Linking Uncultivated Microbial Populations and Benthic Carbon Turnover by Using Quantitative Stable Isotope Probing

ABSTRACTBenthic environments harbor highly diverse and complex microbial communities that control carbon fluxes, but the role of specific uncultivated microbial groups in organic matter turnover is poorly understood. In this study, quantitative DNA stable isotope probing (DNA-qSIP) was used for the first time to link uncultivated populations of bacteria and archaea to carbon turnover in lacustrine surface sediments. After 1-week incubations in the dark with [13C]bicarbonate, DNA-qSIP showed that ammonia-oxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural13C-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with [13C]bicarbonate, followed by extraction and concentration of high-molecular-weight (HMW) (>50-kDa) organic matter. qSIP showed that the labeled organic matter was turned over within 1 week by 823 microbial populations (operational taxonomic units [OTUs]) affiliated primarily with heterotrophicProteobacteria,Chloroflexi,Verrucomicrobia, andBacteroidetes. However, several OTUs affiliated with the candidate microbial taxaLatescibacteria,Omnitrophica,Aminicentantes,Cloacimonates,AC1,Bathyarchaeota, andWoesearchaeota, groups known only from genomic signatures, also contributed to biomass turnover. Of these 823 labeled OTUs, 52% (primarily affiliated withProteobacteria) also became labeled in 1-week incubations with [13C]bicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with13C-labeled HMW organic matter. These taxa consisted primarily of uncultivated populations within theFirmicutes,Bacteroidetes,Verrucomicrobia, andChloroflexi, highlighting their ecological importance. Our study helps define the role of several poorly understood, uncultivated microbial groups in the turnover of benthic carbon derived from “dark” primary production.IMPORTANCELittle is known about the ecological role of uncultivated microbial populations in carbon turnover in benthic environments. To better understand this, we used quantitative stable isotope probing (qSIP) to quantify the abundance of diverse, specific groups of uncultivated bacteria and archaea involved in autotrophy and heterotrophy in a benthic lacustrine habitat. Our results provide quantitative evidence for active heterotrophic and autotrophic metabolism of several poorly understood microbial groups, thus demonstrating their relevance for carbon turnover in benthic settings. Archaeal ammonia oxidizers were significant drivers ofin situ“dark” primary production supporting the growth of heterotrophic bacteria. These findings expand our understanding of the microbial populations within benthic food webs and the role of uncultivated microbes in benthic carbon turnover.

Download Full-text

Linking Specific Heterotrophic Bacterial Populations to Bioreduction of Uranium and Nitrate in Contaminated Subsurface Sediments by Using Stable Isotope Probing

Applied and Environmental Microbiology ◽

10.1128/aem.05247-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 8197-8200 ◽

Cited By ~ 14

Author(s):

Denise M. Akob ◽

Lee Kerkhof ◽

Kirsten Küsel ◽

David B. Watson ◽

Anthony V. Palumbo ◽

...

Keyword(s):

Stable Isotope ◽

Nitrate Reduction ◽

Contaminated Sediments ◽

Stable Isotope Probing ◽

Microbial Populations ◽

Bacterial Populations ◽

Content Type ◽

Reducing Conditions ◽

Subsurface Sediments ◽

Microbial Groups

ABSTRACTShifts in terminal electron-accepting processes during biostimulation of uranium-contaminated sediments were linked to the composition of stimulated microbial populations using DNA-based stable isotope probing. Nitrate reduction preceded U(VI) and Fe(III) reduction in [13C]ethanol-amended microcosms. The predominant, active denitrifying microbial groups were identified as members of theBetaproteobacteria, whereasActinobacteriadominated under metal-reducing conditions.

