Archaeal 16S rRNA Gene Copies Research Articles

Hyperarid soil microbial community response to simulated rainfall.

The exceptionally long and protracted aridity in the Atacama Desert (AD), Chile, provides an extreme, terrestrial ecosystem that is ideal for studying microbial community dynamics under hyperarid conditions. Our aim was to characterize the temporal response of hyperarid soil AD microbial communities to ex situ simulated rainfall (5% g water/g dry soil for 4 weeks) without nutrient amendment. We conducted replicated microcosm experiments with surface soils from two previously well-characterized AD hyperarid locations near Yungay at 1242 and 1609 masl (YUN1242 and YUN1609) with distinct microbial community compositions and average soil relative humidity levels of 21 and 17%, respectively. The bacterial and archaeal response to soil wetting was evaluated by 16S rRNA gene qPCR, and amplicon sequencing. Initial YUN1242 bacterial and archaeal 16S rRNA gene copy numbers were significantly higher than for YUN1609. Over the next 4 weeks, qPCR results showed significant increases in viable bacterial abundance, whereas archaeal abundance decreased. Both communities were dominated by 10 prokaryotic phyla (Actinobacteriota, Proteobacteria, Chloroflexota, Gemmatimonadota, Firmicutes, Bacteroidota, Planctomycetota, Nitrospirota, Cyanobacteriota, and Crenarchaeota) but there were significant site differences in the relative abundances of Gemmatimonadota and Chloroflexota, and specific actinobacterial orders. The response to simulated rainfall was distinct for the two communities. The actinobacterial taxa in the YUN1242 community showed rapid changes while the same taxa in the YUN1609 community remained relatively stable until day 30. Analysis of inferred function of the YUN1242 microbiome response implied an increase in the relative abundance of known spore-forming taxa with the capacity for mixotrophy at the expense of more oligotrophic taxa, whereas the YUN1609 community retained a stable profile of oligotrophic, facultative chemolithoautotrophic and mixotrophic taxa. These results indicate that bacterial communities in extreme hyperarid soils have the capacity for growth in response to simulated rainfall; however, historic variations in long-term hyperaridity exposure produce communities with distinct putative metabolic capacities.

Excellent excrement? Frass impacts on a soil's microbial community, processes and metal bioavailability

The commercial rearing of insects is a growing economic sector. Therefore, an assessment was made of the potential of its by-product, frass, to be a soil improver. Essential plant nutrients were extracted (using 0.01 M CaCl2 or Mehlich 3) from frass of mealworms (Tenebrio molitor), black soldier flies (Hermetia illucens) and buffalo worms (Alphitobius diaperinus). A 28-day incubation, in which frass was added to a sandy loam soil at application rates of 2.5% or 5% (w/w), assessed its effects on soil microbial biomass, abundance of bacteria, archaea and fungi, carbon mineralisation and nitrification. In a separate 56-day incubation, the impact of frass on heavy metal bioavailability in an artificially contaminated, carbon-poor substrate was tested. All frass types featured high electrical conductivity, a mildly acidic to neutral pH and C: N ratios between 11 and 16. Black soldier fly frass (BSFF) was richer in extractable ammonium, phosphorus, potassium and magnesium than mealworm frass (MWF) and buffalo worm frass (BWF) but poorer in extractable calcium. All frass types stimulated carbon mineralisation, nitrification, bacterial and archaeal 16S rRNA gene copy numbers, and fungal biomass as determined by ergosterol concentrations. Bacterial and particularly fungal abundances were stimulated by the 5% frass application rate whereas archaeal abundances were greater in the 2.5% application rate regimes. The 2.5% application rate of MWF and BWF led to a profound build-up of soil extractable nitrite. Correspondingly, these treatments featured the highest 16S rRNA gene copy numbers of archaea, a domain encompassing organisms which oxidise ammonium to nitrite. No nitrite was detectable in soil amended with BSFF. The 5% application rates induced microbial biomass growth (as determined by extractable DNA concentrations) only when BSFF was applied. This was possibly due to differences in the frass types' extractable nutrient or labile carbon contents. BSFF and BWF amendment led to significantly higher microbial biomass in a metal-contaminated substrate. This was likely due to frass providing nutrients, energy and reduced metal bioavailability: extractable zinc, copper, cadmium and nickel concentrations fell due to increased metal sorption and complexation. All frass types could be used as ameliorants in metal-contaminated soils, while BSFF shows most promise as an organic fertiliser as its use did not cause soil nitrite build-up.

