Source: Agricultural Research Service, Southern Regional Research Ctr submitted to
INCREASING THE VALUE OF COTTONSEED
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0428791
Grant No.
(N/A)
Project No.
6054-41000-103-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 2, 2015
Project End Date
Jun 1, 2020
Grant Year
(N/A)
Project Director
DOWD M K
Recipient Organization
Agricultural Research Service, Southern Regional Research Ctr
1100 Robert E. Lee Blvd.
New Orleans,LA 70124-4305
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5011810104080%
7121899200020%
Goals / Objectives
The overall goal of the project is to improve the postharvest utilization of cottonseed and thereby increase the value of the U.S. cotton crop through improved understanding of cottonseed composition, properties and processing of the seedâ¿¿s components. There are five total objectives in the project. Three objectives focus on studying and modifying the oil, protein, and hull components of the seed. One objective is directed toward the study of processing operations to improve the separation of these components and the last objective is directed toward isolation of minor components that may exhibit beneficial bioactivity. Objective 1) Enable the development of new, commercial cotton varieties which express high levels of oleic acid in the seed. Sub-objective 1a) Study FAD2 structure in naturally high oleic acid cotton accessions. Sub-objective 1b) Use genes and other DNA regulatory elements associated with cyclopropyl fatty acid synthesis to silence production of these fatty acids in developing cottonseed. Sub-objective 1c) Determine the compositional and functional property differences between naturally high oleic acid and normal cottonseed oils. Objective 2) Enable new commercial process technologies that maximize the profitability of converting low-gossypol cotton seed into oil and meal products. Sub-objective 2a) Determine conditions that result in low-color oils from the processing of glandless cottonseed. Sub-objective 2b) Physically refine crude cottonseed oil from glandless cottonseed to produce commercial grade oil. Objective 3) Enable the commercial production of new products from the protein fraction of cottonseed meal. Sub-objective 3a) Improve water resistance of cottonseed protein meals, concentrates and isolates used as wood adhesives. Sub-objective 3b) Explore the use of cottonseed proteins as functional additives in non-food commercial products. Sub-objective 3c) Explore the use of cottonseed protein fractions to improve non-food product properties. Objective 4) Enable the commercial production of new bioactive food ingredients from glandless (no gossypol) cottonseed. Sub-objective 4a) Identify minor bioactive phenolic components from glandless cottonseed. Sub-objective 4b) Identify bioactive peptides and proteins from glandless cottonseed. Objective 5) Enable the commercial production of new products from the carbohydrate components in cottonseed burrs, hulls and kernels. Sub-objective 5a) Isolate, characterize, and study the functionality of hemicellulosic components from seed processing byproducts. Sub-objective 5b) Exploit the potential use of hull and other seed byproducts as fillers in composite materials.
Project Methods
Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. To alter cottonseed oil composition, a combination of genetic manipulation and classical breeding will be used. Various physical and chemical techniques will be employed at the laboratory level to mimic processing steps and to fractionate meal (i.e., protein) and hull components. Chemical, enzymatic, and physical techniques will be used to modify these isolated components and to characterize the resulting products. Performance of these fractions for different potential applications will be achieved through a series of physical testing methods. Isolation of seed minor components will be achieved for bioactivity studies through chemical fractionation and chromatographic methods and several cell-based assays will be used to test for activity.

Progress 06/02/15 to 06/01/20

Outputs
Progress Report Objectives (from AD-416): The overall goal of the project is to improve the postharvest utilization of cottonseed and thereby increase the value of the U.S. cotton crop through improved understanding of cottonseed composition, properties and processing of the seed⿿s components. There are five total objectives in the project. Three objectives focus on studying and modifying the oil, protein, and hull components of the seed. One objective is directed toward the study of processing operations to improve the separation of these components and the last objective is directed toward isolation of minor components that may exhibit beneficial bioactivity. Objective 1) Enable the development of new, commercial cotton varieties which express high levels of oleic acid in the seed. Sub-objective 1a) Study FAD2 structure in naturally high oleic acid cotton accessions. Sub-objective 1b) Use genes and other DNA regulatory elements associated with cyclopropyl fatty acid synthesis to silence production of these fatty acids in developing cottonseed. Sub-objective 1c) Determine the compositional and functional property differences between naturally high oleic acid and normal cottonseed oils. Objective 2) Enable new commercial process technologies that maximize the profitability of converting low-gossypol cotton seed into oil and meal products. Sub-objective 2a) Determine conditions that result in low-color oils from the processing of glandless cottonseed. Sub-objective 2b) Physically refine crude cottonseed oil from glandless cottonseed to produce commercial grade oil. Objective 3) Enable the commercial production of new products from the protein fraction of cottonseed meal. Sub-objective 3a) Improve water resistance of cottonseed protein meals, concentrates and isolates used as wood adhesives. Sub-objective 3b) Explore the use of cottonseed proteins as functional additives in non-food commercial products. Sub-objective 3c) Explore the use of cottonseed protein fractions to improve non-food product properties. Objective 4) Enable the commercial production of new bioactive food ingredients from glandless (no gossypol) cottonseed. Sub-objective 4a) Identify minor bioactive phenolic components from glandless cottonseed. Sub-objective 4b) Identify bioactive peptides and proteins from glandless cottonseed. Objective 5) Enable the commercial production of new products from the carbohydrate components in cottonseed burrs, hulls and kernels. Sub-objective 5a) Isolate, characterize, and study the functionality of hemicellulosic components from seed processing byproducts. Sub-objective 5b) Exploit the potential use of hull and other seed byproducts as fillers in composite materials. Approach (from AD-416): Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. To alter cottonseed oil composition, a combination of genetic manipulation and classical breeding will be used. Various physical and chemical techniques will be employed at the laboratory level to mimic processing steps and to fractionate meal (i.e., protein) and hull components. Chemical, enzymatic, and physical techniques will be used to modify these isolated components and to characterize the resulting products. Performance of these fractions for different potential applications will be achieved through a series of physical testing methods. Isolation of seed minor components will be achieved for bioactivity studies through chemical fractionation and chromatographic methods and several cell-based assays will be used to test for activity. The objectives of the cottonseed project fall under National Program 306 and contain elements of both the Food and Non-food components. Under the Food component, the work addresses Problems 1A, to define, measure, and preserve/enhance/reduce attributes that impact quality and marketability; Problem 1B, to develop new bioactive ingredients and functional foods; and Problem 1C, to develop new and improved food processing technologies. Under the Non-food component, the work relates to Problem 2B to enable technologies for producing new marketable non-food biobased products derived from agricultural products and byproducts and estimate the potential economic value of the new products. The progress described will first discuss efforts made during the final year of the project followed by a summary of the accomplishments achieved over the course of the five- year project cycle. To increase cottonseed oleic acid levels (Objective 1) , a back-cross was made between a nematode-resistant plant variety that had been previously identified as having higher than normal levels of oleic acid and plants of an early generation (F2) of a cross made between GB713, a wild plant that contains high levels of oleic acid, and SG747, a publicly available cotton line. This was done to try to increase the seed oil oleic acid level beyond the levels in the recently released germplasm lines (named HOa1-HOa4), which were around 33-35% oleic acid. Prodigy seeds of these crosses were self-pollinated to yield seed from the first segregating generation. These seeds were then field planted in Starkville, Mississippi. One-hundred and fifty-one plants were obtained, and seeds of each plant produced oleate levels between 32 and 56%. This range indicates that significantly higher oleic acid levels are possible beyond that obtained from the nematode resistant-released lines, and justifies the continuation of this breeding strategy. In addition, DNA was extracted from the lowest (32-34%) and highest oleic acid plants (51-56%) and is being sequenced to look for the genetic differences that contribute to the elevated oleic acid trait. The highest oleic acid plants (53-56%) will undergo a second back-cross with the HOa1 line to further the breeding process.Cottonseed oil also contains small amounts of cyclopropane and cyclopropene fatty acids (a class of unsaturated cyclic fatty acids) that may have detrimental and beneficial physiological effects but their synthesis is poorly understood. ARS reseachers at New Orleans, Louisiana, have made DNA strands by gene editing and these will be used to support editing the cotton genome to answer questions about the synthesis of the acids and to determine how best to tailor the levels of these acids toward the beneficial forms. Collaborative projects with Texas A&M University and University of North Texas to further this work are being established. The transformation of cotton with the first constructs will begin soon. Experiments were conducted to look at the effects of hull material on cottonseed oil extraction and color. Gossypol has been blamed for causing crude cottonseed oils to be difficult to bleach to a low color. Extractions with seed of different gossypol levels has not shown the effect, leading to the thought that the variable presence of hulls during extraction might be the cause of the problem. Adding different levels of hulls to kernel tissue that was carefully cleaned of all hull and other debris did not exhibit increased oil color after oil extraction, or after refining and bleaching of the oil. However, extraction of glandless seeds and seeds of the recently developed ultra-low gossypol plant lines showed dramatic color differences despite having separated all of the hulls prior to solvent extraction and both samples having very low and similar gossypol levels (about 200 ppm). The difference indicates that it is the glands themselves, which are absent in the glandless seed but present in the ultra-low gossypol seed, that are responsible for the dark oil color and bleaching difficulties. Work is continuing to identify the components that contribute to the effect. Experiments on cottonseed proteins (Objective 3) focused on the adhesive performance of different protein fractions. Cottonseed protein was separated into water- and alkali- soluble fractions. Strength testing showed better bonding with alkali- soluble proteins than with the water-soluble proteins. Infrared spectroscopy of the glue line indicated better wettability for the alkali- protein formulations than for the water-protein formulations. In addition, adhesive experiments blending cottonseed protein and polyester plus different promoters are in progress. The polyester/cottonseed protein blends showed good results, with some formulations improving the dry adhesive strength by 70% and hot water adhesive strength by 50%. To improve water resistance, adding phosphoric acid and calcium salts to protein formulations was tested, and the results showed that combination enhanced the water resistance of the adhesive in comparison with other modifiers, due to calcium ion presenting a better synergy with the acid. A modest amount of work was also conducted to develop uses for gin trash and cotton derived xylan (Objective 5). When 10 and 20% weights of modified gin trash were added to polypropylene, the composites gave the same or slightly higher tensile strength and higher modulus, but lower elongation than the pure polymer. Up to 60% weight of gin trash could be added to a slightly different polymer. These materials showed reduced tensile strength and elongation at break but enhanced stiffness compared with the polymer without the gin trash addition. No significant improvement in mechanical properties was observed when the gin trash was modified with either acetic or succinate acid to improve its compatibility with the polymer. Additionally, acetate and succinate derivatives of xylan were prepared. By changing the catalyst, dosage, and temperature, the derivatives could be prepared with different degree of substitution. Progress was also made on a few satellite efforts. Under a reimbursable cooperative agreement (#58-6054-9-007) to identify genes that limit high level production of industrially useful fatty acids, six candidate genes have been identified. These will be over expressed in cotton to test the gene activity in modified plants. Work also continues on a reimbursable agreement (#58-6054-9-009) to evaluate cottonseed genotypes for differences in cyclopropyl fatty acid content and to evaluate the fatty acid profiles of commercial seed known to have germination difficulties. Finally, cottonseed strength studies were started due to recent concerns that the ginning of weak seed is leading to greater levels of seed coat neps, i.e., fibers with attached hull fragments. A genetics-environment study is continuing, as well as a study comparing the seed strengths of Upland (Gossypium hirsutum L.) and Pima (Gosspyium barbadense L.) varieties. The Australian cotton industry has expressed some interest in this work, as they are also having regional ginning difficulties. Summarizing the accomplishments over the five year project cycle, substantial progress was made on the development of cottonseed lines with higher levels of seed oil oleic acid (Objective 1). The proposed approach for this was initially delayed because of the identification of germplasm (bred to have nematode resistance) that also contained higher than normal levels of oleic acid. This material ultimately resulted in a germplasm release of plant lines (HOa1-HOa4) with double the normal levels of oleic acid. Additionally, the use of some of the nematode resistant parental material helped overcome the limited flowering of the wild high oleic cotton accessions, which helped re-establish the schedule for the originally planned breeding effort. Our first back-cross made from the planned breeding process resulted in plants with oleate levels considerably higher than expected (discussed above). Continuing with the breeding strategy should yield stable upland germplasm with 50% oleic acid or more, i.e., greater than our initial goal of 42%. Toward the second cottonseed oil effort, work to better understand the synthesis of the cyclopropyl fatty acids in cotton is progressing, although there is still much to learn. The molecular tools needed for these studies were developed, which was what was to be accomplished in this project cycle. Regarding our cottonseed processing objective (Objective 2), it was learned that the high oleate lines have different physical properties that suggest some niche market uses. Also, it was found that the bleaching problems that occur with some crude cottonseed oils are due to components from the cottonseed pigment glands and not the hulls or gossypol itself. However, attempts to refine low-gossypol crude oils with physical methods were not successful due to the presence of an unknown phosphorus component that has proved difficult to remove. The protein adhesive work of Objective 3 identified a number of formulation additives that improved cottonseed protein adhesive strength and water resistance. Two patent applications have been filed; to date, one patent has been issued and the second application is currently being evaluated. Efforts (Objective 4) to identify additional bioactivity compounds has been less successful. While some crude ethanol extracts exhibited some bioactivity; no specific compounds were identified, and the degree of activity was insufficient to warrant additional consideration. The final objective of the project (Objective 5) was to investigate the hemicellulose components from the seed. This material could only be isolated in small yield (6%) from cottonseed hulls and was more difficult to purify than are hemicelluloses from other woody materials. It would not be economic to recover this material at a practical scale. Record of Any Impact of Maximized Teleworking Requirement: Maximum telework has delayed a number of laboratory activities planned for the year. Some of the crosses needed for the seed oil oleate work were not completed, which will delay the breeding effort by one growth cycle. Efforts to identify the pigmented components of gland walls that contribute to cottonseed oil color have not been able to proceed. Additionally, some of the bioactivity studies associated with the identification of potentially useful bioactivity cottonseed components that were to be completed this summer have also been delayed. Elements of various agreements and projects with stakeholders have also been delayed, and the collaborators and stakeholders have been contacted to discuss extensions. Technology transfer and conference presentations and attendance have also been largely curtailed. While manuscripts for the current year are largely in progress, delays in laboratory activities will delay manuscript preparation for the upcoming year. Accomplishments 01 Improved cottonseed based adhesive formulations. The elimination of formaldehyde from the synthetic adhesives used in the wood product industry is highly desired for environment and worker safety. One approach for this is the use of bio-based protein adhesives, but these preparations have a number of limitations. ARS researchers at New Orleans, Louisiana, have found specific compounds containing catechol and carboxylic functionalities that enhance the adhesive performance of cottonseed protein. The use of these compounds improved the water resistance of the adhesive joints. A provisional patent application has been filed, and the work is of interest to the wood products industry as a method to help eliminate formaldehyde from their processes. 02 Development of higher oleic acid cottonseed oils. Vegetable oils with higher levels of oleic acid tend to be more oxidatively stable at elevated temperatures than oils with higher level linoleic acid. These oils are favored as frying oils, as the improved temperature stability allows the oils to be used for longer periods. ARS researchers at New Orleans, Louisiana, have conducted breeding experiments and have found that seed oil oleic acid levels can be increased to levels greater than what is currently available. This will be useful for developing cottonseed oils with higher levels of oleic acid than are currently available and will have improved stability when used for deep fat frying. These oils are important to the fats and oil communities. 03 Use of calcium salts and phosphates for improved cottonseed based adhesive formulations. To improve adhesive water resistance, ARS researchers at New Orleans, Louisiana, have been exploring the addition of additives to protein adhesive formulations to improve their water resistance. The addition of phosphoric acid and calcium salts showed that the combinations enhanced the water resistance of cottonseed protein based adhesives, due to calcium ion release from the chloride presenting a better synergy with the acid. The work is a step toward expanding the potential use of proteins as wood adhesives in place of current synthetic formulations.

