An optimized crop-livestock system can achieve a safe and just planetary boundary for phosphorus at the sub-basin level in China.

The contribution of crop and livestock production to the exceedance of the planetary boundary for phosphorus (P) in China is still unclear, despite the country's well-known issues with P fertilizer overuse and P-related water pollution. Using coupled models at sub-basin scales we estimate that livestock production increased the consumption of P fertilizer fivefold and exacerbated P losses twofold from 1980 to 2017. At present, China's crop-livestock system is responsible for exceeding what is considered a 'just' threshold for fertilizer P use by 30% (ranging from 17% to 68%) and a 'safe' water quality threshold by 45% (ranging from 31% to 74%) in 25 sub-basins in China. Improving the crop-livestock system will keep all sub-basins within safe water quality and just multigenerational limits for P in 2050.

Microbial life-history strategies mediate microbial carbon pump efficacy in response to N management depending on stoichiometry of microbial demand.

The soil microbial carbon pump (MCP) is increasingly acknowledged as being directly linked to soil organic carbon (SOC) accumulation and stability. Given the close coupling of carbon (C) and nitrogen (N) cycles and the constraints imposed by their stoichiometry on microbial growth, N addition might affect microbial growth strategies with potential consequences for necromass formation and carbon stability. However, this topic remains largely unexplored. Based on two multi-level N fertilizer experiments over 10 years in two soils with contrasting soil fertility located in the North (Cambisol, carbon-poor) and Southwest (Luvisol, carbon-rich), we hypothesized that different resource demands of microorganism elicit a trade-off in microbial growth potential (Y-strategy) and resource-acquisition (A-strategy) in response to N addition, and consequently on necromass formation and soil carbon stability. We combined measurements of necromass metrics (MCP efficacy) and soil carbon stability (chemical composition and mineral associated organic carbon) with potential changes in microbial life history strategies (assessed via soil metagenomes and enzymatic activity analyses). The contribution of microbial necromass to SOC decreased with N addition in the Cambisol, but increased in the Luvisol. Soil microbial life strategies displayed two distinct responses in two soils after N amendment: shift toward A-strategy (Cambisol) or Y-strategy (Luvisol). These divergent responses are owing to the stoichiometric imbalance between microbial demands and resource availability for C and N, which presented very distinct patterns in the two soils. The partial correlation analysis further confirmed that high N addition aggravated stoichiometric carbon demand, shifting the microbial community strategy toward resource-acquisition which reduced carbon stability in Cambisol. In contrast, the microbial Y-strategy had the positive direct effect on MCP efficacy in Luvisol, which greatly enhanced carbon stability. Such findings provide mechanistic insights into the stoichiometric regulation of MCP efficacy, and how this is mediated by site-specific trade-offs in microbial life strategies, which contribute to improving our comprehension of soil microbial C sequestration and potential optimization of agricultural N management.

mRNA Display Pipeline for Protein Biosensor Construction.

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

Updated loss factors and high-resolution spatial variations for reactive nitrogen losses from Chinese rice paddies.

Anthropogenic reactive nitrogen (Nr) loss has been a critical environmental issue. However, due to the limitations of data availability and appropriate methods, the estimation of Nr loss from rice paddies and associated spatial patterns at a fine scale remain unclear. Here, we estimated the background Nr loss (BNL, i.e., Nr loss from soils without fertilization) and the loss factors (the percentage of Nr loss from synthetic fertilizer, LFs) for five loss pathways in rice paddies and identified the national 1 × 1 km spatial variations using data-driven models combined with multi-source data. Based on established machine learning models, an average of 23.4% (15.3-34.6%, 95% confidence interval) of the synthetic N fertilizer was lost to the environment, in the forms of NH3 (17.4%, 10.9-26.7%), N2O (0.5%, 0.3-0.8%), NO (0.2%, 0.1-0.4%), N leaching (3.1%, 0.8-5.7%), and runoff (2.3%, 0.6-4.5%). The total Nr loss from Chinese rice paddies was estimated to be 1.92 ± 0.52 Tg N yr-1 in 2021, in which synthetic fertilizer-induced Nr loss accounted for 69% and BNL accounted for the other 31%. The hotspots of Nr loss were concentrated in the middle and lower regions of the Yangtze River, an area with extensive rice cultivation. This study improved the estimation accuracy of Nr losses and identified the hotspots, which could provide updated insights for policymakers to set the priorities and strategies for Nr loss mitigation.

