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INVESTIGATOR: Tharayil, N.

PERFORMING INSTITUTION:
CLEMSON UNIVERSITY
CLEMSON, SOUTH CAROLINA 29634

ENHANCING THE NUTRIENT USE EFFICIENCY IN CROP PLANTS BY TAILORING THE NITROGEN AND PHOSPHOROUS RELEASE RATES FROM RENDERED ANIMAL MATERIALS

NON-TECHNICAL SUMMARY: Faced with a steadily dwindling cropping area, current agricultural practices overly rely on chemical fertilizer inputs to boost crop productivity. Present-day agriculture relies heavily on nutrient rich inorganic and chemical forms of nitrogen (N), including urea, ammonium salts, and nitrate salts. This approach results in high nitrogen delivery over a short time that exceeds the capacity for plant uptake, thus resulting in diminished nutrient use efficiency and greater environmental pollution. The reserve of phosphate rock, which currently accounts for 75% of the phosphorous (P) fertilizers, is dwindling at an alarming rate due to the burgeoning demand for P fertilizers. Moreover, the current production practices of nitrogen and phosphorous are highly energy intensive and not sustainable. The proposed approach is to revisit a historically employed fertilizer matrix; rendered animal proteins. The proposed research will develop new formulations of fertilizers, focusing on maximizing the nutrient use efficiency while employing economically feasible source materials. The proposed approach will utilize rendered animal proteins that are unsuitable for feed applications as a base matrix for supplying nitrogen and phosphorous to crops, thus recapture and reuse the nutrients from a resource which otherwise will be discarded. The project will employ various natural plant-based amendments that are spent-waste, to tailor the nutrient release rate from these RM so as to match the nutrient uptake rate of the crops, thus facilitating higher nutrient use efficiency. The project team proposes to produce a pelletized material of varying formulations of RM and amendments and test the nitrogen and phosphorous use efficiency on food crops in greenhouse and field conditions. The proposed recycling of reactive forms of nutrients in animal and plant byproducts will enhance the long-term sustainability of crop production systems by promoting the biological health of soils that is diminished by long-term, exclusive use of inorganic fertilizers. Results from this work will be applicable to other organic fertilizers that include agricultural and municipal wastes.

OBJECTIVES: The production of inorganic fertilizers is highly energy intensive and not sustainable; however, the current agricultural practices greatly dependent on the use of chemical fertilizers to meet the ever burgeoning demand for food production. Because of the fixed nitrogen production capacity of the US, more than 50% of the nitrogen (N) fertilizer demand is met through imports. Furthermore, while the US is the largest exporter of phosphorous (P) fertilizers, the reserve of phosphate rock, which currently accounts for 75% of the P fertilizers, is predicted to be depleted by 2050. In many production systems less than 50% of applied N and 20% of the applied P is taken up by plants and converted to biomass. The high application rate of synthetic fertilizers, combined with low plant utilization results in severe environmental pollution. The current adoption of organic manures is plagued by N loss during storage leading to inconsistent nutrient delivery, and the consequential environmental pollution. The proposed project will utilize various natural amendments to tailor the nutrient release rate from rendered animal proteins (RM) in order to match the nutrient uptake rate of the crops, thus facilitating higher nutrient use efficiency, better soil health, reduced negative environmental impact, and produce quality.The major goal of this project is to substantially improve N and P use efficiency in managed agroecosystems through the efficient and economical recapture and reuse of nutrients in proteinaceous organic amendments, which would otherwise be landfilled, and would potentially be a source of environmental pollution. This will be achieved through 1) modeling the mechanisms and rates of nutrient mineralization and release from RM and 2) modulating the rate of nutrient release from RM by using synthetic and natural compounds that regulate specific steps in the N and P mineralization process, so as to match the nutrient uptake rates of the crop plants.Our specific objectives are to:Characterize the fundamental mechanisms and rates of nutrient mineralization processes from RMTailor the N and P release rate from RM using various synthetic and natural compounds that regulate different steps in the mineralization process,Measure efficiency of N and P use of food crops from RM in greenhouse and field trials.Monitor changes in soil health and quality of produce following partial substitution of traditional fertilizer regime with the optimized RM fertilizer formulations.The fact that the RM can be better characterized and is not time sensitive like other N-rich organic amendments makes it a viable candidate for studying the fundamentals of nutrient release from nitrogenous organic amendments and their influence on soil microbial communities that sustain crop production. Results from this work will be applicable to other organic fertilizers that include agricultural and municipal wastes.

