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Growth time of ten methanogenic archaea strains growing in culture medium SAB-medium or the standard DSMZ media. (Triplicate experiment). doi:10.1371/journal.pone.0061563.g001
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Background:
Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis and Methanobrevibacter arboriphilicus have been cultured from human digestive microbiota. Each one of these fastidious methanogenic archaea requires a specific medium for its growth, hampering their routine isolation and t...
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Context 1
... observation disclosed organisms with a morphology compatible with the studied archaea organism in each essay and no contaminant. Significant differences were observed in the time required for detecting growth of each methanogenic archaea (Figure 1). M. smithii DSMZ 2374, M. smithii DSMZ 2375 and M. smithii ATCC 35061 T grew after 24-hour incubation with a 3-hour doubling time in SAB medium versus 72-hour incubation and a 9- hour doubling time in standard 119 DSMZ medium (P,0.05, ...
Context 2
... the sludge superna- tant as a PABA source, it was necessary to increase the concentration of yeast extract and add 1 g/L of peptone to the SAB medium, in addition to a mixture of volatile fatty acids and vitamin solution. This change led to a considerable reduction in the growth time for all of the tested strains (Figure 1). ...
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... At the time of sampling, the temperature of the landfill was 30 • C; therefore, we conducted all the enrichment and physiological experiments at 30 • C to mimic the site temperature. To enrich methanogenic archaea and anaerobic bacteria, basal carbonate yeast trypticase (BCYT) medium (Touzel and Albagnac, 1983), BCYT + 30% rumen fluid, SAB medium (Khelaifia et al., 2013), and anaerobic groundwater medium were used. Basal medium was fortified with different substrates according to the targeted type of methanogens. ...
The landfill is a cheap way of solid waste management in developing countries. The majority of landfills are non-sanitary and work as open garbage dumping sites and pose threats to public and environmental health. Therefore, an in-depth understanding of the chemistry and microbiology of landfills is imperative to develop the right policies for landfill management. In the current study, we investigated the chemistry and microbiology of three Indian landfill sites using culture-based and culture-independent molecular approaches. Our data indicate that the nature of landfills varies from site to site in terms of chemistry, pollutants, and pathogens. We also enriched and cultivated three methanogens using an optimized medium and constructed two high-quality draft genomes from enriched microbiomes using metagenome-assembled genome approaches. The phylogenomic study of one draft genome showed the highest 93% sequence similarity with members of Methanomassiliicoccaceae and was always enriched with Acholoplasma and Anaerohalosphaera lusitana . Despite all the efforts, we did not isolate it in pure culture and hypothesized that for the cultivation of some not-yet-cultured methanogen, the presence of other organisms plays an important role, and their syntrophic interaction must be discerned for its successful cultivation in the future. Co-cultivation of amino acid-degrading organisms indicates that their co-culture can assist in boosting the growth of methanogens. In addition, our data indicated that landfill leachate contains a heavy load of pollutants and treatment is a must before discharge in nature or use in irrigation or biofertilizer.
... Dental pulps which tested positive by PCR sequencing for M. massiliense were cultured in a Hungate tube containing 5 mL of SAB medium [33] consisting of 100 mg/L amoxicillin, 100 mg/L vancomycin, 100 mg/L imipenem, 50 mg/L daptomycin and 50 mg/L amphotericin B (Sigma Aldrich, Saint Quentin Fallavier, France), in a 5% hydrogen, 10% CO 2 , 85% nitrogen atmosphere at 37 • C for 15 days. Culture was monitored by methane detection with gas chromatography using a Clarus 580 chromatograph (Perkin Elmer, Waltham, MA, USA), as previously described [33]. ...