Co-benefits for net carbon emissions and rice yields through improved management of organic nitrogen and water.

Returning organic nutrient sources (for example, straw and manure) to rice fields is inevitable for coupling crop-livestock production. However, an accurate estimate of net carbon (C) emissions and strategies to mitigate the abundant methane (CH4) emission from rice fields supplied with organic sources remain unclear. Here, using machine learning and a global dataset, we scaled the field findings up to worldwide rice fields to reconcile rice yields and net C emissions. An optimal organic nitrogen (N) management was developed considering total N input, type of organic N source and organic N proportion. A combination of optimal organic N management with intermittent flooding achieved a 21% reduction in net global warming potential and a 9% rise in global rice production compared with the business-as-usual scenario. Our study provides a solution for recycling organic N sources towards a more productive, carbon-neutral and sustainable rice-livestock production system on a global scale.

City-level livestock methane emissions in China from 2010 to 2020.

Livestock constitute the world's largest anthropogenic source of methane (CH4), providing high-protein food to humans but also causing notable climate risks. With rapid urbanization and increasing income levels in China, the livestock sector will face even higher emission pressures, which could jeopardize China's carbon neutrality target. To formulate targeted methane reduction measures, it is crucial to estimate historical and current emissions on fine geographical scales, considering the high spatial heterogeneity and temporal variability of livestock emissions. However, there is currently a lack of time-series data on city-level livestock methane emissions in China, despite the flourishing livestock industry and large amount of meat consumed. In this study, we constructed a city-level livestock methane emission inventory with dynamic spatial-temporal emission factors considering biological, management, and environmental factors from 2010 to 2020 in China. This inventory could serve as a basic database for related research and future methane mitigation policy formulation, given the population boom and dietary changes.

Development of Lyophilized Eukaryotic Cell-Free Protein Expression System Based on Leishmania tarentolae.

Eukaryotic cell-free protein expression systems enable rapid production of recombinant multidomain proteins in their functional form. A cell-free system based on the rapidly growing protozoan Leishmania tarentolae (LTE) has been extensively used for protein engineering and analysis of protein interaction networks. However, like other eukaryotic cell-free systems, LTE deteriorates at ambient temperatures and requires deep freezing for transport and storage. In this study, we report the development of a lyophilized version of LTE. Use of lyoprotectants such as poly(ethylene glycol) and trehalose during the drying process allows retention of 76% of protein expression activity versus nonlyophilized controls. Lyophilized LTE is capable of withstanding storage at room temperature for over 2 weeks. We demonstrated that upon reconstitution the lyophilized LTE could be used for in vitro expression of active enzymes, analysis of protein-protein interactions by AlphaLISA assay, and functional analysis of protein biosensors. Development of lyophilized LTE lowers the barriers to its distribution and opens the door to its application in remote areas.

City-level decoupling between livestock and crop production and its effect on fertilizer usage: Evidence from China.

China is one of the largest producers of livestock production and also with tremendous fertilizer consumption in crop production, regional decoupling between livestock and crop production often results in fertilizer overuse and environmental pollution. However, city-level coupling analysis between livestock and crop production is rare, and its impact on fertilizer usage also remains unclear. Here, we evaluated the nitrogen (N) nutrient supply from the livestock breeding sector and the N nutrient demand of cropland during the 2007-2020 period in a typical agricultural region in China. The city-level coupling degree of livestock and crop production and the effect on fertilizer usage were explored with spatial analysis and regression methods. Our results show that the province level has a relatively high coupling degree. However, significant spatial heterogeneity was found at the city level, especially in western Sichuan Province due to the highly unbalanced distribution of livestock and crop production, and this decoupling phenomenon may hinder fertilizer reduction. Furthermore, we reveal that technological development is not an effective way to achieve sustainable agriculture without other policy instruments, such as livestock spatial relocation, which must be considered when formulating crop-livestock integration policies. The findings expand city-level knowledge of the livestock-crop system and provide important implications for adjusting agricultural practices to realize sustainable agricultural development.

Biofortification of different maize cultivars with zinc, iron and selenium by foliar fertilizer applications.