Impacts
(N/A)

Publications

  • Cao, H., Sethumadhavan, K. 2020. Regulation of cell viability and anti- inflammatory tristetraprolin family gene expression in mouse macrophages by cottonseed extracts. Scientific Reports. 10:775.
  • He, Z., Cheng, H.N., Klasson, K.T., Ford, C., Barroso, V.A.B. 2019. Optimization and practical application of cottonseed meal-based wood adhesive formulations for small wood item bonding. International Journal of Adhesion and Adhesives. 95:102448.
  • Dowd, M.K., McCarty Jr, J.C., Shockey, J., Jenkins, J.N. 2020. Registration of four upland cotton germplasm lines with elevated levels of seed oil oleic acid. Journal of Plant Registrations. 14(1):64-71.
  • Cao, H., Sethumadhavan, K. 2019. Gossypol but not cottonseed extracts or lipopolysaccharides stimulates HuR gene expression in mouse cells. Journal of Functional Foods. 59:25-29.
  • Ge, C., Cheng, H.N., Miri, M.J., Hailstone, R.K., Francis, J.B., Demyttenaere, S.M., Alharbi, N.A. 2020. Preparation and evaluation of composites containing polypropylene and cotton gin trash. Journal of Applied Polymer Science. 137(38):1-13.
  • Li, J., Pardyawong, S., He, Z., Sun, X. S., Wang, D., Cheng, H. N., Zhong, J. 2019. Assessment and application of phosphorus/calcium-cottonseed protein adhesive for plywood production. Journal of Cleaner Production. 229:454-462.
  • Dowd, M.K., Manandhar, R., Delhom, C.D. 2019. Effect of seed orientation, acid delinting, moisture level, and sample type on cottonseed fracture resistance. Transactions of the ASABE. 62(4):1045-1053.
  • Dowd, M.K. 2020. Stability of the gossypol-amine adducts used for chromatographic measurement of total and isomeric gossypol. Journal of the American Oil Chemists' Society. 97(6):671-675.
  • He, Z., Zhang, D., Olanya, O.M. 2020. Antioxidant activities of the water- soluble fractions of glandless and glanded cottonseed protein. Food Chemistry. 325:126907.
  • Cheng, H.N., Girolami, G.S. 2020. H.S. Gutowsky and the use of nuclear magnetic resonance in chemistry. In: Strom, E.T., Mainz, V.V., editors. Pioneers of Magnetic Resonance. American Chemical Society Symposium Series Volume 1349. Washington, D.C.: American Chemical Society. p. 21-31.
  • He, Z., Olk, D.C., Tewolde, H., Zhang, H., Shankle, M. 2019. Carbohydrate and amino acid profiles of cotton plant biomass products. Agriculture. 10(1):2.


Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): The overall goal of the project is to improve the postharvest utilization of cottonseed and thereby increase the value of the U.S. cotton crop through improved understanding of cottonseed composition, properties and processing of the seed⿿s components. There are five total objectives in the project. Three objectives focus on studying and modifying the oil, protein, and hull components of the seed. One objective is directed toward the study of processing operations to improve the separation of these components and the last objective is directed toward isolation of minor components that may exhibit beneficial bioactivity. Objective 1) Enable the development of new, commercial cotton varieties which express high levels of oleic acid in the seed. Sub-objective 1a) Study FAD2 structure in naturally high oleic acid cotton accessions. Sub-objective 1b) Use genes and other DNA regulatory elements associated with cyclopropyl fatty acid synthesis to silence production of these fatty acids in developing cottonseed. Sub-objective 1c) Determine the compositional and functional property differences between naturally high oleic acid and normal cottonseed oils. Objective 2) Enable new commercial process technologies that maximize the profitability of converting low-gossypol cotton seed into oil and meal products. Sub-objective 2a) Determine conditions that result in low-color oils from the processing of glandless cottonseed. Sub-objective 2b) Physically refine crude cottonseed oil from glandless cottonseed to produce commercial grade oil. Objective 3) Enable the commercial production of new products from the protein fraction of cottonseed meal. Sub-objective 3a) Improve water resistance of cottonseed protein meals, concentrates and isolates used as wood adhesives. Sub-objective 3b) Explore the use of cottonseed proteins as functional additives in non-food commercial products. Sub-objective 3c) Explore the use of cottonseed protein fractions to improve non-food product properties. Objective 4) Enable the commercial production of new bioactive food ingredients from glandless (no gossypol) cottonseed. Sub-objective 4a) Identify minor bioactive phenolic components from glandless cottonseed. Sub-objective 4b) Identify bioactive peptides and proteins from glandless cottonseed. Objective 5) Enable the commercial production of new products from the carbohydrate components in cottonseed burrs, hulls and kernels. Sub-objective 5a) Isolate, characterize, and study the functionality of hemicellulosic components from seed processing byproducts. Sub-objective 5b) Exploit the potential use of hull and other seed byproducts as fillers in composite materials. Approach (from AD-416): Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. To alter cottonseed oil composition, a combination of genetic manipulation and classical breeding will be used. Various physical and chemical techniques will be employed at the laboratory level to mimic processing steps and to fractionate meal (i.e., protein) and hull components. Chemical, enzymatic, and physical techniques will be used to modify these isolated components and to characterize the resulting products. Performance of these fractions for different potential applications will be achieved through a series of physical testing methods. Isolation of seed minor components will be achieved for bioactivity studies through chemical fractionation and chromatographic methods and several cell-based assays will be used to test for activity. The objectives of the cottonseed project fall under National Program 306 and contain elements of both the Food and Non-food components. Under the Food component, the work addresses Problems 1A, to define, measure, and preserve/enhance/reduce attributes that impact quality and marketability; Problem 1B, to develop new bioactive ingredients and functional foods; and Problem 1C, to develop new and improved food processing technologies. Under the Non-food component, the work relates to Problem 2B to enable technologies for producing new marketable non-food biobased products derived from agricultural products and byproducts and estimate the potential economic value of the new products. Vegetable oils with high levels of oleic acid are desirable because they are considered heart healthy and because these oils tend to be more stable to oxidation and useful for applications like deep fat frying. As part of Objective 1a, work continued to develop cotton germplasm with elevated levels of oleic acid in the seed oil. Continuing last year⿿s progress, thirteen high oleic acid germplasm lines, initially selected from nematode resistant plants found to contain the trait, were grown in duplicate field plots. Testing of the seeds from these plants indicated that the high oleate trait was genetically stable. Levels in these plants were between 30 and 35%, essentially double the level found in normal cottonseed oil. The four lines with the highest levels have been approved as a germplasm release. While these levels are lower than found in some modified oilseeds (sunflower, soybean), these plants represent a step in the right direction for cottonseed. Continuing work started in 2018, an improved DNA marker that was developed to detect a mutated gene (called fad2-1d) was tested on the plant lines with different levels of oleic acid in their seed oils. All lines with over 30% oleic acid tested positive for the marker, confirming the involvement of this mutated gene in the trait. Additional testing conducted on the population of 2017 field-grown nematode-resistant plants revealed oleic acid levels between 13 to 38%. Plants with greater than 27% oleic acid tested positive only for the mutated gene and plants with less than 20% oleic acid appeared not to contain the mutated gene. Plants with intermediate levels of oleic acid appear to have a copy of both the mutated and non-mutated forms. Because the upland lines do not have the levels of oleic acid observed in the wild plants, other genetic differences are likely contributing to the oleate level in the wild germplasm. Markers representing several other candidate mutant genes are being developed and tested. Larger scale genome sequencing and comparisons between high- and low-oleic acid individual plants within the breeding populations generated during the development of the high oleic acid lines (referred to as HOa lines) is also planned to search for the contributing genes. Cottonseed oil also contains small amounts of cyclopropylene fatty acids, which is a class of cyclic unsaturated fatty acids that may be associated with detrimental effects when fed or consumed at large levels. Cottonseed lacking these fatty acids could be more valuable. Two members of a family of genes responsible for producing these fatty acids (called cyclopropane synthases, or CPS) are currently being targeted for inactivation using genome editing technologies. As part of Objective 1b, the necessary DNA constructs are being prepared to test the effectiveness of CPS elimination on cyclopropyl fatty acid synthesis. Work continues under a reimbursable cooperative agreement to identify genes in host crops that limit engineering plants to produce high levels of industrially useful fatty acids. Plant germplasm with targeted mutations in four different genes have been completed, and these lines are being analyzed for changes in oil content and composition. One line, containing a combination of two mutated genes, is nearly complete. Four other lines with different gene targets are in development. This work is being conducted in Arabidopsis but the results will be applicable to cotton and other oil seeds. Work also continued on understanding differences in the processing of low-gossypol seed (Objective 2). Extraction of oil from these seeds was not problematic and can be achieved by either pressing or solvent extraction. The color of the oil is lighter, which may help solve color development issues that can occur with cottonseed oil. Physical refining of the oil, however, has proven difficult as the phosphorus in the crude oil has been difficult to remove. Degumming reduced the phosphorus level from about 300 ppm to 50 ppm, but the level needs to be 5 ppm in order to continue with this refining strategy. The results suggest that there is a non-phospholipid phosphorus component in the crude oil. To try to determine the nature of this compound, phosphorus nuclear magnetic resonance (NMR) testing is being conducted on the degummed oil samples and the degummed residual. Additionally, a number of physical adsorbents are being tested to see if the phosphorus can be removed by an absorbent. As a component of Objective 2a, properties of the low-gossypol meals were investigated to understand how cooking protocols will need to be altered to maximize the value of the protein for different purposes (e.g., aquaculture feeds, or food uses). Meals were prepared from low-gossypol seed by dehulling and milling to yield particles that would cook more evenly. Cooking protocols were applied using steam and dry heat at varying times and temperatures followed by hexane extraction under a standard protocol. The meals are being tested this summer for protein, residual oil, color, and protein digestibility. A number of compounds were tested to improve cottonseed protein adhesive formulations (Objective 3). Catechin, gallic acid, and caffeic acid (phenolic compounds) were evaluated with cottonseed protein isolate (CPI). The addition of gallic or caffeic acid improved dry adhesive performance, hot water resistance, and the soak test, while catechin only showed marginal improvement. Thus, the carboxylic acid components of these compounds seem to be a factor for improving adhesive strength. Additionally, phosphoric acid, potassium phosphate, calcium oxide, and calcium hypophosphate were also added to CPI formulations and tested. These compounds also improved the dry, wet, and soak strengths of protein adhesive formulations. The best performance was obtained with phosphoric acid, which improved water resistance by 88%. Also, an optimized cottonseed protein isolate formulation was applied to pine plywood, and the water resistance of these samples passed industrial soak tests as Type II (interior) plywood. Water-washed cottonseed meal, cottonseed oil and polycaprolactone (a type of biodegradable polymer) were used to formulate a bio-based plastic (Objective 3b). These blended materials showed satisfactory mechanical and adhesive properties. The findings suggest that cottonseed protein can be a viable raw material for the formulation of bioplastics, and possibly can be used as a hot melt adhesive. As part of Objective 4a, extracts of cottonseed were prepared and tested for their activity on cell growth and gene expression with different cancer cells, mouse macrophages (white blood cells), and adipocytes (fat cells). The extracts decreased mitochondrial activity of some cancer cells but no activity was observed in macrophages or adipocytes. The extracts also increased gene expression in macrophages. For Objective 4b, cottonseed proteins were treated with trypsin, an enzyme that degrades protein to form peptides, but these peptides did not exhibit biological activity. Other proteases are being explored to produce different peptides for further testing. In collaboration with scientists from the Rochester Institute of Technology (Rochester, New York), composites made from polypropylene and gin trash have been prepared and tested. Together with a coupling agent, (i.e., maleic anhydride-modified polypropylene [MAPP]), and ethylene- vinyl acetate copolymer, the mixtures were extruded into films. In general, the addition of gin trash reduced tensile strength and elongation but enhanced Young⿿s modulus (stiffness). Films were also made from gin trash that had been washed with toluene solvent and derivatized with MAPP, which improved mechanical properties (Objective 5). The advantages of gin trash as a filler include low cost, ease in absorbing dyes and pigments, greater gas permeability, and greater affinity for water. Thus, the composites may be useful where reduced cost or additional material stiffness are desirable. Finally, a number of stakeholder initiatives have been either started or completed. Cottonseed oil high in tocotrienols has been extracted for further testing. These genetically-modified oils will likely be more stable to oxidation. Work was also started to produce an oil with an elevated level of dihydrosterculic acid (one of the cyclopropyl fatty acids in cottonseed oil). The oil will be used at the University of Georgia, Athens, Georgia, to confirm earlier tests of the benefits of this fatty acid in slowing the onset of fatty liver disease. Cottonseed oil naturally has small levels of this acid (0.2-0.3%) and this work may suggest a new direction for cottonseed oil research. Finally, an initial study was conducted on cottonseed hull strength. Ginners have been reporting problems with small and weak seed, which appears to have occurred because of decades of breeding cotton for maximal fiber yield. Damaged seed is detrimental to oil and fiber processors. The initial study found that a wide range in hull strengths exist within current commercial lines. Accomplishments 01 Release of cotton germplasm with elevated seed oil oleate levels. Vegetable oils with elevated levels of oleic acid are desirable as these oils tend to last longer in deep fat fryers. ARS researchers from New Orleans, Louisiana, working with ARS researchers from Starkville, Mississippi, have identified cotton plants with levels of oleic acid in the seed oil approximately double the level of this fatty acid in normal commercial cottonseed oil. The trait has been found to be genetically stable. These plant lines will be released to allow cotton breeders to use the germplasm. Based on prior usage of cottonseed oil for frying, regaining this market would represent a substantial gain for the industry. Assuming a 25% higher premium price and a 25% market penetration, these elevated oleic acid oils would be worth $110 million more than standard oil. 02 Domestic production of industrial oils. Because of severe allergic reactions to the proteins of the caster plant, castor oil production is prohibited in the United States. Because the oil contains unusual fatty acids that make it an important industrial lubricant, large amounts of castor oil are imported into the United States. Working with University collaborators, ARS researchers in New Orleans, Louisiana, produced oilseed lines containing some of the highest reported seed levels of these fatty acids outside the caster seeds. These lines were engineered to express a combination of two genes from the oil synthesis pathway of the castor bean plant. The work is a step in the direction of being able to produce a castor-like lubricating oil within the United States. 03 Biodegradable plastics from cottonseed proteins. Biodegradable polymers are of current interest because of the need for sustainability, the environmental concerns of plastic contamination and disposal, and toxic hazards involved in the production and degradation of synthetic polymers. ARS researchers at New Orleans, Louisiana, have taken water- washed cottonseed meal and oil and polycaprolactone (a biodegradable polyester) to form a bioplastic. These blends showed mechanical and adhesive properties that will be useful for some applications. The findings suggest that cottonseed protein can be a viable raw material for the formulation of bioplastics, and a possible application as a hot melt adhesive. The market for global biodegradable plastics is growing and expected to reach $6 billion by 2025. 04 Additives improve adhesive performance of cottonseed proteins. Bio- based protein adhesives suffer from being less water resistance than petroleum-based adhesives. To improve the water resistance of protein based adhesive formulations, ARS researchers in New Orleans, Louisiana, studied the adhesive properties of cottonseed proteins mixed with different additives. Additives with a phosphate or carboxylic component improved the water resistance of the formulations. The work is a step toward the development of bio-based adhesives that are as water resistant as petroleum-based adhesives.

