Effects of whole-body vibration on bone properties in aged rats.

OBJECTIVE: This study aimed to explore optimal conditions of whole-body vibration (WBV) for improving bone properties in aged rats. METHODS: Eighty-week-old rats were divided into baseline control (BC), age-matched control (CON) and experimental groups, which underwent WBV (0.5 g) at various frequencies (15, 30, 45, 60 or 90 Hz) or WBV (45 Hz) with various magnitudes (0.3, 0.5, 0.7 or 1.0 g) for 7 weeks. After interventions, femur bone size, bone mechanical strength and circulating bone formation/resorption markers were measured, and trabecular bone microstructure (TBMS) and cortical bone geometry (CBG) of femurs were analyzed by micro-CT. RESULTS: Several TBMS parameters and trabecular bone mineral content were significantly lower in the 15 Hz WBV (0.5 g) group than in the CON group, suggesting damage to trabecular bone. On the other hand, although frequency/magnitude of WBV did not influence any CBG parameters, the 0.7 g and 1.0 g WBV (45 Hz) group showed an increase in tissue mineral density of cortical bone compared with the BC and CON groups, suggesting the possibility of improving cortical bone properties. CONCLUSION: Based on these findings, it should be noted that WBV conditions are carefully considered when applied to elderly people.

Parasympathetic nervous activity is associated with oxytocin in multiparous, but not primiparous, women during the perinatal period.

Oxytocin (OXT) is thought to have antidepressant/anxiolytic effects in postpartum women. Primiparous women tend toward an attenuated lactation compared with multiparous women. However, so far, little is known about the relationship between OXT and autonomic nervous activity (ANA) in perinatal women and whether it may be different in primiparous and multiparous women. Therefore, the objective of this study was to answer this question by determining both ANA and salivary OXT levels in primiparous and multiparous perinatal women. In 18 primiparous and 18 multiparous women, who underwent a physical and physiological examination, ANA measurement by heart rate variability and saliva sampling were performed during the perinatal period. Saliva OXT concentration was determined by a highly sensitive ELISA. OXT release into saliva was obtained from multiplying saliva OXT concentration by saliva flow rate. In the postpartum period, multiparous women had higher parasympathetic nervous activity (PNA) and lower physical stress index (PSI) compared with primiparous women. Furthermore, multiparous postpartal women had higher OXT compared with primiparous or multiparous prepartal women. In addition, in multiparous perinatal women, OXT correlated positively with PNA, but negatively with PSI. These results suggest that after parturition, multiparous mothers may switch over to the "feed and breed" system more quickly due to increased OXT compared with primiparous mothers. Our findings support antidepressant/anxiolytic and anti-stress effects of OXT. In postpartal women exposed to synthetic OXT, ANA measurement may provide a clue to clarify the effects of exogenous OXT on postpartum psychiatric disorders.

Whole body vibration at low-frequency can increase trabecular thickness and width in adult rats.

OBJECTIVE: This study aimed to examine whether WBV becomes a possible modality for the primary prevention of osteoporosis by exploring WBV frequency that has positive effects on bone properties in adult rats. METHODS: Thirty-six 24-week-old rats were divided into one control and 5 experimental groups, which underwent WBV at various frequencies (15, 30, 45, 60 or 90 Hz), with a magnitude of 0.5 g, for 15 min/day, 5 days/week, for 8 weeks. Bone size, muscle weight and bone mechanical strength were measured at the end of experimental period. Bone mass, trabecular bone microarchitecture (TBMA) and cortical bone geometry were analyzed by micro-CT. Circulating bone formation/resorption markers were determined by ELISA. RESULTS: Body weight-corrected soleus weight in all experimental groups and body weight-corrected extensor digitorum longus muscle weight in the 15 and 30 Hz groups were significantly higher than those of the control group, respectively. Femur trabecular thickness and width were significantly higher in the 15 Hz group than in the control group. However, there was no difference in bone mechanical strength or bone formation/resorption markers among all groups. CONCLUSION: These results suggest that WBV at low-frequencies may become a potent modality for the primary prevention of osteoporosis in adults.

