Horonobe Underground Research Laboratory Research Articles

Biofilm Formation on Excavation Damaged Zone Fractures in Deep Neogene Sedimentary Rock.

Deep underground galleries are used to access the deep biosphere in addition to mining and other engineering applications, such as geological disposal of radioactive waste. Fracture networks developed in the excavation damaged zone (EDZ) are concerned with accelerating mass transport, where microbial colonization might be possible due to the availability of space and nutrients. In this study, microbial biofilms at EDZ fractures were investigated by drilling from a 350-m-deep gallery and subsequent borehole logging at the Horonobe Underground Research Laboratory (URL). By using microscopic and spectroscopic techniques, the dense colonization of microbial cells was demonstrated at the surfaces of the EDZ fractures with high hydraulic conductivity. 16S rRNA gene sequence analysis revealed the dominance of gammaproteobacterial lineages, the cultivated members of which are aerobic methanotrophs. The near-complete genomes from Horonobe groundwater, affiliated with the methanotrophic lineages, were fully equipped with genes involved in aerobic methanotrophy. Although the mediation of aerobic methanotrophy remains to be demonstrated, microbial O2 production was supported by the presence of genes in the near-complete genomes, such as catalase and superoxide dismutase that produce O2 from reactive oxygen species and a nitric oxide reductase gene with the substitutions of amino acids in motifs. It is concluded that the EDZ fractures provide energetically favorable subsurface habitats for microorganisms.

Relationship between fault transmissivity, flow dimensions and effective hydraulic conductivity in siliceous mudstone of the Wakkanai Formation around the Horonobe Underground Research Laboratory in Japan

Effective hydraulic conductivity is typically used to evaluate groundwater flow in faulted/fractured media. Estimating effective hydraulic conductivity relies on modelling the transmissivity and hydraulic connectivity of faults/fractures using a large dataset. As an alternative to evaluating hydraulic connectivity, flow dimension is an easily available and quantitative indicator of fault/fracture hydraulic connectivity. However, the flow dimension has not been used as much quantitatively to evaluate the effective hydraulic conductivity. This study demonstrates how flow dimensions in poorly self-sealed faults directly relate to effective hydraulic conductivities. Effective hydraulic conductivities of massive siliceous mudstone of the Wakkanai Formation around the Horonobe Underground Research Laboratory (URL) in Japan were analysed by simulation with long-term (>10 years) data of hydraulic pressure and inflow during the construction and operation of the URL. The estimated effective hydraulic conductivities varied from c . 10 −8 to c . 10 −11 m s −1 with an increase in depth from c . 250 to c . 600 m. The fault local transmissivities and flow dimensions from packer tests varied from c . 10 −6 to c . 10 −10 m 2 s −1 and from c . 2.0 or more to c . 1.0 with increasing depth, respectively. When applying the Landau–Lifshitz–Matheron formula to calculate the fault effective transmissivities, these variations correlate well and the estimated effective hydraulic conductivities are sound.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

Numerical simulations for describing generation of excavation damaged zone: Important case study at Horonobe underground research laboratory

The aim of the present research was to establish a case study for the prediction of the unknown EDZ (Excavation Damaged Zone) distribution using a numerical analysis calibrated by replicating the trends in the EDZ observed from one of the representative underground research fields in Japan (Horonobe URL). In this study, a 2D numerical analysis using a damage model, which can determine rock deformation and fracturing simultaneously, is presented. It was calibrated to reproduce the excavation of the gallery at the Horonobe URL at a depth of 350 ​m. Simulated results show an excellent agreement with the extent of the measured EDZ and capture the failure modes of EDZ fractures suggested by the in-situ observations. Finally, the calibrated numerical analysis was used to realistically estimate the EDZ formation for the geological disposal of high-level radioactive waste (HLW) under the same environment as that of the above-mentioned galley at the Horonobe URL. Consequently, it was shown that the tensile/shear hybrid fractures dominantly constituted the EDZ and propagated to a maximum extent of about 0.3 ​m from the cavity wall during the cavity excavation for the HLW disposal. Overall, the calibrated numerical analysis and resulting estimations, targeted for the environment at the depth of 350 ​m at the Horonobe URL, where mudstone is located, should be useful for predicting the trends in the EDZ distribution expected in the implementation of HLW disposal projects under deep geological conditions, such as those that exist in Japan, which are dominated by sedimentary rocks, including mudstone.

