David D. Myrold

Evidence for Different Contributions of Archaea and Bacteria to the Ammonia-Oxidizing Potential of Diverse Oregon Soils

ABSTRACT A method was developed to determine the contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to the nitrification potentials (NPs) of soils taken from forest, pasture, cropped, and fallowed (19 years) lands. Soil slurries were exposed to acetylene to irreversibly inactivate ammonia monooxygenase, and upon the removal of acetylene, the recovery of nitrification potential (RNP) was monitored in the presence and absence of bacterial or eukaryotic protein synthesis inhibitors. For unknown reasons, and despite measureable NPs, RNP did not occur consistently in forest soil samples; however, pasture, cropped, and fallowed soil RNPs commenced after lags that ranged from 12 to 30 h after acetylene removal. Cropped soil RNP was completely prevented by the bacterial protein synthesis inhibitor kanamycin (800 μg/ml), whereas a combination of kanamycin plus gentamicin (800 μg/ml each) only partially prevented the RNP (60%) of fallowed soils. Pasture soil RNP was completely insensitive to either kanamycin, gentamicin, or a combination of the two. Unlike cropped soil, pasture and fallowed soil RNPs occurred at both 30°C and 40°C and without supplemental NH4+ (≤10 μM NH4+ in solution), and pasture soil RNP demonstrated ∼50% insensitivity to 100 μM allyl thiourea (ATU). In addition, fallowed and pasture soil RNPs were insensitive to the fungal inhibitors nystatin and azoxystrobin. This combination of properties suggests that neither fungi nor AOB contributed to pasture soil RNP and that AOA were responsible for the RNP of the pasture soils. Both AOA and AOB may contribute to RNP in fallowed soil, while RNP in cropped soils was dominated by AOB.

Dynamics of ammonia-oxidizing archaea and bacteria populations and contributions to soil nitrification potentials

It is well known that the ratio of ammonia-oxidizing archaea (AOA) and bacteria (AOB) ranges widely in soils, but no data exist on what might influence this ratio, its dynamism, or how changes in relative abundance influences the potential contributions of AOA and AOB to soil nitrification. By sampling intensively from cropped-to-fallowed and fallowed-to-cropped phases of a 2-year wheat/fallow cycle, and adjacent uncultivated long-term fallowed land over a 15-month period in 2010 and 2011, evidence was obtained for seasonal and cropping phase effects on the soil nitrification potential (NP), and on the relative contributions of AOA and AOB to the NP that recovers after acetylene inactivation in the presence and absence of bacterial protein synthesis inhibitors. AOB community composition changed significantly (P⩽0.0001) in response to cropping phase, and there were both seasonal and cropping phase effects on the amoA gene copy numbers of AOA and AOB. Our study showed that the AOA:AOB shifts were generated by a combination of different phenomena: an increase in AOA amoA abundance in unfertilized treatments, compared with their AOA counterparts in the N-fertilized treatment; a larger population of AOB under the N-fertilized treatment compared with the AOB community under unfertilized treatments; and better overall persistence of AOA than AOB in the unfertilized treatments. These data illustrate the complexity of the factors that likely influence the relative contributions of AOA and AOB to nitrification under the various combinations of soil conditions and NH4+-availability that exist in the field.

Measuring aerobic respiration in stream ecosystems using the resazurin-resorufin system

[1]xa0The use of smart tracers to study hydrologic systems is becoming more widespread. Smart tracers are compounds that irreversibly react in the presence of a process or condition under investigation. Resazurin (Raz) is a smart tracer that undergoes an irreversible reduction to resorufin (Rru) in the presence of cellular metabolic activity. We quantified the relationship between the transformation of Raz and aerobic bacterial respiration in pure culture experiments using two obligate aerobes and two facultative anaerobes, and in colonized surface and shallow (<10 cm) hyporheic sediments using reach-scale experiments. We found that the transformation of Raz to Rru was nearly perfectly (minr2 = 0.986), positively correlated with aerobic microbial respiration in all experiments. These results suggest that Raz can be used as a surrogate to measure respiration in situ and in vivoat different spatial scales, thus providing an alternative to investigate mechanistic controls of solute transport and stream metabolism on nutrient processing. Lastly, a comparison of respiration and mass-transfer rates in streams suggests that field-scale respiration is controlled by the slower of respiration and mass transfer, highlighting the need to understand both biogeochemistry and physics in stream ecosystems.

