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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Estimating the CO 2 Fertilization Effect on Extratropical Forest Productivity From Flux‐Tower Observations

Abstract The land sink of anthropogenic carbon emissions, a crucial component of mitigating climate change, is primarily attributed to the CO 2 fertilization effect on global gross primary productivity (GPP). However, direct observational evidence of this effect remains scarce, hampered by challenges in disentangling the CO 2 fertilization effect from other long‐term confounding drivers, particularly climatic changes. Here, we introduce a novel statistical approach to separate the CO 2 fertilization effect on photosynthetic carbon uptake using eddy covariance (EC) records across 38 extratropical forest sites. We find the median stimulation rate of GPP to be 3.2 ± 0.9 gC m −2 yr −1 ppm −1 (or 16.4 ± 4.2% per 100 ppm) under increasing atmospheric CO 2 across these sites, respectively. To validate the robustness of our findings, we test our statistical method using factorial simulations of an ensemble of process‐based land surface models. We address additional factors, including nitrogen deposition and land management, that may impact plant productivity, potentially confounding the attribution to the CO 2 fertilization effect. Assuming these site‐specific effects offset to some extent across sites as random factors, the estimated median value still reflects the strength of the CO 2 fertilization effect. However, disentanglement of these long‐term effects, often inseparable by timescale, requires further causal research. Our study provides direct evidence that the photosynthetic stimulation is maintained under long‐term CO 2 fertilization across multiple EC sites. Such observation‐based quantification is key to constraining the long‐standing uncertainties in the land carbon cycle under rising CO 2 concentrations.

Environmental Sciences & Ecology↗
DOE OSTI2025

Aboveground Rather Than Belowground Productivity Drives Variability in Miscanthus × giganteus Net Primary Productivity

Quantifying the carbon (C) uptake of Miscanthus × giganteus ( M × g ) in both aboveground and belowground structures (e.g., net primary productivity (NPP)) and differences among methodological approaches is crucial. Our objectives were to directly measure Mxg NPP and evaluate the effects of nitrogen application, location, and belowground biomass sampling methods. We hypothesize that increased nitrogen application increases the overall NPP of M × g and that quantifying rhizome biomass using excavations will produce the lowest variability between replicates. We collected biomass from mature M × g stands from three locations in Iowa with three nitrogen application rates and one site in Illinois. We destructively sampled at two time points, when rhizome mass is anticipated to be at a minimum (initial) and anticipated to be at its maximum (peak). Biomass was collected from 1 × 1 m quadrats in which one in-clump and one beside-clump cores were collected and then excavated to 30 cm depth to extract all rhizomes. We found that aboveground M × g NPP ranged from 15.4 Mg DM ha –1 year –1 to 36.4 Mg DM ha –1 year–1 and belowground M × g NPP ranged from 4.4 Mg DM ha –1 year –1 to 19.6 Mg DM ha –1 year –1 . M × g NPP varied across sites, fertilization, and calculation assumptions. Aboveground NPP (yield) was on average 68.7% of the total NPP. Root-to-shoot ratios at peak biomass decreased with nitrogen application rate, from an average of 1.9 for 0 N plots to 0.89 for 224 N fertilized plots. There was more variation in core data than from excavations; however, when in-clump and beside-clump cores were averaged together, core and excavation averages were not different. Overall, these results show that the range of mature M × g NPP is driven by aboveground productivity, influenced by nitrogen application and site. Our results provide useful data to constrain agro-ecosystem models and provide crucial insights for future perennial belowground sampling.