... Dental pulps which tested positive by PCR sequencing for M. massiliense were cultured in a Hungate tube containing 5 mL of SAB medium [33] consisting of 100 mg/L amoxicillin, 100 mg/L vancomycin, 100 mg/L imipenem, 50 mg/L daptomycin and 50 mg/L amphotericin B (Sigma Aldrich, Saint Quentin Fallavier, France), in a 5% hydrogen, 10% CO 2 , 85% nitrogen atmosphere at 37 • C for 15 days. Culture was monitored by methane detection with gas chromatography using a Clarus 580 chromatograph (Perkin Elmer, Waltham, MA, USA), as previously described [33]. M. massiliense-positive cultures were subcultured using two conditions: in one condition, 200 µL was inoculated in a Hungate tube containing 5 mL of SAB medium incubated under a 5% hydrogen, 10% CO 2 , 85% nitrogen atmosphere, and in the other condition, 200 µL was inoculated in liquid GG medium under a nitrogen atmosphere [34]. ...
... Tubes were incubated for five days at 37 • C. In an attempt to isolate M. massiliense in pure culture, the M. massiliense-P. piscolens co-culture Q8282 was subcultured (in duplicate) in five distinct conditions: 100 µL of a growing culture was inoculated in GG medium as a positive control; in GG medium supplemented with 100 mg tetracycline; in GG medium supplemented with 100 mg tetracycline and 3 g/L of acetate; in GG medium supplemented with 100 mg tetracycline with volatile fatty acids solution [33]; and in GG medium supplemented with 100 mg tetracycline with 3 g/L of acetate and volatile fatty acids solution. All tubes were incubated for three weeks at 37 • C. Negative controls encompassed the same five conditions, but with the inoculation of 100 µL of DPBS 1× water. ...
Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1–2-µm-long and 0.6–0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.
... A 0.5 g 16S rRNA RT-PCR-positive stool sample was suspended in 10 mL of Dulbecco's phosphatebuffered saline (DPBS) 1× water (Thermo Fisher Scientific, Waltham, MA, USA) and vortexed for a few seconds. A 200 µL volume of this suspension was inoculated into a Hungate tube containing GG medium (29, patent FR 23 01404) that is derived from SAB medium (42), containing acetate, formate, and methanol as standards, previously degassed for 3 minutes with 2 bar nitrogen. The inoculated tube was incubated for 7 days at 37°C. ...
... The inoculated tube was incubated for 7 days at 37°C. On day 7, methane was detected by gas chromatography using a Clarus 580 chromatograph (Perkin Elmer, Waltham, MA, USA) confirming methanogen growth as previously described (42) and optical density was measured by putting the Hungate tubes in a spectrophotometer cell density meter model 40 (Thermo Fisher Scientific, Waltham, MA, USA). Then, 200 µL of growing culture was inoculated into a fresh GG medium containing fresh antibiotics, 100 mg/L amoxicillin, 100 mg/L vancomycin, 100 mg/L imipenem, 50 mg/L daptomycin, and 50 mg/L amphotericin B (Sigma Aldrich, Saint Quentin Fallavier, France) and supplemented with ethanol at 2% (vol/vol). ...
Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense ( Ca . M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca . M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca . M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca . M. massiliense and possible transfer between hosts.
IMPORTANCE
Methanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca . M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.
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Since Carl Woese’s discovery of archaea as a third domain of life, numerous archaeal species have been discovered, yet archaeal diversity is poorly characterized. Culturing archaea is complicated, but several queries about archaeal cell biology, evolution, physiology, and diversity need to be solved by culturing and culture-dependent techniques. Increasing interest in demand for innovative culturing methods has led to various technological and methodological advances. The current review explains frequent hurdles hindering uncultured archaea isolation and discusses features for more archaeal cultivation. This review also discusses successful strategies and available media for archaeal culturing, which might be helpful for future culturing practices.