Use of Aliphatic n-Alkynes To Discriminate Soil Nitrification Activities of Ammonia-Oxidizing Thaumarchaea and Bacteria

ABSTRACT Ammonia (NH3)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH3 oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤20 μM concentrations of 1-alkynes C8 and C9. In contrast, NH3 oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C8 (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH4 +-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH4 +-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH4 +-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.

Altered precipitation regime affects the function and composition of soil microbial communities on multiple time scales

Climate change models predict that future precipitation patterns will entail lower-frequency but larger rainfall events, increasing the duration of dry soil conditions. Resulting shifts in microbial C cycling activity could affect soil C storage. Further, microbial response to rainfall events may be constrained by the physiological or nutrient limitation stress of extended drought periods; thus seasonal or multiannual precipitation regimes may influence microbial activity following soil wet-up. We quantified rainfall-driven dynamics of microbial processes that affect soil C loss and retention, and microbial community composition, in soils from a long-term (14-year) field experiment contrasting Ambient and Altered (extended intervals between rainfalls) precipitation regimes. We collected soil before, the day following, and five days following 2.5-cm rainfall events during both moist and dry periods (June and September 2011; soil water potential = -0.01 and -0.83 MPa, respectively), and measured microbial respiration, microbial biomass, organic matter decomposition potential (extracellular enzyme activities), and microbial community composition (phospholipid fatty acids). The equivalent rainfall events caused equivalent microbial respiration responses in both treatments. In contrast, microbial biomass was higher and increased after rainfall in the Altered treatment soils only, thus microbial C use efficiency (CUE) was higher in Altered than Ambient treatments (0.70 +/- 0.03 > 0.46 +/- 0.10). CUE was also higher in dry (September) soils. C-acquiring enzyme activities (beta-glucosidase, cellobiohydrolase, and phenol oxidase) increased after rainfall in moist (June), but not dry (September) soils. Both microbial biomass C:N ratios and fungal:bacterial ratios were higher at lower soil water contents, suggesting a functional and/or population-level shift in the microbiota at low soil water contents, and microbial community composition also differed following wet-up and between seasons and treatments. Overall, microbial activity may directly (C respiration) and indirectly (enzyme potential) reduce soil organic matter pools less in drier soils, and soil C sequestration potential (CUE) may be higher in soils with a history of extended dry periods between rainfall events. The implications include that soil C loss may be reduced or compensated for via different mechanisms at varying time scales, and that microbial taxa with better stress tolerance or growth efficiency may be associated with these functional shifts.

FOAM (Functional Ontology Assignments for Metagenomes): A Hidden Markov Model (HMM) database with environmental focus

A new functional gene database, FOAM (Functional Ontology Assignments for Metagenomes), was developed to screen environmental metagenomic sequence datasets. FOAM provides a new functional ontology dedicated to classify gene functions relevant to environmental microorganisms based on Hidden Markov Models (HMMs). Sets of aligned protein sequences (i.e. ‘profiles’) were tailored to a large group of target KEGG Orthologs (KOs) from which HMMs were trained. The alignments were checked and curated to make them specific to the targeted KO. Within this process, sequence profiles were enriched with the most abundant sequences available to maximize the yield of accurate classifier models. An associated functional ontology was built to describe the functional groups and hierarchy. FOAM allows the user to select the target search space before HMM-based comparison steps and to easily organize the results into different functional categories and subcategories. FOAM is publicly available at http://portal.nersc.gov/project/m1317/FOAM/.

Back to the Future of Soil Metagenomics

Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek (Table 1). The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?

Bacterial and archaeal amoA gene distribution covaries with soil nitrification properties across a range of land uses.

