Source: SPERO ENERGY, INC. submitted to
HIGH-PERFORMING BIORENEWABLE MICRONUTRIENTS FOR AGRICULTURAL APPLICATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1012624
Grant No.
2017-33610-26995
Cumulative Award Amt.
$100,000.00
Proposal No.
2017-00583
Multistate No.
(N/A)
Project Start Date
Sep 1, 2017
Project End Date
Apr 30, 2019
Grant Year
2017
Program Code
[8.8]- Biofuels and Biobased Products
Recipient Organization
SPERO ENERGY, INC.
3400 HAMILTON ST
WEST LAFAYETTE,IN 47906
Performing Department
(N/A)
Non Technical Summary
Spero Energy seeks to increase the availability and competitiveness of biobased products. One area which could benefit greatly from a biobased and biodegradable product is the agricultural chelate market. Chelates are molecules which bind micronutrients essential for plant growth, and efficiently deliver the micronutrients to the plant. The main chelates used today are ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). In the agricultural sector, EDTA and DTPA are used to deliver essential metals such as iron, copper, manganese, and zinc to plants. Despite the widespread use of EDTA and DTPA, growing bodies of evidence suggest that use of these chelates is leading to a host of environmental problems including contamination of domestic and global water sources. Beyond the agricultural sector, these chelates are used extensively in domestic and industrial applications, further contributing to the water pollution problem.Through this project, Spero Energy, Inc. is developing chelates which are modeled after natural plant materials and designed to be biodegradable. Spero Energy will produce these biodegradable chelates in our chemistry laboratory located in Thousand Oaks, CA. Spero plans to produce chelates which can bind micronutrients, deliver the micronutrients to plants, then biodegrade quickly into non-toxic components. The chelates will be tested to measure how well they can bind the micronutrients and to ensure biodegradability using industry standard methods. After Spero has selected its highest performing and biodegradable chelate, we will work with industry experts to bring this much needed technology to mass production. Through commercialization of high performing, biodegradable chelates, Spero Energy seeks to help the agricultural community grow the foods we need without causing water pollution.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
10252102000100%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
5210 - Fertilizers;

Field Of Science
2000 - Chemistry;
Goals / Objectives
With the mission of developing 'green' and sustainable processes for the conversion of abundant non-food biomass into high value chemicals, Spero Energy seeks to increase the availability and competitiveness of biobased products. One area which could benefit greatly from a biobased and biodegradable product is the agricultural chelate market. Currently, the metal chelating aminocarboxylates ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are used extensively in industrial, domestic, and agricultural applications. In the agricultural sector, EDTA and DTPA are used to deliver soluble forms of essential metals such as iron, copper, manganese, and zinc to plants. Despite the widespread use of EDTA and DTPA, growing bodies of evidence suggest that use of these chelates is leading to a host of environmental problems including contamination of domestic and global water sources.Through this Phase I research project, Spero Energy aims to provide a foundation from which high performing, biodegradable, and cost competitive chelates will be commercialized during Phase II of the project. Spero Energy plans to develop an expanded chelate library to include a range of biodegradable chelates, and select the optimal chelate for commercialization. Experiments conducted through Phase I research will quantify the binding constants of the chelates to bind key metal micronutrients and quantify the biodegradability of the chelates. Phase II work will include toxicology testing, and further greenhouse studies to provide a complete data set for potential customers. Additionally, Phase II will be used to improve the solubility of lignin based biodegradable chelates in preparation for launch of a totally biobased renewable chelate product line.The objectives of this Phase I SBIR project are as follows:Expansion of the library of biodegradable chelates produced by Spero Energy. This objective will be accomplished using proprietary methods to produce metal chelating molecules. Successful synthesis of the new chelates will be quantified by evaluation of reaction yields and purity. The metal-ligand binding modes of the newly synthesized chelates will be evaluated through x-ray crystallography.Determine binding constants for chelates under agriculturally relevant conditions. Initial tests of chelates previously synthesized by Spero Energy, have shown promising results in their ability to bind iron and effectively deliver iron to plants in a greenhouse setting. To compare these promising new chelates directly to the commercial chelates EDTA, DTPA, and EDDHA additional binding constant testing must be completed. During this key objective, the binding constants of Spero produced chelates will be measured with the metals most commonly chelated for agricultural applications. These include Fe2+/3+, Cu2+, Zn2+, and Mn2+. Completion of this objective will answer the following technical question: (1) How do the metal binding constants of chelates synthesized by Spero Energy compare to commercially available products?Measure biodegradability of chelates. In this objective the biodegradability of the most promising chelates will be quantified by standard methods detailed in the Organization for Economic Co-operation and Development (OECD) guideline for testing chemicals. Following completion of Key Objectives 1 and 2 the most promising chelates will have been evaluated in terms of favorability of binding constants, formation constants over a range of pH values, solubility, and cost. The most promising chelates will then be subjected to biodegradability testing. Biodegradability testing will be carried out by a 3rd party, so that the results will be considered unbiased for regulatory purposes. Completion of this objective will answer the following technical questions (1) Which of our most promising chelates meet the regulatory specifications to be labeled as biodegradable?
Project Methods
This project will be conducted beginning with a series of chemical reactions used to produce biodegradable chelates. This work will be carried out using synthetic organic chemistry techniques in the laboratory of Spero Energy. Reaction products will be characterized by NMR spectroscopy and elemental analysis. X-ray crystallography will be used to evaluate the binding modes of the chelates when bound to metals such as iron.The stability constants of newly synthesized chelates will be determined by potentiometric techniques to allow for direct comparison of their ability to bind relevant metals with industry standard chelates. Additionally, the biodegradability of synthesized chelates will be measured with standard laboratory methods such as the OECD CO2 evolution test.Chelates synthesized by Spero Energy will undergo constant evaluation throughout the course of this project. Initial evaluation will include determination of product yield and purity. Further evaluation will include determination of stability constants when key micronutrients are bound by the chelates. Determination of the stability constants will allow for direct comparison with industry standard chelates such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminesuccinic acid (EDDS) and iminodisuccinic acid (IDS). Biodegradability testing will also be used to evaluate Spero's hypothesis that chelates modeled after lignin moieties will be biodegradable.