Download Full-text

High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol

Applied and Environmental Microbiology ◽

10.1128/aem.05973-11 ◽

2011 ◽

Vol 78 (1) ◽

pp. 163-169 ◽

Cited By ~ 7

Author(s):

Peter Andeer ◽

Stuart E. Strand ◽

David A. Stahl

Keyword(s):

Stable Isotope ◽

Restriction Fragment Length Polymorphism ◽

Length Polymorphism ◽

Fragment Length Polymorphism ◽

Buoyant Density ◽

Stable Isotope Probing ◽

Microbial Populations ◽

Bacterial Isolates ◽

Content Type ◽

Restriction Fragment Length

ABSTRACTStable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)–TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli,Rhodococcus,Variovorax, andMicrobacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Download Full-text

DNA Stable-Isotope Probing Delineates Carbon Flows from Rice Residues into Soil Microbial Communities Depending on Fertilization

Applied and Environmental Microbiology ◽

10.1128/aem.02151-19 ◽

2020 ◽

Vol 86 (7) ◽

Cited By ~ 3

Author(s):

Yali Kong ◽

Yakov Kuzyakov ◽

Yang Ruan ◽

Junwei Zhang ◽

Tingting Wang ◽

...

Keyword(s):

Organic Matter ◽

Stable Isotope ◽

High Throughput Sequencing ◽

C Sequestration ◽

Stable Isotope Probing ◽

Plant Residue ◽

Mineral Fertilizers ◽

Microbial Groups ◽

Rice Residues ◽

Fungal Genus

ABSTRACT Decomposition of crop residues in soil is mediated by microorganisms whose activities vary with fertilization. The complexity of active microorganisms and their interactions utilizing residues is impossible to disentangle without isotope applications. Thus, 13C-labeled rice residues were employed, and DNA stable-isotope probing (DNA-SIP) combined with high-throughput sequencing was applied to identify microbes active in assimilating residue carbon (C). Manure addition strongly modified microbial community compositions involved in the C flow from rice residues. Relative abundances of the bacterial genus Lysobacter and fungal genus Syncephalis were increased, but abundances of the bacterial genus Streptomyces and fungal genus Trichoderma were decreased in soils receiving mineral fertilizers plus manure (NPKM) compared to levels in soils receiving only mineral fertilizers (NPK). Microbes involved in the flow of residue C formed a more complex network in NPKM than in NPK soils because of the necessity to decompose more diverse organic compounds. The fungal species (Jugulospora rotula and Emericellopsis terricola in NPK and NPKM soils, respectively) were identified as keystone species in the network and may significantly contribute to residue C decomposition. Most of the fungal genera in NPKM soils, especially Chaetomium, Staphylotrichum, Penicillium, and Aspergillus, responded faster to residue addition than those in NPK soils. This is connected with the changes in the composition of the rice residue during degradation and with fungal adaptation (abundance and activity) to continuous manure input. Our findings provide fundamental information about the roles of key microbial groups in residue decomposition and offer important cues on manipulating the soil microbiome for residue utilization and C sequestration in soil. IMPORTANCE Identifying and understanding the active microbial communities and interactions involved in plant residue utilization are key questions to elucidate the transformation of soil organic matter (SOM) in agricultural ecosystems. Microbial community composition responds strongly to management, but little is known about specific microbial groups involved in plant residue utilization and, consequently, microbial functions under different methods of fertilization. We combined DNA stable-isotope (13C) probing and high-throughput sequencing to identify active fungal and bacterial groups degrading residues in soils after 3 years of mineral fertilization with and without manure. Manuring changed the active microbial composition and complexified microbial interactions involved in residue C flow. Most fungal genera, especially Chaetomium, Staphylotrichum, Penicillium, and Aspergillus, responded to residue addition faster in soils that historically had received manure. We generated a valuable library of microorganisms involved in plant residue utilization for future targeted research to exploit specific functions of microbial groups in organic matter utilization and C sequestration.

Download Full-text

Identification of Microbial Populations Assimilating Nitrogen from RDX in Munitions Contaminated Military Training Range Soils by High Sensitivity Stable Isotope Probing

Environmental Science & Technology ◽

10.1021/es401729c ◽

2013 ◽

pp. 130904161051002 ◽

Cited By ~ 7

Author(s):

Peter Andeer ◽

David A. Stahl ◽

Lorraine Lillis ◽

Stuart E. Strand

Keyword(s):

Stable Isotope ◽

Military Training ◽

High Sensitivity ◽

Stable Isotope Probing ◽

Microbial Populations

Download Full-text

Identifying the Active Microbiome Associated with Roots and Rhizosphere Soil of Oilseed Rape

Applied and Environmental Microbiology ◽

10.1128/aem.01938-17 ◽

2017 ◽

Vol 83 (22) ◽

Cited By ~ 40

Author(s):

Konstantia Gkarmiri ◽

Shahid Mahmood ◽

Alf Ekblad ◽

Sadhna Alström ◽

Nils Högberg ◽

...

