Stabilization of microbial network by co-digestion of swine manure and organic wastes.

The production of biogas from organic waste has attracted considerable interest as a solution to current energy and waste management challenges. This study explored the methane (CH4) production potential of swine manure (SM), food waste (FW), and tomato waste (TW) and the changes in the microbial community involved in the anaerobic digestion process. The results revealed that the CH4 production potentials of the four kinds of SM samples were influenced by the characteristics of SM (e.g., age and storage period). Among the four kinds of SM samples, the CH4 yield from the manure directly sampled from primiparous sows (SM3) was the highest. The CH4 yield was significantly improved when SM3 was co-digested with FW, but not with TW. The addition of SM fostered a stable CH4 production community by enhancing the interaction between methanogens and syntrophic bacteria. Furthermore, the addition of FW as a co-substrate may improve the functional redundancy structure of the methanogenesis-associated network. Overall, the characteristics of SM must be considered to achieve consistent CH4 yield efficiency from anaerobic digestion since CH4 production potentials of SM can be different. Also, the contribution of co-substrate to the synergistic relationship between methanogens and syntrophic bacteria can be considered when a co-substrate is selected in order to enhace CH4 yield from SM.

A simple protocol for cultivating the bacterivorous soil nematode Caenorhabditis elegans in its natural ecology in the laboratory.

The complex interplay between an animal and its surrounding environment requires constant attentive observation in natural settings. Moreover, how ecological interactions are affected by an animal's genes is difficult to ascertain outside the laboratory. Genetic studies with the bacterivorous nematode Caenorhabditis elegans have elucidated numerous relationships between genes and functions, such as physiology, behaviors, and lifespan. However, these studies use standard laboratory culture that does not reflect C. elegans true ecology. C. elegans is found growing in nature and reproduced in large numbers in soils enriched with rotting fruit or vegetation, a source of abundant and diverse microbes that nourish the thriving populations of nematodes. We developed a simple mesocosm we call soil-fruit-natural-habitat that simulates the natural ecology of C. elegans in the laboratory. Apples were placed on autoclaved potted soils, and after a soil microbial solution was added, the mesocosm was subjected to day-night, temperature, and humidity cycling inside a growth chamber. After a period of apple-rotting, C elegans were added, and the growing worm population was observed. We determined optimal conditions for the growth of C. elegans and then performed an ecological succession experiment observing worm populations every few days. Our data showed that the mesocosm allows abundant growth and reproduction of C. elegans that resembles populations of the nematode found in rotting fruit in nature. Overall, our study presents a simple protocol that allows the cultivation of C. elegans in a natural habitat in the laboratory for a broad group of scientists to study various aspects of animal and microbial ecology.

Determinants of microbial colonization on microplastics through wastewater treatment processes: The role of polymer type and sequential treatment.

This study examines the microbial colonization characteristics of microplastics (MPs) in wastewater treatment plants (WWTPs), focusing on polymer types (High-Density Polyethylene (HDPE) and Polyethylene Terephthalate (PET)) and various stages of wastewater treatments. Through individual and sequential deployment approaches, the research aimed to identify the determinants of bacterial colonization on MPs, whether they were introduced at each stage of treatment individually or in sequence from primary to tertiary stages. The study revealed that the stage of wastewater treatment profoundly influenced bacterial colonization on the polymer types MPs, with bacterial attachment being largely niche-specific. HDPE showed increased sensitivity to wastewater composition, leading to selective biofilm formation. For instance, in HDPE, Firmicutes accounted for 25.1 ± 0.04 % during primary treatment, while Alphaproteobacteria increased significantly in the tertiary treatment to 19.8 ± 0.1 %. Conversely, PET exhibited a stochastic pattern of bacterial colonization due to differences in surface hydrophilicity. Additionally, in sequential deployments, a notable shift towards stochastic bacterial attachment on MPs, particularly with HDPE was observed. The Shannon diversity values for MP biofilms were consistently higher than those for wastewater across all stages, with PET showing an increase in diversity in sequential deployments (Shannon diversity: 5.01 ± 0.03 for tertiary stage). These findings highlight the critical role of MPs as carriers of diverse bacteria, emphasizing the necessity for strategies to mitigate their impact in WWTPs. This study presents a significant advancement in our understanding of the interactions between MPs and microbial populations in WWTP environments.