APPROACH: To achieve the proposed objectives, the project team will develop new RM-based fertilizer formulations, focusing on maximizing the nutrient use efficiency while employing economically feasible agricultural byproducts that are recycled to recapture the reactive form of nutrients, which would otherwise form a source of environmental pollution. The experimental approach of this project is dived into 1) Formulation, 2) Lab-scale Testing, 3) Greenhouse Trials, and 4) Field Trials. The proposed approach will utilize rendered animal proteins or rendered materials (RM) as a base matrix for fertilizer formulations to augment traditional fertilizers in a tailored organic formulation. Triglyceride and fatty acid fractions from the rendered material will be removed to produce extracted rendered material (ERM). The project team will study the nutrient supplying capacity, and the metabolic processes facilitating nutrient release from ERM through lab and greenhouse experiments. The rate of release of N and P from the RM will be fine-tuned through the incorporation of natural amendments that regulate microbial metabolism to attain a synchronicity between the nutrient supply rate of the RM formulations and the nutrient demand rate of the crops. Through these optimizations, the team plans to achieve a slow and sustained release of N and P so as to substitute RM for up to 50% of the recommended pre-plant application of mineral fertilizers for row crops. The formulations will be pelletized in order to simplify field application and to further control the nutrient release. The formulations will be applied as pre-plant to substitute 50% of the recommended nitrogen application to corn over two-year field trial. The performance of this fertilizer regime will be compared against the traditional cultivation practices that rely solely on chemical fertilizers. The N and P inputs and outputs will be measured including crop biomass, leaching and volatilization losses and the overall N and P budget will be computed. Along with the yield performance of the RM substituted fertilizer regime, the research team would evaluate crop nutrient use efficiency, ability to improve soil microbial biomass and activity, soil aggregation and stability. Nutritional quality of the produce will be captured by measuring oil, starch proteins and phytochemicals including beta-carotenes and phytosterols.

PROGRESS: 2019/05 TO 2020/05
Target Audience:The results from this study were conveyed to agriculture producers, educators, academic peers and students through presentations during field days and at American Society of Agronomy and Crop Science Society of America. Changes/Problems:A No-Cost Extension was requested, and was granted. This would allow us to replicate the field study for another year. What opportunities for training and professional development has the project provided?Two PhD students and two undergraduate students were trained on this project. Graduate students working on this project was able to interact with scientists at various research organizations, and received valuable feedback on the project. Both undergraduate and graduate students practiced communication of technical results to scientific and non-scientific audience and stakeholders.The graduate students were trained in how to write a research proposal and on various analysis associated with this project. How have the results been disseminated to communities of interest?The results from the study were conveyed to agriculture producers, educators, academic peers and students through three presentations at American Society of Agronomy, Crop Science Society of America and Soil Science Society of America and at the Piedmont Research and Education Center field day. The results were also shared with Industry and other agencies through multiple presentations. What do you plan to do during the next reporting period to accomplish the goals?We will collect the observations, other soil nutrient status, yield and quality parameters from the field plots established in April 2020. We will measure and analyze the strawberry fruit phyto-nutrient content and chemistry of the strawberry leaves and determine the improvement in the fruit quality of the strawberries under different forms of N fertilizers. We will publish the results from the current studies in the peer reviewed journals.