Hartman, Theodore [Univ. of Illinois at Urbana-Cha↗
DOE OSTI2021

Data from: Niche differentiation of bacterial versus archaeal soil nitrifiers induced by ammonium inhibition along a management gradient

Soil nitrification, mediated mainly by ammonia oxidizing archaea (AOA) and bacteria (AOB), converts ammonium (NH4+) to nitrite (NO2−) and thence nitrate (NO3−). To better understand ecological differences between AOA and AOB, we investigated the nitrification kinetics of AOA and AOB under eight replicated cropped and unmanaged ecosystems (including two fertilized natural systems) along a long-term management intensity gradient in the upper U.S. Midwest. For five of eight ecosystems, AOB but not AOA exhibited Haldane kinetics (inhibited by high NH4+ additions), especially in perennial and successional systems. In contrast, AOA predominantly exhibited Michaelis-Menten kinetics, suggesting greater resistance to high nitrogen inputs than AOB. These responses suggest the potential for NH4+-induced niche differentiation between AOA and AOB. Additionally, long-term fertilization significantly enhanced maximum nitrification rates (Vmax) in the early successional systems for both AOA and AOB, but not in the deciduous forest systems. This was likely due to pH suppression of nitrification in the acidic forest soils, corroborated by a positive correlation of Vmax with soil pH but not with amoA gene abundance. Results also demonstrated that soil nitrification potentials were relatively stable, as there were no seasonal differences. Overall, results suggest that (1) NH4+ inhibition of AOB but not AOA could be another factor contributing to niche differentiation between AOA and AOB in soil, and (2) nitrification by both AOA and AOB can be significantly promoted by long-term nitrogen inputs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rising CO 2 and warming reduce global canopy demand for nitrogen

Nitrogen (N) limitation has been considered as a constraint on terrestrial carbon uptake in response to rising CO 2 and climate change. By extension, it has been suggested that declining carboxylation capacity (V cmax ) and leaf N content in enhanced-CO 2 experiments and satellite records signify increasing N limitation of primary production. We predicted Vcmax using the coordination hypothesis and estimated changes in leaf-level photosynthetic N for 1982–2016 assuming proportionality with leaf-level V cmax at 25°C. The whole-canopy photosynthetic N was derived using satellite-based leaf area index (LAI) data and an empirical extinction coefficient for V cmax , and converted to annual N demand using estimated leaf turnover times. The predicted spatial pattern of V cmax shares key features with an independent reconstruction from remotely sensed leaf chlorophyll content. Predicted leaf photosynthetic N declined by 0.27% yr -1 , while observed leaf (total) N declined by 0.2–0.25% yr -1 . Predicted global canopy N (and N demand) declined from 1996 onwards, despite increasing LAI. Leaf-level responses to rising CO 2 , and to a lesser extent temperature, may have reduced the canopy requirement for N by more than rising LAI has increased it. This finding provides an alternative explanation for declining leaf N that does not depend on increasing N limitation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Niche Differentiation of Bacterial Versus Archaeal Soil Nitrifiers Induced by Ammonium Inhibition Along a Management Gradient

Soil nitrification, mediated mainly by ammonia oxidizing archaea (AOA) and bacteria (AOB), converts ammonium (NH 4 + ) to nitrite (NO 2 – ) and thence nitrate (NO 3 – ). To better understand ecological differences between AOA and AOB, we investigated the nitrification kinetics of AOA and AOB under eight replicated cropped and unmanaged ecosystems (including two fertilized natural systems) along a long-term management intensity gradient in the upper U.S. Midwest. For five of eight ecosystems, AOB but not AOA exhibited Haldane kinetics (inhibited by high NH 4 + additions), especially in perennial and successional systems. In contrast, AOA predominantly exhibited Michaelis-Menten kinetics, suggesting greater resistance to high nitrogen inputs than AOB. These responses suggest the potential for NH 4 + -induced niche differentiation between AOA and AOB. Additionally, long-term fertilization significantly enhanced maximum nitrification rates (V max ) in the early successional systems for both AOA and AOB, but not in the deciduous forest systems. This was likely due to pH suppression of nitrification in the acidic forest soils, corroborated by a positive correlation of V max with soil pH but not with amoA gene abundance. Results also demonstrated that soil nitrification potentials were relatively stable, as there were no seasonal differences. Overall, results suggest that (1) NH 4 + inhibition of AOB but not AOA could be another factor contributing to niche differentiation between AOA and AOB in soil, and (2) nitrification by both AOA and AOB can be significantly promoted by long-term nitrogen inputs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Collaborative Research Proposal: Time Resolved Optical Emissions Spectroscopy and Laser Induced Fluorescence Spectroscopy of Nanosecond Pulsed Discharges in a Gas-Liquid Water Film Reactor