Biochar derived from rice husk: Impact on soil enzyme and microbial dynamics, lettuce growth, and toxicity.

This study was set to investigate the effects of rice husk biochar (RHB) on soil characteristics and growth of lettuce (Lactuca sativa). A comprehensive research approach was employed to examine the effect of different RHB concentrations (i.e., 0-1.5%) on soil pH, soil enzyme activities (i.e., alkaline phosphatase, beta-glucosidase, and dehydrogenase), soil microbial community, lettuce growth, and earthworm toxicity. The results showed that, within the studied RHB concentration range, the RHB application did not have significant effects on the soil pH. However, the enzyme activities were increased with increasing RHB concentration after the 28 d-lettuce growth period. The RHB application also increased the abundances of the bacterial genera Massilia and Bacillus and fungal genus Trichocladium having the plant growth promoting abilities. Furthermore, the study revealed that the root weight and number of lettuce leaves were significantly increased in the presence of the RHB, and the growth was dependent on the RHB concentration. The improved lettuce growth can be explained by the changes in the enzyme and microbial dynamics, which have resulted from the increased nutrient availability with the RHB application. Additionally, the earthworm toxicity test indicated that the tested RHB concentrations can be safely applied to soil without any significant ecotoxicity. In conclusion, this study underscores the potential of RHB as a soil amendment with positive effects on crop growth, highlighting the utilization of agricultural byproducts to enhance soil biological quality and plant growth through biochar application.

Identification of Three Human POLH Germline Variants Defective in Complementing the UV- and Cisplatin-Sensitivity of POLH-Deficient Cells.

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1-432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3'-T and 5'-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants-substantially reduced in TLS activity-failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may increase the individual susceptibility to UV irradiation and cisplatin chemotherapy.

Continuous operation of fungal wheel reactor based on solid-state fermentation for the removal of pharmaceutical and personal care products.

Wood-rotting fungi and their enzymatic systems represent promising biocatalysts for the removal of pharmaceuticals and personal care products (PPCPs) from wastewater. We designed a fungal wheel reactor (FWR) based on solid-state fermentation (SSF) of Trametes versicolor and a lignocellulosic substrate, which was used as an immobilization carrier for fungal biomass and the sole initial nutrient source for producing fungal oxidative enzymes. Three pharmaceutical and personal care products, acetaminophen, bisphenol A and carbamazepine, were spiked into the synthetic wastewater and the treatment was carried out under non-sterile conditions. Acetaminophen was completely removed from the FWR until laccase was observed. The acetaminophen removal efficiency was retrieved by replacing the fungal wheel with fresh SSF products. Bisphenol A and carbamazepine were removed via enzymatic activity and adsorption. When the fungal wheel was replaced, acetaminophen began to be completely removed, even after laccase depletion. The microbial community analysis indicated that the continuous removal of acetaminophen was mainly due to the high proportion of T. versicolor. The relative abundance of the co-occurring microbial community might be responsible for the divergence in acetaminophen removal between two of fungal wheel-replaced reactors. Overall, FWRs are promising tools for the removal of PPCPs by highly reactive enzymatic mechanisms as well as adsorption on the carrier surface. By replacing SSF and settled microbial communities, FWRs may continuously contribute to bioremediation over a long-term period.

Phenotypic convergence of bacterial adaption to sub-lethal antibiotic treatment.

Microorganisms can adapt quickly to changes in their environment, leading to various phenotypes. The dynamic for phenotypic plasticity caused by environmental variations has not yet been fully investigated. In this study, we analyzed the time-series of phenotypic changes in Staphylococcus cells during adaptive process to antibiotics stresses using flow cytometry and Raman spectroscopy. The nine antibiotics with four different mode of actions were treated in bacterial cells at a sub-lethal concentration to give adaptable stress. Although the growth rate initially varied depending on the type of antibiotic, most samples reached the maximum growth comparable to the control through the short-term adaptation after 24 h. The phenotypic diversity, which showed remarkable changes depending on antibiotic treatment, converged identical to the control over time. In addition, the phenotype with cellular biomolecules converted into a bacterial cell that enhance tolerance to antibiotic stress with increases in cytochrome and lipid. Our findings demonstrated that the convergence into the phenotypes that enhance antibiotic tolerance in a short period when treated with sub-lethal concentrations, and highlight the feasibility of phenotypic approaches in the advanced antibiotic treatment.