IMPACT: 2019/05 TO 2020/05
What was accomplished under these goals? PHOSPHORUS: Meat and bone meal (MBM), a byproduct of the animal rendering industry, has excellent potential to supplement the inorganic fertilizers in crop production due to its balanced availability of nutrients. Along with 8% nitrogen, MBM also contains 11.3% P. Initial lab incubation studies showed that >90% of the total P in the MBM is bound as hydroxyapatite (an insoluble form) and is not mineralized within first 36 days after incubation. Follow up greenhouse studies confirmed the low availability of P from MBM, where P use efficiency of corn in MBM treatments were ~ 10%, which is 50% lower than that of inorganic fertilizers. Due to the high content, yet low bioavailable to P in MBM, the application of MBM to supplement the N requirement of the crops could result in a substantial buildup of residual P in soil, which, when transported to water bodies through erosion results in P pollution. Thus, there is a critical need to improve the mobilization and plant uptake of P from MBM for its sustainable, efficient, and economical use as fertilizer. To optimize the P solubilization from MBM, lab incubation and greenhouse experiments were conducted to test for the effectiveness of plant-microbe interactions to enhance the solubility of P in MBM. Penicillium bilaiae (P. bilaiae), which is a phosphorus solubilizing fungus and commercially available under the product name Jumpstart, was tested in lab incubation study for its efficacy to mobilize the P from MBM. We observed that P. bilaiae increased the P mobilization from MBM by 200% during the initial 7 days of incubation. Different mycorrhizae species (Glomus intraredices and Gigaspora gigentia) were tested for their compatibility with MBM and corn. In MBM treatments, while Glomus intraredices had a positive effect on the P uptake of corn, Gigaspora gigentia decreased the shoot P content and shoot biomass of corn. Based on these results mixture of Glomus species (Glomus intraradices, Glomus mosseae, Glomus aggregatum, Glomus etunicatum) and phosphorus solubilizing fungus P. bilaiae were tested for their the potential to improve the P mobilization and uptake from MBM. Mycorrhizae mixture and P. bilaiae was mixed in with the MBM before incorporating into the soils, and experiment was set up in 3-gallon pots. Corn was harvested at 40 days after sowing and soil P at harvest was fractionated into different chemical forms and tissue P content was measured. Penicillium bilaiae with and without mycorrhizae significantly increased (~60%) the plant available fraction of soil P, and decreased the calcium bound P fraction (~30%) of soil P. Moreover, microbial biomass P was higher under P. bilaiae application along with MBM. However, P. bilaiae did not have any effect on the Fe/Al bound P in the soils. Mycorrhizae alone did not affect the soils P fractions except for a higher microbial bound P fraction. Mycorrhizae with and without P. bilaiae increased the uptake of P from MBM by 200-400%. Highest shoot and root P uptake from MBM was observed with a mixture of mycorrhizae and P. bilaiae resulting in a synergistic relationship with respect to P uptake from MBM. Moreover, all the P mobilizers with MBM significantly increased the soil and root enzymatic activity mainly the activity of acid phosphomonoesterase (30-40%). NITROGEN: The first year of field trial (2019 season) that tested the effectiveness of MBM as a viable supplement to inorganic fertilizer produced promising results. The basal dose of N recommendation is split into two parts with 1:1 and 1.5:1 ratio applied through MBM and CaNO3, respectively (integrated nutrient management). The basal dose of 1:1 is applied for the three-split application of N, as three-split application is recommended common practice for the irrigated corn in SC. We also tested the two-split application of N fertilizers with the MBM supplement at 1.5:1 ratio under irrigated conditions. We found that major form of N under MBM supplement is NH4+, whereas N form under the inorganic fertilizer alone is mostly NO3- in the soils, at 30 days after sowing. Basal dose supplemented with MBM retained 40.3% and 21.7% higher nitrogen in the plow zone of soil at 30 days after sowing for two split and three split application, respectively, as compared to inorganic fertilizers alone. MBM supplement of the inorganic fertilizers met the plant N demands as assessed by the statistical similar plant height and SPAD-chlorophyll index. MBM supplement of inorganic fertilizers significantly lowered the downward movement of the N below the root zone, which was 3-fold lower NO3- movement into the leachate below the crop root zone during the crop growth period. Integrated application of MBM and inorganic fertilizers had 200% higher soil protease (microbial enzyme) activity as compared to 100% CaNO3. Corn grain yield under integrated fertilizers (2 and 3 split) application was similar to 100% CaNO3 application (3 split), but was significantly higher to 100% CaNO3 (2 split) application. Overall the results were promising on multiple fronts: While recycling an easily available agricultural byproduct as fertilizer, our integrated nutrient management approach reduced the nutrient leaching losses by seven times, reduced the frequency of farm operations by one-third, reduced the amount of chemical fertilizer usage by 25%, and improved the soil and microbial health up to five times, all while producing similar yield as that of conventional chemical fertilizer-based corn production system. Plots were set up for the second year to test the reproducibility of these results as influenced by edaphic and climatic conditions. HIGH-VALUE CROPS: This rate of mineralization of N from MBM, and soil accumulation of N forms (NO3- vs NH4+) can be closely regulated by the co-application of Karanja oil, an agricultural by product that inhibits the nitrification. The rate and form of nitrogen influence the phytochemical concentration in plants. Thus, the above approach of coapplying MBM with Karanja could be used to enhance the nutritive quality of produce. To observe the effect of MBM, karanja oil coated MBM and different forms of N fertilizers on the fruit quality of high value crops such as strawberries, a greenhouse experiment was conducted. Two alpine cultivars (French alpine and Pineapple crush) of strawberries were transplanted into 1.5-gallon pots. Organic fertilizers (MBM, and Karanja coated MBM) were applied before transplanting the strawberries and NH4+, NO3- and NH4NO3 treatments were applied as Hoagland solution a regular intervals so that all the treatments received the same amount of N. We observed higher shoot biomass under MBM, karanja coated MBM, NO3- and NH4NO3 as compared to NH4+ form of N. The fruit yield was statistically similar among the karanja coated MBM, NO3-, NH4NO3 and NH4+ form of N as compared to MBM alone. We measured the volatile profile and phytonutrient content of the strawberry fruits. Preliminary data analysis showed that fruits under NO3- form of nitrogen has higher amino acid content and whereas fruits under NH4+ from of N in pineapple crush cultivar has higher sugars such as glucose, fructose and sucrose. We are currently analyzing the secondary metabolites profile such as phenolic, flavonoids and tannins in the produce to determine the fruit quality of the strawberries. Based on the results MBM + Karanja seems to facilitate a higher phytochemical content compared to other forms of N treatments.