Long-Term Intake of Green Tea Extract Causes Mal-Conformation of Trabecular Bone Microarchitecture in Growing Rats.

The purpose of this study was to examine the effects of green tea extract (GTE) intake on bone structural and physiological properties, such as bone mass, trabecular bone microarchitecture, cortical bone geometry, and bone mechanical strength, in growing rats. Four-week-old male Wistar rats were divided into the following four groups: standard diet feeding for 85 days (S-CON) or 170 days (L-CON), and GTE diet feeding for 85 days (S-GTE) or 170 days (L-GTE). At the end of the experiment, in addition to measurement of circulating bone formation/resorption markers, bone mass, trabecular bone microarchitecture, and cortical bone geometry were analyzed in the left femur, and bone mechanical strength of the right femur was measured. There was no difference in all bone parameters between the S-CON and S-GTE groups. On the other hand, the L-GTE group showed the decrease in some trabecular bone mass/microarchitecture parameters and no change in cortical bone mass/geometry parameters compared with the L-CON group, and consequently the reduction in bone weight corrected by body weight. There was no difference in bone formation/resorption markers and bone mechanical strength between the S-CON and S-GTE groups and also between the L-CON and L-GTE groups. However, serum leptin levels were significantly lower in the L-GTE group than in the L-CON group. Thus, the long-term GTE intake had negative effects on bone, especially trabecular bone loss and microarchitecture mal-conformation, in growing rats.

Petrothermobacter organivorans gen. nov., sp. nov., a thermophilic, strictly anaerobic bacterium of the phylum Deferribacteres isolated from a deep subsurface oil reservoir.

A novel thermophilic, anaerobic, chemoheterotrophic, acetate-oxidizing and iron(III)-, manganese(IV)-, nitrate- and sulfate-reducing bacterium, designated strain ANAT, was isolated from a deep subsurface oil field in Japan (Yabase oil field, Akita Pref.). Cells of strain ANAT were Gram-stain-negative, non-motile, non-spore forming and slightly curved or twisted rods (1.5-5.0 µm long and 0.6-0.7 µm wide). The isolate grew at 25-60 °C (optimum 55 °C) and pH 6.0-8.0 (optimum pH 7.0). The isolate was capable of reducing iron(III), manganese(IV), nitrate and sulfate as an electron acceptor. The isolate utilized a limited range of electron donors such as acetate, lactate, pyruvate and yeast extract for iron reduction. Strain ANAT also used pyruvate, fumarate, succinate, malate, yeast extract and peptone for fermentative growth. The major respiratory quinones were menaquinone-7(H8) and menaquinone-8. The strain contained C18 : 0, iso-C18 : 0 and C16 : 0 as the major cellular fatty acids. The G+C content of the genomic DNA was 34.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ANAT was closely related to Calditerrivibrio nitroreducens in the phylum Deferribacteres with low sequence similarities (89.5 %), and formed a distinct clade within the family Deferribacteraceae. In addition, the isolate is the first sulfate-reducing member of the phylum Deferribacteres. Based on phenotypic, chemotaxonomic and phylogenetic properties, a novel genus and species, Petrothermobacter organivorans gen. nov., sp. nov., is proposed for the isolate (type strain=ANAT= NBRC 112621T=DSM 105015T).

Methanomicrobium antiquum sp. nov., a hydrogenotrophic methanogen isolated from deep sedimentary aquifers in a natural gas field.