... Nevertheless, several media were standardized and optimized so far, and studies are still in progress to suggest novel media and appropriate culture conditions for specific archaeal strains. For instance, Balch medium (Balch and Wolfe 1976), basal culture medium (Scherer and Sahm 1981), BSM medium (Kurr et al. 1991), MJYPGS medium (Nakagawa and Takai 2006), SAB medium (Khelaifa et al. 2013), NHA medium (Shih et al. 2015), DSM 282 medium (Topçuoğlu et al. 2016), McF medium (Long et al. 2017), etc. for the specific growth of Methanobacterium ruminantium, Methanosarcina barkeri, Methanopyrus kandleri, Thermococcales, Methanobrevibacter smithii, Haloarchaea, Methanothermococcus thermolithotrophicum and Methanococcus maripaludis, respectively, under specific controlled culture conditions. Archaeal culture techniques can be improvised as suggested by Sun et al. (2019) by refining the selective media based on DNA and RNA level, testing with different media composition and chemical parameters and combining different techniques like co-culture and high-throughput culturing together. ...
The success of aquaculture production greatly depends on the health status of aquatic species and the proper management of the water quality. The significance of microbes in their contribution to the healthy aquatic environment, disease control and proper waste management is incredible. Among these, the role of bacteria in developing probiotics, vaccines, immunostimulants, bioremediators, quorum quenchers, etc. has been well established. Nevertheless, the importance of archaea, the third domain of life, in aquaculture applications is least explored. However, the advances in culture-independent methods revealed the archaeal diversity and their ubiquitous occurrence in various aquaculture systems such as recirculating aquaculture systems (RAS), aquaculture ponds, aquaponics systems and biofloc systems. Most of the studies on archaea in aquaculture systems were concentrated on nitrification and the associated ammonia oxidizing archaea (AOA). Most archaea detected from the aquaculture systems belong to the phylum Thaumarchaeota and Euryarchaeota, and they play a vital in the nitrogen and sulphur cycles. Thus, the archaea are proposed as potent bioaugmentors for mitigating the nitrogen and sulphur toxicity in cultured system. Besides nutrient cycling, recent studies highlight the role of archaea in aquaculture nutrition and pave the way for aquafeed development as probiotics. Several factors such as temperature, pH, salinity and dissolved oxygen affect the archaeal diversity and abundance in aquaculture. Challenges in the isolation and maintenance of pure archaeal cultures limit our understanding and applications of archaea in aquaculture. Considering all the limitations and lack of collective information on archaeal aquaculture, this review summarizes the significance and diversity of archaea in various aquaculture systems, factors affecting their diversity, detection methods, applications and advantage over bacteria, challenges and future prospects.
... Fastidious methanogens such as Mbt. formicicum, which needs certain growth factors, amino acids, and co-enzymes, were , Unhavare (f), Chopda 1 (g) and Chopda 2 (h) hot springs isolated solely on BY medium (Khelaifia et al. 2013). In our earlier study, the BY medium was also found to support the growth of diverse hydrogenotrophic methanogens (Joshi et al. 2018). ...
Mesophilic and thermophilic methanogens belonging to the hydrogenotrophic, methylotrophic, and acetotrophic groups were isolated from Indian hot spring environments using BY and BCYT growth media. Following initial Hinf I-based PCR–RFLP screening, 70 methanogens were sequenced to ascertain their identity. These methanogens were phylogenetically and physiologically diverse and represented different taxa distributed across three physiological groups, i.e., hydrogenotrophs (53), methylotrophs (14) and acetotrophs (3). Overall, methanogens representing three families, five genera, and ten species, including two putative novel species, were recognized. The highest number and diversity of methanogens was observed at 40 ℃, dominated by Methanobacterium (10; 3 species), Methanosarcina (9; 3 species), Methanothermobacter (7; 2 species), Methanomethylovorans (5; 1 species) and Methanoculleus (3; 1 species). Both putative novel methanogen species were isolated at 40 ℃ and belonged to the genera Methanosarcina and Methanobacterium. At 55 ℃, limited diversity was observed, and resulted in the isolation of only two genera of methanogens, i.e., Methanothermobacter (28; 2 species) and Methanosarcina (4; 1 species). At 70 ℃, only members of the genus Methanothermobacter (5; 2 species) were isolated, whereas no methanogen could be cultured at 85 ℃. Ours is the first study that documents the extensive range of cultivable methanogenic archaea inhabiting hot springs across various geothermal provinces of India.