PUBLICATIONS (not previously reported): 2019/05 TO 2020/05
1. Type: Journal Articles Status: Published Year Published: 2020 Citation: Jatana, B.S., Kitchens, C., Ray, C., Tharayil, N., (2020). Regulating the nutrient release rates from proteinaceous agricultural byproducts using organic amendments and its effect on soil chemical and biological properties. Biology and Fertility of Soils. 56, 747â758
2. Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Jatana, B.S., Kitchens, C., Ray, C., Tharayil, N., (2020) Microbiota-mediated mobilization of phosphorus from rendered animal materials and its effect on plant physiology, root morphology, soil chemical and microbiological properties. Soil Biology & Biocemistry.
3. Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Jatana, B.S., Kitchens, C., Ray, C., Tharayil, N., (2019). Modification of nitrogen and phosphorus mineralization from rendered animal materials for extended plant nutrient availability. Graduate student government research symposium, Clemson University (Poster)
4. Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Jatana, B.S., Kitchens, C., Ray, C., Tharayil, N., (2020) Integrated Use of Organic and Inorganic N Fertilizers to Improve the Crop N Use Efficiency and to Reduce the N Leaching Losses. Southern ASA meeting-January 31,2020 Kentucky (Oral Talk).

PROGRESS: 2016/05/15 TO 2017/05/14
Target Audience:The project is utilizing the rendering material produced by Central Bi-products Inc. (Redwood Falls, MN) and Valley Proteins Inc. (ward, SC). The objective and the potential outcome of the study was communicated with the industry through site visits. The results of the ongoing study will be presented at the upcoming SSSA annual meeting in FL (Nov 2017). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Project provided interdisciplinary training to two PhD students. One student was trained on the chemical engneering aspect of the project involving formulation of the fertilizer. A second student was provided training on conducting lab incubation studies and measurement of fluxes of nitrogen and P in soils. How have the results been disseminated to communities of interest?The project is utilizing the rendering material produced by Central Bi-products Inc. (Redwood Falls, MN) and Valley Proteins Inc. (ward, SC). The objective and the potential outcome of the study was communicated with the industry through site visits. The results of the ongoing study will be presented at the upcoming SSSA annual meeting in FL (Nov 2017). What do you plan to do during the next reporting period to accomplish the goals?Overall, the result from the first yearindicate that MBM is a promising source of mineral N and P for both of the soils, and S sulfur amendment, up to 8% by weight of MBM, could counteract the drastic increase in pH that was observed when MBM was applied alone. The rate of mineralization of MBM in both soils were higher with ~40% of N released within two weeks. Naturally occurring plant amendments that can regulate the ammonification and nitrification process, including neem, karanja and tannins, will be tested to reduce the initial rapid rate of mineralization of MBM so as to extend the window of N and P availability to the crop plants. Further, greenhouse studies will be conducted with corn using the optimized and pelletized formulations of MBM, and the total N budget will be calculated.