Prokaryotic communities of technozems of the spoil heaps of Kursk magnetic anomaly

Background. Spoil heaps chronosequences are convenient models to analyze the succession of microbiome during restoration of anthropogenically disturbed landscapes. The investigation of the heavy metal content in lands with mining activity, can be used as an indicator of ecosystem recovery.
 Materials and methods. Objects were technozems of 1-year, 25- and 50-year-old embryonic soils, and control soil under forest. Quantitative polymerase chain reaction (qPCR) and NGS-sequencing of V4 region of 16S rRNA gene were applied. Results. During the soil-forming process, an increase organic carbon and nitrogen, as well as a gradual increase archaeal 16S rRNA gene copies and in the number of Bradyrhizobiaceae, Blastocatellaceae, Xantobacteriaceae. Although we found a number of taxa that increased during soil-forming process (Thaumarchaeota, Bradyrhizobiaceae, Blastocatellaceae, Xantobacteriaceae), technozems of different ages had a similar structure and diversity of prokaryotic communities, differing from a nature soil. Biodiversity analysis revealed that technozems generally had a similar structure and diversity of prokaryotic communities, significantly differing from the mature soil a specific clusterization of microbiomes. The HM contents and bacterial abundances remained at the same level in chronosequence.
 Conclusions. The 50 years of soil development on overburden spoil heaps is not enough for the recovery from HM contamination and restoration of soil ecosystem functioning.

Spatial variations in bacterial and archaeal abundance and community composition in boreal forest pine mycorrhizospheres

Mycorrhizal fungi have a strong impact on soil biota. In this study, bacterial and archaeal populations in different parts of Suillus bovinus - Pinus sylvestris mycorrhizospheres in boreal forest were quantified and identified by DNA analysis. The numbers of bacterial and archaeal 16S rRNA gene copies were highest in uncolonized humus and lowest in fruiting bodies. The numbers of bacterial 16S rRNA gene copies varied from 1.3 × 107 to 3.1 × 109 copies g−1 fw and archaeal copies from 4.1 × 107 to 9.6 × 108 copies g−1 fw. The relatively high number of archaeal 16S rRNA gene copies was likely due to the cold and highly organic habitat. The presence of hyphae appeared to further promote archaeal numbers and the archaea:bacteria ratio was over one in samples containing only fungal material. Most detected archaea belonged to terrestrial Thaumarchaeota. Proteobacteria, Actinobacteria and Acidobacteria were predictably the dominating bacterial taxa in the samples with clear trend of Betaproteobacteria preferring the pine root habitats.

Response of freshwater sediment archaeal community to metal spill

Archaea play an important role in the biogeochemical cycling of elements in the environment. Heavy metals are ubiquitous pollutants in the environment. Previous studies have revealed a considerable influence of metal pollution on the archaeal community, but the short-term response of the archaeal community to metal pollution remains unclear. Hence, the present study investigated the short versus long-term responses of overall archaeal communities in freshwater sediments after exposure to accidental metal pollution caused by the discharge of heavy metal-containing wastewater from an indium-producing factory. Quantitative PCR was used to determine the archaeal abundance, while Illumina MiSeq sequencing was applied to characterize the diversity and structure of the archaeal community. The abundance (2.47 × 105–1.55 × 108 archaeal 16S rRNA gene copies per gram dry sediment), diversity (Shannon diversity index = 2.49–4.45) and structure of overall archaeal community illustrated a drastic temporal change. The archaeal communities mainly comprised the phyla Euryarchaeota, Thaumarchaeota and Bathyarchaeota. The exposure to metal pollution induced an increase in the proportion of Euryarchaeota but lowered the proportion of Thaumarchaeota. The accidental metal pollution exerted a profound impact on the archaeal community in freshwater sediment. This study could contribute our understanding of the short versus long-term response of archaeal communities to metal pollution.

Archaea are prominent members of the prokaryotic communities colonizing common forest mushrooms.