Impacts
(N/A)

Publications

  • Cheng, H.N., Wyckoff, W., Dowd, M.K., He, Z. 2019. Evaluation of adhesion properties of blends of cottonseed protein and anionic water-soluble polymers. Journal of Adhesion Science and Technology. 33(1):66-78.
  • He, Z., Guo, M., Sleighter, R.L., Zhang, H., Chanel, F., Hatcher, P.G. 2018. Characterization of defatted cottonseed meal-derived pyrolysis bio- oil by ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Journal of Analytical & Applied Pyrolysis. 136:96-106.
  • Pradyawong, S., Li, J., He, Z., Sun, X.S., Wang, D., Cheng, H.N., Klasson, K.T. 2018. Blending cottonseed meal products with different protein contents for cost-effective wood adhesive performances. Industrial Crops and Products. 126:31-37.
  • Cheng, H.N., Gross, R.A., Smith, P.B. 2018. Green polymer chemistry: pipelines toward new products and processes. In: Cheng, H.N., Gross, R.A., Smith, P.B., Editors. Green Polymer Chemistry: New Products, Processes, and Applications. ACS Symposium Series. Washington, DC: American Chemical Society. p. 1-11.
  • Cao, H., Sethumadhavan, K., Bland, J.M. 2018. Isolation of cottonseed extracts that affect human cancer cell growth. Scientific Reports. 8:10458.
  • Cao, H., Sethumadhavan, K. Cottonseed extracts and gossypol regulate diacylglycerol acyltransferase gene expression in mouse macrophages. Journal of Agricultural and Food Chemistry. 66(24):6022-6030. Epub 2018 Jun 8.
  • Cheng, H.N. 2019. Enzymatic modification of polymers. In: Kobayashi S., Uyama H., Kadokawa J. (eds). Enzymatic Polymerization towards Green Polymer Chemistry. Green Chemistry and Sustainable Technology. Singapore: Springer. p. 357-385.
  • Cheng, H.N., Kilgore, K., Ford, C., Fortier, C., Dowd, M.K., He, Z. 2019. Cottonseed protein-based wood adhesive reinforced with nanocellulose. Journal of Adhesion Science and Technology. 33(12):1357-1368.
  • Cheng, H.N., Ford, C.V., He, Z. 2019. Evaluation of polyblends of cottonseed protein and polycaprolactone plasticized by cottonseed oil. International Journal of Polymer Analysis and Characterization. 24(5):389- 398.
  • Ling, Z., Wang, T., Makerem, M., Santiago Cintron, M., Cheng, H.N., Kang, X., Bacher, M., Porthast, A., Rosenau, T., King, H.A., Delhom, C.D., Nam, S., Edwards, J.V., Kim, S., Xu, F., French, A.D. 2019. Effects of ball milling on the structure of cotton cellulose. Cellulose. 26(1):305-328.


Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): The overall goal of the project is to improve the postharvest utilization of cottonseed and thereby increase the value of the U.S. cotton crop through improved understanding of cottonseed composition, properties and processing of the seed¿s components. There are five total objectives in the project. Three objectives focus on studying and modifying the oil, protein, and hull components of the seed. One objective is directed toward the study of processing operations to improve the separation of these components and the last objective is directed toward isolation of minor components that may exhibit beneficial bioactivity. Objective 1) Enable the development of new, commercial cotton varieties which express high levels of oleic acid in the seed. Sub-objective 1a) Study FAD2 structure in naturally high oleic acid cotton accessions. Sub-objective 1b) Use genes and other DNA regulatory elements associated with cyclopropyl fatty acid synthesis to silence production of these fatty acids in developing cottonseed. Sub-objective 1c) Determine the compositional and functional property differences between naturally high oleic acid and normal cottonseed oils. Objective 2) Enable new commercial process technologies that maximize the profitability of converting low-gossypol cotton seed into oil and meal products. Sub-objective 2a) Determine conditions that result in low-color oils from the processing of glandless cottonseed. Sub-objective 2b) Physically refine crude cottonseed oil from glandless cottonseed to produce commercial grade oil. Objective 3) Enable the commercial production of new products from the protein fraction of cottonseed meal. Sub-objective 3a) Improve water resistance of cottonseed protein meals, concentrates and isolates used as wood adhesives. Sub-objective 3b) Explore the use of cottonseed proteins as functional additives in non-food commercial products. Sub-objective 3c) Explore the use of cottonseed protein fractions to improve non-food product properties. Objective 4) Enable the commercial production of new bioactive food ingredients from glandless (no gossypol) cottonseed. Sub-objective 4a) Identify minor bioactive phenolic components from glandless cottonseed. Sub-objective 4b) Identify bioactive peptides and proteins from glandless cottonseed. Objective 5) Enable the commercial production of new products from the carbohydrate components in cottonseed burrs, hulls and kernels. Sub-objective 5a) Isolate, characterize, and study the functionality of hemicellulosic components from seed processing byproducts. Sub-objective 5b) Exploit the potential use of hull and other seed byproducts as fillers in composite materials. Approach (from AD-416): Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. To alter cottonseed oil composition, a combination of genetic manipulation and classical breeding will be used. Various physical and chemical techniques will be employed at the laboratory level to mimic processing steps and to fractionate meal (i.e., protein) and hull components. Chemical, enzymatic, and physical techniques will be used to modify these isolated components and to characterize the resulting products. Performance of these fractions for different potential applications will be achieved through a series of physical testing methods. Isolation of seed minor components will be achieved for bioactivity studies through chemical fractionation and chromatographic methods and several cell-based assays will be used to test for activity. Progress was made on all five of the project objectives, all of which fall under National Program 306 and contains elements of both the Food and Non-food components. Under the Food component, the work addresses Problems 1A, to define, measure, and preserve/enhance/reduce attributes that impact quality and marketability; Problem 1B, to develop new bioactive ingredients and functional foods; and Problem 1C, to develop new and improved food processing technologies. Under the Non-food component, the work relates to Problem 2B to enable technologies for producing new marketable non-food biobased products derived from agricultural products and byproducts and estimate the potential economic value of the new products. To promote the use of cottonseed oil as a frying oil, higher levels of oleic acid than are normally present in the oil (~17%) are desirable. As part of Objective 1, the wild GB-713 cotton plant that we had previously identified as having a high oleic acid trait was also found to exhibit nematode resistance. Because of this other trait, the GB-713 genotype has been used to transfer nematode resistance into an agronomic short-staple cotton variety (SG-747). Seeds from different steps in this breeding program were tested for oil oleic acid level, and elevated levels (30-40%) were found throughout the breeding sequence. Four series of plants from this program were planted in the summer of 2017 and harvested in the fall. Two of the plant lines were finished lines (i.e., the plants are considered genetically stable), and two lines were intermediate in the breeding process. Testing these plants indicated elevated levels of oleic acid in some plants from all four plant lines. Seeds from plants of the two finished lines exhibiting the highest oleic acid levels were planted in the greenhouse and harvested to increase the available seed. These seeds were then used to plant field rows this summer. If these plants are found to be uniformly high in oleic acid (around 30% is expected for the one finished plant line and ~35% is expected for the second plant line), then the genetic components that contribute to these increased levels will be considered stable, and these plant lines will be made available in germplasm releases for commercialization. These will represent the first stable agronomic Upland cotton cultivars with elevated seed oil oleic acid levels. Also in support of achieving Objective 1, an improved DNA marker was developed for a mutant desaturase gene (called fad2-1d) present in the wild GB-713 plants, because our first marker exhibited false positives. This gene is responsible for at least part of the high oleic acid trait and is useful for helping to select individual plants for the high oleic acid trait. The improved marker will be used to select field plants for the mutant desaturase. As the marker was found to be present in all of the plants from one of the finished lines, which all had about 30% seed oil oleic acid, the gene appears to account for about half of the elevated oleate trait present in GB-713 plants. This suggests that additional mutations exist in the wild plant. A list of candidate mutant genes has been determined. Marker testing protocols for the top candidates in this list are being developed, and testing will begin soon. In order to study the properties of cottonseed oils with high levels of oleic acid, bulk seeds from GB-713 plants (which were produced at the ARS Winter Nursery) were extracted to recover a high-oleate cottonseed oil. The oil was then refined and bleached. This oil will be used in the upcoming year for solvent fractionation experiments to compare the fractionation properties of the two oil types. Reducing the level of cyclopropyl fatty acids in cottonseed is also desirable as these acids contribute unwanted biological activity to the oil. Using the genome sequence of the cotton wildtype plant (GB-713), the family of cyclopropyl genes (called cps) has been identified, and the DNA elements that control the expression of these genes have been cloned. These elements have been attached to a reporter gene that produces a brightly colored chemical in the plant tissues where it is active. The DNA fusion molecules are being prepared for expression in cotton hairy root cultures to study which of the cps genes likely contributes the most to cyclopropyl fatty acid production. During oil refining, cottonseed oil can sometimes develop high color, a process referred to as color set, which can affect its value. Glandless cottonseed genotypes have reduced levels of phenolic compounds. Because phenolic compounds can oxidize to form dark oxidation products, oil from glandless plants may exhibit reduced color formation during refining. To better understand these effects, crude cottonseed oils from glandless plants were refined by different techniques (Objective 2). Several batches of crude oil were prepared by dehulling, flaking, cooking and extruding the kernel tissue followed by extraction with hexane, a commercial solvent. These crude oil samples were used to study different refining strategies. The normal chemical refining with sodium hydroxide (a base that reacts with some oil impurities) produced oils with very low Lovibond red color (0.7 to 0.9 red values). Although not as low as the color of cottonseed oils prepared by pressing in earlier experiments, these red color levels are still well below the standard for prime cottonseed oil (2.5 red value). Hence, crude oils from glandless plants when processed by chemical refining should yield refined oils of much lower color, and the color set problems that affected cottonseed oil in the past would be eliminated. The physical refining of vegetable oils is an alternative refining process that purifies the oil without sodium hydroxide. This refining process, however, does not appear to work with cottonseed oil. Repeated attempts have been made, but the initial degumming of the oil did not reduce phosphorus levels sufficiently to allow for continuation of the refining process. The physical oil refining process of cottonseed oil might be easier with pressed oils, which have less impurities than solvent extracted oils. At present, obtaining pressed oil from glandless seed has been difficult due to the unavailability of the processing equipment, which is owned by an outside group. It is unclear when we will obtain access to this processing equipment. Under Objective 3, new uses for cottonseed protein, which at present is used solely as low-value feed ingredient, are being evaluated. With the help of researchers at the ARS National Center of Agricultural Utilization Research in Peoria, Illinois, large-scale (10 Kg) production of cottonseed protein isolate (>90% protein) was achieved. This material was needed to support collaborative work with the Department of Biorenewable Resources, Mississippi State University at Starkville, Mississippi, to produce bio-based termite-resistant plywood boards. The first boards were prepared late in 2017, and an optimization process is underway to determine the best conditions for adhering the veneer layers. The first stage of the optimization process analyzed boards made with different amounts of protein and water. This experiment indicated that a range of both protein and water levels can be used to produce boards. Experiments to determine the best pressing temperature and time are in progress. Several additional protein adhesive experiments have been conducted utilizing either cottonseed protein isolate or cottonseed meal. Experiments on the preparation of cottonseed protein meals for use as wood adhesives showed that the drying method employed during meal preparation had an impact on adhesive performance. High-temperature oven drying in adhesive formulations requires the use of higher temperatures during pressing to form adhesive joints with adhesive strength equal to that developed from protein meals dried at lower temperature. The effect of smaller particle sizes on the adhesive properties of the protein meal was found to be minimal but some decrease in water resistance was noted with the smaller particles. Replacing up to 40% of the urea-formaldehyde adhesive with cottonseed meal showed doubled water soaked bonding strength, compared to either pure synthetic resin or cottonseed meal adhesives alone. Additionally, cottonseed protein isolate, when added to nonwoven cotton products, increased the dry strength of the products. Eleven percent protein tripled the tear strength and burst strength compared to untreated nonwoven fabric. As part of Objective 4, protocols were developed to isolate bioactive extracts from cottonseed, and these extracts were tested for their activity on different human cancer cells. Gossypol (a polyphenolic metabolite present in cottonseed) and ethanol extracts of glanded and glandless cottonseed kernels significantly decreased the mitochondrial activity (energy production) of breast cancer cells and pancreas cancer cells. In addition, cottonseed extracts and gossypol affected the expression of DGAT enzymes (an enzyme associated with lipid biosynthesis). Results suggest that this is an inducible gene that responds to stimulators, such as polyphenols, whose enzyme product plays an important role in fat biosynthesis. The results indicate that gossypol and ethanol extracts from glanded cottonseed kernels are stimulators of DGAT2 gene expression and that they may be novel agents for intervention of lipid-related diseases. Different xylan (a carbohydrate polymer) preparations were isolated from cotton plant byproducts, including cottonseed hulls and cotton burrs (Objective 5). This complex carbohydrate was treated to make positively and negatively charged polymers, and these novel products were characterized by a number of analytical techniques. Mixed solutions of the differently charged polymers were found to increase the dry strength of paper by 75%. Accomplishments 01 Cottonseed oil with desirable elevated levels of oleic acid. Vegetable oils with elevated levels of oleic acid are desirable as these oils tend to last longer in deep fat fryers. ARS researchers from New Orleans, Louisiana, working with ARS researchers from Starkville, Mississippi, have identified cotton plants that combine two beneficial traits: elevated (30%) oleic acid in seed oil and broad spectrum root resistance to soil worm pests. Additional agronomic and chemical tests are ongoing but so far the traits appear to be genetically stable, and this plant line will be submitted for germplasm release in a year. These will be the first agronomic cotton plants to exhibit elevated levels of oleic acid in the seed oil, and the oil from these plants should be better able to compete with other vegetable oils used for frying. Based on prior usage of cottonseed oil for frying, regaining this market would represent a substantial market for the industry. Assuming a 25% higher premium price and a 25% market penetration, these elevated oleic acid oils would be worth $110 million more than standard oil. 02 Use of cottonseed protein as a strength additive for nonwoven cotton. Nonwoven fabrics are being used in a wide range of consumer products. Cotton-based nonwovens are of interest because of their ability to be recycled, resulting in more environmentally friendly products compared with petroleum-based counterparts. ARS researchers in New Orleans, Louisiana, have used cottonseed protein as an additive to increase the dry strength of cotton-based nonwovens. If these strengthened products increase the use of cotton in nonwoven materials by 10%, the value of the additional cotton fabric used in would be around $30 million dollars. These results will be helpful to enhance the usage of cotton nonwovens and cottonseed protein in several applications. 03 Improved protein-based wood adhesive formulations. Researchers in New Orleans, Louisiana, have developed improved wood adhesive formulations by including different chemical additives. The inclusion of small amounts of polymer additives, for example, improves both the dry adhesive strength and the hot water resistance of the protein-based formulations. The results will be of interest to the wood products industry that is seeking to eliminate formaldehyde from current wood adhesive formulations. 04 Low color cottonseed oils. Dark oil color is a sporadic problem associated with cottonseed oil refining that reduces the value of the oil. For some seed, oils have to be handled that bleach to less than ideal color levels and these oils often can only be managed by blending with light color oils. ARS researchers in New Orleans, Louisiana, have been studying the refining and bleaching of cottonseed oils prepared from new seed varieties that do not have the pigment glands of regular cottonseed. The bleachable color of the oil extracted from these seed is much lower than the color of oil from regular glanded seed. The result should eliminate this occasional processing complication and will be of interest to cottonseed oil processors.