Keyword(s):

Brassica Napus ◽

Stable Isotope ◽

Oilseed Rape ◽

High Throughput ◽

High Throughput Sequencing ◽

Rhizosphere Soil ◽

Stable Isotope Probing ◽

Biofuel Production ◽

Content Type ◽

Bacteria And Fungi

ABSTRACT RNA stable isotope probing and high-throughput sequencing were used to characterize the active microbiomes of bacteria and fungi colonizing the roots and rhizosphere soil of oilseed rape to identify taxa assimilating plant-derived carbon following 13CO2 labeling. Root- and rhizosphere soil-associated communities of both bacteria and fungi differed from each other, and there were highly significant differences between their DNA- and RNA-based community profiles. Verrucomicrobia, Proteobacteria, Planctomycetes, Acidobacteria, Gemmatimonadetes, Actinobacteria, and Chloroflexi were the most active bacterial phyla in the rhizosphere soil. Bacteroidetes were more active in roots. The most abundant bacterial genera were well represented in both the 13C- and 12C-RNA fractions, while the fungal taxa were more differentiated. Streptomyces, Rhizobium, and Flavobacterium were dominant in roots, whereas Rhodoplanes and Sphingomonas (Kaistobacter) were dominant in rhizosphere soil. “Candidatus Nitrososphaera” was enriched in 13C in rhizosphere soil. Olpidium and Dendryphion were abundant in the 12C-RNA fraction of roots; Clonostachys was abundant in both roots and rhizosphere soil and heavily 13C enriched. Cryptococcus was dominant in rhizosphere soil and less abundant, but was 13C enriched in roots. The patterns of colonization and C acquisition revealed in this study assist in identifying microbial taxa that may be superior competitors for plant-derived carbon in the rhizosphere of Brassica napus. IMPORTANCE This microbiome study characterizes the active bacteria and fungi colonizing the roots and rhizosphere soil of Brassica napus using high-throughput sequencing and RNA-stable isotope probing. It identifies taxa assimilating plant-derived carbon following 13CO2 labeling and compares these with other less active groups not incorporating a plant assimilate. Brassica napus is an economically and globally important oilseed crop, cultivated for edible oil, biofuel production, and phytoextraction of heavy metals; however, it is susceptible to several diseases. The identification of the fungal and bacterial species successfully competing for plant-derived carbon, enabling them to colonize the roots and rhizosphere soil of this plant, should enable the identification of microorganisms that can be evaluated in more detailed functional studies and ultimately be used to improve plant health and productivity in sustainable agriculture.

Download Full-text

Barium stable isotopes as a fingerprint of biological cycling in the Amazon River basin

Biogeosciences ◽

10.5194/bg-17-5989-2020 ◽

2020 ◽

Vol 17 (23) ◽

pp. 5989-6015

Author(s):

Quentin Charbonnier ◽

Julien Bouchez ◽

Jérôme Gaillardet ◽

Éric Gayer

Keyword(s):