Electrical discharge plasma formed in contact with liquid water is of interest for a wide range of applications in chemical, biomedical, agricultural, electrical, and materials science and engineering. Such plasma reactors are of very timely importance since they also have significant disinfection capability by inactivating bacteria, viruses and other pathogens. Many types of plasma sources including those driven by AC, DC, RF, microwave, and pulsed electrical power supplies coupled to a wide range of different electrode configurations and reactor designs have been developed and explored which contact the plasma with liquid water. Recent roadmaps have recommended that further work is needed to develop our understanding of the fundamental chemical and physical process which occur at the interface of non-thermal plasma with liquid water solutions in order to advance this large diversity of applications which ultimately depend upon efficient production of key reactive chemical species. There is a wide range of interacting factors that affect the chemical reactions that occur in the plasma, in the liquid phase, and at the interface. These factors ultimately govern the key reactive chemical species formed and used in the various applications and they include a) the reactor design and input parameters, b) the discharge and transport processes, c) the plasma properties, and d) the resulting chemical reactions. For pulsed discharges, the power supply design and output parameters control the applied voltage, frequency, rise time, and width (duration) of the applied pulses. The reactor design involves specification of the gas-liquid contacting methods, electrode gap distance, reactor volume and shape, and gas and liquid flow rates achievable. The gas and liquid compositions as well as the liquid properties such as pH and conductivity are also of key importance in determination of the resulting chemical reactions. In addition, the important plasma properties include plasma gas temperature, electron density, electron energy (distribution), and size of the plasma channels which are all affected by the discharge and transport properties. Many studies have focused on specific aspects of these various processes. Of particular important and relevance to the proposed work is the utilization of nanosecond pulses to generate plasma in gas-liquid systems, liquid bubbles, underwater, and in gases. Recent advances in nanosecond pulses provide significant advantages in utilization with liquid water. For example, fast rise time and short pulses are less sensitive to water conductivity, such short pulses may provide advantages in fast temporal quenching of the plasma, and fundamental analysis of pulse properties, including pulse shape and width, may be facilitated by investigation of single filamentary fast pulses. Many studies have also dealt with the role of the gas composition on formation of reactive oxygen species (ROS) (i.e., hydroxyl radicals – ·OH, hydrogen peroxide – H 2 O 2 , various atomic oxygen species, ozone-O 3 , hydroperoxyl radicals HO 2 ·) and reactive nitrogen species (RNS) (i.e., nitrogen oxides – NO, NO 2 , N 2 O - collectively termed NO X , nitrite-NO 2 - , nitrate-NO 3 - , peroxynitrite-ONOO - ). The hydroxyl radical is the critical species in many chemical oxidation reactions for chemical degradation of toxic compounds in water and gases and for synthesis of some compounds. The mixture of various nitrogen oxide species is important for many biochemical and biological processes involved in biomedical and agricultural applications including disinfection and fertilizer production. The present proposal focuses on determination of the effects of time resolved electron density and hydroxyl radicals on plasma chemical reactions through collaboration with the Princeton Collaborative Low Temperature Plasma Research Facility (PCRF) at the Princeton Plasma Physics Laboratory (PPPL). In order to further investigate the role of the plasma generated electrons and hydroxyl radicals on the overall formation of the key species including hydrogen peroxide, hydroxyl radicals, and nitrogen oxides, the proposed work, thus seeks to determine high resolution time resolved electron density by optical emissions spectroscopy and time resolved hydroxyl radicals using laser induced fluorescence in the nanosecond discharge reactor. The combination of data on electron density and hydroxyl radical concentration will be utilized in the present work to more fully characterize the chemical reaction processes in this system and to advance the design, development, and operation of such chemical reactors for a wide range of applications.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2020

Associative nitrogen fixation linked with three perennial bioenergy grasses in field and greenhouse experiments