In this study, the abundance and composition of prokaryotic communities associated with the inner tissue of fruiting bodies of Suillus bovinus, Boletus pinophilus, Cantharellus cibarius, Agaricus arvensis, Lycoperdon perlatum, and Piptoporus betulinus were analyzed using culture-independent methods. Our findings indicate that archaea and bacteria colonize the internal tissues of all investigated specimens and that archaea are prominent members of the prokaryotic community. The ratio of archaeal 16S rRNA gene copy numbers to those of bacteria was >1 in the fruiting bodies of four out of six fungal species included in the study. The largest proportion of archaeal 16S rRNA gene sequences belonged to thaumarchaeotal classes Terrestrial group, Miscellaneous Crenarchaeotic Group (MCG), and Thermoplasmata. Bacterial communities showed characteristic compositions in each fungal species. Bacterial classes Gammaproteobacteria, Actinobacteria, Bacilli, and Clostridia were prominent among communities in fruiting body tissues. Bacterial populations in each fungal species had different characteristics. The results of this study imply that fruiting body tissues are an important habitat for abundant and diverse populations of archaea and bacteria.

Comparative analyses of microbial structures and gene copy numbers in the anaerobic digestion of various types of sewage sludge

Anaerobic co-digestion of various sewage sludges is a promising approach for greater recovery of energy, but the process is more complicated than mono-digestion of sewage sludge. The applicability of microbial structure analyses and gene quantification to understand microbial conditions was evaluated. The results show that information from gene analyses is useful in managing anaerobic co-digestion and damaged microbes in addition to conventional parameters like total solids, pH and biogas production. Total bacterial 16S rRNA gene copy numbers are the most useful tools for evaluating unstable anaerobic digestion of sewage sludge, rather than mcrA and total archaeal 16S rRNA gene copy numbers, and high-throughput sequencing. First order decay rates of gene copy numbers during pH failure were higher than typical decay rates of microbes in stable operation. The sequencing analyses, including multidimensional scaling, showed very different microbial structure shifts, but the results were not consistent.

Comparison of Archaeal Populations in Soil and Their Encapsulated Iron-Manganese Nodules in Four Locations Spanning from North to South China

ABSTRACTTo characterize the archaeal community composition in soil originating iron-manganese nodules, four types of soils—brown soil, yellow-cinnamon soil, yellow brown soil and red soil—and their associated iron-manganese nodules were collected from Queyu (QY), Zaoyang (ZY), Wuhan (WH) and Guiyang (GY), China, respectively, and subjected to quantitative polymerase chain reaction, cloning and sequencing analyses. The results showed that the archaeal 16S rRNA gene copy numbers in nodules, ranging between 3.59 × 102 and 4.17 × 103 copies g−1 dry nodule, were about 50–1000 times lower than those in their corresponding soils (1.87 × 105 to 1.08 × 106 copies g−1 dry soil), correlating with the low organic matter in the nodules, while archaea accounted for a relatively higher proportion of total prokaryote in nodules than in soils. Community composition analysis suggested that the archaeal diversity in both soils and nodules were much lower than bacterial, but archaeal community structures were similar to each other among the soils and nodules from the same location but varied among four locations, converse to the previous observation that bacterial community shifted markedly between nodules and soils as the result of habitat filtering. The archaeal communities in both soils and nodules were predominated by Thaumarchaeota Group I.1b with the relative abundance ranging between 73.88 and 94.17%, except that Euryarchaeota dominated the archaeal community in one nodule sample (WHn) developed from lake sediment. The finding shed new light on the archaeal diversity and their ecophysiology in different habitats, and further supported the opinion that archaea are more adaptable to stress and unfavorable conditions.

Influence of land use on bacterial and archaeal diversity and community structures in three natural ecosystems and one agricultural soil.