Ammonia-oxidizing bacteria and ammonia-oxidizing archaea are commonly found together in soils, yet the factors influencing their relative distribution and activity remain unclear. We examined archaeal and bacterial amoA gene distribution, and used a novel bioassay to assess archaeal and bacterial contributions to nitrification potentials in soils spanning a range of land uses (forest, pasture, cultivated and long-term fallowed cropland) along a 10u2003km transect. The assay, which quantifies the extent to which acetylene-inactivated soil nitrification potential recovers (RNP) in the presence of bacterial protein synthesis inhibitors, indicated a significant archaeal contribution to the nitrification potentials of the pasture and long-term fallowed soils. Archaeal amoA gene abundance did not vary significantly among the soils, but bacterial amoA gene abundance did, resulting in archaealu2003:u2003bacterial amoA abundance ratios ranging from 1.1u2003±u20030.8 in cultivated soils to 396u2003±u2003176 in pasture soils. Both archaeal and bacterial amoA gene compositions were heterogeneous across the landscape, but differed in their patterns of variability. Archaeal amoA gene distributions were distinct among each of the three main land-use types: forest, pasture and cropland soils. In contrast, bacterial amoA gene composition was distinct in forest and in cultivated cropland, while pasture and long-term fallowed cropland soils were similar. In both pasture and long-term fallowed cropland soils, one phylotype of Nitrosospira cluster 3a was highly abundant. This distinct bacterial amoA gene fingerprint correlated with significant contributions of archaea to RNP of both soils, despite differences in archaeal amoA gene composition between the pasture and fallowed soils. This observation suggests that the factors driving the development of ammonia-oxidizing bacteria community composition might influence the extent of archaeal contribution to soil nitrification.

Soil Microbe Active Community Composition and Capability of Responding to Litter Addition after 12 Years of No Inputs

ABSTRACT One explanation given for the high microbial diversity found in soils is that they contain a large inactive biomass that is able to persist in soils for long periods of time. This persistent microbial fraction may help to buffer the functionality of the soil community during times of low nutrients by providing a reservoir of specialized functions that can be reactivated when conditions improve. A study was designed to test the hypothesis: in soils lacking fresh root or detrital inputs, microbial community composition may persist relatively unchanged. Upon addition of new inputs, this community will be stimulated to grow and break down litter similarly to control soils. Soils from two of the Detrital Input and Removal Treatments (DIRT) at the H. J. Andrews Experimental Forest, the no-input and control treatment plots, were used in a microcosm experiment where Douglas-fir needles were added to soils. After 3 and 151 days of incubation, soil microbial DNA and RNA was extracted and characterized using quantitative PCR (qPCR) and 454 pyrosequencing. The abundance of 16S and 28S gene copies and RNA copies did not vary with soil type or amendment; however, treatment differences were observed in the abundance of archaeal ammonia-oxidizing amoA gene abundance. Analysis of ∼110,000 bacterial sequences showed a significant change in the active (RNA-based) community between day 3 and day 151, but microbial composition was similar between soil types. These results show that even after 12 years of plant litter exclusion, the legacy of community composition was well buffered against a dramatic disturbance.

Ecological consequences of the expansion of N2-fixing plants in cold biomes

Research in warm-climate biomes has shown that invasion by symbiotic dinitrogen (N2)-fixing plants can transform ecosystems in ways analogous to the transformations observed as a consequence of anthropogenic, atmospheric nitrogen (N) deposition: declines in biodiversity, soil acidification, and alterations to carbon and nutrient cycling, including increased N losses through nitrate leaching and emissions of the powerful greenhouse gas nitrous oxide (N2O). Here, we used literature review and case study approaches to assess the evidence for similar transformations in cold-climate ecosystems of the boreal, subarctic and upper montane-temperate life zones. Our assessment focuses on the plant genera Lupinus and Alnus, which have become invasive largely as a consequence of deliberate introductions and/or reduced land management. These cold biomes are commonly located in remote areas with low anthropogenic N inputs, and the environmental impacts of N2-fixer invasion appear to be as severe as those from anthropogenic N deposition in highly N polluted areas. Hence, inputs of N from N2 fixation can affect ecosystems as dramatically or even more strongly than N inputs from atmospheric deposition, and biomes in cold climates represent no exception with regard to the risk of being invaded by N2-fixing species. In particular, the cold biomes studied here show both a strong potential to be transformed by N2-fixing plants and a rapid subsequent saturation in the ecosystem’s capacity to retain N. Therefore, analogous to increases in N deposition, N2-fixing plant invasions must be deemed significant threats to biodiversity and to environmental quality.