Abstract Associative nitrogen (N 2 )‐fixation (ANF) by bacteria in the root‐zone of perennial bioenergy grasses has the potential to replace or supplement N fertilizer and support sustainable production of biomass, but its application in marginal ecosystems requires further evaluation. In this study, we first combined both greenhouse and field experiments, to explore the N 2 fixation effects of three temperate feedstocks Miscanthus × giganteus (giant miscanthus, Freedom), Panicum virgatum (switchgrass, Alamo), and Saccharum sp. (energycane, Ho 02‐147). In field studies across three growing seasons, plant and soil pools of candidate feedstocks were partially composed of N derived from the atmosphere (Ndfa). Energycane, giant miscanthus, and switchgrass were estimated to derive >30%, %Ndfa. Greenhouse studies were also performed to trace isotopically labeled 15 N 2 into plant biomass and soil pools. Evidence for Ndfa was detected in all three feedstock grasses (using reference 15 N of soil, chicory, and sorghum, δ 15 N~+7.0). Isotopically labeled 15 N 2 was traced into biomass (during grass elongation stage) and soil pools. Extrapolation of rates during the 24 hr labeling period to 50 days estimated 30%–55% of plant Ndfa, with the greatest Ndfa for energycane. The findings of the field natural abundance and greenhouse 15 N 2 feeding experiments provided complementary evidence that perennial bioenergy grasses have the potential to support relatively high rates of ANF, and accumulate diazotroph‐derived N into biomass when grown on non‐fertilized soil.

09 BIOMASS FUELS↗
DOE OSTI2020

Electrolyte Engineering for Efficient Electrochemical Nitrate Reduction to Ammonia on a Titanium Electrode

Nitrates from agricultural runoff and industrial waste streams are a notorious waste product and hazardous pollutant. Traditional electrochemical water remediation approaches aim to solve this problem by converting nitrates to environmentally benign N 2 while minimizing the production of environmentally hazardous side products such as ammonia and nitrous oxide in a process known as “denitrification”. We modify this concept and outline an opportunity to optimize the conversion of nitrates into ammonia, which is also a key commodity product used as a fertilizer, potential fuel, and chemical precursor. The electrochemical conversion of nitrates to ammonia recycles the fixed nitrogen and offers an appealing and supplementary alternative to the energy- and resource-intensive Haber-Bosch process. In this study, we investigated the effect of varying electrochemical conditions (pH, nitrate concentration, and applied potential) on the selective reduction of nitrate to ammonia at a titanium cathode. We observed that high concentrations of both protons and nitrate ions are needed to achieve high selectivity, reaching a peak of 82% Faradaic efficiency to ammonia at an applied potential of -1 V versus RHE and a partial current density to NH 3 of -22 mA/cm 2 , using 0.4 M [NO 3 – ] at pH ~0.77. The Ti electrode, as a poor hydrogen evolution catalyst with notable corrosion resistance, provides a large window of operating conditions to achieve high selectivity in the reduction of nitrate anions. Stability of the system was evaluated, and we found a high Faradaic efficiency throughout the course of an 8 h experiment. After electrochemical testing, titanium hydride was observed at the cathode surface. We also show a preliminary technoeconomic study, indicating that it may be feasible to employ an electrochemical strategy for the production of ammonium nitrate.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Mucilage produced by aerial roots hosts diazotrophs that provide nitrogen in Sorghum bicolor

Sorghum ( Sorghum bicolor ) is an important food, feed, and fodder crop worldwide and is gaining popularity as an energy crop due to its high potential for biomass production. Some sorghum accessions develop many aerial roots and produce an abundant carbohydrate-rich mucilage after rain. This aerial root mucilage is similar to that observed in landraces of maize ( Zea mays ) from southern Mexico, which have been previously shown to host diazotrophs. In this study, we characterized the aerial root development of several sorghum accessions and the impact of humidity on this trait. We conducted a microbiome study of the aerial root mucilage of maize and sorghum and isolated numerous diazotrophs from field sorghum mucilage. We observed that the prevailing phyla in the mucilage were Pseudomonadota, Bacteroidota, and Bacillota. However, bacterial abundances varied based on the genotype and the location. Using acetylene reduction, 15 N 2 gas feeding, and 15 N isotope dilution assays, we confirmed that these sorghum accessions can acquire about 40% of their nitrogen from the atmosphere through these associations on aerial roots. Nitrogen fixation in sorghum aerial root mucilage offers a promising avenue to reduce reliance on synthetic fertilizers and promote sustainable agricultural practices for food, feed, fodder, and bioenergy production.

