Archaea-An International Microbiological Journal最新文献

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability of Methanosarcina thermophila to use carbon dioxide (CO₂) for catabolic and anabolic metabolism was not proven until now. Here, we show that M. thermophila used CO₂ to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO₂ consecutively, resulting in a biphasic methane production. Growth exclusively from CO₂ occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from ¹³CO₂ into ¹³CH₄ molecules was measured to validate the obtained data. New insights into the physiology of M. thermophila can serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

Microorganisms play an essential role in the performance of constructed wetlands (CWs) for wastewater treatment. However, there has been limited discussion on the characteristics of microbial communities in CWs for treatment of effluents from marine recirculating aquaculture systems (RAS). This study is aimed at characterizing the microbial communities of pilot-scale CWs with Salicornia bigelovii for treatment of saline wastewater from a land-based Atlantic salmon RAS plant located in Northern China. Illumina high-throughput sequencing was employed to identify the profile of microbial communities of three CWs receiving wastewater under different total ammonia nitrogen (TAN) concentrations. Results of this study showed remarkable spatial variations in diversity and composition of microbial communities between roots and substrates in three CWs, with distinct response to different TAN concentrations. In particular, Proteobacteria, Firmicutes, Cyanobacteria, and Bacteroidetes were predominant in roots, while Cyanobacteria, Proteobacteria, Firmicutes, Verrucomicrobia, and Bacteroidetes were prevalent in substrates. Moreover, redundancy analysis indicated that specific functional genera, such as Nitrosopumilus, Vibrio, Pseudoalteromonas, Nitrospina, and Planctomyces, played key roles in the removal of nitrogen/phosphorus pollutants and growth of wetland plants. From a microorganism perspective, the findings of this study could contribute to better understanding of contaminants' removal mechanism and improved management of CWs for treatment of effluents from land-based marine aquaculture.

An integrated anaerobic fluidized-bed membrane bioreactor (IAFMBR) was investigated to treat synthetic high-strength benzothiazole wastewater (50 mg/L) at a hydraulic retention time (HRT) of 24, 18, and 12 h. The chemical oxygen demand (COD) removal efficiency (from 93.6% to 90.9%), the methane percentage (from 70.9% to 69.27%), and the methane yield (from 0.309 m³ CH₄/kg·COD_removed to 0.316 m³ CH₄/kg·COD_removed) were not affected by decreasing HRTs. However, it had an adverse effect on membrane fouling (decreasing service period from 5.3 d to 3.2 d) and benzothiazole removal efficiency (reducing it from 97.5% to 82.3%). Three sludge samples that were collected on day 185, day 240, and day 297 were analyzed using an Illumina® MiSeq platform. It is striking that the dominant genus of archaea was always Methanosaeta despite of HRTs. The proportions of Methanosaeta were 80.6% (HRT 24), 91.9% (HRT 18), and 91.2% (HRT 12). The dominant bacterial genera were Clostridium in proportions of 23.9% (HRT 24), 16.4% (HRT 18), and 15.3% (HRT 12), respectively.

Polyphosphates (PolyP) are linear polymers of orthophosphate residues that have been proposed to participate in metal resistance in bacteria and archaea. In addition of having a CopA/CopB copper efflux system, the thermoacidophilic archaeon Metallosphaera sedula contains electron-dense PolyP-like granules and a putative exopolyphosphatase (PPX _Msed , Msed_0891) and four presumed pho84-like phosphate transporters (Msed_0846, Msed_0866, Msed_1094, and Msed_1512) encoded in its genome. In the present report, the existence of a possible PolyP-based copper-resistance mechanism in M. sedula DSM 5348^T was evaluated. M. sedula DSM 5348^T accumulated high levels of phosphorous in the form of granules, and its growth was affected in the presence of 16 mM copper. PolyP levels were highly reduced after the archaeon was subjected to an 8 mM CuSO₄ shift. PPX _Msed was purified, and the enzyme was found to hydrolyze PolyP in vitro. Essential residues for catalysis of PPX _Msed were E111 and E113 as shown by a site-directed mutagenesis of the implied residues. Furthermore, M. sedula ppx, pho84-like, and copTMA genes were upregulated upon copper exposure, as determined by qRT-PCR analysis. The results obtained support the existence of a PolyP-dependent copper-resistance system that may be of great importance in the adaptation of this thermoacidophilic archaeon to its harsh environment.

