2006 Annual Report
While on the one hand there is a need to develop new uses for the abundant supply of fermentable FOCs, an equally urgent need exists on the other hand to overcome challenges to the commercialization of biobased materials. In an era of ever increasing global awareness for the needs of environmental responsibility and natural-resources management, there is a strong push toward the adoption of a biobased economy. Among the key challenges to the realization of this goal are the needs to lower the costs and to improve the properties of the biobased materials and fuels. This project uniquely seeks to develop a fermentation-based technology platform to utilize these useful, renewable, inexpensive and structurally versatile FOCs as feedstocks to produce and subsequently modify selected environmentally friendly biomaterials, including biopolymers [i.e., poly(hydroxyalkanoates) (PHAs), poly(gamma-glutamic acid) (PGA) and cyanophycin (CP)], biosurfactants [i.e., sophorolipids (SLs) and rhamnolipids (RLs)], and platform chemicals [i.e., itaconic acid, diols]. Accordingly, organisms capable of producing these biobased products from FOCs will be identified by screening or constructed by genetic engineering techniques. Product yields will be optimized through adoption of new fermentation methodologies such as fed-batch or continuous cultures. New structures and varied compositions of the products will be variously achieved through genetic, protein and metabolic engineering methods; selective use of FOC feedstocks and other additives during fermentation; and (bio)chemical/physical modification and blending of the native bioproducts. It is anticipated that the multidisciplinary approach to bioconvert FOCs to bioproducts will lead to the development of cost-competitive and environmentally friendly consumer items and industrial products.
The work is relevant to ARS National Program Action Plan - NP#306, Quality and Utilization of Agricultural Products, and to its Research Component, New Processes, New Uses and Value-Added Foods and Biobased Products. The work specifically addresses its mission to enhance the economic viability and competitiveness of U.S. agriculture by maintaining the quality of harvested agricultural commodities or otherwise enhancing their marketability, meeting consumer needs, developing environmentally friendly and efficient processing concepts, and expanding domestic and global market opportunities through the development of value-added food and nonfood products and processes.
The potential impact of the work is enormous. If the economics and properties of the targeted bioproducts were improved through this research project, then these ecologically advantageous materials would indeed become viable alternatives for their petroleum-based counterparts in many applications. The potential volumes and values of their markets could be gleaned from the market sizes of their comparable petro-based materials. For example, the annual output of the global plastics industry is estimated at 300 billion pounds, with a value of over $150 billion. The specialty chemicals, 1,2- and 1,3-propanediols, have an estimated market volume of 300 million pounds per year. The 2002 global surfactants demand was calculated as 11.2 million metric tons, while the projected market penetration by biosurfactants in various applications ranges from 8-25% in the year 2010. These estimates underscore the potential increased demand for fats, oils, and their derivatives if they could be used as the feedstocks for these biobased products. On the end-user side, various biodegradable and biocompatible polymers, surfactants, and specialty chemicals will be produced for use in the manufacturing or formulation of end-use consumer and industrial products, such as adhesives, coalescing agents, films, cleaning solutions, containers, fibers, thickening agents, metalworking fluids, and composites. In summary, the expected results of this research should ultimately impact the following groups: (1) The processors and renderers of fats/oils that benefit from the market opportunity created by the new uses of their products, (2) the producers of biopolymers/biosurfactants and the manufacturers of biodegradable and ecologically friendly consumer products that adopt our technology to improve the cost/performance factor of their goods, and (3) the consumer public when they have ready access to environmentally friendly and affordable plastics, surfactants and their derived products.
Objective 2, Post-fermentative Modification: Explore the modification of the microbially produced biomaterials such as surfactants and biopolymers to improve physical and chemical properties for alternative end uses and/or conversion to secondary value-added coproducts. Develop and characterize end-use consumer products using as ingredients native or modified biomaterials - Initiate synthesis of derivatives of PHA biopolymers by introducing cross-linkable and other functional groups into their unsaturated bonds using electrophilic or radical reagents. - Initiate preparation of modified glycolipid biosurfactants (e.g., SL) to improve physical and/or surfactant properties by evaluating suitable catalytic or synthetic approaches - Initiate work to isolate value-added FAs and carbohydrates of glycolipid biosurfactants (e.g., SL) for subsequent synthesis of new chemicals and/or polymers.
