at high Cu(2+) concentration, most probably catalyzed by pMMO.

Interactions between benthic copepods, bacteria and diatoms promote nitrogen retention in intertidal marine sediments.

The present study aims at evaluating the impact of diatoms and copepods on microbial processes mediating nitrate removal in fine-grained intertidal sediments. More specifically, we studied the interactions between copepods, diatoms and bacteria in relation to their effects on nitrate reduction and denitrification. Microcosms containing defaunated marine sediments were subjected to different treatments: an excess of nitrate, copepods, diatoms (Navicula sp.), a combination of copepods and diatoms, and spent medium from copepods. The microcosms were incubated for seven and a half days, after which nutrient concentrations and denitrification potential were measured. Ammonium concentrations were highest in the treatments with copepods or their spent medium, whilst denitrification potential was lowest in these treatments, suggesting that copepods enhance dissimilatory nitrate reduction to ammonium over denitrification. We hypothesize that this is an indirect effect, by providing extra carbon for the bacterial community through the copepods' excretion products, thus changing the C/N ratio in favour of dissimilatory nitrate reduction. Diatoms alone had no effect on the nitrogen fluxes, but they did enhance the effect of copepods, possibly by influencing the quantity and quality of the copepods' excretion products. Our results show that small-scale biological interactions between bacteria, copepods and diatoms can have an important impact on denitrification and hence sediment nitrogen fluxes.

Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at -80 °C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Cyclic lipodepsipeptides produced by Pseudomonas spp. naturally present in raw milk induce inhibitory effects on microbiological inhibitor assays for antibiotic residue screening.

Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

The more, the merrier: heterotroph richness stimulates methanotrophic activity.

Although microorganisms coexist in the same environment, it is still unclear how their interaction regulates ecosystem functioning. Using a methanotroph as a model microorganism, we determined how methane oxidation responds to heterotroph diversity. Artificial communities comprising of a methanotroph and increasing heterotroph richness, while holding equal starting cell numbers were assembled. We considered methane oxidation rate as a functional response variable. Our results showed a significant increase of methane oxidation with increasing heterotroph richness, suggesting a complex interaction in the cocultures leading to a stimulation of methanotrophic activity. Therefore, not only is the methanotroph diversity directly correlated to methanotrophic activity for some methanotroph groups as shown before, but also the richness of heterotroph interacting partners is relevant to enhance methane oxidation too. In this unprecedented study, we provide direct evidence showing how heterotroph richness exerts a response in methanotroph-heterotroph interaction, resulting in increased methanotrophic activity. Our study has broad implications in how methanotroph and heterotroph interact to regulate methane oxidation, and is particularly relevant in methane-driven ecosystems.

The nitrate-ammonifying and nosZ-carrying bacterium Bacillus vireti is a potent source and sink for nitric and nitrous oxide under high nitrate conditions.

Several Gram-positive bacteria carry genes for anaerobic reduction of NO3(-) via NO2(-) to NH4(+) or gaseous nitrogen compounds, but the processes are understudied for these organisms. Here, we present results from a whole-genome analysis of the soil bacterium Bacillus vireti and a phenotypic characterization of intermediate and end-products, formed under anoxic conditions in the presence of NO3(-). Bacillus vireti has a versatile metabolism. It produces acetate, formate, succinate and lactate from fermentation and performs dissimilatory nitrate reduction via NO2(-) to ammonium (DNRA) using NrfA, while NirB may detoxify NO2(-) in the cytoplasm. Moreover, it produces NO from an unknown source and reduces it via N2O to N2 using two enzymes connected to denitrification: an unusual NO reductase, qCuA Nor encoded by cbaA, and a z-type N2O reductase, encoded by nosZ. In batch cultures, B. vireti reduced all NO3(-) to NO2(-) before the NO2(-) was reduced further. The quantities of all products varied with the initial NO3(-) concentration. With 5 mM NO3(-) , 90% was reduced to NH4 (+) while with ≥ 20 mM NO3(-), 50% was reduced to NO, N2O and N2. This organism is thus an aggressive NO2(-) accumulator and may act as a net source and sink of NO and N2O.

Niche differentiation in nitrogen metabolism among methanotrophs within an operational taxonomic unit.

BACKGROUND: The currently accepted thesis on nitrogenous fertilizer additions on methane oxidation activity assumes niche partitioning among methanotrophic species, with activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure Unfortunately, widely applied tools for microbial community assessment only have a limited phylogenetic resolution mostly restricted to genus level diversity, and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments. RESULTS: We demonstrated that fourteen genotypically different Methylomonas strains, thus distinct below the level at which most techniques assign operational taxonomic units (OTU), show a versatile physiology in their nitrogen metabolism. Differential responses, even among strains with identical 16S rRNA or pmoA gene sequences, were observed for production of nitrite and nitrous oxide from nitrate or ammonium, nitrogen fixation and tolerance to high levels of ammonium, nitrate, and hydroxylamine. Overall, reduction of nitrate to nitrite, nitrogen fixation, higher tolerance to ammonium than nitrate and tolerance and assimilation of nitrite were general features. CONCLUSIONS: Differential responses among closely related methanotrophic strains to overcome inhibition and toxicity from high nitrogen loads and assimilation of various nitrogen sources yield competitive fitness advantages to individual methane-oxidizing bacteria. Our observations proved that community structure at the deepest phylogenetic resolution potentially influences in situ functioning.

