David S. Duncan

Nitrous oxide emissions during establishment of eight alternative cellulosic bioenergy cropping systems in the North Central United States

Greenhouse gas (GHG) emissions from soils are a key sustainability metric of cropping systems. During crop establishment, disruptive land‐use change is known to be a critical, but under reported period, for determining GHG emissions. We measured soil N2O emissions and potential environmental drivers of these fluxes from a three‐year establishment‐phase bioenergy cropping systems experiment replicated in southcentral Wisconsin (ARL) and southwestern Michigan (KBS). Cropping systems treatments were annual monocultures (continuous corn, corn–soybean–canola rotation), perennial monocultures (switchgrass, miscanthus, and poplar), and perennial polycultures (native grass mixture, early successional community, and restored prairie) all grown using best management practices specific to the system. Cumulative three‐year N2O emissions from annuals were 142% higher than from perennials, with fertilized perennials 190% higher than unfertilized perennials. Emissions ranged from 3.1 to 19.1 kg N2O‐N ha−1 yr−1 for the annuals with continuous corn > corn–soybean–canola rotation and 1.1 to 6.3 kg N2O‐N ha−1 yr−1 for perennials. Nitrous oxide peak fluxes typically were associated with precipitation events that closely followed fertilization. Bayesian modeling of N2O fluxes based on measured environmental factors explained 33% of variability across all systems. Models trained on single systems performed well in most monocultures (e.g., R2 = 0.52 for poplar) but notably worse in polycultures (e.g., R2 = 0.17 for early successional, R2 = 0.06 for restored prairie), indicating that simulation models that include N2O emissions should be parameterized specific to particular plant communities. Our results indicate that perennial bioenergy crops in their establishment phase emit less N2O than annual crops, especially when not fertilized. These findings should be considered further alongside yield and other metrics contributing to important ecosystem services.

Comprehensive Biothreat Cluster Identification by PCR/Electrospray-Ionization Mass Spectrometry

Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.

Nitrogen Fertilization Effects on Productivity and Nitrogen Loss in Three Grass-Based Perennial Bioenergy Cropping Systems

Nitrogen (N) fertilization can greatly improve plant productivity but needs to be carefully managed to avoid harmful environmental impacts. Nutrient management guidelines aimed at reducing harmful forms of N loss such as nitrous oxide (N2O) emissions and nitrate (NO3-) leaching have been tailored for many cropping systems. The developing bioenergy industry is likely to make use of novel cropping systems, such as polycultures of perennial species, for which we have limited nutrient management experience. We studied how a switchgrass (Panicum virgatum) monoculture, a 5-species native grass mixture and an 18-species restored prairie responded to annual fertilizer applications of 56 kg N ha-1 in a field-scale agronomic trial in south-central Wisconsin over a 2-year period. We observed greater fertilizer-induced N2O emissions and sub-rooting zone NO3- concentrations in the switchgrass monoculture than in either polyculture. Fertilization increased aboveground net primary productivity in the polycultures, but not in the switchgrass monoculture. Switchgrass was generally more productive, while the two polycultures did not differ from each other in productivity or N loss. Our results highlight differences between polycultures and a switchgrass monoculture in responding to N fertilization.

Bouncing Back: Plant-Associated Soil Microbes Respond Rapidly to Prairie Establishment

It is well established that soil microbial communities change in response to altered land use and land cover, but less is known about the timing of these changes. Understanding temporal patterns in recovering microbial communities is an important part of improving how we assess and manage reconstructed ecosystems. We assessed patterns of community-level microbial diversity and abundance in corn and prairie plots 2 to 4 years after establishment in agricultural fields, using phospholipid fatty acid biomarkers. Principal components analysis of the lipid biomarkers revealed differing composition between corn and prairie soil microbial communities. Despite no changes to the biomass of Gram-positive bacteria and actinomycetes, total biomass, arbuscular mycorrhizal fungi biomass, and Gram-negative bacteria biomass were significantly higher in restored prairie plots, approaching levels found in long-established prairies. These results indicate that plant-associated soil microbes in agricultural soils can shift in less than 2 years after establishment of perennial grasslands.