Fertilizer-based biofortification is a strategy for combating worldwide malnutrition of zinc (Zn), iron (Fe) and selenium (Se). Field experiments were conducted to investigate the effects of foliar treatments on concentrations of Zn, Fe, Se, N and bioavailability of Zn and Fe in grains of three maize cultivars grown at three locations. We compared the efficacy of ZnO nanoparticles (ZnO-NPs), Zn complexed chitosan nanoparticles (Zn-CNPs), conventional ZnSO4 and a cocktail solution (containing Zn, Fe and Se). All treatments were foliar-applied at rate of 452 mg Zn L-1, plus urea. Applying ten-fold less Zn (at rate of 45.2 mg Zn L-1) plus urea in the form of ZnO-NPs, Zn-CNPs, or ZnSO4 resulted in no increase, or a negligible increase, in grain Zn concentration compared with deionized water. By contrast, among the different Zn sources plus urea applied by foliar sprays, conventional ZnSO4 was the most efficient in improving grain Zn concentration. Furthermore, foliar application of a cocktail solution effectively improved grain concentrations of Zn, Fe, Se and N simultaneously, without a grain yield trade-off. For example, the average grain concentrations were simultaneously increased from 13.8 to 22.1 mg kg-1 for Zn, from 17.2 to 22.1 mg kg-1for Fe, from 21.4 to 413.5 ug kg-1 for Se and from 13.8 to 14.7 g kg-1 for N by foliar application of a cocktail solution. Because grain yield was significantly negatively correlated with grain nutrient concentrations, the magnitude of increase in grain concentrations of Zn and Fe was most pronounced in the maize cultivar with the lowest grain yield (Zhengdan958 grown in Linyi). Foliar application of a cocktail solution also significantly decreased the phytic acid (PA) concentration, ratios of PA/Fe and PA/Zn in grains, indicating an increased bioavailability of Fe and Zn for human health. In conclusion, we found that a foliar application of a cocktail solution including Zn, Fe, Se and N was most effective for biofortification, but that the grains with the lowest yield contained the greatest concentration of these elements. This finding highlights the need to breed maize varieties that are capable of achieving both high grain yield and high grain nutritional quality to address food security and human health challenges.

Ammonia mitigation campaign with smallholder farmers improves air quality while ensuring high cereal production.

Reducing cropland ammonia (NH3) emissions while improving air quality and food supply is a challenge, particularly in China where there are millions of smallholder farmers. We tested the effectiveness of a tailored nitrogen (N) management strategy applied to wheat-maize cropping systems in 'demonstration squares' across Quzhou County in the North China Plain. The N-management techniques included optimal N rates, deep fertilizer placement and application of urease inhibitors, implemented through cooperation between government, researchers, businesses and smallholders. Compared with conventional local smallholder practice, our NH3 mitigation campaign reduced NH3 volatilization from wheat and maize by 49% and 39%, and increased N-use efficiency by 28% and 40% and farmers' profitability by 25% and 19%, respectively, with no detriment to crop yields. County-wide atmospheric NH3 and fine particulate matter (with aerodynamic diameter <2.5 µm) concentrations decreased by 40% and 8%, respectively. County-wide net benefits were estimated at US$7.0 million. Our demonstration-square approach shows that cropland NH3 mitigation and improved air quality and farm profitability can be achieved simultaneously by coordinated actions at the county level.

In Vitro Reconstitution and Analysis of SARS-CoV-2/Host Protein-Protein Interactions.

The emergence of viral threats such as Ebola, ZIKA, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires a rapid and efficient approach for elucidating mechanisms of pathogenesis and development of therapeutics. In this context, cell-free protein synthesis (CFPS) holds a promise to resolve the bottlenecks of multiplexed protein production and interaction analysis among host and pathogen proteins. Here, we applied a eukaryotic CFPS system based on Leishmania tarentolae extract (LTE) protein expression in combination with AlphaLISA proximity-based protein interaction technology to identify intraviral and viral-human protein interactions of SARS-CoV-2 virus that can potentially be targeted by the existing or novel antiviral therapeutics. We produced and tested 54 putative human-viral protein pairs in vitro and identified 45 direct binary protein interactions. As a casing example of the assay's suitability for drug development applications, we analyzed the effect of a putative biologic on the human angiotensin-converting enzyme 2/receptor-binding domain (hACE2/RBD) interaction. This suggests that the presented pathogen characterization platform can facilitate the development of new therapeutic agents.

Data-driven estimation of nitric oxide emissions from global soils based on dominant vegetation covers.