Methyloparacoccus murrellii gen. nov., sp. nov., a methanotroph isolated from pond water.

Two novel methanotrophic strains, R-49797(T) and OS501, were isolated from pond water in South Africa and Japan, respectively. Strains R-49797(T) and OS501 shared 99.7% 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, non-motile cocci with a diplococcoid tendency and contained type I methanotroph intracytoplasmic membranes. The pmoA gene encoding particulate methane monooxygenase was present. Soluble methane monoooxygenase (sMMO) activity, the mmoX gene encoding sMMO and the nifH gene encoding nitrogenase were not detected. Methane and methanol were utilized as sole carbon source. The strains grew optimally at 25-33 °C (range 20-37 °C) and at pH 6.3-6.8 (range 5.8-9.0). The strains did not support growth in media supplemented with 1% (w/v) NaCl. For both strains, the two major fatty acids were C(16 : 1)ω7c and C(16 : 0) and the DNA G+C content was 65.6 mol%. The isolates belong to the family Methylococcaceae of the class Gammaproteobacteria and cluster most closely among the genera Methylocaldum, Methylococcus and Methylogaea, with a 16S rRNA gene sequence similarity of 94.2% between strain R-49797(T) and its closest related type strain (Methylocaldum gracile VKM 14L(T)). Based on the low 16S rRNA gene sequence similarities with its nearest phylogenetic neighbouring genera, the formation of a separate lineage based on 16S rRNA and pmoA gene phylogenetic analysis, and the unique combination of phenotypic characteristics of the two isolated strains compared with the genera Methylocaldum, Methylococcus and Methylogaea, we propose to classify these strains as representing a novel species of a new genus, Methyloparacoccus murrellii gen. nov., sp. nov., within the family Methylococcaceae. The type strain of Methyloparacoccus murrellii is R-49797(T) ( = LMG 27482(T) = JCM 19379(T)).

Pathway of nitrous oxide consumption in isolated Pseudomonas stutzeri strains under anoxic and oxic conditions.

The microbial consumption of nitrous oxide (N2O) has gained great interest since it was revealed that this process could mitigate the greenhouse effect of N2O. The consumption of N2O results from its reduction to dinitrogen gas (N2) as part of the denitrification process. However, there is ongoing debate regarding an alternative pathway, namely reduction of N2O to NH4(+), or assimilatory N2O consumption. To date, this pathway is poorly investigated and lacks unambiguous evidence. Enrichment of denitrifying activated sludge using a mineral nitrogen-free medium rendered a mixed culture capable of anoxic and oxic N2O consumption. Dilution plating, isolation and deoxyribonucleic acid fingerprinting identified a collection of Pseudomonas stutzeri strains as dominant N2O consumers in both anaerobic and aerobic enrichments. A detailed isotope tracing experiment with a Pseudomonas stutzeri isolate showed that consumption of N2O via assimilatory reduction to NH4(+) was absent. Conversely, respiratory N2O reduction was directly coupled to N2 fixation.

Methylomonas lenta sp. nov., a methanotroph isolated from manure and a denitrification tank.

Two methanotrophic bacteria, strains R-45377(T) and R-45370, were respectively isolated from a slurry pit of a cow stable and from a denitrification tank of a wastewater treatment plant in Belgium. The strains showed 99.9 % 16S rRNA gene sequence similarity. Cells were Gram-negative, motile rods containing type I methanotroph intracytoplasmic membranes. Colonies and liquid cultures appeared white to pale pink. The pmoA gene encoding particulate methane monooxygenase (pMMO) and the nifH gene encoding nitrogenase were present. Soluble methane monooxygenase (sMMO) activity, the presence of the mmoX gene encoding sMMO and the presence of the pxmA gene encoding a sequence-divergent pMMO were not detected. Methane and methanol were utilized as sole carbon sources. The strains grew optimally at 20 °C (range 15-28 °C) and at pH 6.8-7.3 (range pH 6.3-7.8). The strains grew in media supplemented with up to 1.2 % NaCl. The major cellular fatty acids were C16 : 1ω8c, C16 : 1ω5c, C16 : 1ω7c, C14 : 0, C15 : 0 and C16 : 0 and the DNA G+C content was 47 mol%. 16S rRNA gene- and pmoA-based phylogenetic analyses showed that the isolates cluster among members of the genus Methylomonas within the class Gammaproteobacteria, with pairwise 16S rRNA gene sequence similarities of 97.5 and 97.2 % between R-45377(T) and the closest related type strains, Methylomonas scandinavica SR5(T) and Methylomonas paludis MG30(T), respectively. Based on phenotypic characterization of strains R-45377(T) and R-45370, their low 16S rRNA gene sequence similarities and the formation of a separate phylogenetic lineage compared with existing species of the genus Methylomonas, we propose to classify these strains in a novel species, Methylomonas lenta sp. nov., with R-45377(T) ( = LMG 26260(T) = JCM 19378(T)) as the type strain.

Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas.

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.