First-arrival traveltime tomography for monitoring the excavation damaged zone in the Horonobe Underground Research Laboratory

Subsurface excavation results in the formation of a zone called excavation damaged zone (EDZ) around the tunnel wall. An EDZ is a major concern in the field of high-level radioactive waste disposal because it may act as a flow path after the closure of a repository. In this study, first-arrival traveltime tomography was repeatedly conducted on the EDZ at a depth of 350 ​m in the Horonobe Underground Research Laboratory. However, the acquired data was highly affected by the support structure on the drift wall. For proper visualization of the EDZ, information about the structure was incorporated into the inversion by modifying the model constraint. The synthetic study showed that the approach reproduced the EDZ in the model without the artifacts. The method was applied to field data, and the EDZ around the drift was detected. The inversion was extended to a time-lapse inversion to trace the changes in P-wave velocity in the EDZ. The synthetic study demonstrated that temporal changes in the P-wave velocity distribution could be detected. Data obtained from 12 surveys under open-drift conditions were analyzed by time-lapse inversion. The results indicated that the EDZ did not undergo sealing or evolution at the site for approximately seven years.

Effects of flow dimension in faulted or fractured rock on natural reductions of inflow during excavation: a case study of the Horonobe Underground Research Laboratory site, Japan

Major inflows of groundwater can occur during excavation in faulted or fractured rock masses, even if pre-excavation grouting is applied; postexcavation grouting may then be required to reduce these inflows. However, the diffusion equation for fluid pressure suggests that inflows may reduce naturally by 50–90% or more within days or weeks when the dimension of the flow in faults or fractures that feed the inflow (flow dimension) is close to 1, but inflow reduction is minimal when the flow dimension is close to 3. Therefore, if the flow dimension is close to 1, the natural reduction in inflow may obviate countermeasures. Nevertheless, this natural reduction being dependent on flow dimension is seldom considered explicitly when planning excavations or countermeasures. To verify the applicability of the relationship between natural changes of inflow and the flow dimension, this study measured changes in inflow at six locations at the Horonobe site, Japan, where major inflows occurred during excavations of tunnels or shafts in faulted or fractured siliceous rocks. The flow dimension at each location was assessed using pre-excavation packer tests in surface-based boreholes. The results confirm that changes in the inflows during the days and weeks immediately after their commencement depended consistently on the assessed flow dimension. Natural reductions in inflow during excavation are predictable based on the flow dimension, which can be estimated using pre-excavation borehole investigations or from the initial changes in inflow during the first several days. This approach may be helpful for improving the efficiency of excavations.

Anaerobic methane-oxidizing activity in a deep underground borehole dominantly colonized by Ca. Methanoperedenaceae.

The family Ca. Methanoperedenaceae archaea mediates the anaerobic oxidation of methane (AOM) in different terrestrial environments. Using a newly developed high-pressure laboratory incubation system, we investigated 214- and 249-m deep groundwater samples at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Ca. Methanoperedenaceae archaea have been shown by genome-resolved metagenomics, respectively. The groundwater samples amended with 13 C-labelled methane and amorphous Fe(III) were incubated at a pressure of 1.6MPa. After 3-7 days of incubation, the AOM rate was 45.8 ± 19.8nM/day in 214-m groundwater. However, almost no activity was detected from 249-m groundwater. Based on the results from 16S rRNA gene analysis, the abundance of Ca. Methanoperedenaceae archaea was high in the 214-m deep groundwater sample, whereas Ca. Methanoperedenaceae archaea was undetected in the 249-m deep groundwater sample. These results support the in situ AOM activity of Ca. Methanoperedenaceae archaea in the 214-m deep subsurface borehole interval. Although the presence of Fe-bearing phyllosilicates was demonstrated in the 214-m deep groundwater, it needs to be determined whether Ca. Methanoperedenaceae archaea use the Fe-bearing phyllosilicates as in situ electron acceptors by high-pressure incubation amended with the Fe-bearing phyllosilicates.