Venado, Rafael E.↗
DOE OSTI2020

Rice NIN-LIKE PROTEIN 4 plays a pivotal role in nitrogen use efficiency

Nitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavour in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the rice NIN-like protein 4 (OsNLP4) significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 plays a pivotal role in rice NUE and sheds light on crop NUE improvement.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Increased leaf area index and efficiency drive enhanced production under elevated atmospheric [ CO 2 ] in a pine‐dominated stand showing no progressive nitrogen limitation

Abstract Enhancement of net primary production (NPP) in forests as atmospheric [CO 2 ] increases is likely limited by the availability of other growth resources. The Duke Free Air CO 2 Enrichment (FACE) experiment was located on a moderate‐fertility site in the southeastern US, in a loblolly pine ( Pinus taeda L.) plantation with broadleaved species growing mostly in mid‐canopy and understory. Duke FACE ran from 1994 to 2010 and combined elevated [CO 2 ] (eCO 2 ) with nitrogen (N) additions. We assessed the spatial and temporal variation of NPP response using a dataset that includes previously unpublished data from 6 years of the replicated CO 2 × N experiment and extends to 2 years beyond the termination of enrichment. Averaged over time (1997–2010), NPP of pine and broadleaved species were 38% and 52% higher under eCO 2 compared to ambient conditions. Furthermore, there was no evidence of a decline in enhancement over time in any plot regardless of its native site quality. The relation between spatial variation in the response and native site quality was suggested but inconclusive. Nitrogen amendments under eCO 2 , in turn, resulted in an additional 11% increase in pine NPP. For pine, the eCO 2 ‐induced increase in NPP was similar above‐ and belowground and was driven by both increased leaf area index ( L ) and production efficiency (PE = NPP/ L ). For broadleaved species, coarse‐root biomass production was more than 200% higher under eCO 2 and accounted for the entire production response, driven by increased PE. Notably, the fraction of annual NPP retained in total living biomass was higher under eCO 2 , reflecting a slight shift in allocation fraction to woody mass and a lower mortality rate. Our findings also imply that tree growth may not have been only N‐limited, but perhaps constrained by the availability of other nutrients. The observed sustained NPP enhancement, even without N‐additions, demonstrates no progressive N limitation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The rhizosphere and cropping system, but not arbuscular mycorrhizae, affect ammonia oxidizing archaea and bacteria abundances in two agricultural soils

Arbuscular mycorrhizal fungi (AMF) form symbioses with roots that can enhance plant nutrition. While AMF have been shown to have a role in soil nitrogen (N) cycling, it is unclear whether AMF affect N cycling microbes such as ammonia-oxidizing bacteria (AOB) and archaea (AOA), which convert ammonium into nitrite in the first step of nitrification. In this study, we examined the effects of AMF on AOA and AOB abundances within the corn rhizosphere and bulk soil of conventional (corn-soybean rotation with inorganic fertilizer) and diversified (cornsoybean- oats/alfalfa-oats rotation with composted manure) systems. We hypothesized that AMF would decrease AOA and AOB abundances in a cropping-system dependent manner, possibly due to competition for ammonium. We grew corn deficient or proficient in AMF symbiosis in microcosms for 10 weeks. At the end of the experiment, both soils planted with the AMF-proficient corn genotype had higher ammonium and lower nitrate pool sizes compared to the same soils planted with the AMF-deficient corn genotype. Likewise, total plant N was higher in the AMF-proficient genotype compared to the AMF-deficient genotype. Despite changes in soil inorganic N pool sizes, AOA and AOB abundances were unaffected by plant AMF-proficiency. Instead, AOA abundance was greater in the rhizosphere than in the bulk soil regardless of cropping system, and AOB abundance was greater in the conventional than the diversified cropping system soil regardless of proximity to the root. These data indicate that 1) AMF did not affect AOA or AOB abundance in these N-rich soils but other factors such as root proximity and inorganic fertilization did and 2) AOA and AOB have differing ecological niches within rhizosphere and bulk soil that should be considered when managing for nitrogen losses.