FY2006, from milestone chart for project 1935-41000-067-00D, 27 months (10/05 - 9/06): Objective 1. Develop fermentation-based bioconversion systems Sub-objective 1.1, Exploring New Targeted Products - Characterize physical, chemical, or material properties of FOC-derived RL, vlc-SL or gamma-PGA. - Initiate optimization to increase cell growth and/or product yields. - Continue to identify/secure organisms that bioconvert FOC to other targeted products (e.g., cyanophycin (CP), itaconic acid (IA), diols). Sub-objective 1.2, Strain Improvement - Continue cloning and characterization of genes for biosynthesis of SL and other targeted products such as RL. - Start construction of gene-knockout strains to affect metabolic flow of the biosynthesis of targeted bioproducts. - Construct modified genes to obtain PHA synthases with altered substrate or product specificity. Sub-objective 1.3, Fermentation Manipulation - Evaluate the use of branched-chain or functionalized fatty acids to produce mcl-PHA with these starting materials incorporated. - Continue the evaluation of various coproduct streams (including meat & bone meal hydrolysate; cf. CRIS 1935-41440-015, Obj. 3, Apply in Fermentation Media) for the production of PHA, biosurfactants and other target products. - Optimize yields of SLs containing C14 or under or C20 or over FA. - Continue to evaluate genetically altered organisms (see Sub-Obj. 1.2 Strain Improvement) for their capabilities to produce target products.
Objective 2, Post-fermentative Modification - Continue investigation to produce modified PHA polymers and initiate the evaluation of their chemical, physical and material properties toward their use as films, adhesives, or elastomers. - Continue investigation to prepare and determine the properties of modified glycolipid biosurfactants (e.g., SL) aimed to improve physical or surfactant properties. - Determine suitable routes to isolate value-added fatty acids from glycolipid biosurfactants on a large scale. Initiate investigation to derivatize the value-added fatty acids from glycolipid biosurfactants to obtain polymer precursors or novel polymers.
FY2007, from milestone chart for project 1935-41000-067-00D, 39 months (10/06 - 9/07): Objective 1. Develop fermentation-based bioconversion systems Sub-objective 1.1, Exploring New Targeted Products - Evaluate the production of CP, IA, or diols from FOC, and initiate characterization of these fat-/oil-derived products in their intended applications. Sub-objective 1.2, Strain Improvement - Begin the construction and characterization of next-generation of metabolically and genetically modified strains, and protein-engineered enzymes to obtain new bioproducts with high yields and altered properties. Sub-objective 1.3, Fermentation Manipulation - Scale up gamma-PGA and CP fermentations (from Sub-Obj. 1.1 Strain Improvement) to benchtop scale. - Develop fermentation conditions or chemical means by which polyol molecules (i.e., polyethylene glycol, hydroxy fatty acids) can be esterified onto the alpha-carboxyl group of gamma-PGA. - Determine physical and mechanical properties of branched chain and functionalized PHA and vlc- and other mcl-SLs.
Objective 2, Post-fermentative Modification - Continue to determine properties of modified PHA polymers for their intended uses. - Continue to prepare and determine properties of next-generation modified biosurfactants and other derived products, and prepare sufficient quantities in preparation for evaluation by potential industrial partners in applications such as cleaning solutions, cosmetics, cast films or containers.
FY2008, from milestone chart for project 1935-41000-067-00D, 51 months (10/07 - 9/08): Objective 1. Develop fermentation-based bioconversion systems Sub-objective 1.1, Exploring New Targeted Products - Initiate production and characterization of 2nd generation byproducts via feedstock manipulation, molecular biology, post-synthesis modification (see Sub-objectives 1.2 Strain Improvement; 1.3 Fermentation Manipulation; and Objective 2 Post-fermentative Modification.) Sub-objective 1.2, Strain Improvement - Continue the construction, characterization and evaluation of next-generation molecularly engineered strains and enzymes. Sub-objective 1.3 Fermentation Manipulation - Evaluate tandem batch fermentation approach to synthesize PHB/V from crude glycerol. - Determine the properties of bioengineered products [from Sub-Obj. 1.2]. - Continue scale up production on all biomaterials [including bioengineered products from Sub-Obj. 1.2] that show potential for industrial use.