A mesophilic, hydrogenotrophic methanogen, designated strain MobHT, was isolated from sediments derived from deep sedimentary, natural-gas-bearing aquifers in Japan. Strain MobHT utilized H2/CO2 or formate, but not ethanol, 1-propanol, 2-propanol, 2-butanol or cyclopentanol, for growth and methane production. In addition, acetate and tungsten were required for growth. Yeast extract stimulated the growth, but was not required. The cells were weakly motile with multiple flagella, presented as a curved-rod-shaped (0.8×2.0 µm) and occurred singly or in pairs. Strain MobHT grew at 15-40 °C (optimum 35 °C) and at pH 5.9-7.9 (optimum pH 7.0-7.5). The sodium chloride range for growth was 0-5.8 % (optimum 2 %). The G+C content of the genomic DNA was 37.6 mol%. In the phylogenetic tree based on the 16S rRNA gene sequences, strain MobHT clustered together with Methanomicrobium mobile (95.4 % in sequence similarity), and formed a distinct clade from Methanolacinia petrolearia SEBR 4847T (95.6 %) and Methanolacinia paynteri G-2000T (95.4 %). The two species of the genus Methanolacinia utilized 2-propanol, whereas strain MobHT and Methanomicrobium mobile, the sole species of the genus Methanomicrobium, do not. Based on phenotypic and phylogenetic features, we propose a novel species for the isolate with the name, Methanomicrobiumantiquum sp. nov. The type strain is MobHT (=DSM 21220T=NBRC 104160T).

Tepidicaulis marinus gen. nov., sp. nov., a marine bacterium that reduces nitrate to nitrous oxide under strictly microaerobic conditions.

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium,Parvibaculum, and Rhodoligotrophos (92-93 % similarity) within the class Alphaproteobacteria. Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16-48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

Tidal volume and diaphragm muscle activity in rats with cervical spinal cord injury.

[Purpose] The purpose of this study was to make an experimental model of cervical spinal cord injury (CSCI) using Wistar rats, in order to analyze the influence of CSCI on the respiratory function. [Subjects] Thirty-two male 12-week-old Wistar rats were used. [Methods] The CSCI was made at the levels from C3 to C7, and we performed pneumotachography and electromyography (EMG) on the diaphragm. Computed tomography was used to determine the level of spinal cord damage. [Results] After the operation, the tidal volume of the rats with a C3 level injury decreased to approximately 22.3% of its pre-injury value. In addition, in the same rats, the diaphragmatic electromyogram activity decreased remarkably. Compared with before CSCI, the tidal volume decreased to 78.6% of its pre-injury value in CSCI at the C5 level, and it decreased to 94.1% of its pre-injury value in CSCI at the C7 level. [Conclusion] In the rats that sustained a CSCI in this study, the group of respiratory muscles that receive innervation from the thoracic spinal cord was paralyzed. Therefore, the EMG signal of the diaphragm increased. These results demonstrate that there is a relationship between respiratory function and the level of CSCI.

Methylocaldum marinum sp. nov., a thermotolerant, methane-oxidizing bacterium isolated from marine sediments, and emended description of the genus Methylocaldum.

An aerobic, methane-oxidizing bacterium (strain S8(T)) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain is closely related to members of the genus Methylocaldum (97.6-97.9 % similarity) within the class Gammaproteobacteria. Strain S8(T) was a Gram-staining-negative, non-motile, coccoid or short rod-shaped organism. The temperature range for growth of strain S8(T) was 20-47 °C (optimum growth at 36 °C). It required NaCl (>0.5 %), tolerated up to 5 % NaCl and utilized methane and methanol. The major cellular fatty acid and major respiratory quinone were C16 : 0 and 18-methylene ubiquinone 8, respectively. The DNA G+C content was 59.7 mol%. Strain S8(T) possessed mmoX, which encodes soluble methane monooxygenase, as well as pmoA, which encodes the particulate methane monooxygenase. On the basis of this morphological, physiological, biochemical and genetic information, the first marine species in the genus Methylocaldum is proposed, with the name Methylocaldum marinum sp. nov. The type strain is S8(T) ( = NBRC 109686(T) = DSM 27392(T)). An emended description of the genus Methylocaldum is also provided.

Methanohalophilus levihalophilus sp. nov., a slightly halophilic, methylotrophic methanogen isolated from natural gas-bearing deep aquifers, and emended description of the genus Methanohalophilus.

A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed.

Methyloceanibacter caenitepidi gen. nov., sp. nov., a facultatively methylotrophic bacterium isolated from marine sediments near a hydrothermal vent.