IMPACT: 2016/05/15 TO 2017/05/14
What was accomplished under these goals? Faced with a steadily dwindling cropping area, current agricultural practices overly rely on chemical fertilizer inputs to boost crop productivity. Present-day agriculture relies heavily on nutrient rich inorganic and chemical forms of nitrogen (N), including urea, ammonium salts, and nitrate salts. This approach results in high nitrogen delivery over a short time that exceeds the capacity for plant uptake, thus resulting in diminished nutrient use efficiency and greater environmental pollution. The reserve of phosphate rock, which currently accounts for 75% of the phosphorous (P) fertilizers, is dwindling at an alarming rate due to the burgeoning demand for P fertilizers. Moreover, the current production practices of nitrogen and phosphorous are highly energy intensive and not sustainable. The proposed approach is to revisit a historically employed fertilizer matrix; rendered animal proteins. The focus of the project is to employ various natural plant-based amendments that are spent-waste, to tailor the nutrient release rate from these rendering material so as to match the nutrient uptake rate of the crops, thus facilitating higher nutrient use efficiency. Meat and bone meal (MBM) was chosen for the study as the nitrogenous raw material because it is a lower cost material as compared to feed grade poultry biproduct meal and other rendered materials but possesses greater composition consistency compared to other agricultural biproducts, such as litter or manure. Furthermore, along with N, MBM contains a rich source of phosphorous and other minerals, which allows for a better balanced fertilization. The MBM was obtained from two independent suppliers Central Bi-products Inc. (Redwood Falls, MN) and Valley Proteins Inc. (Ward, SC). Nutrient variability among suppliers and batches was less than 10%. Lipid extractions were optimized and performed by hexane extraction prior to formulation. The lipid fractions are valued at 2-3 times that of the MBM and do not provide significant nutrient value to the fertilizer, but are valuable in alternative products such as biodiesel or lubricants. Furthermore, removal of the lipids increases the material consistency and lowers the carbon to nitrogen ratio; both of which are beneficial. Complete lipid extraction was carried out for the MBM samples using a hexane Soxhlet extractor. The elemental N, P, K content of the hexane-extracted MBM was 8.3 ± 0.3 %, 11.4 ± 0.4 %, 1 ± 0.01 %, respectively and the secondary nutrient content was 2.6% Ca, 0.5% Mg, and 0.7% S. The C/N ratio of the extracted material was 3.60, and ash content was 40%. Since the sodium content of the MBM was 0.8%, the hexane-extracted material was washed sequentially twice with deionized water to reduce sodium content. The washing reduced the Na content by 50%, and the washed MBM material (WMBM) had a similar N content as that of non-washed MBM, however the P and K content in the WMBM also decreased by ~50% and 80%, respectively. Four lab incubation experiments were conducted to assess and optimize the N and P release rates from both MBM and WMBM. The rate of mineralization in these experiments was tested in two soils that have different organic matter and clay content and cropping histories- Cecil clay soils (OM- 1.5%; clay-13%; pH 5.9; conventional farming) and loamy sand (OM- 4.5%; clay-5%; pH 4.8; organic farming). MBM and WMBM was applied at a rate of 500 mg per 50g soil and the incubation was conducted at 75% field capacity at 23 ± 1 °C. The form and rate of release of N and P and soil chemistry were monitored at three to seven day internals. The pH of both soils increased >8.5 six days into the incubation, with the rate of pH increase plateauing off after 9 days. This increase in pH was similar for both MBM and WMBM, and the pH increase could be partly