... The samples of SRS were diluted in a culture medium to a final concentration of 20 g/L VS. The culture medium was formulated according to previous independent work [31], with the following modification: 1.5 g/L NaCl, 18 O. pH was adjusted to 7, and after autoclaving the medium, the following filtered compounds were added: 2 g/L NaHCO 3 , 27.7 mM butyric acid, 68 mM methanol, and 25.5 mM CH 3 COONa. ...
In this paper, sediments from the Santiago River were characterized to look for an alternative source of inoculum for biogas production. A proteomic analysis of methane-processing archaea present in these sediments was carried out. The Euryarchaeota superkingdom of archaea is responsible for methane production and methane assimilation in the environment. The Santiago River is a major river in México with great pollution and exceeded recovery capacity. Its sediments could contain nutrients and the anaerobic conditions for optimal growth of Euryarchaeota consortia. Batch bioreactor experiments were performed, and a proteomic analysis was conducted with current database information. The maximum biogas production was 266 NmL·L −1 ·g VS −1 , with 33.34% of methane, and for proteomics, 3206 proteins were detected from 303 species of 69 genera. Most of them are metabolically versatile members of the genera Methanosarcina and Methanosarcinales, both with 934 and 260 proteins, respectively. These results showed a diverse euryarcheotic species with high potential to methane production. Although related proteins were found and could be feeding this metabolism through the methanol and acetyl-CoA pathways, the quality obtained from the biogas suggests that this metabolism is not the main one in carbon use, possibly the sum of several conditions including growth conditions and the pollution present in these sediments
... The distinction between pathogenic and commensal bacteria is becoming an increasingly complex and ambiguous task, especially when the microorganisms usually found in healthy states are involved in complex interactions with their host leading to a state of disease or infection [15]. Recent years have been marked by a paradigm shift in the study of the human microbiota, with a re-emergence of culture-dependent approaches [16][17][18][19][20][21]. Recent studies have been mainly devoted to cultivating the gut microbiota and studies on the oral microbiota remain very limited. ...
... Antioxidants have long been used to establish an anaerobic atmosphere or to contribute to its improvement [43,44]. Indeed, antioxidants such as sodium sulphide (Na 2 S), or amino acids such as cysteine, are found in old culture media optimised for the culture of anaerobic bacteria but also for the culture of methanogenic archaea for which sensitivity to oxygen is widely demonstrated [17,43,44]. Since then, many studies have focused on the beneficial effect of antioxidants for the culture of fastidious anaerobes. ...
... The culture of methanogenic archaea is fastidious and requires an external supply of H 2 and CO 2 required for their growth [17]. In 2016, Khelaifia et al. succeeded in substituting the gaseous supply of H 2 and CO 2 by a co-culture with Bacteroides thetaiotaomicron used as the sole source of hydrogen and CO 2 in a dual chamber culture device. ...
Recent years have been marked by a paradigm shift in the study of the human microbiota, with a re-emergence of culture-dependent approaches. Numerous studies have been devoted to the human microbiota, while studies on the oral microbiota still remain limited. Indeed, various techniques described in the literature may enable an exhaustive study of the microbial composition of a complex ecosystem. In this article, we report different methodologies and culture media described in the literature that can be applied to study the oral microbiota by culture. We report on specific methodologies for targeted culture and specific culture techniques and selection methodologies for cultivating members of the three kingdoms of life commonly found in the human oral cavity, namely, eukaryota, bacteria and archaea. This bibliographic review aims to bring together the various techniques described in the literature, enabling a comprehensive study of the oral microbiota in order to demonstrate its involvement in oral health and diseases.
