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Tungsten-enhanced growth of Methanosphaera stadtmanae
© Dridi et al.; licensee BioMed Central Ltd. 2012
Received: 16 November 2011
Accepted: 25 April 2012
Published: 15 May 2012
The methanogenic Archaea Methanosphaera stadtmanae has been detected in the human gut microbiota by both culture and culture-independent methods. Its growth reaches an exponential phase after 5 to 7-day culture in medium 322 (10% vol). Our recent successful isolation of Methanomassiliicoccus luminyensis, a tungstate-selenite-requiring Archaea sharing similar metabolism characteristics with M. stadtmanae prompted us to study the effects of tungsten and selenium on M. stadtmanae growth.
Addition of 0.2 mg/L sodium tungstate to medium 322 yielded, 48 hours after inoculation, a growth rate equivalent to that obtained after 6 days with control culture as measured by methane monitoring and optical density measurement. Addition of 50 μg/mL sodium selenate had no effect on M. stadtmanae growth. Quantitative real-time PCRs targeting the M. stadtmanae 16S rRNA confirmed these data.
These data provide new information regarding the poorly known nutritional requirements of the human gut colonizing organisms M. stadtmanae. Adding sodium tungstate to basal medium may facilitate phenotypic characterization of this organism and additionally aid the isolation of new Archaea from complex host microbiota.
Methanosphaera stadtmanae is a spherical-shaped, non-motile archaeon initially isolated from human feces . M. stadtmanae was the first human Archaea to be genome sequenced and analysis of the genome confirmed that M. stadtmanae belonged to Methanobacteriales. PCR-based analyses further indicated that M. stadtmanae-specific sequences could be detected in stool specimen in up to 30% of individuals . However, M. stadtmanae is a fastidious organism, with only one M. stadtmanae isolate reported and accordingly only one M. stadtmanae strain available in public collections. M. stadtmanae oxidizes hydrogen to reduce methanol into methane [1, 2]. This metabolic trait has been already reported for M. stadtmanae, and more recently for members of the genus Methanobacterium (e.g. M. veterum and M. lacus; [5, 6]) within the order Methanobacteriales. We recently isolated Methanomassiliicoccus luminyensis, the first cultured representative of new order of methanoarchaea . This archaeon exhibits a metabolic trait similar to that of M. stadtmanae by using hydrogen as electron donor and methanol as electron acceptor . Unexpectedly, we observed that addition of tungstate-selenite to culture medium had been a key factor for successful isolation of M. luminyensis and that this archaeon indeed required tungstate-selenite as an essential element for growth. We therefore tested the hypothesis that the addition of sodium tungstate or sodium selenate or both to basal culture medium would also enhance the growth of M. stadtmanae.
M. stadtmanae DSMZ 3091 T (ATCC 43021T) purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) was grown on medium 322 (http://www.dsmz.de) incubated at 37°C in Hungate tubes (Dutscher, Issy-les-Moulineaux, France) under 2-bar pressure of a H2/CO2 (80–20) atmosphere. The inoculated medium (10% vol) was incubated at 37°C with shaking. On the exponential phase of this first culture, a second inoculation was performed by 10% vol. in the same basal medium modified or not by the addition of Na2O4W (0.2 mg/L) and/or Na2O4Se (50 μg/L) (Sigma, Saint-Quentin Fallavier, France). Non-inoculated media were used as negative controls and each experiment was repeated ten times.
Growth was assessed by optical microscope observation, parallel methane production measurement and measurement of the optical density of the medium. Methane production measurement used a GC-8A gas chromatograph (Shimadzu, Champs-sur-Marne, France) equipped with a thermal conductivity detector and a Chromosorb WAW 80/100 mesh SP100 column (Alltech, Carquefou, France). N2 at a pressure of 100 kPa was used as the carrier gas. The detector and the injector temperatures were 200°C, and the column temperature was 150°C. H2 consumption and CH4 production were measured every 6 hours for 24 hours and then every 12 hours for 6 days. The optical density at 580 nm was measured by inserting Hungate tubes into the spectrophotometer (Varian Cary50; Agilent Technologies, Massy, France). Experiment was done in triplicate and average optical density value for the three replicates was calculated.
M. stadtmanae DNA extraction, quantification and sequencing were performed as previously described based on specific quantitative real-time PCR targeting 16S rRNA gene .
M. stadtmanae 16S rDNA gene copy number after 48-hour culture with Na 2 O 4 W + Na 2 O 4 Se or only Na 2 O 4 W and a 6-day culture with no Na 2 O 4 W + Na 2 O 4 Se or only with Na 2 O 4 Se (Mean and standard deviation were calculated for 10 independent culture tests for each condition)
Na2O4W + Na2O4Se
Na2O4W + Na2O4Se
Na2O4W + Na2O4Se
2.13E + 10
4.42E + 12
3.93E + 12
4.02E + 12
3.22E + 12
5.56E + 09
1.84E + 11
3.67E + 11
2.23E + 11
1.53E + 11
Requirement of tungsten or/and selenium for growth of methanogens as reported in bibliography
In the absence of tungstate, M. stadtmanae exhibited a growth delay of 5–7 days which is long for testing in vitro susceptibility to antibiotics . As we now observed that tungsten enhances the growth of two taxonomically unrelated methanogens, M. stadtmanae and M. luminyensis, we suggest that tungsten-containing media could be incorporated into the panel of media used for the isolation and culture of new methanogens from clinical and environmental specimens, and for testing their in-vitro susceptibility to antibiotics.
Methanogenic Archaea recently emerged as normal components of the human gastrointestinal and oral microbial ecosystems, where they could play important roles in health and diseases . However, the isolation of such organisms requires long incubation times and strict anoxic atmosphere and is hampered by the incomplete knowledge of their nutritional requirements . In fact, the result obtained in the present study may prompt further phenotypic characterization including extended antibiotic susceptibility testing  and even allowing isolation of new Archaea in order to assess understanding their contribution in the physiology of complex human microbiomes and their potential role in the course of infections.
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