attributed to the deamination reactions that occur during the initial stages of ammonification. To counteract the increase in pH caused during the deamination, in subsequent experiments, elemental sulfur was used as an amendment. The elemental S was applied at 6 different rate- 0, 1, 2, 4, 8, 16, 25% weight of MBM. The highest rate of application of S is well within the recommended rate of S amendment to decrease soil pH. The experimental setup was similar to as that of above. At 14 days of incubation, the 1 and 2% S was instrumental in maintaining the pH of the organic soils to the background pH, but was less effective in curbing the increase in pH of the conventional soil. The increase of S content to 8% was instrumental in maintaining the pH of the conventional soils to background levels. Compared to the respective background, the ammonium content of the loamy sand soil increased 8 folds, where as that of the Cecil clay soils increased ~12 folds with the application of MBM. By 14 days of incubation 40 ± 1.5 % of the organic N in the added MBM was mineralized to ammonium and nitrate. The addition of S influenced this conversion only at a higher rate of application, where 16% S addition resulted in 55 ± 2.1 % conversation of organic N in MBM to mineral forms at 14 days of incubation. Ammonium dominated the form of mineral N in both soils after MBM application, with ammonium-to-nitrate ratio exceeding 25 across all rate of S application. The influence of S on content of ammonium in soils was linear, with increasing percent of S marginally increasing the amount of ammonium in soils. However, the influence of S on nitrate content exhibited a nonlinear trend, with a maximum amount of nitrate accumulation in 4 and 8% of S, and a decreases with a lower and higher S application rates. The orthophosphate content of both the soils also increased ~4 folds at 14 days after the application of MBM, and this P mineralization decreased with an increase in S addition in organic soil. Both ammonification and nitrification in soils are carried out by bacterial species, and hence it is important to track the changes in abundance of bacterial species during incubation. Bacterial abundance can be reliably quantified using muramic acids, which is a marker for bacterial peptidoglycans. The literature reported methods of muramic acid quantitation were further optimized for convenience (shorter incubation time) and sensitivity (tandem mass spectrometric analysis) using the soils that were retrieved after the above incubation experiments. Samples incubated with MBM for 15 days were hydrolyzed in 6, 9 and 12N HCl for 3, 4.5 and 6 h at 105 °C. Further, a solid phase extraction (SPE) was employed to clean up the hydrolysates before mass spectrometric analysis using a triple-quadrupole mass spectrometer. Percent recovery and limits of detection were calculated for the new method. Increasing the acid concentration resulted in a 33% increase of extraction of muramic acid from the incubated soils at 3 h hydrolysis time. Hydrolysis for 6 h resulted in similar extraction of muramic acid across different acid concentrations. Thus, increasing the acid concentration helped in reducing the time of hydrolysis by half. Muramic acid recovery was more than 90% with SPE clean up. The sensitivity of mass spectrometric analysis after the sample cleanup was two times higher than that of the current literature reported values. This optimized method will be used for monitoring the changes in soil bacterial biomass during the greenhouse and field experiments.

PUBLICATIONS: 2016/05/15 TO 2017/05/14
Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Bhupinder, J; Kitchens, C; Ray, C; Tharayil, N. 2017. Optimization of nitrogen and phosphorous release rates from rendered animal products using natural amendments. Soil Science Society of America Annual Meeting, Tampa FL, Nov 3-6.