A moderately thermophilic, methanol-oxidizing bacterium (strain Gela4(T)) was isolated from methane-utilizing mixed-culture originating from marine sediment near a hydrothermal vent. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Gela4(T) was closely related to members of the genus 'Methyloligella' (94.7% similarity) within the class Alphaproteobacteria. Strain Gela4(T) was a Gram-staining-negative and aerobic organism. Cells were rod-shaped and non-motile. The temperature range for growth of strain Gela4(T) was 19-43 °C (optimal growth at 35 °C). Strain Gela4(T) tolerated up to 9% NaCl with an optimum at 1%. The organism was a facultative methylotroph that could utilize methanol, methylamine, trimethylamine and a variety of multi-carbon compounds. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63.9 mol%. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Methyloceanibacter caenitepidi is proposed, with Gela4(T) ( = NBRC 109540(T) = DSM 27242(T)) as the type strain.

Cultivation and biogeochemical analyses reveal insights into methanogenesis in deep subseafloor sediment at a biogenic gas hydrate site.

Gas hydrates deposited in subseafloor sediments are considered to primarily consist of biogenic methane. However, little evidence for the occurrence of living methanogens in subseafloor sediments has been provided. This study investigated viable methanogen diversity, population, physiology and potential activity in hydrate-bearing sediments (1-307 m below the seafloor) from the eastern Nankai Trough. Radiotracer experiments, the quantification of coenzyme F430 and molecular sequencing analysis indicated the occurrence of potential methanogenic activity and living methanogens in the sediments and the predominance of hydrogenotrophic methanogens followed by methylotrophic methanogens. Ten isolates and nine representative culture clones of hydrogenotrophic, methylotrophic and acetoclastic methanogens were obtained from the batch incubation of sediments and accounted for 0.5-76% of the total methanogenic sequences directly recovered from each sediment. The hydrogenotrophic methanogen isolates of Methanocalculus and Methanoculleus that dominated the sediment methanogen communities produced methane at temperatures from 4 to 55 °C, with an abrupt decline in the methane production rate at temperatures above 40 °C, which is consistent with the depth profiles of potential methanogenic activity in the Nankai Trough sediments in this and previous studies. Our results reveal the previously overlooked phylogenetic and metabolic diversity of living methanogens, including methylotrophic methanogenesis.

Evidence for syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the high-temperature petroleum reservoir of Yabase oil field (Japan).

The methanogenic communities and pathways in a high-temperature petroleum reservoir were investigated through incubations of the production water and crude oil, combined with radiotracer experiments and molecular biological analyses. The incubations were conducted without any substrate amendment and under high-temperature and pressurized conditions that mimicked the in situ environment (55°C, 5 MPa). Changes in methane and acetate concentrations during the incubations indicated stoichiometric production of methane from acetate. Rates of hydrogenotrophic methanogenesis measured using [(14)C]-bicarbonate were 42-68 times those of acetoclastic methanogenesis measured using [2-(14) C]-acetate, implying the dominance of methane production by syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the environment. 16S rRNA gene sequence analyses of the incubated production water showed bacterial communities dominated by the genus Thermacetogenium, known as a thermophilic syntrophic acetate-oxidizing bacterium, and archaeal communities dominated by thermophilic hydrogenotrophic methanogens belonging to the genus Methanothermobacter. Furthermore, group-specific real-time PCR assays revealed that 16S rRNA gene copy numbers of the hydrogenotrophic methanogens affiliated with the order Methanobacteriales were almost identical to those of archaeal 16S rRNA genes. This study demonstrates that syntrophic acetate oxidation is the main methanogenic pathway in a high-temperature petroleum reservoir.

A distinct freshwater-adapted subgroup of ANME-1 dominates active archaeal communities in terrestrial subsurfaces in Japan.