Objective 2, Post-fermentative Modification - Initiate communication with appropriate industrial sector to establish collaboration for further evaluation of all promising modified products in their intended applications.
FY2009, from milestone chart for project 1935-41000-067-00D, 60 months (10/08 - 7/1/09): Objective 1. Develop fermentation-based bioconversion systems Sub-objective 1.1, Exploring New Targeted Products - (None) Sub-objective 1.2, Strain Improvement - Complete the construction, characterization and evaluation of next-generation molecularly engineered strains and enzymes. Sub-objective 1.3, Fermentation Manipulation - Produce the targeted products and their derivatives [including bioengineered products from Sub-Obj. 1.2] under optimized fermentation conditions and initiate technology transfer.
Objective 2, Post-fermentative Modification - Complete the communication of technology and evaluation by industry on the more promising products.
Itaconic acid (IA) is a potentially important industrial dicarboxylic acid. The esters of IA are possible biobased replacements for methyl methylacrylate used in coatings applications and produced at about 2 billion pounds annually in the U.S. CWU researchers, in collaboration with an industrial partner, are investigating production of itaconic esters by esterification of IA obtained from fermentation of agricultural feedstocks. Work in progress includes setting up fermentation processes for IA production by two fungal strains. Separately, these researchers are also testing reaction conditions and catalysts for converting IA to its esters.
Bio-glycerol and soy molasses continue to be the major coproduct streams that project researchers are investigating as feedstocks for the production of biobased products targeted in this CRIS project. They continue to study the kinetics of utilization of the substrates (e.g., glycerol, fatty acids and esters, carbohydrates) in these feedstocks to better design and optimize fermentation conditions for increased cell growth and product yields. They continue to perform genetic modification of strains to enable better utilization of the sugars in soy molasses. In their constant search for inexpensive feedstocks, they have initiated work in collaboration with WRRC/ARS scientists to evaluate coproducts of the Alaskan fishing industry, i.e., crude pollack oil and fish hydrolysates, as fermentation substrates for PHA production. In collaboration with scientists from CRIS 1935-41000-015-00D of this management unit, CWU researchers are also investigating meat & bone meal (MBM) as a fermentation substrate on an exploratory basis in order to assess our ability to effectively remove the lipidic material from the MBM, and to utilize that lipidic material for the production of bioproducts such as PHA
A perennial issue in PHA production is to obtain in high yields the desired biopolymer with certain specifications of monomer composition and molecular weight. Industry has claimed the ability to economically produce certain PHAs such as PHB/V and PHBHx, but research still strives to achieve the same for the other PHA types. Project researchers have previously demonstrated the use of chimeric genes, feedstock manipulation, or additives (e.g., glycerol and PEG) to vary the molecular weight and monomer composition of mcl- and scl-PHA. The scientists had constructed additional chimeric PHA synthase genes that proved inactive, but will continue to vary and fine-tune the chimeric components of the genes to achieve active constructs. CWU researchers are now studying the production of mcl-PHA polymers with methyl-branched side chains using as feedstocks the mixtures of branched fatty acids synthesized by researchers from CRIS 1935-41000-066-00D. GC/MS data show that as the PHA monomer chain lengths increase, a larger number of structural isomers are formed. For example, results have shown 3 structural isomers of 3-hydroxyoctanoate, 5 structural isomers of 3-hydroxydecanoate, 7 structural isomers of 3-hydroxydodecanoate and 9 structural isomers of 3-hydroxytetradecanoate. This increase in the number of structural isomers presumably is the result of the presence of methyl-branched side chains where the branching is present at different points along the chain. The researchers are presently in the process of verifying branching by NMR analysis. Yields of the branched-chain mcl-PHA are low, and efforts to improve yields are ongoing.
Material with new properties may result from the blending of the highly crystalline short-chain-length (scl-) PHA with the highly amorphous medium-chain-length (mcl-) PHA. Project researchers have continued to investigate ways to produce scl-/mcl-PHA polymer blends. They had experimented with solution casting and melt casting as possibilities for blend film formation. Both procedures have their drawbacks. Ongoing attempts are using chemical additives to reduce the interfacial tension between the two polymers and promote their miscibility to realize scl-/mcl-PHA blends.