Raman-Deuterium Isotope Probing and Metagenomics Reveal the Drought Tolerance of the Soil Microbiome and Its Promotion of Plant Growth.

Drought has become a major agricultural threat leading crop yield loss. Although a few species of rhizobacteria have the ability to promote plant growth under drought, the drought tolerance of the soil microbiome and its relationship with the promotion of plant growth under drought are scarcely studied. This study aimed to develop a novel approach for assessing drought tolerance in agricultural land by quantitatively measuring microbial phenotypes using stable isotopes and Raman spectroscopy. Raman spectroscopy with deuterium isotope probing was used to identify the Raman signatures of drought effects from drought-tolerant bacteria. Counting drought-tolerant cells by applying these phenotypic properties to agricultural samples revealed that 0% to 52.2% of all measured single cells had drought-tolerant properties, depending on the soil sample. The proportions of drought-tolerant cells in each soil type showed similar tendencies to the numbers of revived pea plants cultivated under drought. The phenotype of the soil microbiome and plant behavior under drought conditions therefore appeared to be highly related. Studying metagenomics suggested that there was a reliable link between the phenotype and genotype of the soil microbiome that could explain mechanisms that promote plant growth in drought. In particular, the proportion of drought-tolerant cells was highly correlated with genes encoding phytohormone production, including tryptophan synthase and isopentenyl-diphosphate delta-isomerase; these enzymes are known to alleviate drought stress. Raman spectroscopy with deuterium isotope probing shows high potential as an alternative technology for quantitatively assessing drought tolerance through phenotypic analysis of the soil microbiome. IMPORTANCE Soil microbiome has played a critical role in the plant survival during drought. However, the drought tolerance of soil microbiome and its ability to promote plant growth under drought is still scarcely studied. In this study, we identified the Raman signature (i.e., phenotype) of drought effects from drought-tolerant bacteria in agricultural soil samples using Raman-deuterium isotope probing (Raman-DIP). Moreover, the number of drought-tolerant cells measured by Raman-DIP was highly related to the survival rate of plant cultivation under drought and the abundance of genes encoding phytohormone production alleviating drought stress in plant. These results suggest Raman-DIP is a promising technology for measuring drought tolerance of soil microbiome. This result give us important insight into further studies of a reliable link between phenotype and genotype of soil microbiome for future plant-bacteria interaction research.

Flow cytometric monitoring of the bacterial phenotypic diversity in aquatic ecosystems.

Flow cytometry is a promising tool used to identify the phenotypic features of bacterial communities in aquatic ecosystems by measuring the physical and chemical properties of cells based on their light scattering behavior and fluorescence. Compared to molecular or culture-based approaches, flow cytometry is suitable for the online monitoring of microbial water quality because of its relatively simple sample preparation process, rapid analysis time, and high-resolution phenotypic data. Advanced statistical techniques (e.g., denoising and binning) can be utilized to successfully calculate phenotypic diversity by processing the scatter data obtained from flow cytometry. These phenotypic diversities were well correlated with taxonomic-based diversity computed using next-generation 16S RNA gene sequencing. The protocol provided in this paper should be a useful guide for a fast and reliable flow cytometric monitoring of bacterial phenotypic diversity in aquatic ecosystems.

Microbial phenomics linking the phenotype to fonction: The potential of Raman spectroscopy.

Raman spectroscopy is a promising tool for identifying microbial phenotypes based on single cell Raman spectra reflecting cellular biochemical biomolecules. Recent studies using Raman spectroscopy have mainly analyzed phenotypic changes caused by microbial interactions or stress responses (e.g., antibiotics) and evaluated the microbial activity or substrate specificity under a given experimental condition using stable isotopes. Lack of labelling and the nondestructive pretreatment and measurement process of Raman spectroscopy have also aided in the sorting of microbial cells with interesting phenotypes for subsequently conducting physiology experiments through cultivation or genome analysis. In this review, we provide an overview of the principles, advantages, and status of utilization of Raman spectroscopy for studies linking microbial phenotypes and functions. We expect Raman spectroscopy to become a next-generation phenotyping tool that will greatly contribute in enhancing our understanding of microbial functions in natural and engineered systems.