Organic Matter ◽

Stable Isotope ◽

Trace Element ◽

Isotope Composition ◽

Silicate Weathering ◽

Biological Cycling ◽

Biological Uptake ◽

Dissolved Load ◽

Silicate Rocks

Abstract. The biological cycle of rock-derived nutrients on the continents is a major component of element transfer between the Earth's surface compartments, but its magnitude currently remains elusive. The use of the stable isotope composition of rock-derived nutrients, which can be fractionated during biological uptake, provides a promising path forward with respect to quantifying biological cycling and its overall contribution to global element cycling. In this paper, we rely on the nutrient-like behaviour of the trace element barium (Ba) and use its elemental and stable isotope compositions in dissolved and sediment load river samples to investigate biological cycling in the Amazon Basin. From these measurements, we show that dissolved Ba mainly derives from silicate rocks, and a correlation between dissolved Ba and K abundances suggests that biological cycling plays a role in the Ba river budget. Furthermore, the isotope composition of Ba (δ138Ba) in the dissolved load was found to be significantly different from that of the parent silicate rocks, implying that dissolved Ba isotopic signatures are affected by (i) the precipitation of soil-forming secondary phases as well as (ii) biological uptake and release from dead organic matter. Results from an isotope mass balance method applied to the river dissolved load data indicate that, after its release to solution by rock weathering, Ba is partitioned between the river dissolved load, secondary weathering products (such as those found in soils and river sediments), and the biota. In most sub-catchments of the Amazon, river Ba abundances and isotope compositions are significantly affected by biological cycling. Relationships between estimates of Ba cycled through biota and independent metrics of ecosystem dynamics (such as gross primary production and terrestrial ecosystem respiration) allow us to discuss the role of environmental parameters such as climate or erosion rates on the biological cycling of Ba and, by extension, the role of major rock-derived nutrients. In addition, catchment-scale mass and isotope budgets of Ba show that the measured riverine export of Ba is lower than the estimated delivery of Ba to the Earth surface through rock alteration. This indicates the existence of a missing Ba component, which we attribute to the formation of Ba-bearing particulate organics (possibly accumulating as soil organic matter or currently growing biomass within the catchments) and to organic-bound Ba exported as “unsampled” river particulate organic matter. Given our findings on the trace element Ba, we explore whether the river fluxes of most major rock-derived nutrients (K, Mg, Ca) might also be significantly affected by biological uptake or release. A first-order correction of river-derived silicate weathering fluxes from biological cycling shows that the carbon dioxide (CO2) consumption by silicate weathering at the mouth of the Amazon could be several times higher than the previously reported value of 13 × 109 mol CO2 yr−1 (Gaillardet et al., 1997). Overall, our study clearly shows that the chemical and isotope compositions of rivers in the Amazon – and most likely in other large river basins – bear a biological imprint, thereby challenging common assumptions made in weathering studies.

Download Full-text

Stable isotope probing of hypoxic toluene degradation at the Siklós aquifer reveals prominent role of Rhodocyclaceae

FEMS Microbiology Ecology ◽

10.1093/femsec/fiy088 ◽

2018 ◽

Vol 94 (6) ◽

Cited By ~ 11

Author(s):

András Táncsics ◽

Anna Róza Szalay ◽

Milan Farkas ◽

Tibor Benedek ◽

Sándor Szoboszlay ◽

...

Keyword(s):

Stable Isotope ◽

Stable Isotope Probing ◽

Toluene Degradation

Download Full-text

Section 8 update: Stable isotope probing of nucleic acids to identify active microbial populations

10.1007/springerreference_76361 ◽

2012 ◽

Keyword(s):

Stable Isotope ◽

Nucleic Acids ◽

Stable Isotope Probing ◽

Microbial Populations ◽

Section 8

Download Full-text

Identification of Benzo[a]pyrene-Metabolizing Bacteria in Forest Soils by Using DNA-Based Stable-Isotope Probing

Applied and Environmental Microbiology ◽

10.1128/aem.01983-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7368-7376 ◽

Cited By ~ 58

Author(s):

Mengke Song ◽

Chunling Luo ◽

Longfei Jiang ◽

Dayi Zhang ◽

Yujie Wang ◽

...

Keyword(s):

Stable Isotope ◽

Forest Soils ◽

Contaminated Soils ◽

Stable Isotope Probing ◽

Soil Microcosms ◽

Content Type ◽

The Family ◽

Polycyclic Aromatic ◽

Uncultivated Bacteria ◽

Different Soils

ABSTRACTDNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genusTerrimonas(familyChitinophagaceae, orderSphingobacteriales), whereasBurkholderiaspp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the familyOxalobacteraceae(genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP13C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils.

Download Full-text