Impacts
(N/A)

Publications

  • Zheng, L., Shockey, J., Bian, F., Chen, G., Shan, L., Li, X., Wan, S., Peng, Z. 2017. Variant amino acid residues alter the enzyme activity of peanut type 2 diacylglycerol acyltransferases. Frontiers in Plant Science. 8(1751):1-15.
  • Zheng, L., Shockey, J., Guo, F., Shi, L., Li, X., Shan, L., Wan, S., Peng, Z. 2017. Discovery of a new mechanism for regulation of plant triacylglycerol metabolism: The peanut diacylglycerol acyltransferase-1 gene family transcriptome is highly enriched in alternative splicing variants. Journal of Plant Physiology. 219:62-70.
  • He, Z., Cheng, H.N., Olanya, O.M., Uknalis, J., Zhang, X., Koplitz, B.D., He, J. 2018. Surface characterization of cottonseed meal products by SEM, SEM-EDS, XRD and XPS analysis. Journal of Materials Science Research. 7(1) :28-40.
  • Cheng, H.N., Villalpando, A., Easson, M.W., Dowd, M.K. 2017. Characterization of cottonseed protein isolate as a paper additive. International Journal of Polymer Analysis and Characterization. 22(8):699- 708.
  • Alam, M.S., Watanabe, W.O., Carroll, P.M., Gabel, J.E., Corum, M.A., Seaton, P., Wedegaertner, T.C., Rathore, K.S., Dowd, M.K. 2018. Evaluation of genetically-improved (glandless) and genetically-modified low-gossypol cottonseed meal as alternative protein sources in the diet of juvenile southern flounder Paralichthys lethostigma reared in a recirculating aquaculture system. Aquaculture. 489:36-45.
  • He, Z., Cheng, H.N., Klasson, K.T., Olanya, O.M., Uknalis, J. 2017. Effects of particle size on the morphology and water-and thermo-resistance of washed cottonseed meal-based wood adhesives. Polymers.
  • Liu, M., Wang, Y., Wu, Y., He, Z., Wan, H. 2018. "Greener" adhesives composed of urea-formaldehyde resin and cottonseed meal for wood-based composites. Journal of Cleaner Production. 187:361-371.
  • Cheng, H.N., Doemeny, L.J., Geraci, C.L., Schmidt, D.G. 2016. Nanotechnology overview: Opportunities and challenges. In: Cheng, H.N., Doemeny, L.J., Geraci, C.L., Schmidt, D.G., editors. Nanotechnology: Delivering the Promise Volume 2. ACS Symposium Series, Washington, DC: American Chemical Society. p. 1-12.
  • Klasson, K.T. 2018. QXLA: Adding upper quantiles for the studentized range to Excel for multiple comparison procedures. Journal of Statistical Software. Journal of Statistical Software, Code Snippets. Vol(85):1-9.
  • Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2017. Effects of phosphorus- containing additives on soy and cottonseed protein as wood adhesives. International Journal of Adhesion and Adhesives. 77:51-57.
  • Paton, C.M., Vaughan, R.A., Alpergin, E.S.S., Assadi-Porter, F., Dowd, M.K. 2017. Dihydrosterculic acid from cottonseed oil suppresses desaturase activity and improves liver metabolomic profiles of high-fat-fed mice. Nutrition Research. 45:52-62.
  • He, Z., Cheng, H.N. 2017. Evaluation of wood bonding performance of water- washed cottonseed meal-based adhesives with high solid contents and low press temperatures. Journal of Adhesion Science and Technology. 31(23) :2620-2629
  • Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2017. Wood adhesive properties of cottonseed protein with denaturant additives. Journal of Adhesion Science and Technology. 31(24):2657¿2666.
  • Li, N., Prodyawong, S., He, Z., Sun, X.S., Wang, D. 2017. Effect of drying methods on the physicochemical properties and adhesion performance of water-washed cottonseed meal. Industrial Crops and Products. 109:281-287.
  • He, Z., Chiozza, F. 2017. Adhesive strength of pilot-scale-produced water- washed cottonseed meal in comparison with a synthetic glue for non- structural interior application. Journal of Materials Science Research. 6(3):20-26.
  • Dowd, M.K., Pelitire, S.M., Delhom, C.D. 2018. Seed-fiber ratio, seed index, and seed tissue and compositional properties of current cotton cultivars. Journal of Cotton Science. 22:60-74.


Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): The overall goal of the project is to improve the postharvest utilization of cottonseed and thereby increase the value of the U.S. cotton crop through improved understanding of cottonseed composition, properties and processing of the seed�s components. There are five total objectives in the project. Three objectives focus on studying and modifying the oil, protein, and hull components of the seed. One objective is directed toward the study of processing operations to improve the separation of these components and the last objective is directed toward isolation of minor components that may exhibit beneficial bioactivity. Objective 1) Enable the development of new, commercial cotton varieties which express high levels of oleic acid in the seed. Sub-objective 1a) Study FAD2 structure in naturally high oleic acid cotton accessions. Sub-objective 1b) Use genes and other DNA regulatory elements associated with cyclopropyl fatty acid synthesis to silence production of these fatty acids in developing cottonseed. Sub-objective 1c) Determine the compositional and functional property differences between naturally high oleic acid and normal cottonseed oils. Objective 2) Enable new commercial process technologies that maximize the profitability of converting low-gossypol cotton seed into oil and meal products. Sub-objective 2a) Determine conditions that result in low-color oils from the processing of glandless cottonseed. Sub-objective 2b) Physically refine crude cottonseed oil from glandless cottonseed to produce commercial grade oil. Objective 3) Enable the commercial production of new products from the protein fraction of cottonseed meal. Sub-objective 3a) Improve water resistance of cottonseed protein meals, concentrates and isolates used as wood adhesives. Sub-objective 3b) Explore the use of cottonseed proteins as functional additives in non-food commercial products. Sub-objective 3c) Explore the use of cottonseed protein fractions to improve non-food product properties. Objective 4) Enable the commercial production of new bioactive food ingredients from glandless (no gossypol) cottonseed. Sub-objective 4a) Identify minor bioactive phenolic components from glandless cottonseed. Sub-objective 4b) Identify bioactive peptides and proteins from glandless cottonseed. Objective 5) Enable the commercial production of new products from the carbohydrate components in cottonseed burrs, hulls and kernels. Sub-objective 5a) Isolate, characterize, and study the functionality of hemicellulosic components from seed processing byproducts. Sub-objective 5b) Exploit the potential use of hull and other seed byproducts as fillers in composite materials. Approach (from AD-416): Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. To alter cottonseed oil composition, a combination of genetic manipulation and classical breeding will be used. Various physical and chemical techniques will be employed at the laboratory level to mimic processing steps and to fractionate meal (i.e., protein) and hull components. Chemical, enzymatic, and physical techniques will be used to modify these isolated components and to characterize the resulting products. Performance of these fractions for different potential applications will be achieved through a series of physical testing methods. Isolation of seed minor components will be achieved for bioactivity studies through chemical fractionation and chromatographic methods and several cell-based assays will be used to test for activity. The genome of a wild Gossypium barbadense cotton accession (GB-713) was sequenced as part of Objective 1a. This accession has a higher than normal level high-oleic acid and a lower than normal level of linoleic acid in its seed oil. From the genomic data, a gene was identified (referred to as FAD-2d1) that appears to be abnormal. The abnormality appears to be adjacent to the gene, which we believe prevents correct localization of the enzyme within the cell leading to reduced activity. A DNA (deoxyribonucleic acid) construct was generated of this region to help identify this sequence in breeding experiments. Additionally, although GB-713 cotton is photoperiodic (i.e., it does not readily flower outside its native environment), plants of this accession did flower for a short period in our greenhouse, and the pollen was used to make crosses to two G. barbadense varieties (long-stable cotton) and two G. hirsutum varieties (short-staple cotton). The seed from these crosses can be used to start a breeding effort to move the high oleic acid trait into agronomic cotton. However, not enough seed was generated to be sure that we will get the desired combination of traits from both parents. Fortunately, another avenue now appears more promising. During the preparation of these crosses, it was learned that GB-713 had previously been used in another ARS breeding effort in the Genetics and Sustainable Agriculture Research Unit (Starkville, Mississippi). In discussing this work with the researchers involved, a few seed of their finished lines and their initial crosses were obtained for testing. Variable numbers of seeds of the finished lines showed levels of oleic acid greater than 30% compared with typical agronomic varieties having values about 17-18%. Although not as high as seed from the initial GB-713 plants (>40%), this represents an increase worth investigating. Additionally, a number of the plants of these seeds were positive for our gene mutation. This material may shorten the time needed to breed the higher oleic acid trait, and a series of field tests have been designed to further explore this possibility. Cottonseed oil contains three cyclopropyl fatty acids (or CPFAs) that may be less desirable for some whole seed feeding applications. Cotton contains several cyclopropane synthase (CPS) genes but it is currently unknown which of these contributes to CPFA production in seed oil. As part of Objective 1b, a series of gene promoters associated with CPS activity were identified. These regions were cloned and will be used for functional testing in cultured cotton roots and other model plant systems to identify which genes should be engineered to lower the level of seed CPFA acids. A relatively new gene knockout technique for making changes to specific sequences of DNA is being developed in pursuit of this objective and as part of other collaborative efforts. The Cotton Fiber Bioscience Research Unit (New Orleans, Louisiana) has produced one stable cotton transgenic plant using the components of this first-generation system. Ongoing collaborations between our two groups will continue to develop cotton lines that have stable, heritable gene knockout pedigrees that may lead to improved insect or drought resistance or seed property changes. As part of Objective 2, efforts were made to show that crude oils from glandless cottonseed would be easier to refine than crude oils from glanded seed. Using the traditional chemical refining technique, bleached oils from glandless seed were obtained with a Lovibond red value of 0.3, compared with a Lovibond red value of 2.5 that is normally acceptable for cottonseed oil. However, physically refining oils (without sodium hydroxide) resulted in higher red values (2.6) that would be borderline acceptable. Experiments with solvent-extracted oils are in progress but a similar trend is apparent. Deodorization of the oils is also planned and is part of a Cotton, Inc. reimbursable agreement that is discussed below. Work on the use of cottonseed protein as wood adhesives also continues as part of Objective 3. By adding small levels of additives to cottonseed protein isolate-based adhesives, improved adhesion was found. The addition of acidic amino acids or one of several phosphate compounds resulted in increased tensile strength and water resistance, suggesting that charge-charge interactions are important for adhesion. The addition of small amounts of protein denaturants or anionic polymers resulted in variable improvement. Washed cottonseed meal was also evaluated in some studies. Formulation pH, protein storage time, and the rheological properties of the formulations were evaluated. Electron micrographs were obtained to evaluate the structures of the wood-adhesive interface. As part of a recently developed CRADA agreement with Mississippi State University to develop bio-based boards with protein adhesives and termite inhibitory properties, our first plywood and particle boards were prepared with cottonseed protein isolate. While the properties of these boards are inferior to those prepared from petroleum-based adhesives, improvement is expected as many variables have not been optimized. Dispersion of the protein appears to become more difficult with increased scale of adhesion, i.e., getting a uniform covering of the wood particles with the adhesive formulation, and this problem will need further study. The properties of guayule plant resin, which is known to impede termite activity, are also being studied. Understanding how the viscosity is affected by temperature and its solubility in solvents is currently being determined to help formulate methods of application. Glandless cottonseed extracts were used to treat mouse macrophages and cancer cells and cytotoxicity and key gene expression of immunological responses were monitored as a component of Objective 4a. No toxicity was observed from the extracts, but cells treated with gossypol present in the glanded cottonseed extracts did show effects. Additionally, proteins were extracted and separated and the bioactivity of any novel proteins will be tested. Xylan, a natural polymer of xylose, has been isolated from cottonseed hulls and cotton burrs as part of Objective 5, and anionic and cationic derivatives of these polymers have been made. The properties of these derivatives have been determined, including rheological behavior and viscosity. When these preparations were applied to the surface of paper, paper strength was found to increase appreciably. In response to stakeholder issues, a few additional efforts were furthered. First, the Unified Methods Committee of the American Oil Chemists� Society (AOCS) has approved an update of the gossypol-HPLC method (AOCS Ba 8-93) and has requested that we work with AOCS staff on an inter-laboratory study to upgrade the assay to an AOCS Official Method. Second, a three year survey of commercial cottonseed-fiber properties was completed this year. Seed-fiber ratio, seed index, the percentage of linter, hull, and kernel components and the oil, protein, and gossypol composition of the seed were studied to better understand the changes that have occurred to seed properties because of decades of intensive breeding for fiber yield. Seed-fiber ratio and seed index have decreased appreciably compared with early reports (before 1950), indicating that seed has become smaller with the breeding changes. Small differences in the percentage of linters were apparent, which may have occurred because of the combination of breeding and improved ginning. In contrast, the relative percentage of hulls, which might relate to weaker seeds that are currently an issue with ginners, does not appear to have been affected. Third, work on the use of cotton gin trash as a filler in polymer preparation, which is a collaborative effort with researchers at the National Center of Crops Utilization Research (Peoria, Illinois), is continuing. Progress report for Agreement 6054-41000-103-14R. The targeted gene knockout technology commonly known as �CRISPR� was incorporated into an existing gene overexpression/gene silencing toolbox. Promoter and terminator regulatory elements that drive activity of the two components of the system have been identified and shown to lead to reasonable rates of rapid, heritable gene knockouts, which is essential for breeding pure knockout lines within reasonable times. This system has been applied to model plant lines, and first-generation knockout lines targeting each of at least four different genes that might affect novel fatty acid production are being analyzed. Progress report for Agreement 6054-41000-103-11N. The opening and closing of rice florets takes 6 to 10 days. The regulation of this process is very important for hybrid rice seed production. However, little is known about the effects of plant hormones on this process. Previous studies have shown that the chemicals jasmonatic acid and methyl jasmonate promote rice floret opening. In this study, the effects of six plant hormones were investigated. Most of the treatments increased the percentages of open florets and increased the time that the floret were open. Progress report for Agreement 58-6054-7-0012. Construction of a laboratory-scale vegetable oil deodorizer has begun for studying cottonseed oil refining. An initial design has been prepared, and a glassblower from the Chemistry Department of the Louisiana State University in Baton Rouge, Louisiana, has been contracted to fabricate the three pieces of glassware needed. Two pieces are complete and the third piece is in preparation. The equipment for the refining and bleaching operation is in place and 10 gallons of pressed glandless cottonseed oil has been generated for the effort. Accomplishments 01 Discovery of high oleic acid trait in the seed oil of previously breed cotton cultivars. ARS researchers in New Orleans, Louisiana, have seeds from recently identified and bred cotton varieties that have higher than normal levels of oleic acid in the seed oil. While higher levels of oil oleic acid are known in wild cotton germplasm, this finding should shorten the breeding time that is needed for developing cottonseed oils with elevated levels of oleic acid. This should help with the development of cottonseed oil to complete with other vegetable- based oils for some market applications. Oils with high levels of oleic acid are more stable in deep fat frying applications and are nutritionally more beneficial. 02 Use of carbohydrate polymers as promoters for cottonseed protein in wood adhesive application. There is ongoing interest in using agro- based materials (like proteins) in wood adhesives, but cost, adhesive strength, and water resistance are the major issues. ARS researchers in New Orleans, Louisiana, showed that the addition of a few specific carbohydrate polymers to cottonseed protein adhesive formulations increased the tensile strength of glued wood veneers by 50% and increased their water resistance by 30%. The improved performance should make cottonseed protein more competitive as an ingredient in wood adhesive formulations and it should lower the costs of formulations. The work will help develop cottonseed proteins as bio- based wood adhesives. 03 New interpenetrating polymer involving cottonseed protein. ARS researchers in New Orleans, Louisiana, have earlier shown cottonseed protein (CSP) to be a viable ingredient in wood adhesives. They now came up with a new approach to further improve the adhesive performance of CSP. This involved the in situ polymerization of a polyester in the presence of CSP. In an optimal case, the new CSP-derived polymer increased the dry tensile strength by 100% compared with CSP alone. The hot water resistance test was increased 50-70% increase over CSP alone. These new composite adhesive formulation may lead to new ideas and direction that further promote the use of agricultural products in value-added applications. 04 Xylan isolated and characterized from cotton byproducts. Hemicellulose, a common plant polymer, is an abundant material in nature but is underutilized. Xylan, a common form of hemicellulose was isolated from cotton burrs (outer boll husks) and cottonseed hulls. Anionic and cationic derivatives of these xylans were synthesized by ARS researchers in New Orleans, Louisiana. Whereas the viscous properties of anionic and cationic xylan by themselves were not interesting, when the cationic and anionic modified xylans were combined the solution the viscosity dropped indicating interaction between the two polymer forms. When these were applied to the surface of paper, the paper�s dry strength increased significantly. Thus, the combination of cationic/ anionic xylan may have promising properties that should have industrial and product applications.