Three Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H2O2 Sensitivity in POLI-Deficient Cells.

Human Y-family DNA polymerase (pol) ι is involved in translesion DNA synthesis (TLS) and base excision repair (BER) of oxidative DNA damage. Genetic variations may alter the function of pol ι and affect cellular susceptibility to oxidative genotoxic agents, but their effects remain unclear. We investigated the impacts of 10 human missense germline variations on pol ι function by biochemical and cell-based assays. Both polymerase and deoxyribose phosphate (dRP) lyase activities were determined utilizing recombinant pol ι (residues 1-445) proteins. The K209Q, K228I, and Q386R variants showed 4- to 53-fold decreases in specificity constants (kcat/Km) for dCTP insertion opposite G and 8-oxo-7,8-dihydroguanine compared to the wild-type. The R126C and K345E variants showed wild-type-like polymerase activity, although these two variants (as well as the R209Q, K228I, and Q386R variants) showed greater than 6-fold decreases in dRP lyase activity compared to the wild-type. A CRISPR/Cas9-mediated POLI knockout conferred higher sensitivity to H2O2 in human embryonic kidney (HEK293) cells. Exogenous expression of the full-length wild-type, R126C, and K345E variants fully rescued the H2O2 sensitivity in POLI-deficient cells, while full-length R209Q, K228I, and Q386R variants did not rescue the sensitivity. Our results indicate that the R126C and K345E variants (having wild-type-like polymerase activity, albeit impaired in dRP lyase activity) could fully rescue the H2O2 sensitivity in POLI-deficient cells, while the R209Q, K228I, and Q386R variants, all impaired in polymerase and dRP lyase activity, failed to rescue the sensitivity, indicating the relative importance of TLS-related polymerase function of pol ι rather than its BER-related dRP lyase function in protection from oxidative stress. The possibility exists that the hypoactive pol ι variants increase the individual susceptibility to oxidative genotoxic agents.

Sequential biowashing-biopile processes for remediation of crude oil contaminated soil in Kuwait.

The application of biological processes for remediation of the aged crude oil-contaminated soil of Kuwait can be an inefficient way, thus, this study developed 20 d-sequential biowashing and biopile processes where the biowashing step uses an enrichment culture of the indigenous soil bacterial community and the biopile step includes hemoglobin-catalyzed oxidation (HCO). The residual total petroleum hydrocarbons (TPH) concentrations and CO2 generation were measured to determine the removal efficiency, and the bacterial community changes were studied to investigate the effect of the sequential processes on the soil indigenous bacterial community. The enrichment culture grown on hemoglobin showed an increased surface activity, and this promoted desorption and emulsification of crude oil from the soil sample in the biowashing step resulting in 75% TPH removal. Potential surfactant-producing bacterial species were observed in the soil sample after biowashing. The HCO in the beginning of the biopile step removed 21% of the residual TPH, and further TPH removal was observed with a longer biopile period. Overall, the sequential biowashing and biopile processes removed 86% TPH. The results show that the developed sequential biowashing and biopile processes can be used to efficiently remediate the aged crude oil-contaminated soil of Kuwait.

Interaction among soil physicochemical properties, bacterial community structure, and arsenic contamination: Clay-induced change in long-term arsenic contaminated soils.

Pyrosequencing analyses to determine soil bacterial communities were conducted with forty-two soil samples collected from rice paddy and forest/farmland soils (Group A and B, respectively) at a long-term As-contaminated site. Soil physicochemical properties, such as the concentrations of As, Fe, Al, and Mn, pH, organic matter content, and clay content, were found to be significantly different with land use, and more importantly, strongly affected the bacterial community structure of the soil samples. When fitting the soil properties onto a nonmetric multidimensional scale plot of soil bacterial communities, clay content was found to be the most important factor in clustering the bacterial communities (R2 = 0.4831, p-value = 0.001). Phylum Chloroflexi (-1.03 of bioplot score) and Planctomycetes (1.31 of bioplot score) showed a significant relationship with clay content in soil samples. Interestingly, thebacterial phylotypes linked to clay content were only found in the soil samples of group B with low clay content, and had a strong relationship to As contamination in the redundancy analysis and the correlation analysis.Our results suggest that clay content seems to be negatively related to As contamination in soils, which, in turn, strongly influences the structure of bacterial communities in As-contaminated soil.