Tetramethylammonium-degrading methanogenic consortia from a complete-mixing suspended sludge (CMSS) and an upflow anaerobic sludge blanket (UASB) reactors were studied using multiple PCR-based molecular techniques and shotgun proteomic approach. The prokaryotic 16S rRNA genes of the consortia were analyzed by quantitative PCR, high-throughput sequencing, and DGGE-cloning methods. The results showed that methanogenic archaea were highly predominant in both reactors but differed markedly according to community structure. Community and proteomic analysis revealed that Methanomethylovorans and Methanosarcina were the major players for the demethylation of methylated substrates and methane formation through the reduction pathway of methyl-S-CoM and possibly, acetyl-CoA synthase/decarbonylase-related pathways. Unlike high dominance of one Methanomethylovorans population in the CMSS reactor, diverse methylotrophic Methanosarcina species inhabited in syntrophy-like association with hydrogenotrophic Methanobacterium in the granular sludge of UASB reactor. The overall findings indicated the reactor-dependent community structures of quaternary amines degradation and provided microbial insight for the improved understanding of engineering application.

Many hydrogenotrophic methanogens use either H₂ or formate as the major electron donor to reduce CO₂ for methane production. The conventional cultivation of these organisms uses H₂ and CO₂ as the substrate with frequent replenishment of gas during growth. H₂ is explosive and requires an expensive gassing system to handle safely. Formate is as an ideal alternative substrate from the standpoints of both economy and safety but leads to large changes in the culture pH during growth. Here, we report that glycylglycine is an inexpensive and nontoxic buffer suitable for growth of Methanococcus maripaludis and Methanothermococcus okinawensis. This cultivation system is suitable for growth on liquid as well as solid medium in serum bottles. Moreover, it allows cultivation of liter scale cultures without expensive fermentation equipment. This formate cultivation system provides an inexpensive and flexible alternative for the growth of formate-utilizing, hydrogenotrophic methanogens.

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC from Methanobrevibacter millerae SM9 was cloned and expressed in Escherichia coli and biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen for α-ketoglutarate. Optimal activity was observed at pH 6.5. The apparent K_M for coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate the V_max was 93.9 μmol min^-1 mg^-1 and k_cat was 62.8 s^-1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

DNA sequence analysis of the human gut revealed the presence a seventh order of methanogens referred to as Methanomassiliicoccales. Methanomassiliicoccus luminyensis is the only member of this order that grows in pure culture. Here, we show that the organism has a doubling time of 1.8 d with methanol + H₂ and a growth yield of 2.4 g dry weight/mol CH₄. M. luminyensis also uses methylamines + H₂ (monomethylamine, dimethylamine, and trimethylamine) with doubling times of 2.1-2.3 d. Similar cell yields were obtained with equimolar concentrations of methanol and methylamines with respect to their methyl group contents. The transcript levels of genes encoding proteins involved in substrate utilization indicated increased amounts of mRNA from the mtaBC2 gene cluster in methanol-grown cells. When methylamines were used as substrates, mRNA of the mtb/mtt operon and of the mtmBC1 cluster were found in high abundance. The transcript level of mtaC2 was almost identical in methanol- and methylamine-grown cells, indicating that genes for methanol utilization were constitutively expressed in high amounts. The same observation was made with resting cells where methanol always yielded the highest CH₄ production rate independently from the growth substrate. Hence, M. luminyensis is adapted to habitats that provide methanol + H₂ as substrates.

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 10⁴ ± 6.9 × 10³ colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

Groundwater environments provide habitats for diverse microbial communities, and although Archaea usually represent a minor fraction of communities, they are involved in key biogeochemical cycles. We analysed the archaeal diversity within a mixed carbonate-rock/siliciclastic-rock aquifer system, vertically from surface soils to subsurface groundwater including aquifer and aquitard rocks. Archaeal diversity was also characterized along a monitoring well transect that spanned surface land uses from forest/woodland to grassland and cropland. Sequencing of 16S rRNA genes showed that only a few surface soil-inhabiting Archaea were present in the groundwater suggesting a restricted input from the surface. Dominant groups in the groundwater belonged to the marine group I (MG-I) Thaumarchaeota and the Woesearchaeota. Most of the groups detected in the aquitard and aquifer rock samples belonged to either cultured or predicted lithoautotrophs (e.g., Thaumarchaeota or Hadesarchaea). Furthermore, to target autotrophs, a series of ¹³CO₂ stable isotope-probing experiments were conducted using filter pieces obtained after filtration of 10,000 L of groundwater to concentrate cells. These incubations identified the SAGMCG Thaumarchaeota and Bathyarchaeota as groundwater autotrophs. Overall, the results suggest that the majority of Archaea on rocks are fixing CO₂, while archaeal autotrophy seems to be limited in the groundwater.