Impacts
(N/A)

Publications

  • Bacigalupe, A., He, Z., Escobar, M.M. 2017. Effects of rheology and viscosity of biobased adhesives on bonding performance. In: He, Z., editor. Bio-based Wood Adhesives: Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. 293-309.
  • Liu, S., Zhu, Y., Wu, F., Meng, W., Wang, H., He, Z., Guo, W., Song, F., Geisy, J.P. 2017. Using solid 13C NMR coupled with solution 31P NMR spectroscopy to investigate molecular species and lability of organic carbon and phosphorus from aquatic plants in Tai Lake, China. Environmental Science and Pollution Research. 24:1880-1889.
  • He, Z., Wan, H. 2017. Bio-based wood adhesives research: Advances and outlooks. In: He, Z., editor. Bio-based Wood Adhesives: Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. 340-353.
  • He, Z. 2017. Bio-based wood adhesives--preparation, characterization, and testing. In: He, Z., editor. Bio-based Wood Adhesives: Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. v-ix.
  • Farina, M., Mauri, M., Patriarca, G., Simonutti, R., Klasson, K.T., Cheng, H.N. 2016. 129Xe NMR studies of morphology and accessibility in porous biochar from almond shells. RSC Advances. 6(105):103803-103810.
  • Mao, A., He, Z., Wan, H., Li, Q. 2017. Preparation, properties, and bonding utilization of pyrolysis bio-oil. In: He, Z., editor. Bio-based Wood Adhesives: Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. 250-279.
  • Zhan, Z., Chen, Y., Shockey, J., Han, X., Wang, Y. 2016. Proteomic analysis of tung tree (Vernicia fordii) oilseeds during the developmental stages. Molecules. 21:1486.
  • Cao, H., Sethumadhavan, K., Rajasekaran, K. 2016. Identification of an Mg2+-independent soluble phosphatidate phosphatase in cottonseed (Gossypium hirsutum L.). Advances in Biological Chemistry. 6:169-179.
  • He, Z., Zhang, H., Tewolde, H., Shankle, M. 2017. Chemical characterization of cotton plant parts for multiple uses. Agricultural and Environmental Letters. 2:110044-110049. doi:10.2134/ael2016.11.0044.
  • Moulana, M., Taylor, E., Edholm, E., Quiniou, S., Wilson, M., Bengten, E. 2014. Identification and characterization of TCRgamma and TCRdelta chains in channel catfish, Ictalurus punctatus. Immunogenetics. 66(9-10):545-561.
  • Zhou, L., He, H., Li, M.-C., Song, K., Cheng, H.N., Wu, Q. 2016. Morphological influence of cellulose nanoparticles (CNs) from cottonseed hulls on rheological properties of polyvinyl alcohol/CN suspensions. Carbohydrate Polymers. 153:445-454.
  • He, Z., Chapital, D.C., Cheng, H.N. 2016. Effects of pH and storage time on the adhesive and rheological properties of cottonseed meal-based products. Journal of Applied Polymer Science. 13:43637. doi:10.1002/APP. 43637.
  • Siccardi,III, A.J., Richardson, C.M., Dowd, M.K., Wedegaertner, T.C., Holmes, K.A., Samocha, T.M. 2016. Digestibility of glandless cottonseed protein in diets for pacific white shrimp, litopenaeus vannamei. Journal of the World Aquaculture Society. 47(1):97-106.
  • He, Z., Uchimiya, S.M., Guo, M. 2016. Production and characterization of biochar from agricultural by-products: Overview and use of cotton biomass residues. In: Guo, M., He, Z., Uchimiya, S.M., editors. Agricultural and Environmental Applications of Biochar: Advances and Barriers. SSSA Special Publication 63. Madison, WI: Soil Science Society of America, Inc. p. 63- 86.
  • Dowd, M.K. 2015. Seed. In: Fang, D.D., Percy, R.G., editors. Cotton, Agronomy Monograph 57. Madison, WI: American Society of Agronomy, Inc., Crop Science Society of America, Inc., and Soil Science Society of America, Inc. p. 745-781.
  • Cheng, H.N., Rau, M.W., Dowd, M.K., Easson, M.W., Condon, B.D. 2014. Comparison of soybean and cottonseed oils upon hydrogenation with nickel, palladium and platinum catalysts. Journal of the American Oil Chemists' Society. 91:1461-1469.
  • Xin, P., Huang, Y., Hse, C., Cheng, H.N., Huang, C., Huang, Pan, H. 2017. Modification of cellulose with succinic anhydride in TBAA/DMSO mixed solvent under catalyst-free conditions. Materials. 10(5):526.
  • He, Z., Chapital, D.C., Cheng, H.N., Modesto, O.O. 2016. Adhesive properties of water washed cottonseed meal on four types of wood. Journal of Adhesion Science and Technology. 30(19):2109-2119.
  • Cao, H. 2015. Genome-wide analysis of oleosin gene family in 22 tree species: An accelerator for metabolic engineering of biofuel crops and agrigenomics industrial applications? Omics - A Journal Of Integrative Biology. 19(9):521-541.
  • Van Erp, H., Shockey, J., Zhang, M., Adhikari, N.D., Browse, J. 2015. Reducing isozyme competition increases target fatty acid accumulation in seed triacylglycerols of transgenic Arabidopsis. Plant Physiology. 168:36- 46.
  • Li, M.-C., Wu, Q., Song, K., Cheng, H.N., Suzuki, S., Lei, T. 2016. Chitin nanofibers as reinforcing and antimicrobial agents in carboxymethyl cellulose films: Influence of partial deacetylation. ACS Sustainable Chemistry & Engineering. 4:4385-4395.
  • Anderson, A.D., Alam, M.S., Watanabe, W.O., Carroll, P.M., Wedegaertner, T. C., Dowd, M.K. 2016. Full replacement of menhaden fish meal protein by low- gossypol cottonseed flour protein in the diet of juvenile black sea bass Centropristis striata. Aquaculture. 464:618-628.
  • Feng, Y., Zhang, L., Fu, J., Li, F., Wang, L., Tan, X., Mo, W., Cao, H. 2016. Characterization of glycolytic pathway genes using RNA-Seq in developing seeds of Eucommia ulmoides. Journal of Agricultural and Food Chemistry. (64):3712-3731.
  • He, Z., Cheng, H.N. 2017. Preparation and utilization of water washed cottonseed meal as wood adhesives. In: He, Z., editor. Bio-based Wood Adhesives: Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. 156-178.
  • Fein, W., Zhu, Y., Wu, F., He, Z., Zhang, C., Geisy, J.P. 2016. Forms and lability of phosphorus in algae and aquatic macrophytes characterized by solution 31P NMR coupled with enzymatic hydrolysis. Scientific Reports. 6:37164-37174. doi:10.1038/srep37164.
  • He, Z., Pagliari, P.H., Waldrip, H.M. 2016. Applied and environmental chemistry of animal manure: A review. Pedosphere. 26:779-816.
  • He, Z., Umemura, K. 2017. Utilization of citric acid in wood bonding. In: He,Z., editor. Bio-based Wood Adhesives-Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. 221-238.
  • Cheng, H.N., He, Z. 2017. Wood adhesives containing proteins and carbohydrates. In: He, Z., editor. Bio-based Wood Adhesives: Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. 140-155.
  • Wan, H., He, Z., Mao, A., Liu, X. 2017. Synthesis of polymers from liquefied biomass and their utilization in wood bonding. In: He, Z., editor. Bio-based Wood Adhesives: Preparation, Characterization, and Testing. Boca Raton, Florida: CRC Press. pp. 239-259.


Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): The overall goal of the project is to improve the postharvest utilization of cottonseed and thereby increase the value of the U.S. cotton crop through improved understanding of cottonseed composition, properties and processing of the seed�s components. There are five total objectives in the project. Three objectives focus on studying and modifying the oil, protein, and hull components of the seed. One objective is directed toward the study of processing operations to improve the separation of these components and the last objective is directed toward isolation of minor components that may exhibit beneficial bioactivity. Objective 1) Enable the development of new, commercial cotton varieties which express high levels of oleic acid in the seed. Sub-objective 1a) Study FAD2 structure in naturally high oleic acid cotton accessions. Sub-objective 1b) Use genes and other DNA regulatory elements associated with cyclopropyl fatty acid synthesis to silence production of these fatty acids in developing cottonseed. Sub-objective 1c) Determine the compositional and functional property differences between naturally high oleic acid and normal cottonseed oils. Objective 2) Enable new commercial process technologies that maximize the profitability of converting low-gossypol cotton seed into oil and meal products. Sub-objective 2a) Determine conditions that result in low-color oils from the processing of glandless cottonseed. Sub-objective 2b) Physically refine crude cottonseed oil from glandless cottonseed to produce commercial grade oil. Objective 3) Enable the commercial production of new products from the protein fraction of cottonseed meal. Sub-objective 3a) Improve water resistance of cottonseed protein meals, concentrates and isolates used as wood adhesives. Sub-objective 3b) Explore the use of cottonseed proteins as functional additives in non-food commercial products. Sub-objective 3c) Explore the use of cottonseed protein fractions to improve non-food product properties. Objective 4) Enable the commercial production of new bioactive food ingredients from glandless (no gossypol) cottonseed. Sub-objective 4a) Identify minor bioactive phenolic components from glandless cottonseed. Sub-objective 4b) Identify bioactive peptides and proteins from glandless cottonseed. Objective 5) Enable the commercial production of new products from the carbohydrate components in cottonseed burrs, hulls and kernels. Sub-objective 5a) Isolate, characterize, and study the functionality of hemicellulosic components from seed processing byproducts. Sub-objective 5b) Exploit the potential use of hull and other seed byproducts as fillers in composite materials. Approach (from AD-416): Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. To alter cottonseed oil composition, a combination of genetic manipulation and classical breeding will be used. Various physical and chemical techniques will be employed at the laboratory level to mimic processing steps and to fractionate meal (i.e., protein) and hull components. Chemical, enzymatic, and physical techniques will be used to modify these isolated components and to characterize the resulting products. Performance of these fractions for different potential applications will be achieved through a series of physical testing methods. Isolation of seed minor components will be achieved for bioactivity studies through chemical fractionation and chromatographic methods and several cell-based assays will be used to test for activity. Work continued on the development of cotton varieties with different oil properties. Transferring a high oleic acid (a desirable monounsaturated fatty acid) trait from wild plants to cultivated varieties has not been successful. Alternative approaches will now be tried. These will involve either applying a virus known to induce flowering or growing the plants in a different environment. Costa Rica, which may be soon available as a replacement to the closed Cotton Winter Nursery, may be an option. Understanding the molecular basis for high oleic acid levels in cottonseed oil is progressing. In conjunction with ARS collaborators at the Genomics and Bioinformatics Research Unit (Stoneville, Mississippi) and the Food and Feed Safety Unit (New Orleans, Louisiana), the complete DNA (deoxynucleic acid) sequence was determined for a cotton plant that has unusually high levels of oleic acid in its oil. The entire gene family for an enzyme important in regulating the oleic acid level was identified. This gene family is being studied closely for changes in its DNA that could account for the high oleic acid character. This data set is also helping to identify genes that contribute to the unwanted production of cyclic fatty acids. The data is also helping identify regulatory genes that control oil synthesis in oilseed plants. The basic components of a new molecular technique (called cRISPER) for selectively targeting gene activities have been assembled. These components are undergoing functional testing in model plants and microbes. Successful preliminary findings with these tools have encouraged collaborators in the Cotton Fiber Bioscience Unit (New Orleans, Louisiana) to begin the process of creating cotton plants with a specific gene that helps to direct the location of gene changes. This will be useful for developing plants with specific genes blocked. Successful combinations of these modified plants, paired with the other gene components, will be tested in a hairy root tissue culture (which can be conveniently grown in the laboratory) for their suppressive effects on cyclic fatty acid production. Several small phosphate compounds were added to cottonseed proteins adhesive formulations and were tested for their adhesive properties. Several of these compounds increased adhesive strength and water resistance. The results appear to be specific to formulations composed of cottonseed proteins, as similar improvements were not found with soybean proteins. This difference may be related to the different levels of arginine (one of several amino acids found in proteins) in the two protein types. In addition, a re-examination of the use of protein denaturants (chemicals that cause proteins to unfold) has also found adhesive improvement at conditions not previously studied. Cottonseed meal was also studied as an adhesive. The effects of pH and storage time were considered. Under a completed Reimbursable Agreement with Kansas State University (Manhattan, Kansas), the production of water-washed cottonseed meal was scaled up to a 20 lb. pilot-plant procedure. This work provided a quantity of the washed meal for industrial adhesive applications and research efforts. Chemical analysis of the products provided the fundamental data for quality control of future mill scale production of these products. A CRADA (Cooperative Research and Development Agreement) has been established with the Department of Sustainable Products of Mississippi State University (Starkville, Mississippi) to produce particle boards with cottonseed protein-based adhesives. An initial sample of cottonseed protein isolate was prepared for this work. This material will be used to explore the conditions to make suitable boards. After reasonable pressing conditions are determined, guayule resin (a plant-based insect deterrent) will be added, and the boards will be tested for termite and fungal resistance. In addition, two invention disclosures have been written on the cottonseed adhesives work. The patent review committee has approved the filing of one patent application, which will include claims related to blended protein preparations, and the addition of carboxylic acids and phosphorus-containing chemicals to protein-based adhesives. To determine if cottonseed contained minor compounds that are active in biological systems, defatted glandless cottonseeds were extracted different solutions containing alcohols. Different concentrations of ethanol extracts from the seed coat and kernels were used to treat biological cell cultures. Cell viability assays showed that extracts from cottonseed retained cell survival while the control tests lost significant mitochondrial (energy production) activity over a 24 h period. High concentrations of purified gossypol (a well-known bioactive compound in cottonseed) resulted in significant cell death. RNA (ribonucleic acid) was extracted from the cells to analyze if gene expression was affected by the extracts, with an emphasis on genes related to immune system response. Peptides and proteins were also extracted from glanded and glandless seeds. The fractions were separated into individual components. These protein components are being further characterized by mass spectrometry. The function and bioactivity of these novel proteins are being tested in biological assays. Xylan (a carbohydrate plant polymer) has been isolated from cottonseed hulls (outer part of the seed) and cotton burrs (outer covering of the cotton bowl), and a number of treatments were used to improve the purity and properties of these preparations. The preparations were derivatized with different classes of compounds to alter their properties. Preliminary studies with the modified products have shown that when combined with cellulosic materials useful film-like materials can be prepared. The xylan extracted from the hull was also treated to form a more bio-based polyurethane polymer. The viscosity of these preparations, helpful for some product applications, was also determined. To better understand and control seed damage that occurs during ginning (the process used to separate fiber and seed), seeds have been studied for their mechanical strength. Several seed samples, including different commercial varieties and varieties grown in different conditions, were tested for tensile strength, maximum elongation before breakage, and breakage energy. Results show that significant differences exist among the samples, and both genetic and growing conditions appear to affect this property. This may provide a convenient method to test for the tendency of different seed types to be damaged. Because of the many decades of successful breading cotton varieties for increased fiber yield, it has generally been believed that cottonseed has become smaller. To understand these changes, a series of seed samples were characterized for their fiber-to-seed ratio, seed size, linter (short fibers that remain with the seed after ginning), hulls, and kernel tissue percentages, and kernel seed, protein, and gossypol composition. Results indicate that seed-to-fiber ratio has decreased from around 1.7 in the 1940s to an average of 1.45 today. Similarly, seed size has on average decreased around 20% over a similar time period. Smaller differences were found regarding the percentage of linters, but the amounts of hull and kernel tissue and the amounts of kernel protein and oil do not have appear to have been significantly changed by breeding for fiber yield. Accomplishments 01 Improved protein adhesive formulations. ARS scientists at New Orleans, Louisiana, have found that by including additives in cottonseed based protein adhesives, improved adhesive performance can be obtained. Both adhesive strength and water resistance have been improved by the addition of small amounts of organic acids or phosphorous containing compounds. Improved water resistance is of particular interest, as most protein based adhesives exhibit poor water resistance. 02 Less expensive protein adhesives. ARS scientists at New Orleans, Louisiana, have found that by blending cottonseed proteins with other components in adhesive formulations, improved adhesive formulations can be obtained. Blending some cottonseed protein with soybean protein allowed for improved performance over soybean protein alone. Additionally, cottonseed-based protein adhesives retain much of their properties when blended with relatively cheap fibrous fillers. These blends may provide an opportunity to decrease the amount of protein used in adhesive formulations, thereby reducing cost. 03 Preparation of carbohydrate polymer products from cotton byproducts. ARS scientists at New Orleans, Louisiana, have extracted carbohydrate- based polymer fractions (i.e., hemicelluloses) from cotton and cottonseed processing waste materials, and improved the purity and properties of the resulting material. Biodegradable film materials have been made from these materials. These materials may also be useful as thickeners in a number of water-based product formulations. 04 Mutant cotton genotype sequenced. ARS scientists at New Orleans, Louisiana, and at Stoneville, Mississippi, have completed the genome sequence of a mutant Gossypium barbadense accession with high levels of oleic acid in its seed oil. This will allow the rapid assessment of genetic differences that result in this desirable trait, which may occur because of modification of one or several enzymes plus various regulatory elements. The work will be of particular interest to those working to expand the value and uses for cottonseed oil. 05 Genes identified for desaturating monounsaturated fatty acids. The full complement of cotton fatty acid desaturase 2 genes (also known as FAD2 genes) have been identified by ARS researchers at New Orleans, Louisiana. These proteins are critical during production of seed oils and other lipid components, and they control the balance between monounsaturates (which typically contain the best properties for human and animal health as well as frying stability) and diunsaturates (which are less desirable). Eight separate genes have been identified, and the set represents that largest known FAD2 gene family currently reported in the plant science literature. The work will help guide efforts to improve the properties and utilization of cottonseed oil.

Impacts
(N/A)

Publications

  • Liu, Y., He, Z., Shankle, M., Tewolde, H. 2015. Compositional features of cotton plant biomass fractions characterized by attenuated total reflection Fourier transform infrared spectroscopy. Industrial Crops and Products. 79:283-286.
  • Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2016. Soy and cottonseed protein blends as wood adhesives. Industrial Crops and Products. 85:324-330.
  • He, Z., Chapital, D.C., Cheng, H.N. 2016. Comparison of the adhesive performances of soy meal, water washed meal fractions, and protein isolates. Modern Applied Science. 10(5):112-120.
  • Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2016. Use of additives to enhance the properties of cottonseed protein as wood adhesives. International Journal of Adhesion and Adhesives. 68:156-160.
  • He, Z., Klasson, K.T., Wang, D., Li, N., Zhang, H., Zhang, D., Wedegaertner, T.C. 2015. Pilot-scale production of washed cottonseed meal and co-products. Modern Applied Science. 10(2):25-33.
  • Zhu, L., Krens, F., Smith, M.A., Li, X., Qi, W., Van Loo, E.N., Iven, T., Feussner, I., Nazarenus, T.J., Huai, D., Taylor, D.C., Zhou, X., Green, A. G., Shockey, J., Klasson, K.T., Mullen, R.T., Huang, B., Dyer, J.M., Cahoon, E.B. 2016. Dedicated industrial oilseed crops as metabolic engineering platforms for sustainable industrial feedstock production. Scientific Reports. 6:22181.
  • Shockey, J., Regmi, A., Cotton, K., Adhikari, N., Browse, J., Bates, P.D. 2015. Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in Triacylglycerol Biosynthesis. Plant Physiology. 170:163-179.
  • Liu, S., Zhu, Y., Meng, W., He, Z., Feng, W., Zhang, C., Giesy, J.P. 2015. Release and transformation of carbon and phosphorus from aquatic macrophytes of lakes: Insight from solid-state 13C NMR and solution 31P NMR spectroscopy. Science of the Total Environment. 543:746-756.