Role of hemoglobin in hemoglobin-based remediation of the crude oil-contaminated soil.

This study investigated the changes in the indigenous microbial community structure with hemoglobin (Hb) application to determine the role of Hb in Hb-based remediation of crude oil-contaminated soil. The phylogenetic diversity of the bacterial community showed that the Hb addition selected surfactants-producing species, thereby, promoting TPH degradation. The significant increase in the CO2 generation, which can be related to the increase in the bacterial abundance inferred from the 16S rRNA gene copy number, supports the enhanced TPH degradation with Hb application. The similar residual TPH concentrations in the presence of only hydrogen peroxide (H2O2) and both Hb and H2O2 suggested that the role of Hb as a catalyst was not as significant as the role of Hb as a nutrient. Also, in the presence of H2O2, a greater recovery of the microbial community structure was observed with the double Hb injection than the single Hb injection. Overall, this study shows that the Hb-based remediation strategies via microbial metabolism can be successfully applied to remediate the crude-oil contaminated Kuwaiti soil.

Pedobacter solisilvae sp. nov., isolated from forest soil.

The bacteria strain EN12T was isolated from forest soil in the Republic of Korea. The cells were Gram-negative, non-motile and rod-shaped, and the strain was strictly aerobic. Phylogenetic analysis of its 16S rRNA gene sequences showed that strain EN12T belonged to the class Sphingobacteriia of the phylum Bacteroidetes, and its closest relative is Pedobacter namyangjuensis 5G38T, with a sequence similarity of 95.5 %. The average DNA sequence similarity from validly described species within the genus Pedobacter was 92.5±1.3 %. Chemotaxonomic data including major ubiquinones (menaquinone-7), polar lipids (phosphatidylethanolamine and sphingolipid) and fatty acids (iso-C15 : 0, iso-C17 : 0 3-OH, and C16 : 1ω6c/C16 : 1ω7c) also supported an affiliation of strain EN12T with the genus Pedobacter. Genotypic and phenotypic differentiation of strain EN12T from six published Pedobacter species was revealed through DNA-DNA relatedness and physiological/biochemical tests. Results of these phenotypic, phylogenetic and chemotaxonomic analyses indicated that strain EN12T is a novel species in the genus Pedobacter, for which we propose the name Pedobacter solisilvae sp. nov. (=KCTC 42612T=LMG 28820T).

Chitinimonas lacunae sp. nov., isolated from artificial pond in Korea.

A gram-negative, rod shaped bacterium designated as strain H2T was isolated from an artificial pond in Korea. The strain H2T was able to grow aerobically and anaerobically with optimal growth occurring at 30 °C and pH 7.0 under aerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain H2T belonged to the genus Chitinimonas of the family Burkholderiaceae. Phylogenetic similarity calculated from 16S rRNA gene sequences of strain H2T and valid species belongs to the genus Chitinimonas ranged from 93.2 % (for Chitinimonas taiwanensis cfT) to 94.4 % (for Chitinimonas prasina LY03T), and strain H2T formed a tight monophyletic group with them. Predominant fatty acids were C16 : 0 and summed feature 3, which consisted of C16 : 1ω6c and/or C16 : 1ω7c. The major respiratory quinone of the strain H2T was ubiquinone-8, and DNA G+C content was 60.2 %. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, unidentified aminolipid, and unidentified phospholipid. The biochemical characteristics that distinguished strain H2T from other Chitinimonas species included positive cystine arylamidase activity and lacked α-chymotrypsin and ß-glucosidase (aesculin hydrolysis) activity. In addition, reciprocal DNA-DNA relatedness between H2T and three Chitinimonas strains ranged from 32.0 to 43.7 %. On the basis of its phylogenetic, chemotaxonomic, and genotypic characteristics, strain H2T represents a novel species of the genus Chitinimonas. Chitinimonas lacunae sp. nov. is proposed with the type strain H2T (=KCTC 52574T=LMG 29894T).