Progress 09/01/17 to 04/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided the opportunity for a chemist employed by Spero, Daniel Morgan, to gain valuable training. Daniel received training in UV-vis spectroscopy as well as nuclear magnetic resonance (NMR) spectroscopy. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During Technical Objective 1, biobased chelates were synthesized utilizing Aspartic acid, glutamic acid, and proline. The biobased chelates were synthesized using proprietary reaction conditions. In general, the reactions involved a condensation reaction between 4-phenolsulfonic acid and an amino acid (aspartic acid, glutamic acid, or proline). The chelate which incorporated aspartic acid (SAL) was synthesized in yields of 90%, with a purity of 80%. The chelate incorporating glutamic acid (SAL-G) was synthesized in yields of 32% with 25% purity. The chelate incorporating proline (SAL-P) was synthesized in yields of 50% with a purity of 43%. The SAL, SAL-G, and SAL-P chelates were isolated as solids and the identity and purity of the reaction products were confirmed by 1H- and 13C-NMR. While X-ray crystallography of the synthesized chelates was proposed as Phase I work, Spero was unable to produce crystals of sufficient size and quality of SAL, SAL-G, and SAL-P despite many crystallization attempts. The 4-phenolsulfonic acid incorporated into the SAL, SAL-G, and SAL-P chelates greatly increases the water solubility of the chelates and limits the solubility in other organic solvents. While this quality is highly desirable for micronutrient chelates, it has made crystallization a challenge. Based on the high yield and purity of the SAL chelate and low cost of aspartic acid ($1.76/kg), SAL was selected as the most promising biobased chelate of the three synthesized. During Technical Objective 2, the metal complexation constants of Spero's SAL chelate were measured with Fe+2. Iron is a critical micronutrient required by plants, and chelated iron is commonly applied through foliar or soil applications in agriculture. In this objective, a series of experiments were performed to directly compare the iron binding ability of Spero's SAL chelate to GLDA. GLDA is a leading biodegradable chelate sold by AkzoNobel. In these experiments, an aqueous solution of 0.33 mM Fe-SAL complex was first prepared by dissolving SAL and FeSO4 in water. The absorbance of the resulting Fe-SAL complex was measured using a UV spectrophotometer. The solution of Fe-SAL was titrated with increasing amounts of GLDA chelate, with the absorbance of each solution measured after mixing. This experiment was completed at pH = 5,7,9. The iron complexation constant of Spero's SAL chelate was calculated using the published metal complexation constant for Fe-GLDA and derived equilibrium equations. The calculated metal complexation constants for Spero's SAL chelate and Fe+2 are 8.1 (log K) at pH 5, 9.0 (log K) at pH 7, 13 (log K) at pH 9. Spero's SAL chelate outperforms AkzoNobel's GLDA chelate at pH 9. The high performance of Spero's SAL chelate at elevated pH is significant, as there is interest as the agricultural industry in the ability of a chelate to increase iron uptake by plants in soil with pH higher than 6.5. Ethylenediaminetetraacetic acid (EDTA) itself is not able to effectively chelate iron above pH 6.5 (Pinto et al. 2014, Zekri and Thomas 2009). To chelate iron at higher pH, diethylenetriaminepentaacetic acid (DTPA) can be used up to pH 7.5 and ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA) can be used up to pH 9 (Zekri and Thomas 2009). However, EDDHA is non-biodegradable and expensive to outperform biodegradable chelates. Spero's SAL chelate could be a promising chelate option at elevated pH values. During Technical Objective 3, the biodegradability of Spero's SAL chelate was tested. Spero had hypothesized that the SAL chelate would be biodegradable due to incorporation of biobased materials (aspartic acid) and structural modeling based on the biopolymer lignin. Biodegradability of the SAL chelate was quantified by standard methods detailed in the Organization for Economic Co-operation and Development (OECD) guideline for testing chemicals (Organization for Economic Co-operation and Development 1992). Biodegradability testing of the SAL chelate was completed by RespirTek, Inc. a well-respected and certified environmental testing laboratory. During a 28-day biodegradation study, RespirTek verified that Spero's SAL chelate is biodegradable, and meets the requirements to obtain the status of "Ultimate Biodegradability".

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