One crucial factor in the cradle-to-grave life-cycle analysis of PHA is its biodegradability. Project researchers had previously identified the degradation capabilities of two different bacterial strains (Pseudomonas lemoignei and Comamonas P37C) in the biodegradation of both short-chain-length (scl-) and medium-chain (mcl-) PHA. They have developed new microscopic technology to monitor biodegradation of PHA films, and are in the process of applying this method to samples of melt-extruded scl-PHA film (PHB). In addition, they are initiating study on an exploratory basis to characterize the depolymerase enzymes from the two bacterial strains described here. This will allow us to better understand how the structural differences in PHA depolymerase enzymes affect the degradation process, leading to potentially more efficient biodegradation processes to improve the life-cycle analysis prospects of PHA.
Although sophorolipids (SLs) are valuable biosurfactants produced in copious amounts by some yeast strains, they suffer from lacking certain properties needed for some applications. Project researchers have previously developed a patent-pending synthetic approach that allows the production of a series of modified SLs with increased water solubility. They are currently developing a method that allows for protection from chemical reaction of all but one of the hydroxy groups of the sugar moiety of SL. Conversion of this single free hydroxy group into other groups that affect the water-solubility of SL will be the subject of future work. A molecular-biological approach is also being pursued to effect the production of modified SLs. Toward this end, the genes potentially involved in SL biosynthesis in two yeast strains are either completely or partially sequenced or cloned. The researchers are currently cloning and expressing these genes in a heterologous host to assay for the gene function. These genes will form the basis for construction of mutants capable of producing SLs with the desired disaccharide and fatty acid moieties.
Since SLs can be produced in copious amounts, it follows that their fatty acid side chain, which is predominantly (omega-1)-hydroxyoleic acid, is also abundantly available, after the easy removal of the carbohydrate portion. Project researchers have employed synthetic chemistry to convert this fatty acid into new value-added products. Both amino and hydroxy groups have been added to the fatty acid chain. These groups can be added selectively to specific regions of the chain -- at either end or in the middle. Polyamino and polyhydroxy versions have also been prepared. These compounds should be of interest as novel building blocks for functionalized polymers, as well as for use as lubricants. The C18 fatty acid derived from SL has also been used to prepare its elongated variants by using well-known (Wittig) chemical routes. The particular example prepared to date is the C19 terminal alkene (a class of compounds that has many uses), but this method will be widely applicable to the preparation of a range of very-long chain fatty acids, e.g., C22, C26, which may contain extra functional groups if desired. Very-long chain fatty acids are of interest for their tough mechanical properties and are found in naturally occurring waxes such as jojoba and rice bran.
Project 1935-41000-067-01R - This paragraph serves to document research conducted under a reimbursable agreement between ARS and CSREES-NRI. Project researchers had completed the cloning and expression of a class III PHA synthase gene and its truncated species for subsequent crystallization and structural determination. They had also initiated the cloning of a new class III PHA synthase gene from a photosynthetic bacterium. To this end, the phaC gene coding for one subunit of the PHA synthase now has been cloned and sequenced, and the subcloning of this gene into an expression vector for gene expression and subsequent crystallization attempt is in progress. The researchers also work to clone from this bacterium the phaE gene that codes for the other subunit of the enzyme. On another front, project researchers had previously successfully isolated and identified three partial gene sequences coding proteins that potentially interact with a major class II PHA synthase of Pseudomonas resinovorans, phaC1. Subsequent attempts to clone the complete sequences of the three potential candidate genes by chromosomal walking have proven more challenging than initially thought and is still in progress. Similarly, work is also still in progress to construct mutant strains of P. resinovorans in which each of these candidate gene products was inactivated by gene disruption to evaluate the roles of these proteins in PHA biosynthesis.