Studying shifts in microbial communities under different land use can help in determining the impact of land use on microbial diversity. In this study, we analyzed four different land-use types to determine their bacterial and archaeal diversity and abundance. Three natural ecosystems, that is, wetland (WL), grassland (GL), and forest (FR) soils, and one agricultural soil, that is, tea plantation (TP) soil, were investigated to determine how land use shapes bacterial and archaeal diversity. For this purpose, molecular analyses, such as quantitative polymerase chain reaction (Q-PCR), 16S rRNA gene sequencing, and terminal restriction fragment length polymorphism (T-RFLP), were used. Soil physicochemical properties were determined, and statistical analyses were performed to identify the key factors affecting microbial diversity in these soils. Phylogenetic affiliations determined using the Ribosomal Database Project (RDP) database and T-RFLP revealed that the soils had differing bacterial diversity. WL soil was rich in only Proteobacteria, whereas GR soil was rich in Proteobacteria, followed by Actinobacteria. FR soil had higher abundance of Chloroflexi species than these soils. TP soil was rich in Actinobacteria, followed by Chloroflexi, Acidobacteria, Proteobacteria, and Firmicutes. The archaeal diversity of GL and FR soils was similar in that most of their sequences were closely related to Nitrososphaerales (Thaumarchaeota phylum). In contrast, WL soil, followed by TP soil, had greater archaeal diversity than other soils. Eight different archaeal classes were found in WL soil, and Pacearchaeota class was the richest one. The abundance of bacterial and archaeal 16S rRNA gene copies in WL and GL soils was significantly higher than that in FR and TP soils. Redundancy analysis showed that bacterial diversity was influenced by abiotic factors, e.g., total organic carbon and pH, whereas total nitrogen, pH, and cation exchange capacity (CEC) significantly affected archaeal community composition. Pearson correlation analysis showed that bacterial and archaeal 16S rRNA gene abundance had the highest correlation with clay content (r > 0.905, P < 0.01), followed by total-P, CEC, pH, and silt (%). These results will lead to more comprehensive understanding of how land use affects microbial distribution.

Presence of Archaea in the Indoor Environment and Their Relationships with Housing Characteristics.

Archaea are widespread and abundant in soils, oceans, or human and animal gastrointestinal (GI) tracts. However, very little is known about the presence of Archaea in indoor environments and factors that can regulate their abundances. Using a quantitative PCR approach, and targeting the archaeal and bacterial 16S rRNA genes in floor dust samples, we found that Archaea are a common part of the indoor microbiota, 5.01 ± 0.14 (log 16S rRNA gene copies/g dust, mean ± SE) in bedrooms and 5.58 ± 0.13 in common rooms, such as living rooms. Their abundance, however, was lower than bacteria: 9.20 ± 0.32 and 9.17 ± 0.32 in bedrooms and common rooms, respectively. In addition, by measuring a broad array of environmental factors, we obtained preliminary insights into how the abundance of total archaeal 16S rRNA gene copies in indoor environment would be associated with building characteristics and occupants' activities. Based on the results, Archaea are not equally distributed within houses, and the areas with greater input of outdoor microbiome and higher traffic and material heterogeneity tend to have a higher abundance of Archaea. Nevertheless, more research is needed to better understand causes and consequences of this microbial group in indoor environments.

Effect of 7-year application of a nitrification inhibitor, dicyandiamide (DCD), on soil microbial biomass, protease and deaminase activities, and the abundance of bacteria and archaea in pasture soils

The nitrification inhibitor dicyandiamide (DCD) has been shown to be highly effective in reducing nitrate (NO3 −) leaching and nitrous oxide (N2O) emissions when used to treat grazed pasture soils. However, there have been few studies on the possible effects of long-term DCD use on other soil enzyme activities or the abundance of the general soil microbial communities. The objective of this study was to determine possible effects of long-term DCD use on key soil enzyme activities involved in the nitrogen (N) cycle and the abundance of bacteria and archaea in grazed pasture soils. Three field sites used for this study had been treated with DCD for 7 years in field plot experiments. The three pasture soils from three different regions across New Zealand were Pukemutu silt loam in Southland in the southern South Island, Horotiu silt loam in the Waikato in the central North Island and Templeton silt loam in Canterbury in the central South Island. Control and DCD-treated plots were sampled to analyse soil pH, microbial biomass C and N, protease and deaminase activity, and the abundance of bacteria and archaea. The three soils varied significantly in the microbial biomass C (858 to 542 μg C g−1 soil) and biomass N (63 to 28 μg N g−1), protease (361 to 694 μg tyrosine g−1 soil h−1) and deaminase (4.3 to 5.6 μg NH4 + g−1 soil h−1) activity, and bacteria (bacterial 16S rRNA gene copy number: 1.64 × 109 to 2.77 × 109 g−1 soil) and archaea (archaeal 16S rRNA gene copy number: 2.67 × 107 to 3.01 × 108 g−1 soil) abundance. However, 7 years of DCD use did not significantly affect these microbial population abundance and enzymatic activities. Soil pH values were also not significantly affected by the long-term DCD use. These results support the hypothesis that DCD is a specific enzyme inhibitor for ammonia oxidation and does not affect other non-target microbial and enzyme activities. The DCD nitrification inhibitor technology, therefore, appears to be an effective mitigation technology for nitrate leaching and nitrous oxide emissions in grazed pasture soils with no adverse impacts on the abundance of bacteria and archaea and key enzyme activities.