Constant-head step injection tests to quantify the stress dependence of fracture transmissivity in an excavation damaged zone: A case study from the Horonobe Underground Research Laboratory

In an underground repository for high-level radioactive waste disposal , fracture transmissivity in an excavation damaged zone (EDZ) along tunnels or deposition holes can decrease during the post-closure period via processes such as self-sealing by clay-swelling at the EDZ's fracture surface or an increase in effective normal stress acting on the fractures owing to swelling of backfilling or buffer materials. Hydromechanical coupling models for the stress-dependence of fracture transmissivity are helpful to estimate the change in an EDZ's fracture transmissivity after closure. The applicability of the applied models should be confirmed by in situ tests at the given site; this appears to be facilitated by using constant-head step injection tests. However, injection testing is rarely applied to EDZ fractures . To investigate the applicability of injection tests, the present study performed them on single, tensile EDZ fractures in the Horonobe Underground Research Laboratory hosted by poorly swelling mudstone . Furthermore, the Barton–Bandis normal stress-dependent fracture-closure model quantified the stress-dependence of EDZ facture transmissivity. The fracture's hydraulic aperture increased gradually during injection, and its variation was well reproduced by fitting the model. Although the model requires the normal stress, this parameter was reasonably estimated by the fitting analyses. Constant-head step injection tests coupled with the Barton–Bandis model is believed to be a convenient method for preliminarily quantifying or verifying the stress dependence of EDZ fracture transmissivity, at least for poorly self-sealed, tensile EDZ-fractures. • Assessment combines step injection testing and Barton–Bandis fracture-closure model. • It quantifies fracture transmissivity stress dependence in excavation damaged zone. • The method can also potentially estimate total normal stress acting on a fracture.

Variation in fault hydraulic connectivity with depth in mudstone: An analysis of poroelastic hydraulic response to excavation in the Horonobe URL

Faulted mudstones have low effective hydraulic conductivity if the faults have limited hydraulic connectivity. Therefore, the hydraulic connectivity of faults is a crucial consideration in the geological disposal of high-level radioactive waste. There is a simple method based on single-borehole investigations to classify domains of faults in mudstone as having either high or low hydraulic connectivity. However, the nature of the hydraulic connectivity’s transition with depth around the boundary between domains of faults with high and low hydraulic connectivity remains poorly understood. At the Horonobe Underground Research Laboratory (Japan), three shafts have been excavated in a Neogene siliceous mudstone, and hydraulic pressure has been monitored at boreholes during the laboratory’s construction and operation. This study analyzed long-term hydraulic pressure data to estimate the variation of effective hydraulic conductivity and explore the nature of the variation of fault hydraulic connectivity with depth around the predicted boundary (at ∼400 m depth) between domains of faults with high and low hydraulic connectivity (with less connectivity below the boundary). As the observed hydraulic pressure was greatly affected by the Mandel–Cryer effect, numerical simulations considered poroelastic effects. They showed that the effective hydraulic conductivity gradually decreased from ∼400 to ∼500 m depth, becoming comparable with that of intact rock below ∼500 m. Theoretical analysis of the observed data also indicated the same variation with depth. These results suggest that the hydraulic connectivity of faults does not change abruptly, but instead varies gradually over several tens of meters around the domain boundary.

Sorption of Cs+ and Eu3+ ions onto sedimentary rock in the presence of gamma-irradiated humic acid

The influence of humic acid (HA) and its radiological degradation on the sorption of Cs+ and Eu3+ by sedimentary rock (obtained from the Horonobe Underground Research Laboratory in Japan) was investigated to understand the sorption process of metal ions and humic substances. Aldrich HA solution was gamma-irradiated assuming a strong radiation from a highly radioactive waste to be disposed of in deep geological formations. Batch sorption experiments were performed to evaluate the effect of gamma-irradiated HA on the sorption of Cs+ and Eu3+ ions. The addition of non-irradiated HA weakened the Eu sorption because of the lower sorption of the negatively charged Eu-HA complexes compared with free Eu ions. The sorption of Cs ions was barely affected by the presence of HA and its gamma irradiation. The concentration ratio of metal complexed and non-complexed species in the solid and liquid phases was evaluated by sequential filtration and chemical equilibrium calculations. The ratios were low in both phases for Cs and supported the minimal contribution of HA to Cs sorption. However, the concentration ratio for Eu3+ in the liquid phase was high, indicating that the complexing ability of HA to Eu3+ was higher than that of HA to Cs+.