Project 1935-41000-067-02S - This paragraph serves to document research conducted under Specific Cooperative Agreement between ARS and the University of Georgia. Project researchers had earlier carried out a high throughput crystallization screening study of seleno-methionine-labelled and N-terminal His-tagged fusion proteins of three clones of PHA synthase genes and truncated sequences, but did not observe crystal formation. The researchers are in the process of attempting reductive methylation on the protein samples before another round of crystallization screen. Also in progress are attempts to try different growth conditions, cell hosts or expression vectors to find suitable conditions for getting the soluble form of the one clone that expresses insoluble protein in the original trial. Furthermore, an additional newly isolated class III PHA synthase gene will be subject to similar labeling and methylation procedures for subsequent crystallization screening.
Production of rhamnolipid biosurfactants: A CWU scientist discovered a non-pathogenic bacterium capable of producing rhamnolipids (RL), a class of biosurfactants, under energy-saving bioprocessing conditions. He applied proteomic analysis on this bacterium to successfully identify proteins that were up- and down-regulated during RL synthesis. The genes responsible for RL biosynthesis have been cloned and sequenced. This production process is expected to open up RL applications in areas that are previously off-limit to RL currently obtained from an opportunistic pathogenic bacterium (NP306, Component II).
Fermentation and process technologies for poly(hydroxyalkanoate (PHA) biopolymer production: CWU scientists devised a PHA isolation method that uses less organic solvent and yields cleaner biopolymers compared to the traditional methods. The researchers developed a versatile fermentation platform for the tailored production of PHAs having a desired molecular weight or composition through selective feeding of substrates and additives. A mixed-culture fermentation system for producing blends of soft and hard PHAs was developed. (NP306, Component II)
Sophorolipid (SL) production system: Genetic characterization of the SL biosynthesis pathway of two yeast species by ARS scientists had yielded clones and sequence data on several genes involved in SL biosynthesis. Project researchers successfully produced sophorolipids containing a fatty acid component with 22 carbons by using another yeast species. The new sophorolipids add to the variety of this biosurfactant and expand the potential properties and applications of these materials. (NP306, Component II)
Development of coproduct streams as fermentation feedstocks: To address the generally unfavorable cost factor of producing bioplastics (i.e., PHA) and biosurfactants (i.e., SL), ARS scientists had devised bioprocesses to feed on coproduct streams (e.g., bio-glycerol and soy molasses) generated from industrial production and utilization of animal fats and vegetable oils. An added feature of this process is the ability to tailor the compositional structure of the products, allowing the production of materials suited for specific applications. (NP306, Component II)
Post-harvest modification of PHA and SL: CWU scientists developed chemical and physical methods to strengthen the films cast from PHA. They initiated the formulation of chemical-synthetic procedures for attaching additional functional or chemical groups onto PHA and SL. Valuable components of SL such as the omega-1 fatty acids have been harnessed and further chemically modified to yield new products for use as lubricants and polymer precursors (NP306, Component II).
Overall, the project had generated basic knowledge, valuable materials and important processes that have great impact on the fats and oils industry and the development of a biobased economy. These results have been the subject of many technology transfer activities with partners or collaborators in the public and private sectors.
The three previously filed provisional patent applications have now been re-submitted as regular patent applications: One on the production of rhamnolipid biosurfactants by an energy-saving bioprocess using a non-pathogenic bacterium; the second on the use of bio-glycerol for the fermentative production of open-chain sophorolipids; and the third on the derivatization of sophorolipids by chemical means to improve water-solubility. All of these technologies are available for commercial development and adoption.
PHA and SL samples produced by the fermentation of oils and fats, and some subsequently modified by chemical means, continued to be the subjects of Material Transfer Agreements with industry to evaluate their potential use in products such as metal-working fluids, detergent formulations, lubricants, and structural and lamination adhesives. The constraints to the adoption of these biomaterials remain their high costs, less-than-ideal properties and low yields.
Continued to transfer and provide advice on a PCR-based PHA gene detection technology to researchers worldwide. The technology has been widely adopted to screen for PHA genes in new isolates worldwide.
Solaiman, D.K.Y. (Indianapolis, IN., 14 March, 2006) "Fermentation Studies of Soy Carbohydrates." 1st TAP (Technical Advisory Panel) on Emerging Industrial Opportunities in Bioprocessing, United Soybean Board.
Wyatt, V. (Denver, CO., 9-10 November, 2005) As part of his talk, presented work from this CWU on the fermentation of the glycerol-rich biodiesel coproduct stream to produce sophorolipids. National Biodiesel Board brainstorming meeting.