Changes in the microbial community structure of bacteria, archaea and fungi in response to elevated CO2 and warming in an Australian native grassland soil

The microbial community structure of bacteria, archaea and fungi is described in an Australian native grassland soil after more than 5 years exposure to different atmospheric CO2 concentrations ([CO2]) (ambient, +550 ppm) and temperatures (ambient, + 2°C) under different plant functional types (C3 and C4 grasses) and at two soil depths (0-5 cm and 5-10 cm). Archaeal community diversity was influenced by elevated [CO2], while under warming archaeal 16S rRNA gene copy numbers increased for C4 plant Themeda triandra and decreased for the C3 plant community (P < 0.05). Fungal community diversity resulted in three groups based upon elevated [CO2], elevated [CO2] plus warming and ambient [CO2]. Overall bacterial community diversity was influenced primarily by depth. Specific bacterial taxa changed in richness and relative abundance in response to climate change factors when assessed by a high-resolution 16S rRNA microarray (PhyloChip). Operational taxonomic unit signal intensities increased under elevated [CO2] for both Firmicutes and Bacteroidetes, and increased under warming for Actinobacteria and Alphaproteobacteria. For the interaction of elevated [CO2] and warming there were 103 significant operational taxonomic units (P < 0.01) representing 15 phyla and 30 classes. The majority of these operational taxonomic units increased in abundance for elevated [CO2] plus warming plots, while abundance declined in warmed or elevated [CO2] plots. Bacterial abundance (16S rRNA gene copy number) was significantly different for the interaction of elevated [CO2] and depth (P < 0.05) with decreased abundance under elevated [CO2] at 5-10 cm, and for Firmicutes under elevated [CO2] (P < 0.05). Bacteria, archaea and fungi in soil responded differently to elevated [CO2], warming and their interaction. Taxa identified as significantly climate-responsive could show differing trends in the direction of response ('+' or '-') under elevated CO2 or warming, which could then not be used to predict their interactive effects supporting the need to investigate interactive effects for climate change. The approach of focusing on specific taxonomic groups provides greater potential for understanding complex microbial community changes in ecosystems under climate change.

Archaeal Abundance across a pH Gradient in an Arable Soil and Its Relationship to Bacterial and Fungal Growth Rates

Soil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abundance related to bacterial and fungal growth rates across the same pH gradient. We identified two distinct and opposite effects of pH on the archaeal abundance. In the lowest pH range (pH 4.0 to 4.7), the abundance of archaea did not seem to correspond to pH. Above this pH range, there was a sharp, almost 4-fold decrease in archaeal abundance, reaching a minimum at pH 5.1 to 5.2. The low abundance of archaeal 16S rRNA gene copy numbers at this pH range then sharply increased almost 150-fold with pH, resulting in an increase in the ratio between archaeal and bacterial copy numbers from a minimum of 0.002 to more than 0.07 at pH 8. The nonuniform archaeal response to pH could reflect variation in the archaeal community composition along the gradient, with some archaea adapted to acidic conditions and others to neutral to slightly alkaline conditions. This suggestion is reinforced by observations of contrasting outcomes of the (competitive) interactions between archaea, bacteria, and fungi toward the lower and higher ends of the examined pH gradient.

Distribution and diversity of archaeal communities in selected Chinese soils

To understand the distribution and diversity of archaea in Chinese soils, the archaeal communities in a series of topsoils and soil profiles were investigated using quantitative PCR, T-RFLP combining sequencing methods. Archaeal 16S rRNA gene copy numbers, ranging from 4.96×10(6) to 1.30×10(8) copiesg(-1) dry soil, were positively correlated with soil pH, organic carbon and total nitrogen in the topsoils. In the soil profiles, archaeal abundance was positively correlated with soil pH but negatively with depth profile. The relative abundance of archaea in the prokaryotes (sum of bacteria and archaea) ranged from 0.20% to 9.26% and tended to increase along the depth profile. T-RFLP and phylogenetic analyses revealed that the structure of archaeal communities in cinnamon soils, brown soils, and fluvo-aquic soils was similar and dominated by Crenarchaeota group 1.1b and 1.1a. These were different from those in red soils, which were dominated by Crenarchaeota group 1.3 and 1.1c. Canonical correspondence analysis indicated that the archaeal community was primarily influenced by soil pH.

Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria

The role of ammonia-oxidizing archaea (AOA) in nitrogen cycling in marine sediments remains poorly characterized. In this study, we enriched and characterized AOA from marine sediments. Group I.1a crenarchaea closely related to those identified in marine sediments and "Candidatus Nitrosopumilus maritimus" (99.1 and 94.9% 16S rRNA and amoA gene sequence identities to the latter, respectively) were substantially enriched by coculture with sulfur-oxidizing bacteria (SOB). The selective enrichment of AOA over ammonia-oxidizing bacteria (AOB) is likely due to the reduced oxygen levels caused by the rapid initial growth of SOB. After biweekly transfers for ca. 20 months, archaeal cells became the dominant prokaryotes (>80%), based on quantitative PCR and fluorescence in situ hybridization analysis. The increase of archaeal 16S rRNA gene copy numbers was coincident with the amount of ammonia oxidized, and expression of the archaeal amoA gene was observed during ammonia oxidation. Bacterial amoA genes were not detected in the enrichment culture. The affinities of these AOA to oxygen and ammonia were substantially higher than those of AOB. [(13)C]bicarbonate incorporation and the presence and activation of genes of the 3-hydroxypropionate/4-hydroxybutyrate cycle indicated autotrophy during ammonia oxidation. In the enrichment culture, ammonium was oxidized to nitrite by the AOA and subsequently to nitrate by Nitrospina-like bacteria. Our experiments suggest that AOA may be important nitrifiers in low-oxygen environments, such as oxygen-minimum zones and marine sediments.

Changes in intact membrane lipid content of archaeal cells as an indication of metabolic status

Archaea build their cellular membrane with glycerol dialkyl glycerol tetraether (GDGT) core lipids, bound to polar head groups to form intact GDGTs. These lipids are commonly used as indicators of archaeal biomass in the environment. Here, we measured both intact and core GDGTs and archaeal 16S rRNA gene copies (a proxy for cell abundance) in order to determine cellular GDGT content in a coastal fjord at different locations, seasons and depths. We found that the abundance of both intact and core GDGTs relative to 16S rRNA gene copy numbers varied by two orders of magnitude, were generally higher in surface than deeper water and were elevated during periods associated with seasonal phytoplankton blooms. Core and intact GDGTs followed similar trends, but the ratio of core GDGT/16S rRNA gene copy number reached a maximum in October, following a peak in the ratio of intact GDGT/16S rRNA gene copy number that occurred in August. Our data suggest that spatial and temporal variability in these ratios must be considered when attempting to quantify cell numbers in the environment using either GDGTs or 16S rRNA genes. We used optimized extraction methods for both GDGTs and DNA. However, there is still potential for bias in qPCR methodology that could influence the GDGT/16S rRNA gene copy ratio. The apparent variability in cellular membrane lipid concentration could serve to evaluate the status of archaeal populations as it relates to: (i) varying cell size resulting from changes in either community structure or nutritional status, (ii) population turnover times and (iii) changing environmental factors that affect the lifetime of intact GDGTs after cell death.

Polyphasic analyses of methanogenic archaeal communities in agricultural biogas plants.

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescence in situ hybridization (FISH) was carried out to quantify the methanogenic Archaea in the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophic Methanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order, Methanoculleus was found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic family Methanosaetaceae was determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence of Methanosaetaceae in the biogas reactors and high concentrations of total ammonia (sum of NH(3) and NH(4)(+)) was observed.

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea were 4.59 x 10(9) and 6.68 x 10(5), respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor (Fx), sor (SA), and sor (SB) were identified from metagenomic DNAs of the bioreactors. The sor (Fx) is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor (SA) and sor (SB) showed no significant identity to any genes in GenBank databases. The sor (SB) was cloned and expressed in Escherichia coli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sor (SA) and sor (SB) were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from archaea. Furthermore, with primers specific to the sor (SB) gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document SOR gene in bioleaching reactors and Acidithiobacillus species.