Wattenburger, Cassandra↗
DOE OSTI2025

Switchgrass ( Panicum Virgatum ) and Miscanthus ( Miscanthus × Giganteus ) Long-Term Yield Patterns Reveal Consistent Productivity Declines

Perennial grasses like switchgrass ( Panicum virgatum ) and miscanthus ( Miscanthus × giganteus ) are expected to supply a substantial amount of the United States bioeconomy's feedstock demand. However, uncertainties around their long-term yields challenge the viability of their potential and limit their wider adoption. To resolve their long-term yield patterns, we analyzed over 200 plantings of switchgrass and miscanthus across Michigan and Wisconsin, USA, measured over 5–15 years. We found a consistent two-phase long-term yield dynamic; during a yield-building phase , peak yields occurred within 4–5 years after planting, followed by a yield-decline phase in which switchgrass and miscanthus lost 30%–47% and 14%–40% of peak yields, respectively. Among the potential drivers of this dynamic and the yield decline, we found that weather conditions had little impact, as the variation across years was not large enough to drive the observed yield differences. Added nitrogen increased peak yields by 10%–20% and attenuated the yield decline by 20%–50%. However, since fertilized stands still showed a yield decline, other factors became limiting as stands aged. This conserved long-term yield dynamic has direct implications on management. A farm-to-gate economic analysis suggests replanting switchgrass and miscanthus 5 and 9 years following their peak yields maximizes profit over a 30-year time horizon. Results call for further management and breeding strategies to mitigate the yield-decline phase, and for reparameterization of global bioenergy models with carbon capture and storage, which may overestimate yields and the economic and environmental benefits of crops grown for bioenergy feedstocks.

bioenergy↗
DOE OSTI2021

Fungal community and functional responses to soil warming are greater than for soil nitrogen enrichment

Soil fungi are key regulators of forest carbon cycling and their responses to global change have effects that ripple throughout ecosystems. Global changes are expected to push many fungi beyond their environmental niches, but there are relatively few studies involving multiple, simultaneous global change factors. Here, we studied soil fungal diversity, community composition, co-occurrence patterns, and decomposition gene responses to 10 years of soil warming and nitrogen addition, alone and in combination. We specifically examined whether there were fungal community characteristics that could explain changes in soil carbon storage and organic matter chemistry in chronically warmed and fertilized soil. We found that fungal communities in warmed soils are less diverse and shift in composition. Warming also favored hyperdominance by a few mycorrhizal fungal species and lowered manganese peroxidase but increased hydrolytic enzyme encoding gene potentials. Nitrogen addition did not significantly affect fungal community composition but, like warming, did reduce fungal diversity and favored overdominance by a unique set of mycorrhizal taxa. Warming alone and in combination with nitrogen addition also reduced negative but increased positive fungal co-occurrence probabilities, promoting species coexistence. Negative fungal co-occurrence was positively correlated to soil carbon content, while the proportion of fungal hydrolytic enzyme encoding genes was negatively correlated with soil carbon content. This may reflect fungal life history trade-offs between competition (e.g., reduced negative co-occurrence) and resource acquisition (e.g., higher abundance of hydrolytic enzyme encoding genes) with implications for carbon storage.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Development of modular expression across phylogenetically distinct diazotrophs

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here, we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Soil carbon stocks in temperate grasslands differ strongly across sites but are insensitive to decade-long fertilization