Deep groundwater physicochemical components affecting actinide migration

To better understand the migration behavior of actinides in deep groundwater (GW), the interactions between doped tracers and deep GW components were investigated. La, Sm, Ho, and U tracers (10 or 100 ppb) were doped into sedimentary rock GW samples collected from 250 to 350 m deep boreholes in the experimental gallery of the Horonobe Underground Research Laboratory (URL), Hokkaido, Japan. To evaluate the effect of GW composition on the chemical speciation of actinides, the same tracers were doped into crystalline rock GW samples collected from 300 to 500 m deep boreholes in the experimental gallery at the Mizunami URL, Gifu Prefecture, Japan. Each GW sample was sequentially filtered through a micro-pore filter (0.2 μm) and ultrafilters with a 10 kDa nominal molecular weight limit. Next, the filtrate solutions were analyzed using inductively coupled plasma–mass spectrometry to determine the concentration of tracers retained in solution during each filtration step, and the used filters were analyzed using time-of-flight secondary ion mass spectrometry element mapping and X-ray absorption fine structure spectroscopy to determine the chemical species of the tracers trapped on each filter. It was determined that lanthanide migration was controlled by the amount of phosphates in the Horonobe GW. Therefore, it was expected that the solubility of minor actinides (MAs), which exhibit a similar chemical behavior to that of lanthanides, would be controlled by the formation of phosphates in sedimentary rock GW. Moreover, the data on the Mizunami GW indicated that a fraction of lanthanides and MAs formed hydroxides and/or hydroxocarbonates.

Mudstone redox conditions at the Horonobe Underground Research Laboratory, Hokkaido, Japan: Effects of drift excavation

The mechanical and hydraulic properties of rocks around mine drifts change significantly during the construction and operation of a radioactive-waste repository, with air intrusion typically causing the oxidation of rock and groundwater in excavation-damaged zones (EDZ). Redox conditions in such zones associated with niches excavated in mudstone at the Horonobe Underground Research Laboratory (URL), which is believed to be generally representative of conditions that could exist in the EDZ of a repository, were studied with the aim of improving our understanding of factors that control redox conditions in such rock–groundwater systems. Groundwater Eh values around the niches have reducing values of less than −150 mV. The SO42− concentration, regarded as an oxidation indicator, is consistently as low as 1 μmol L−1. Gas occupies >50% of zone volumes, including CH4 and CO2 with traces of N2 and O2. Cores drilled from host rock around a URL gallery were analyzed, with no pyrite dissolution or precipitation of calcium sulfates being found. It is concluded that oxidizing conditions do not exist in the excavation-damaged zones, which is attributed to the suppression of air intrusion by the release of CH4 and CO2 from groundwater as pressures decreased and their accumulation in fractures. The modeling of oxygen diffusion into host rock further indicates that a reducing environment is maintained around the URL drifts.

Determination of humic substances in deep groundwater from sedimentary formations by the carbon concentration-based DAX-8 resin isolation technique

ABSTRACTConcentrations of humic substance (HS) in deep groundwater from sedimentary formations were determined by the carbon concentration-based DAX-8 resin isolation technique. The groundwater samples were collected from test galleries at depths of 140, 250 and 350 m at the Horonobe Underground Research Laboratory (URL) in Hokkaido, and two observation wells with different depths of 560 and 1050 m in Niigata, Japan. The analytical condition was optimized for the groundwater samples with a high salinity and a high concentration of dissolved organic matters (DOMs). The analytical results showed that the HS concentrations varied from 7.8 to 23.5 mg-C dm−3, depending on the depth and the area. The HS proportions to DOM (%HS) were evaluated to be 58 – 67% and slightly decreased in the deeper groundwater. The regression analysis showed that the HS concentrations are positively correlated with the DOM concentrations. In addition, the low deviation of the %HS values from the slope in the regression equation indicated that the slight variation of %HS can be trivial in the prediction of the concentration of HS. In previous studies, HS concentrations in the groundwater had been determined with a large uncertainty and potential usage of the regression equation for the prediction had not been elucidated. Therefore, the results in this study can provide useful information on the HS concentration and its prediction from the DOM concentration in deep groundwater from sedimentary formations.

