A novel bioprospecting strategy via 13C-based high-throughput probing of active methylotrophs inhabiting oil reservoir surface soil.

Methane-oxidizing bacteria (MOB) have long been considered as a microbial indicator for oil and gas prospecting. However, due to the phylogenetically narrow breath of ecophysiologically distinct MOB, classic culture-dependent approaches could not discriminate MOB population at fine resolution, and accurately reflect the abundance of active MOB in the soil above oil and gas reservoirs. Here, we presented a novel microbial anomaly detection (MAD) strategy to quantitatively identify specific indicator methylotrophs in the surface soils for bioprospecting oil and gas reservoirs by using a combination of 13C-DNA stable isotope probing (SIP), high-throughput sequencing (HTS), quantitative PCR (qPCR) and geostatistical analysis. The Chunguang oilfield of the Junggar Basin was selected as a model system in western China, and type I methanotrophic Methylobacter was most active in the topsoil above the productive oil wells, while type II methanotrophic Methylosinus predominated in the dry well soils, exhibiting clear differences between non- and oil reservoir soils. Similar results were observed by quantification of Methylobacter pmoA genes as a specific bioindicator for the prediction of unknown reservoirs by grid sampling. A microbial anomaly distribution map based on geostatistical analysis further showed that the anomalous zones were highly consistent with petroleum, geological and seismic data, and validated by subsequent drilling. Over seven years, a total of 24 wells have been designed and drilled into the targeted anomaly, and the success rate via the MAD prospecting strategy was 83 %. Our results suggested that molecular techniques are powerful tools for oil and gas prospecting. This study indicates that the exploration efficiency could be significantly improved by integrating multi-disciplinary information in geophysics and geomicrobiology while reducing the drilling risk to a greater extent.

Identifying Active Rather than Total Methanotrophs Inhabiting Surface Soil Is Essential for the Microbial Prospection of Gas Reservoirs.

Methane-oxidizing bacteria (MOB) have long been recognized as an important bioindicator for oil and gas exploration. However, due to their physiological and ecological diversity, the distribution of MOB in different habitats varies widely, making it challenging to authentically reflect the abundance of active MOB in the soil above oil and gas reservoirs using conventional methods. Here, we selected the Puguang gas field of the Sichuan Basin in Southwest China as a model system to study the ecological characteristics of methanotrophs using culture-independent molecular techniques. Initially, by comparing the abundance of the pmoA genes determined by quantitative PCR (qPCR), no significant difference was found between gas well and non-gas well soils, indicating that the abundance of total MOB may not necessarily reflect the distribution of the underlying gas reservoirs. 13C-DNA stable isotope probing (DNA-SIP) in combination with high-throughput sequencing (HTS) furthermore revealed that type II methanotrophic Methylocystis was the absolutely predominant active MOB in the non-gas-field soils, whereas the niche vacated by Methylocystis was gradually filled with type I RPC-2 (rice paddy cluster-2) and Methylosarcina in the surface soils of gas reservoirs after geoscale acclimation to trace- and continuous-methane supply. The sum of the relative abundance of RPC-2 and Methylosarcina was then used as specific biotic index (BI) in the Puguang gas field. A microbial anomaly distribution map based on the BI values showed that the anomalous zones were highly consistent with geological and geophysical data, and known drilling results. Therefore, the active but not total methanotrophs successfully reflected the microseepage intensity of the underlying active hydrocarbon system, and can be used as an essential quantitative index to determine the existence and distribution of reservoirs. Our results suggest that molecular microbial techniques are powerful tools for oil and gas prospecting.

Bioleaching of Heavy Metals from Printed Circuit Boards with an Acidophilic Iron-Oxidizing Microbial Consortium in Stirred Tank Reactors.