Anaerobic methane-oxidizing archaea (ANME) are known to play an important role in methane flux, especially in marine sediments. The 16S rRNA genes of ANME have been detected in terrestrial freshwater subsurfaces. However, it is unclear whether ANME are actively involved in methane oxidation in these environments. To address this issue, Holocene sediments in the subsurface of the Kanto Plain in Japan were collected for biogeochemical and molecular analysis. The potential activity of the anaerobic oxidation of methane (AOM) (0.38-3.54 nmol cm⁻³ day⁻¹) was detected in sediment slurry incubation experiments with a (13) CH(4) tracer. Higher AOM activity was observed in low-salinity treatment compared with high-salinity condition (20‰), which supports the adaptation of ANME in freshwater habitats. The 16S rRNA sequence analysis clearly revealed the presence of a distinct subgroup of ANME-1, designated ANME-1a-FW. Phylogenetic analysis of the mcrA genes also implied the presence of the distinct subgroup in ANME-1. ANME-1a-FW was found to be the most dominant active group in the archaeal communities on the basis of 16S rRNA analysis (75.0-93.8% of total archaeal 16S rRNA clones). Sulfate-reducing bacteria previously known as the syntrophic bacterial partners of ANME-1 was not detected. Our results showed that ANME-1a-FW is adapted to freshwater habitats and is responsible for AOM in terrestrial freshwater subsurface environments.

Development of a preclinical model of ischemic cardiomyopathy in swine.

A number of promising therapies for ischemic cardiomyopathy are emerging, and the role of translational research in testing the efficacy and safety of these agents in relevant clinical models has become important. The goal of this study was to develop a chronic model of ischemic cardiomyopathy in a large animal model. In this study, 40 consecutive pigs were initially enrolled. To induce progressive stenosis, a plastic occluder with a fixed diameter of 1.0 mm fitted with an 18-gauge copper wire was placed around the proximal left anterior descending (LAD) coronary artery. Coronary angiography, hemodynamic measurements, and echocardiography were performed at 2 wk and 1, 2, and 3 mo. Overall mortality was 26% at 3 mo, and up to 80% of the pigs showed total occlusion of LAD at 1 mo. A significant depression of peak LV pressure rate of rise (+dP/dt(max)) was observed in the animals showing total artery occlusion throughout the study. Left ventricular ejection fraction was also impaired, and the left ventricular volumes tended to be larger in the pigs with occlusion. Approximately 10% of scar tissue was found in the LAD occluded pigs, whereas the coronary flow pattern in the rest of the area took the pattern of hibernating myocardium. At the same time, histological and protein analysis established the presence of fibrosis and ongoing apoptosis in the ischemic area. In this model, the timing and incidence of total occlusion and low mortality offer significant advantages over other ischemic cardiomyopathy models in conducting preclinical studies.

Correction: Possible cross-feeding pathway of facultative methylotroph Methyloceanibacter caenitepidi Gela4 on methanotroph Methylocaldum marinum S8.

[This corrects the article DOI: 10.1371/journal.pone.0213535.].

Long-term wheel-running prevents reduction of grip strength in type 2 diabetic rats.

Diabetic skeletal muscles show reduced contractile force and increased fatigability. Hands are a target for several diabetes-induced complications. Therefore, reduced handgrip strength often occurs as a consequence of diabetes. The aim of this study was to examine whether long-term exercise can prevent reduction of grip strength in type 2 diabetes mellitus (T2DM) model OLETF rats, and to explore the mechanisms underlying diabetes-induced grip strength reduction. Ten 5-week-old OLETF rats were used as experimental animals, and five non-diabetic LETO rats as controls of OLETF rats. Half OLETF rats performed daily voluntary wheel-running for 17 months (OLETF + EXE), and the rest of OLETF and LETO rats were sedentary. Grip strength was higher in OLETF + EXE and LETO groups than in OLETF group. OLETF group with hyperglycemia showed an increase in HbA1c, serum TNF-α, and muscle SERCA activity, but a decrease in circulating insulin. Each fiber area, total fiber area, and % total fiber area in type IIb fibers of extensor digitorum longus muscles were larger in OLETF + EXE and LETO groups than in OLETF group. There was a positive correlation between grip strength and the above three parameters concerning type IIb fiber area. Therefore, type IIb fiber atrophy may be the major direct cause of grip strength reduction in OLETF group, although there seems multiple etiological mechanisms. Long-term wheel-running may have blocked the diabetes-induced reduction of grip strength by preventing type IIb fiber atrophy. Regular exercise may be a potent modality for preventing not only the progression of diabetes but muscle dysfunction in T2DM patients.