Genetic survey of indigenous microbial eukaryotic communities, mainly fungi, in sedimentary rock matrices of deep terrestrial subsurface

Culture-independent molecular techniques enable us to analyze microbial communities in various environments. Molecular methods can detect numerous uncultivable prokaryotes in extreme environments including anaerobic, no-light, high-pressure, and high-temperature conditions. Recently, microbial eukaryotes were detected in the deep-sea environments, suggesting that microbial eukaryotes can adapt to a wider range of conditions than previously thought. At the Horonobe Underground Research Laboratory of Horonobe, Japan, we performed a culture-independent analysis of eukaryotes acquired at depths of −250 m and −270 m. Our results indicated that fungi are the dominant eukaryotic component in the deep sedimentary rocks of Horonobe. In the rock matrices, we detected a wide taxonomic range of fungi including Cryptomycota, Basidiomycota, and Ascomycota. This study is the first report on the diversity of eukaryotic species in deep terrestrial subsurface sedimentary rock matrices.

A study of methods to prevent piping and erosion in buffer materials intended for a vertical deposition hole at the Horonobe Underground Research Laboratory

Abstract Piping and erosion phenomena are serious problems affecting the integrity of buffer materials, which are an element of engineered barrier systems in the geological disposal of high-level radioactive waste. In this study, the outflow behaviour and the condition of buffer materials are investigated using a test pit drilled into host rock at the Horonobe Underground Research Laboratory to consider countermeasures to contain the outflow of the buffer material. The results are as follows: (1) Piping and erosion phenomena occur irrespective of the injection flow rate. However, when the rate is small, the buffer material is considered to be self-repairing and the outflow of the buffer material can be suppressed. (2) When the injection water contains large amounts of electrolytes, the surface of the buffer material peels off and precipitates, probably decreasing the waterproof performance. (3) Bentonite pellets are likely to be an effective countermeasure against piping and erosion.

Preliminary results for natural groundwater colloids in sedimentary rocks of the Horonobe Underground Research Laboratory, Hokkaido, Japan

Abstract Colloid concentration is an important parameter in models of colloid-facilitated transport. The purpose of the present study is to characterize colloid concentrations and colloid stability in natural groundwater from the Horonobe Underground Research Laboratory (URL) in Hokkaido, Japan. The particle sizes of colloids in groundwaters from the Horonobe URL range from several nanometres to c. 500 nm, with a mode particle size of c. 120 nm. Evaluation of colloid stability by DLVO theory suggests that larger colloids (i.e. >100 nm in diameter) would be more stable than smaller colloids in some groundwaters. The estimated colloid particle concentrations when considering the results of DLVO calculations ranged from 2.33 × 10 6 to 1.12 × 10 8 particles/ml, and mass concentrations were estimated to range from 45 to 1540 µg l −1 for diameters greater than 100 nm. Colloids in Horonobe groundwaters appear to be less stable, with a moderate potential for transport, than colloids investigated in similar international studies. This reduced stability may be due to relatively higher ionic strengths and moderate dissolved organic concentrations in Horonobe groundwaters compared to their international counterparts.