In this study, bioleaching was carried out for the recovery of metals (copper, zinc, tin, lead, gold and silver) from printed circuit boards residues (PCBs), one of the most important wastes from electrical and electronic equipment, using an acidophilic iron-oxidizing bacterial consortium enriched with minerals from a gold mine in the Arequipa region, Peru. High-throughput sequencing and analysis of the 16S rRNA biomarker revealed that this consortium was predominantly composed of Tissierella, Acidiphilium and Leptospirillum bacteria, from which the latter is known to grow by chemolithotrophy through iron oxidation. After the enrichment process, the acidophilic iron-oxidizing consortium was first tested for its tolerance to different PCBs concentrations, showing best growth up to 10 g/L of PCBs and a tolerance index of 0.383. Based on these results, the bioleaching efficiency of the consortium was investigated for 10 g/L of PCBs in stirred tank reactors coupled to an aeration system, for 18 days. High bioleaching efficiencies were achieved for copper and zinc (69% and 91%, respectively), indicating that these two metals can be easily extracted in this leaching system. Lower extraction efficiencies were achieved for tin (16%) and gold (28%), while for lead and silver only a residual recovery (<0.25%) was detected. These results indicate that the enriched bacterial consortium originating from the Arequipa region, Peru, has a high capacity to recover different metals of economic importance.

Bacterial contribution to 17ß-estradiol mineralization in lake sediment as revealed by 13C-DNA stable isotope probing.

The accumulation of estrogens in aquatic environments has drawn increasing public concern due to their adverse effects on aquatic ecosystems and human health. Bacteria play important roles in eliminating estrogens from the environment, but knowledge of the identity and functions of the microorganisms involved in metabolizing these steroid hormones in the natural microbial communities is lacking. Here, we added 13C-17ß-estradiol (13C-E2) to sediments collected from Zhushan (ZS) Bay, Meiliang (ML) Bay, Gonghu (GH) Bay, and the central area (CA) of the Taihu Lake. The indigenous assimilators of E2 in the sediments were recognized using 13C-DNA stable isotope probing (DNA-SIP), and their effects on 13C-E2 mineralization were studied under aerobic condition. During the 30-day incubation period, ZS Bay had the highest cumulative percentage of 13C-E2 mineralization to 13CO2 (65.5%), while CA presented the lowest (51.4%). Based on DNA-SIP, we saw that Novosphingobium, Ralstonia, Pseudomonas, Sphingomonas, Nitrosomonas, and Alcaligenes were involved in E2-derived 13C assimilation for the entire incubation period. Acinetobacter, Flavobacterium, and Mycobacterium only assimilated 13C for the first half of the incubation. H16 was identified as an E2 assimilator for the first time in this study. In addition, the temporal changes in assimilator abundances during the incubation period indicated that these genera played dominant roles at different stages in the process of E2 biodegradation. The bacteria engaged in the assimilation of E2 in situ were identified, and the rate of increase in the relative abundance of assimilators was significantly (P < 0.05) and positively correlated with the E2 mineralization in sediments. This information enhances our knowledge of in situ E2 biodegradation and provides a potential resource that could be used to eliminate estrogens in sediments.

Grazing weakens competitive interactions between active methanotrophs and nitrifiers modulating greenhouse-gas emissions in grassland soils.

Grassland soils serve as a biological sink and source of the potent greenhouse gases (GHG) methane (CH4) and nitrous oxide (N2O). The underlying mechanisms responsible for those GHG emissions, specifically, the relationships between methane- and ammonia-oxidizing microorganisms in grazed grassland soils are still poorly understood. Here, we characterized the effects of grazing on in situ GHG emissions and elucidated the putative relations between the active microbes involving in methane oxidation and nitrification activity in grassland soils. Grazing significantly decreases CH4 uptake while it increases N2O emissions basing on 14-month in situ measurement. DNA-based stable isotope probing (SIP) incubation experiment shows that grazing decreases both methane oxidation and nitrification processes and decreases the diversity of active methanotrophs and nitrifiers, and subsequently weakens the putative competition between active methanotrophs and nitrifiers in grassland soils. These results constitute a major advance in our understanding of putative relationships between methane- and ammonia-oxidizing microorganisms and subsequent effects on nitrification and methane oxidation, which contribute to a better prediction and modeling of future balance of GHG emissions and active microbial communities in grazed grassland ecosystems.

Expansion of Thaumarchaeota habitat range is correlated with horizontal transfer of ATPase operons.