Koleobacter methoxysyntrophicus gen. nov., sp. nov., a novel anaerobic bacterium isolated from deep subsurface oil field and proposal of Koleobacteraceae fam. nov. and Koleobacterales ord. nov. within the class Clostridia of the phylum Firmicutes.

An anaerobic thermophilic, rod-shaped bacterium possessing a unique non-lipid sheathed-like structure enveloping a single-membraned cell, designated strain NRmbB1T was isolated from at the deep subsurface oil field located in Yamagata Prefecture, Japan. Growth occurred with 40-60°C (optimum, 55°C), 0-2% (2%), NaCl and pH 6.0-8.5 (8.0). Fermentative growth with various sugars was observed. Glucose-grown cells generated acetate, hydrogen, pyruvate and lactate as the main end products. Syntrophic growth occurred with glucose, pyruvate and 3,4,5-trimethoxybenzoate in the presence of an H2-scavenging partner, and growth on 3,4,5-trimethoxybenzoate was only observed under syntrophic condition. The predominant cellular fatty acids were C16:0, iso-C16:0, anteiso-C15:0, and iso-C14:0. Respiratory quinone was not detected. The genomic G+C content was 40.8mol%. Based on 16S rRNA gene phylogeny, strain NRmbB1T belongs to a distinct order-level clade in the class Clostridia of the phylum Firmicutes, sharing low similarity with other isolated organisms (i.e., 87.5% for top hit Moorella thermoacetica DSM 2955T). In total, chemotaxonomic, phylogenetic and genomic characterization revealed that strain NRmbB1T (=KCTC 25035T, =JCM 39120T) represents a novel species of a new genus. In addition, we also propose the associated family and order as Koleobacteraceae fam. nov and Koleobacterales ord. nov., respectively.

Methanogenic archaea use a bacteria-like methyltransferase system to demethoxylate aromatic compounds.

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth's diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.

Unexpected diversity of acetate degraders in anaerobic membrane bioreactor treating organic solid waste revealed by high-sensitivity stable isotope probing.

In anaerobic membrane bioreactor (AnMBR) treating organic solid waste, acetate is one of the most important precursors to CH4. However, the identity and diversity of anaerobic acetate degraders are largely unknown, possibly due to their slow growth rates and low abundances. Here, we identified acetate-degrading microorganisms in the AnMBR sludges by high-sensitivity stable isotope probing. Degradation of the amended 13C-acetate coincided with production of 13CH4 and 13CO2 during the sludge incubation. High-throughput sequencing of RNA density fractions indicated that the aceticlastic and hydrogenotrophic methanogens, i.e., Methanosaeta sp. (acetate dissimilator) and Methanolinea sp. (acetate assimilator), incorporated 13C-acetate significantly. Remarkably, 22 bacterial species incorporating 13C-acetate were identified, whereas their majority was distantly related to the cultured representatives. Only two of them were the class Deltaproteobacteria-affiliated lineages with syntrophic volatile fatty acid oxidation activities. Phylogenetic tree analysis and population dynamics tracing revealed that novel species of the hydrolyzing and/or fermenting taxa, such as the phyla Bacteroidetes, Chloroflexi and Lentisphaerae, exhibited low relative abundances comparable to that of Methanolinea sp. (0.00011%) during the AnMBR operation, suggesting that these bacteria were involved in anaerobic acetate assimilation. Meanwhile, novel species of the phyla Firmicutes, Synergistetes and Caldiserica, the candidate phyla Aminicenantes and Atribacteria and the candidate division GOUTA4-related clade, as well as the known Deltaproteobacteria members, existed at relatively high abundances (0.00031%-0.31121%) in the reactor, suggesting that these bacterial species participated in anaerobic dissimilation of acetate, e.g., syntrophic acetate oxidation. The results of this study demonstrated the unexpected diversity and ecophysiological features of the anaerobic acetate degraders in the AnMBR treating organic solid waste.