We report enhancing soil carbon (C) storage has the potential to offset human-caused increases in atmospheric CO 2 . Rising CO 2 has occurred concurrently with increasing supply rates of biologically limiting nutrients such as nitrogen (N) and phosphorus (P). However, it is unclear how increased supplies of N and P will alter soil C sequestration, particularly in grasslands, which make up nearly a third of non-agricultural land world-wide. Here, we leverage a globally distributed nutrient addition experiment (the Nutrient Network) to examine how a decade of N and P fertilization (alone and in combination) influenced soil C and N stocks at nine grassland sites spanning the continental United States. We measured changes in bulk soil C and N stocks and in three soil C fractions (light and heavy particulate organic matter, and mineral-associated organic matter fractions). Nutrient amendment had variable effects on soil C and N pools that ranged from strongly positive to strongly negative, while soil C and N pool sizes varied by more than an order of magnitude across sites. Piecewise SEM clarified that small increases in plant C inputs with fertilization did not translate to greater soil C storage. Nevertheless, peak season aboveground plant biomass (but not root biomass or production) was strongly positively related to soil C storage at seven of the nine sites, and across all nine sites, soil C covaried with moisture index and soil mineralogy, regardless of fertilization. Overall, we show that site factors such as moisture index, plant productivity, soil texture, and mineralogy were key predictors of cross-site soil C, while nutrient amendment had weaker and site-specific effects on C sequestration. This suggests that prioritizing the protection of highly productive temperate grasslands is critical for reducing future greenhouse gas losses arising from land use change.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Bear Brook Watershed in Maine: Multi-Decadal Whole-Watershed Experimental Acidification

The Bear Brook Watershed in Maine (BBWM) is a long-term research site established to study the response of forest ecosystem function to environmental disturbances of chronic acidic deposition and ecosystem nitrogen enrichment. Starting in 1989, the West Bear (treated) watershed received bimonthly applications of ammonium sulfate [(NH4)2SO4] fertilizer from above the canopy, whereas East Bear (reference) received ambient deposition. The treatments were stopped in 2016, marking the beginning of the recovery phase. Research at the site has focused on soils, streams, and vegetation. Here, we describe data collected over three decades at the BBWM—input and stream output nutrient fluxes, quantitative soil pits and soil chemistry, and soil temperature and moisture.

Patel, Kaizad F.↗
DOE OSTI2021

Long‐term continuous cropping affects ecoenzymatic stoichiometry of microbial nutrient acquisition: a case study from a Chinese Mollisol

Abstract BACKGROUND Soil‐ and plant‐produced extracellular enzymes drive nutrient cycling in soils and are assumed to regulate supply and demand for carbon (C) and nutrients within the soil. Thus, agriculture management decisions that alter the balance of plant and supplemental nutrients should directly alter extracellular enzyme activities (EEAs), and EEA stoichiometry in predictable ways. We used a 12‐year experiment that varyied three major continuous grain crops (wheat, soybean, and maize), each crossed with mineral fertilizer (WCF, SCF and MCF, respectively) or not fertilized (WC, SC and MC, respectively, as controls). In response, we measured the phospholipid fatty acids (PLFAs), EEAs and their stoichiometry to examine the changes to soil microbial nutrient demand under the continuous cropping of crops, which differed with respect to the input of plant litter and fertilizer. RESULTS Fertilizer generally decreased soil microbial biomass and enzyme activity compared to non‐fertilized soil. According to enzyme stoichiometry, microbial nutrient demand was generally C‐ and phosphorus (P)‐limited, but not nitrogen (N)‐limited. However, the degree of microbial resource limitation differed among the three crops. The enzymatic C:N ratio was significantly lower by 13.3% and 26.8%, whereas the enzymatic N:P ratio was significantly higher by 9.9% and 42.4%, in MCF than in WCF and SCF, respectively. The abundances of arbuscular mycorrhizal fungi and aerobic PLFAs were significantly higher in MCF than in WCF and SCF. CONCLUSION These findings are crucial for characterizing enzymatic activities and their stoichiometries that drive microbial metabolism with respect to understanding soil nutrient cycles and environmental conditions and optimizing practices of agricultural management. © 2021 Society of Chemical Industry.

Chen, Xu↗