Thaumarchaeota are responsible for a significant fraction of ammonia oxidation in the oceans and in soils that range from alkaline to acidic. However, the adaptive mechanisms underpinning their habitat expansion remain poorly understood. Here we show that expansion into acidic soils and the high pressures of the hadopelagic zone of the oceans is tightly linked to the acquisition of a variant of the energy-yielding ATPases via horizontal transfer. Whereas the ATPase genealogy of neutrophilic Thaumarchaeota is congruent with their organismal genealogy inferred from concatenated conserved proteins, a common clade of V-type ATPases unites phylogenetically distinct clades of acidophilic/acid-tolerant and piezophilic/piezotolerant species. A presumptive function of pumping cytoplasmic protons at low pH is consistent with the experimentally observed increased expression of the V-ATPase in an acid-tolerant thaumarchaeote at low pH. Consistently, heterologous expression of the thaumarchaeotal V-ATPase significantly increased the growth rate of E. coli at low pH. Its adaptive significance to growth in ocean trenches may relate to pressure-related changes in membrane structure in which this complex molecular machine must function. Together, our findings reveal that the habitat expansion of Thaumarchaeota is tightly correlated with extensive horizontal transfer of atp operons.

Long-term nitrogen fertilization of paddy soil shifts iron-reducing microbial community revealed by RNA-(13)C-acetate probing coupled with pyrosequencing.

Iron reduction is an important biogeochemical process in paddy soils, yet little is known about the microbial coupling between nitrogen and iron reduction. Here, we investigated the shift of acetate-metabolizing iron-reducers under long-term nitrogen fertilization using (13)C-acetate-based ribosomal RNA (rRNA)-stable isotope probing (SIP) and pyrosequencing in an incubation experiment, and the shift of putative iron-reducers in original field samples were investigated by 16S rRNA gene-based pyrosequencing. During SIP incubations, in the presence of iron(III) oxyhydroxides, more iron(II) formation and less methane production were detected in nitrogen-fertilized (N) compared with non-fertilized (NF) soil. In (13)C-rRNA from microcosms amended with ferrihydrite (FER), Geobacter spp. were the important active iron-reducers in both soils, and labeled to a greater extent in N (31% of the bacterial classified sequences) than NF soils (11%). Pyrosequencing of the total 16S rRNA transcripts from microcosms at the whole community level further revealed hitherto unknown metabolisms of potential FER reduction by microorganisms including Pseudomonas and Solibacillus spp. in N soil, Dechloromonas, Clostridium, Bacillus and Solibacillus spp. in NF soil. Goethite (GOE) amendment stimulated Geobacter spp. to a lesser extent in both soils compared with FER treatment. Pseudomonas spp. in the N soil and Clostridium spp. in the NF soil may also be involved in GOE reduction. Pyrosequencing results from field samples showed that Geobacter spp. were the most abundant putative iron-reducers in both soils, and significantly stimulated by long-term nitrogen fertilization. Overall, for the first time, we demonstrate that long-term nitrogen fertilization promotes iron(III) reduction and modulates iron-reducing bacterial community in paddy soils.

Methyl-beta-cyclodextrin enhanced biodegradation of polycyclic aromatic hydrocarbons and associated microbial activity in contaminated soil.

The contamination of soils by polycyclic aromatic hydrocarbons (PAHs) is a widespread environmental problem and the remediation of PAHs from these areas has been a major concern. The effectiveness of many in situ bioremediation systems may be constrained by low contaminant bioavailability due to limited aqueous solubility or a large magnitude of sorption. The objective of this research was to evaluate the effect of methyl-beta-cyclodextrin (MCD) on bioaugmentation by Paracoccus sp. strain HPD-2 of an aged PAH-contaminated soil. When 10% (W/W) MCD amendment was combined with bioaugmentation by the PAH-degrading bacterium Paracoccus sp. strain HPD-2, the percentage degradation of total PAHs was significantly enhanced up to 34.8%. Higher counts of culturable PAH-degrading bacteria and higher soil dehydrogenase and soil polyphenol oxidase activities were observed in 10% (W/W) MCD-assisted bioaugmentation soil. This MCD-assisted bioaugmentation strategy showed significant increases (p < 0.05) in the average well color development (AWCD) obtained by the BIOLOG Eco plate assay, Shannon-Weaver index (H) and Simpson index (lambda) compared with the controls, implying that this strategy at least partially restored the microbiological functioning of the PAH-contaminated soil. The results suggest that MCD-aided bioaugmentation by Paracoccus sp. strain HPD-2 may be a promising practical bioremediation strategy for aged PAH-contaminated soils.