Terrence H. Bell

Linkage between bacterial and fungal rhizosphere communities in hydrocarbon-contaminated soils is related to plant phylogeny

Phytoremediation is an attractive alternative to excavating and chemically treating contaminated soils. Certain plants can directly bioremediate by sequestering and/or transforming pollutants, but plants may also enhance bioremediation by promoting contaminant-degrading microorganisms in soils. In this study, we used high-throughput sequencing of bacterial 16S rRNA genes and the fungal internal transcribed spacer (ITS) region to compare the community composition of 66 soil samples from the rhizosphere of planted willows (Salix spp.) and six unplanted control samples at the site of a former petrochemical plant. The Bray–Curtis distance between bacterial communities across willow cultivars was significantly correlated with the distance between fungal communities in uncontaminated and moderately contaminated soils but not in highly contaminated (HC) soils (>2000 mg kg−1 hydrocarbons). The mean dissimilarity between fungal, but not bacterial, communities from the rhizosphere of different cultivars increased substantially in the HC blocks. This divergence was partly related to high fungal sensitivity to hydrocarbon contaminants, as demonstrated by reduced Shannon diversity, but also to a stronger influence of willows on fungal communities. Abundance of the fungal class Pezizomycetes in HC soils was directly related to willow phylogeny, with Pezizomycetes dominating the rhizosphere of a monophyletic cluster of cultivars, while remaining in low relative abundance in other soils. This has implications for plant selection in phytoremediation, as fungal associations may affect the health of introduced plants and the success of co-inoculated microbial strains. An integrated understanding of the relationships between fungi, bacteria and plants will enable the design of treatments that specifically promote effective bioremediating communities.

Identification of Nitrogen-Incorporating Bacteria in Petroleum-Contaminated Arctic Soils by Using [15N]DNA-Based Stable Isotope Probing and Pyrosequencing

ABSTRACT Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [15N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in 15N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.

Increasing phytoremediation efficiency and reliability using novel omics approaches

Phytoremediation is a cost-effective green alternative to traditional soil remediation technologies, but has experienced varied success in practice. The recent omics revolution has led to leaps in our understanding of soil microbial communities and plant metabolism, and some of the conditions that promote predictable activity in contaminated soils and heterogeneous environments. Combinations of omics tools and new bioinformatics approaches will allow us to understand integrated activity patterns between plants and microbes, and determine how this metaorganism can be modified to maximize growth, appropriate assembly of microbial communities, and, ultimately, phytoremediation activity. Here we provide an overview of how new omics-mediated discoveries can potentially be translated into an effective and reliable environmental technology.

Culture-Dependent and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils.

Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods.

Alteration of microbial community structure affects diesel biodegradation in an Arctic soil.

A wide range of microbial taxa are active in hydrocarbon-contaminated Arctic soils, and many are capable of hydrocarbon metabolism. The most effective hydrocarbon degraders may not naturally dominate following contamination events, so shifts in microbial abundance could potentially increase hydrocarbon biodegradation. In this study, we contaminated an Arctic soil with diesel and used gentamicin and vancomycin to inhibit distinct portions of the microbial community. We measured diesel loss using gas chromatography, bacterial and fungal abundance with qPCR, and assessed bacterial diversity and community composition through Ion Torrent sequencing of 16S rRNA gene amplicons. The combined addition of both antibiotics increased diesel biodegradation significantly relative to the no-antibiotic treatment, despite reduced bacterial and fungal abundance; however, this effect was not observed when nutrients were also added. All treatments produced unique bacterial communities, and both Xanthomonadaceae and Micrococcineae were dominant in the dual antibiotic treatment. The bacterial communities resulting from dual gentamicin and vancomycin addition were similar both with and without nutrients, although nutrient addition produced a much larger fungal population, which may partly explain the differences in biodegradation between these two treatments. These results suggest that the most efficient hydrocarbon-degrading community may not always be promoted naturally in contaminated soils.

Early rhizosphere microbiome composition is related to the growth and Zn uptake of willows introduced to a former landfill.

Although plants introduced for site restoration are pre-selected for specific traits (e.g. trace element bioaccumulation, rapid growth in poor soils), the in situ success of these plants likely depends on the recruitment of appropriate rhizosphere microorganisms from their new environment. We introduced three willow (Salix spp.) cultivars to a contaminated landfill, and performed soil chemical analyses, plant measurements, and Ion Torrent sequencing of rhizospheric fungal and bacterial communities at 4 and 16 months post-planting. The abundance of certain dominant fungi was linked to willow accumulation of Zn, the most abundant trace element at the site. Interestingly, total Zn accumulation was better explained by fungal community structure 4 months post-planting than 16 months post-planting, suggesting that initial microbial recruitment may be critical. In addition, when the putative ectomycorrhizal fungi Sphaerosporella brunnea and Inocybe sp. dominated the rhizosphere 4 months post-planting, Zn accumulation efficiency was negatively correlated with fungal diversity. Although field studies such as this rely on correlation, these results suggest that the soil microbiome may have the greatest impact on plant function during the early stages of growth, and that plant-fungus specificity may be essential.