Associative nitrogen fixation (ANF) in switchgrass (Panicum virgatum) across a nitrogen input gradient

Associative N fixation (ANF), the process by which dinitrogen gas is converted to ammonia by bacteria in casual association with plants, has not been well-studied in temperate ecosystems. We examined the ANF potential of switchgrass (Panicum virgatum L.), a North American prairie grass whose productivity is often unresponsive to N fertilizer addition, via separate short-term 15N2 incubations of rhizosphere soils and excised roots four times during the growing season. Measurements occurred along N fertilization gradients at two sites with contrasting soil fertility (Wisconsin, USA Mollisols and Michigan, USA Alfisols). In general, we found that ANF potentials declined with long-term N addition, corresponding with increased soil N availability. Although we hypothesized that ANF potential would track plant N demand through the growing season, the highest root fixation rates occurred after plants senesced, suggesting that root diazotrophs exploit carbon (C) released during senescence, as C is translocated from aboveground tissues to roots for wintertime storage. Measured ANF potentials, coupled with mass balance calculations, suggest that ANF appears to be an important source of N to unfertilized switchgrass, and, by extension, to temperate grasslands in general.

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition. An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

Conservation implications of the introduction history of meadow fescue (Festuca pratensis Huds.) to the Driftless Area of the Upper Mississippi Valley, USA

Background: Meadow fescue (Festuca pratensis) is an important cold climate pasture grass that faces pressures from climate and land use change, competitive pressure and limited genetic diversity at the species level. Naturalised populations in the Driftless Area of the American Midwest may be beneficial for the conservation and agronomic improvement of the species. Aims: We explored the population structure and genetic diversity of Driftless Area meadow fescue populations, inferring their introduction histories to evaluate their potential contribution to conservation and breeding. Methods: We assigned plants sampled from 71 grasslands throughout the Driftless Area to genetically defined subpopulations based on neutral nuclear (nSSR) markers. Inter-population hybridisation and genetic diversity were combined with chloroplast haplotype scoring to infer introduction history. Results: We identified four subpopulations with distinct geographic distributions and haplotype composition, with 28% of sampled individuals representing admixtures of two or more subpopulations. There was moderate differentiation among subpopulations and sampling sites, with nonetheless high within-site diversity. Three chloroplast haplotypes were detected, occurring in different frequencies among subpopulations. Subpopulations appeared to have been derived from separate introductions of heterogeneous genetic material. Conclusions: Driftless Area meadow fescue populations are unlikely to contribute to conservation of locally adapted European varieties, but represent a valuable resource for the development of novel varieties and for preserving species-level genetic diversity.

Environmental factors function as constraints on soil nitrous oxide fluxes in bioenergy feedstock cropping systems

Nitrous oxide (N2O) is a potent greenhouse gas and major component of the net global warming potential of bioenergy feedstock cropping systems. Numerous environmental factors influence soil N2O production, making direct correlation difficult to any one factor of N2O fluxes under field conditions. We instead employed quantile regression to evaluate whether soil temperature, water‐filled pore space (WFPS), and concentrations of soil nitrate ( NO3− ) and ammonium ( NH4+ ) determined upper bounds for soil N2O flux magnitudes. We collected data over 6 years from a range of bioenergy feedstock cropping systems including no‐till grain crops, perennial warm‐season grasses, hybrid poplar, and polycultures of tallgrass prairie species each with and without nitrogen (N) addition grown at two sites. The upper bounds for soil N2O fluxes had a significant and positive correlation with all four environmental factors, although relatively large fluxes were still possible at minimal values for nearly all factors. The correlation with NH4+ was generally weaker, suggesting it is less important than NO3− in driving large fluxes. Quantile regression slopes were generally lower for unfertilized perennials than for other systems, but this may have resulted from a perpetual state of nitrogen limitation, which prevented other factors from being clear constraints. This framework suggests efforts to reduce concentrations of NO3− in the soil may be effective at reducing high‐intensity periods—”hot moments”—of N2O production.

Aggregate Annual Fluxes

Daily fluxes were aggregated at the plot level over a calendar year. Details of aggregation are given in the paper.