Soils are a major source of global nitric oxide (NO) emissions. However, estimates of soil NO emissions have large uncertainties due to limited observations and multifactorial impacts. Here, we mapped global soil NO emissions, integrating 1356 in-situ NO observations from globally distributed sites with high-resolution climate, soil, and management practice data. We then calculated global and national total NO budgets and revealed the contributions of cropland, grassland, and forest to global soil NO emissions at the national level. The results showed that soil NO emissions were explained mainly by N input, water input and soil pH. Total above-soil NO emissions of the three vegetation cover types were 9.4 Tg N year-1 in 2014, including 5.9 Tg N year-1 (1.04, 95% confidence interval [95% CI]: 0.09-1.99 kg N ha-1 year-1 ) emitted from forest, 1.7 Tg N year-1 (0.68, 95% CI: 0.10-1.26 kg N ha-1 year-1 ) from grassland, and 1.8 Tg N year-1 (0.98, 95% CI: 0.42-1.53 kg N ha-1 year-1 ) from cropland. Soil NO emissions in approximately 57% of 213 countries surveyed were dominated by forests. Our results provide updated inventories of global and national soil NO emissions based on robust data-driven models. These estimates are critical to guiding the mitigation of soil NO emissions and can be used in combination with biogeochemical models.

Fertilization as the most critical factor affecting yield response and agronomic efficiency of phosphorus in Chinese rice production: evidence from multi-location field trials.

BACKGROUND: Efficient utilization of phosphorus (P) has been a major challenge for sustainable agriculture. However, the responses of fertilizer rate, region, soil properties, cropping systems and genotypes to P have not been investigated comprehensively and systematically. RESULTS: A comprehensive analysis of 9863 fertilizer-P experiments on rice cultivation in China showed that rice yield  increased first and then fell down with the addition of P fertilizer, and the highest yield of 7963 kg ha-1 was observed under 100% P treatment. Under 100% P treatment, the yield response of applied P (YRP ) and agronomic efficiency of applied P (AEP ) were 12.8% and 30.1 kg ha-1 , respectively. Lower soil pH (< 5.5) and organic matter (< 30.0 g kg-1 ) were associated with lower YRP and AEP . By contrast, soil available P < 25.0 mg kg-1 resulted in decreased YRP (15.3 to 11.4%) and AEP (32.3 kg kg-1 to 26.2 kg kg-1 ), whereas soil available P > 25.0 mg kg-1 maintained the relatively stable YRP and AEP . Also, the YRP and AEP were significantly higher for single-cropping rice compared to other cropping systems. Moreover, the rice genotypes such as 'Longdun', 'Kendao' and 'Jigeng' had higher YRP and AEP than the average value. Overall, the fertilizer-P rate was the primary factor affecting YRP and AEP , and the recommended P fertilizer rate can be reduced by 9-21 kg P ha-1 compared to existing expert recommendations. CONCLUSION: The present study highlights the role of fertilizer-P rate in maximizing the YRP and AEP , thereby providing a strong basis for future fertilizer management in rice cultivation systems. © 2023 Society of Chemical Industry.

Comprehensive assessment of the utilization of manure in China's croplands based on national farmer survey data.

China's rapid increase in mass excreta and its environmental discharge have attracted substantial attention. However, cropland as a main destination of excreta utilization has not been extensively evaluated. Here, a national survey was used to assess the utilization of manure in croplands across China. The data included the inputs of manure nitrogen (N), phosphorus (P), and potassium (K) for cereals, fruits, vegetables, and other crops, along with the manure proportion of total N, P, and K inputs at the county level. The results showed that the manure N, P, and K inputs were 6.85, 2.14, and 4.65 million tons (Mt), respectively, constituting 19.0%, 25.5%, and 31.1% of the total N, P, and K, respectively. The spatial distribution of the manure proportion of total inputs was lower in Eastern China and higher in Western China. The results provide a detailed description of the utilization of manure nutrients in agricultural areas throughout China, which will serve as basic support for policymakers and researchers involved in future agricultural nutrient management in China.

An enhanced electron transport chain improved astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a carotenoid pigment with antioxidant properties widely used as a feed supplement. Wild-type strains of Phaffia rhodozyma naturally produce low AX yields, but we increased AX yields 50-fold in previous research using random mutagenesis of P. rhodozyma CBS6938 and fermentation optimization. On that study, genome changes were linked with phenotype, but relevant metabolic changes were not resolved. In this study, the wild-type and the superior P. rhodozyma mutant strains were grown in chemically defined media and instrumented fermenters. Differential kinetic, metabolomics, and transcriptomics data were collected. Our results suggest that carotenoid production was mainly associated with cell growth and had a positive regulation of central carbon metabolism metabolites, amino acids, and fatty acids. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites were in abundance and media supplementation of citrate, malate, α-ketoglutarate, succinate, or fumarate increased AX production in the mutant strain. Transcriptomic data correlated with the metabolic and genomic data and found a positive regulation of genes associated with the electron transport chain suggesting this to be the main driver for improved AX production in the mutant strain.