Transplanting Soil Microbiomes Leads to Lasting Effects on Willow Growth, but not on the Rhizosphere Microbiome.

Plants interact closely with microbes, which are partly responsible for plant growth, health, and adaptation to stressful environments. Engineering the plant-associated microbiome could improve plant survival and performance in stressful environments such as contaminated soils. Here, willow cuttings were planted into highly petroleum-contaminated soils that had been gamma-irradiated and subjected to one of four treatments: inoculation with rhizosphere soil from a willow that grew well (LA) or sub-optimally (SM) in highly contaminated soils or with bulk soil in which the planted willow had died (DE) or no inoculation (CO). Samples were taken from the starting inoculum, at the beginning of the experiment (T0) and after 100 days of growth (TF). Short hypervariable regions of archaeal/bacterial 16S rRNA genes and the fungal ITS region were amplified from soil DNA extracts and sequenced on the Illumina MiSeq. Willow growth was monitored throughout the experiment, and plant biomass was measured at TF. CO willows were significantly smaller throughout the experiment, while DE willows were the largest at TF. Microbiomes of different treatments were divergent at T0, but for most samples, had converged on highly similar communities by TF. Willow biomass was more strongly linked to overall microbial community structure at T0 than to microbial community structure at TF, and the relative abundance of many genera at T0 was significantly correlated to final willow root and shoot biomass. Although microbial communities had mostly converged at TF, lasting differences in willow growth were observed, probably linked to differences in T0 microbial communities.

Contrasting the Community Structure of Arbuscular Mycorrhizal Fungi from Hydrocarbon-Contaminated and Uncontaminated Soils following Willow ( Salix spp. L.) Planting

Phytoremediation is a potentially inexpensive alternative to chemical treatment of hydrocarbon-contaminated soils, but its success depends heavily on identifying factors that govern the success of root-associated microorganisms involved in hydrocarbon degradation and plant growth stimulation. Arbuscular mycorrhizal fungi (AMF) form symbioses with many terrestrial plants, and are known to stimulate plant growth, although both species identity and the environment influence this relationship. Although AMF are suspected to play a role in plant adaptation to hydrocarbon contamination, their distribution in hydrocarbon-contaminated soils is not well known. In this study, we examined how AMF communities were structured within the rhizosphere of 11 introduced willow cultivars as well as unplanted controls across uncontaminated and hydrocarbon-contaminated soils at the site of a former petrochemical plant. We obtained 69 282 AMF-specific 18S rDNA sequences using 454-pyrosequencing, representing 27 OTUs. Contaminant concentration was the major influence on AMF community structure, with different AMF families dominating at each contaminant level. The most abundant operational taxonomic unit in each sample represented a large proportion of the total community, and this proportion was positively associated with increasing contamination, and seemingly, by planting as well. The most contaminated soils were dominated by three phylotypes closely related to Rhizophagus irregularis, while these OTUs represented only a small proportion of sequences in uncontaminated and moderately contaminated soils. These results suggest that in situ inoculation of AMF strains could be an important component of phytoremediation treatments, but that strains should be selected from the narrow group that is both adapted to contaminant toxicity and able to compete with indigenous AMF species.

Microbial competition in polar soils: a review of an understudied but potentially important control on productivity.

Intermicrobial competition is known to occur in many natural environments, and can result from direct conflict between organisms, or from differential rates of growth, colonization, and/or nutrient acquisition. It has been difficult to extensively examine intermicrobial competition in situ, but these interactions may play an important role in the regulation of the many biogeochemical processes that are tied to microbial communities in polar soils. A greater understanding of how competition influences productivity will improve projections of gas and nutrient flux as the poles warm, may provide biotechnological opportunities for increasing the degradation of contaminants in polar soil, and will help to predict changes in communities of higher organisms, such as plants.

Plant assemblage composition and soil P concentration differentially affect communities of AM and total fungi in a semi-arid grassland

Adding inorganic P- and N-fixing legumes to semi-arid grasslands can increase forage yield, but soil nutrient concentrations and plant cover may also interact to modify soil fungal populations, impacting short- and long-term forage production. We tested the effect of plant assemblage (seven native grasses, seven native grasses + the domesticated N-fixing legume Medicago sativa, seven native grasses + the native N-fixing legume Dalea purpurea or the introduced grass Bromus biebersteinii + M. sativa) and soil P concentration (addition of 0 or 200 P2O5 kg ha(-1) at sowing) on the diversity and community structure of arbuscular mycorrhizal (AM) fungi and total fungi over two consecutive years, using 454-pyrosequencing of 18S rDNA and ITS amplicons. Treatment effects were stronger in the wet year (2008) than the dry year (2009). The presence of an N-fixing legume with native grasses generally increased AM fungal diversity, while the interaction between soil P concentration and plant assemblage modified total fungal community structure in 2008. Excluding interannual variations, which are likely driven by moisture and plant productivity, AM fungal communities in semi-arid grasslands appear to be primarily affected by plant assemblage composition, while the composition of other fungi is more closely linked to soil P.