... Current protocols for the isolation by culture of M. smithii from clinical specimens of microbiota such as stools (Dridi et al., 2011), oral fluid (Grine et al., 2018), and clinical specimens collected from abscesses such as muscular abscesses and cerebral abscesses (Drancourt et al., 2017), have all relied on supplying hydrogen and carbon dioxide into the culture atmosphere . Hydrogen can be supplied to the culture atmosphere as gaseous hydrogen from an external source (Khelaifia et al., 2013), as chemically produced hydrogen within the culture environment itself (Guindo et al., 2020), or as biotic hydrogen resulting from bacterial fermentation within the culture environment . ...
... M. smithii strain Q5488, a stool isolate preserved in the strains collection of the Unité des Rickettsies (CSUR Q5488 -Supplementary data 1) was inoculated into 30 different Hungate tubes (Dominique Dutscher, Brumath, France), each containing 200 μL of Dulbecco's Phosphate Buffered Saline (DPBS, Fisher Scientific, Waltham, Massachusetts, USA) in order to study six different experimental conditions, as detailed below (in other words, five Hungate tubes per experimental condition). Condition A1 included Hungate tubes seeded with an SAB medium, a versatile medium that allows the growth and the isolation of most of methanogen species as previously reported (Khelaifia et al., 2013), except that it did not contain acetate or formate (medium referred to as SAB 0). These tubes were degassed for three minutes with 2-bar nitrogen, inoculated with 200 μL of M. smithii strain Q5488 and degassed again for 30 s with 2-bar nitrogen. ...
... Condition A2 included five tubes and one control tube, similar to condition A1 with the difference that they were gassed with a mixture of 80% hydrogen and 20% carbon dioxide for 30 s at a pressure of 2 bars. Condition B1 included five Hungate tubes and one control Hungate tube, all containing the SAB medium, as originally described, i.e. containing 1 g/L of acetate and formate (Khelaifia et al., 2013). This SAB medium was prepared from SAB 0 medium by adding 100 μL of a stock solution containing 50 g/L of acetate and formate (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) buffered to pH 7.3 and filtered through a 0.2 μm filter (Merck, Darmstadt, Germany). ...
Methanobrevibacter smithii (M. smithii), the most prevalent and abundant gut methanogen, detoxifies hydrogen into methane and is, therefore, of paramount importance for the equilibrium of the gut microbiota. The isolation by culture of M. smithii has routinely relied upon hydrogen‑carbon dioxide-enriched, oxygen-deprived atmospheres. In this study, we developed a medium referred to as "GG", which allowed for M. smithii growth and isolation by culture in an oxygen-deprived atmosphere, with no supply of either hydrogen or carbon dioxide, making it easier to detect M. smithii by culture in clinical microbiology laboratories.
... One notable exception was methanogenesis, which was substantially underrepresented in RefSeq STIBs for all environments except bioreactors (where the difference was only minor). This later observation is consistent with the fact that methanogens have been historically difficult to culture [38][39][40] , and suggests than methanogenesis is a much more common trait in prokaryotes in natural environments than one would expect based on reference genomes. ...
Common culturing techniques and priorities bias our discovery towards specific traits that may not be representative of microbial diversity in nature. So far, these biases have not been systematically examined. To address this gap, here we use 116,884 publicly available metagenome-assembled genomes (MAGs, completeness ≥80%) from 203 surveys worldwide as a culture-independent sample of bacterial and archaeal diversity, and compare these MAGs to the popular RefSeq genome database, which heavily relies on cultures. We compare the distribution of 12,454 KEGG gene orthologs (used as trait proxies) in the MAGs and RefSeq genomes, while controlling for environment type (ocean, soil, lake, bioreactor, human, and other animals). Using statistical modeling, we then determine the conditional probabilities that a species is represented in RefSeq depending on its genetic repertoire. We find that the majority of examined genes are significantly biased for or against in RefSeq. Our systematic estimates of gene prevalences across bacteria and archaea in nature and gene-specific biases in reference genomes constitutes a resource for addressing these issues in the future.