Fungal necromass increases soil aggregation and organic matter chemical stability under improved cropland management and natural restoration.

The formation and stability of soil organic matter (SOM) is crucial for food security, soil health, and climate change mitigation. Although various SOM stabilization mechanisms have been proposed and investigated, the contribution of plant- and microbial-derived carbon into physical and chemical stabilization processes remain unclear. Therefore, this study investigates lignin phenols, microbial necromass, soil aggregation and SOM chemical composition under three cropland management and two natural restoration strategies: NPK, NPK + manure (NPK + M) and NPK + peat vermiculite (NPK + PV) after 5 years, and natural restoration for 10 and 40 years (NR10 and NR40, respectively). Addition of manure or peat vermiculite and NR40 increased soil organic carbon (SOC) by 86-122 % and 16 %, respectively, compared to the NPK fertilization. Lignin phenols and bacterial necromass-C were the highest under NPK + M, and lignin phenols increased by 0.07 g and microbial necromass-C by 0.44 g with each additional 1 g of SOC. Fungal necromass-C in NPK + PV was 0.14-1.1 times higher than in other treatments. The mean weight diameter of aggregates was the highest, while macroaggregate turnover was the slowest under NPK + PV, indicating increased soil aggregation and physical stability. Natural restoration reduced lignin phenols by 33-40 % and labile O-alkyl C by 4-9 %, but increased resistant alkyl C by 9-15 % compared with other treatments, reflecting the highest chemical stability. High fungal necromass was beneficial to the accumulation of particulate and mineral-associated C and aggregate stability, and decelerated macroaggregate turnover. Aromatic C increased but aliphatic-C/aromatic-C decreased with increasing fungal necromass-C. Consequently, fungal necromass C increases SOM physical stability by slowing aggregate turnover and enhances the chemical stability through the accumulation of recalcitrant C under improved cropland management and natural restoration.

Cropland nitrous oxide emissions exceed the emissions of RCP 2.6: A global spatial analysis.

Nitrous oxide (N2O), as a potent greenhouse gas, must be limited to prevent the global temperature increasing by >2 °C. Cropland is the largest source of anthropogenic N2O emissions; however, earlier estimates for emissions and their exceedances still remain uncertainties. Here, we used a spatially explicit model to estimate cropland N2O emission in 2014 by refined grid-level crop-specific EFs and considered the background emission. We also sought to determine where N2O emissions exceed the "boundary" through analysis of spatial data from representative concentration pathway (RCP) 2.6. The global cropland N2O emission was 2.92 ± 0.59 Tg N yr-1, which far exceeds the 0.82 Tg N yr-1 boundary, over 90 % of cropland areas exceeded the boundary. Western Europe, Southeastern China, Pakistan, and the Ganges Plain exceeded the boundary by >2 kg N ha-1 yr-1. The boundary exceedances showed a positive linear response with respect to total cropland emission and a quadratic response to GDP per capita at the country level. Our study highlights the necessity of accurate estimations of spatial variations in cropland N2O emissions and evaluation of exceedances, to facilitate the development of more effective mitigation measures in different regions.

Bottom-up estimates of reactive nitrogen loss from Chinese wheat production in 2014.

Excessive use of synthetic nitrogen (N) for Chinese wheat production results in high loss of reactive N loss (Nr; all forms of N except N2) into the environment, causing serious environmental issues. Quantifying Nr loss and its spatial variations therein is vital to optimize N management and mitigate loss. However, accurate, high spatial resolution estimations of Nr from wheat production are lacking due to limitations of data generation and estimation methods. Here, we applied the random forest (RF) algorithm to bottom-up N application rate data, obtained through a survey of millions of farmers, to estimate the Nr loss from wheat production in 2014. The results showed that the average total Nr loss was 52.5 kg N ha-1 (range: 4.6-157.8 kg N ha-1), which accounts for 26.1% of the total N applied. The hotspots for high Nr loss are the same as those high applied N, including northwestern Xinjiang, central-southern Hebei, Shandong, central-northern Jiangsu, and Hubei. Our database could guide regional N management and be used in conjunction with biogeochemical models.