Foglia, (Prague, Czech Republic, 25-28 September 2005) Kaufmann Memorial Lecture Award. 26th World Congress and Exhibition of the ISF As part of his talk, presented fermentation work from this CWU on SL and PHA production from fats/oils and coproducts (glycerol and soy molasses).
Marmer, W.N. and Garcia R.A. (Prague, Czech Republic, 25-28 September 2005) "Rendered Products in the Age of TSE's: II. Research on Alternative Applications." 26th World Congress and Exhibition of the ISF. As part of his talk, presented work from this CWU on glycerol fermentation.
Cherry, J.P. (National Chung-Hsing University, Taichung, Taiwan, 19-21 October 2005) Keynote speech: "Advancing the Marketing of Agricultural Commodities by Biobased Technologies." As part of his talk, presented work from this CWU on fermentative production of biopolymers and microbial biosurfactants. International Symposium on Biocatalysis and Biotechnology - Functional Foods and Industrial Products.
Foglia, T.A. "Processing of fats and oils into value-added products." Inform Vol 17 (1), pp 14-15, January 2006. CWU work on the fermentation production of biopolymer and biosurfactants was described.
Solaiman, D., Ashby, R.D., Hotchkiss, A.T., Foglia, T.A. 2006. Biosynthesis of medium-chain-length poly(hydroxyalkanoates) from soy molasses. Biotechnology Letters. 28:157-162.
Zerkowski, J.A., Solaiman, D., Ashby, R.D., Foglia, T.A. 2006. Head group-modified sophorolipids: new cationic, zwitterionic, and anionic surfactants. Journal of Surfactants and Detergents. 9:57-62. Solaiman, D., Ashby, R.D., Foglia, T.A., Marmer, W.N., Hotchkiss, A.T., Kobayashi, H. 2005. Production of biopolymers and biosurfactants from soybean-derived renewable feedstocks. In: Proceedings of the US-Japan Cooperative Program in Natural Resources-Food & Agricultural Panel-34th Annual Meeting, October 23-29, 2005, Susono, Shizuoka. p. 187-191.
Ashby, R.D., Solaiman, D., Foglia, T.A. 2006. New uses for glycerol: fermentation substrates for value-added product synthesis [abstract]. Annual Meeting and Expo of the American Oil Chemists' Society. p. 72.
Ashby, R.D., Solaiman, D., Foglia, T.A. 2006. Macro- and microscopic visual evidence for the biodegradation of poly(hydroxyalkanoates) by pseudomonas lemoignei and comamonas p37c. Bio Environmental Polymer Society. p. 87.
Solaiman, D., Ashby, R.D., Zerkowski, J.A., Hotchkiss, A.T., Foglia, T.A., Marmer, W.N. 2006. Bioconversion of soy-based feedstocks into biopolymers and biosurfactants [abstract]. Annual Meeting and Expo of the American Oil Chemists' Society. p. 14.
Solaiman, D., Gunther, N.W., Ashby, R.D., Foglia, T.A., Kaplan, D.L. 2005. Production of microbial biosurfactants from soy molasses [abstract]. Society of Industrial Microbiology Annual Meeting. p. 75.
Solaiman, D., Ashby, R.D., Foglia, T.A., Marmer, W.N. 2005. Fermentative production of biopolymers and biosurfactants from agricultural feedstocks [abstract]. International Symposium on Biocatalysis and Biotechnology-Functional Foods and Industrial Products. p. 67.
Solaiman, D., Ashby, R.D., Zerkowski, J.A., Foglia, T.A., Marmer, W.N. 2006. The use of fats, oils and coproducts for fermentative production of poly(hydroxyalkanoates) biopolymer and sophorolipid biosurfactants. 3rd World Congress on Industrial Biotechnology and Bioprocessing. p. 47.
Zerkowski, J.A., Solaiman, D. 2006. Synthesis of new multifunctional polyols from an oleic acid derivative [abstract]. Annual Meeting and Expo of the American Oil Chemists' Society. p. 75.
Solaiman, D., Ashby, R.D., Foglia, T.A., Marmer, W.N. 2006. Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates). Applied Microbiology and Biotechnology. 71:783-789.