Correction: Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences.

[This corrects the article DOI: 10.1371/journal.pone.0096197.].

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ.

DNA polymerase (pol) κ efficiently catalyzes error-free translesion DNA synthesis (TLS) opposite bulky N2-guanyl lesions induced by carcinogens such as polycyclic aromatic hydrocarbons. We investigated the biochemical effects of nine human nonsynonymous germline POLK variations on the TLS properties of pol κ, utilizing recombinant pol κ (residues 1-526) enzymes and DNA templates containing an N2-CH2(9-anthracenyl)G (N2-AnthG), 8-oxo-7,8-dihydroguanine (8-oxoG), O6-methyl(Me)G, or an abasic site. In steady-state kinetic analyses, the R246X, R298H, T473A, and R512W variants displayed 7- to 18-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to that of the wild-type, and further showed 5- to 190-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Km for correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to that of the wild-type. These five hypoactive variants also showed similar patterns of attenuation of TLS activity opposite 8-oxoG, O6-MeG, and abasic lesions. By contrast, the T44M variant exhibited 7- to 11-fold decreases in kcat/Km for dCTP insertion opposite N2-AnthG and O6-MeG (as well as for dATP insertion opposite an abasic site) but not opposite both G and 8-oxoG, nor beyond N2-AnthG, compared to that of the wild-type. These results suggest that the R246X, R298H, T473A, R512W, and A471V variants cause a general catalytic impairment of pol κ opposite G and all four lesions, whereas the T44M variant induces opposite lesion-dependent catalytic impairment, i.e., only opposite O6-MeG, abasic, and bulky N2-G lesions but not opposite G and 8-oxoG, in pol κ, which might indicate that these hypoactive pol κ variants are genetic factors in modifying individual susceptibility to genotoxic carcinogens in certain subsets of populations.

Environmental Variables Shaping the Ecological Niche of Thaumarchaeota in Soil: Direct and Indirect Causal Effects.

To find environmental variables (EVs) shaping the ecological niche of the archaeal phylum Thaumarchaeota in terrestrial environments, we determined the abundance of Thaumarchaeota in various soil samples using real-time PCR targeting thaumarchaeotal 16S rRNA gene sequences. We employed our previously developed primer, THAUM-494, which had greater coverage for Thaumarchaeota and lower tolerance to nonthaumarchaeotal taxa than previous Thaumarchaeota-directed primers. The relative abundance estimates (RVs) of Thaumarchaeota (RTHAUM), Archaea (RARCH), and Bacteria (RBACT) were subjected to a series of statistical analyses. Redundancy analysis (RDA) showed a significant (p < 0.05) canonical relationship between RVs and EVs. Negative causal relationships between RTHAUM and nutrient level-related EVs were observed in an RDA biplot. These negative relationships were further confirmed by correlation and regression analyses. Total nitrogen content (TN) appeared to be the EV that affected RTHAUM most strongly, and total carbon content (TC), which reflected the content of organic matter (OM), appeared to be the EV that affected it least. However, in the path analysis, a path model indicated that TN might be a mediator EV that could be controlled directly by the OM. Additionally, another path model implied that water content (WC) might also indirectly affect RTHAUM by controlling ammonium nitrogen (NH4+-N) level through ammonification. Thus, although most directly affected by NH4+-N, RTHAUM could be ultimately determined by OM content, suggesting that Thaumarchaeota could prefer low-OM or low-WC conditions, because either of these EVs could subsequently result in low levels of NH4+-N in soil.

Novel PCR primers for the archaeal phylum Thaumarchaeota designed based on the comparative analysis of 16S rRNA gene sequences.

Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb) archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7%) belonged to the MG-I and SCG, which together contained most (93.9%) of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer's specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and <1% tolerance to non-target taxa, indicating high specificity. To validate this result experimentally, PCRs were performed with THAUM-494 in combination with a universal archaeal primer (ARC917R or 1017FAR) and DNAs from five environmental samples to construct clone libraries. THAUM-494 showed a satisfactory specificity in empirical studies, as expected from the in silico results. Phylogenetic analysis of 859 cloned sequences obtained from 10 clone libraries revealed that >95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and ecological niche of Thaumarchaeota.