A Method for Estimating the Highest Potential Hydraulic Conductivity in the Excavation Damaged Zone in Mudstone

Excavation of deep underground openings induces permeable fractures around the opening due to stress redistribution. Such a zone is called excavation damaged zone (EDZ). In a high-level radioactive waste disposal project, the EDZ might provide pathways for the migration of radionuclides around the facility. Thus, this study focused on the development of a method for estimating the highest potential hydraulic conductivity in the EDZ around a gallery in the Horonobe Underground Research Laboratory, Japan. Borehole televiewer surveys, rock core observations, and hydraulic tests were undertaken to investigate the extent and magnitude of hydraulic conductivity in the EDZ around the gallery. The observed extent (sidewall ≤ 0.6 m; floor ≤ 1.6 m) of the EDZ shows good agreement with the EDZ estimated from hydro-mechanical coupling analysis. The measured hydraulic conductivities of the EDZ (3.5 × 10−6 to 1.24 × 10−8 m/s) are within the range of those based on the Mean Stress Index (MSI), which is defined as the ratio of the effective mean stress derived from numerical analyses to the tensile strength of intact rock. Given that the rock mass is relatively homogeneous and artificial damage (e.g., blasting-induced damage) can be neglected, as in the Horonobe Underground Research Laboratory, the MSI model is likely to be applicable in estimating the highest potential hydraulic conductivity in the EDZ.

Rock mass property evaluation based on the borehole wall images taken by using an ultrasonic scanner (USS)

Discontinuities, such as faults and fractures exist even in Neogene soft sedimentary rocks. These discontinuities play an important role for determination of physical, mechanical and hydraulic properties of rock mass. For geological disposal of high-level radioactive waste, these data should be obtained for designs of the layout of repository, engineered barrier system and safety assessment. Rock mass properties are important information for quantitative rock mass classification as well as for design and construction of underground facilities and assessment of safe waste disposal. Ultrasonic wave reflection intensity of the wall of the borehole drilled from bottom of East Access Shaft in Horonobe Underground Research Laboratory Center, Hokkaido, Japan, was obtained by using the ultrasonic scanner (USS). In this paper, we compared results of USS observation with core logging data including the core observation, the optical digital scanner (ODS) observation, and result of Needle Penetration Index (NPI) tests. These results indicated that ultrasonic wave reflection intensity had a good correlation with other observation results, and USS observation and NPI test are useful techniques for the determination of the detailed rock mass classification.

A Proposed Method to Estimate In Situ Dissolved Gas Concentrations in Gas‐Saturated Groundwater

Gas-saturated groundwater forms bubbles when brought to atmospheric pressure, preventing precise determination of its in situ dissolved gas concentrations. To overcome this problem, a modeling approach called the atmospheric sampling method is suggested here to recover the in situ dissolved gas concentrations of groundwater collected ex situ under atmospheric conditions at the Horonobe Underground Research Laboratory, Japan. The results from this method were compared with results measured at the same locations using two special techniques, the sealed sampler and pre-evacuated vial methods, that have been developed to collect groundwater under its in situ conditions. In gas-saturated groundwater cases, dissolved methane and inorganic carbon concentrations derived using the atmospheric sampling method were mostly within ±4 and ±10%, respectively, of values from the sealed sampler and pre-evacuated vial methods. In gas-unsaturated groundwater, however, the atmospheric sampling method overestimated the in situ dissolved methane concentrations, because the groundwater pressure at which bubbles appear (Pcritical ) was overestimated. The atmospheric sampling method is recommended for use where gas-saturated groundwater can be collected only ex situ under atmospheric conditions.

The Succession of Bacterial Community Structure in Groundwater from a 250-m Gallery in the Horonobe Underground Research Laboratory

ABSTRACTWe investigated the change in bacterial community structure after drilling boreholes, 09-V250-M02 and 09-V250-M03, in the 250-m deep research gallery of the Horonobe Underground Research Laboratory. In the 09-V250-M02 borehole, ϵ-Proteobacteria were predominantly detected in the clone library analyses of the groundwater samples conducted immediately after drilling. All the ϵ-Proteobacteria clones were closely related to Arcobacter spp., which are known to be sulfide-oxidizing chemoautotrophic bacteria. After 4 years, the microbial structure drastically changed, and most detected operational taxonomic units were uncultured species such as candidate division OP9 and Chloroflexi relatives, which are frequently detected in deep sea sediments. The results indicated that the microbial community structure was drastically affected by borehole drilling and was concomitant with oxidation perturbation. However, these disturbed microbial communities changed within a few years to a microbial community composed of uncultivated species such as OP9 and Chloroflexi.