2005 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.
Sophorolipid biosurfactant preparations with tailored open-chain to lactone ratios:
Project researchers established the ability of C. bombicola to use the soy-based glycerol-rich biodiesel coproduct stream (BCS) as well as methyl, ethyl and propyl esters of soy oil to produce sophorolipids (SL) with varying open-chain to lactone ratios. This accomplishment addresses 2 problems:.
Bio-glycerol and soy molasses are presently the major coproduct streams that project researchers are investigating as feedstocks for the production of biobased products targeted in this CRIS project. The researchers have demonstrated the usefulness of these feedstocks in the production of PHA and SL, and are starting to evaluate them in RL production. Their current efforts include analyzing the time-course 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 have initiated 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.
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.
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: A) solution casting in chloroform results in a heterogeneous film as the chloroform is allowed to evaporate, thus causing films with holes in them, B) casting blend films from the melt has not proven viable due to the large difference in melting points for scl-PHA (> 140ºC) and mcl-PHA (~ 40-50ºC), causing the mcl-PHA to break down prior to the melting of scl-PHA. Ongoing attempts will use chemical compatibilizers to realize scl-/mcl-PHA blends.
One crucial factor in the cradle-to-grave life-cycle analysis of PHA is its biodegradability. Project researchers have 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 determined that P. lemoignei produces an extracellular depolymerase enzyme that will degrade sc-PHA (PHB), and Comamonas P37C produces an enzyme that will degrade both mc-PHA and sc-PHA. In addition, they determined that a copolymer of poly(3-hydroxybutyrate-co-10% 3-hydroxyhexanoate) (NODAX™) seems to enhance depolymerase activity by increasing the rate of depolymerization of sc-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 developed a synthetic approach that allows the production of a series of modified SLs with increased water solubility. Appending an amino acid to the carbohydrate introduces positive and/or negative charges, so the new SL can be up to 10-fold more water-soluble than the precursor. Work is in progress to develop synthesis methods to attach different functional groups to SLs to generate derivatives less prone to untimely degradation. 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. 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.
1935-41000-067-01R-This report serves to document research conducted under a reimbursable cooperative agreement between ARS and CSREES-NRI. Project researchers initiated the cloning and expression of class III PHA synthase gene and its truncated species for subsequent cryatallization and structural determination study. Project researchers has also investigated novel intracellular interactions between PHA synthase enzymes and other proteins involved in the biosynthesis of PHA to elucidate the functional properties of the structures of PHA polymerizing enzymes. Initial focus was on a major class II PHA synthase of Pseudomonas resinovorans, phaC1. Project investigators utilized a yeast two-hybrid technique to screen for genes of proteins potentially interacting with PhaC1 enzyme. Accordingly, the full-length P. resinovorans phaC1 gene was used as the bait to survey the P. resinovorans genomic library. The researchers successfully isolated and identified three gene sequences partially coding for potential PhaC1-interacting proteins. BLAST search of the sequenced data resulted in the identification of the three potential PHA-interacting proteins. The cloning of the complete sequences of the three potential candidate genes by chromosomal walking is in progress. Work is also in progress to construct mutant strains of P. resinovorans in which each of the candidate gene products was inactivated by gene disruption to evaluate the roles of these proteins in PHA biosynthesis. Furthermore, the investigators plan to utilize the yeast two-hybrid technique to screen for additional proteins potentially interacting with other PHA synthase enzymes.
1935-41000-067-02S-This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Georgia. Project researchers had carried out preliminary expression trials on 4 clones of PHA synthase genes and truncated sequences. The solubility test results showed that three clones had good expression of soluble proteins. Large-scale isolation of seleno-methionine labelled and N-terminal His-tagged fusion proteins of these three clones had been carried out. Protein purification was achieved by chromatography on a Nickel affinity column, followed by gel filtration chromatography. All three purified protein samples were screened against 384 reagent mixtures made up from 7 commercial sparse matrix screens containing 48 conditions. Initial screening was carried out by the sitting drop vapor diffusion method with Greiner Crystalquick plates setup by a crystallization robot. The plates were moved to the CrystalFarm incubator for storage, imaging and scoring. Unfortunately, no crystal was observed in the first round of crystallization screen. Project researchers plan to carry out reductive methylation on the protein samples before another round of crystallization screen. The reductive methylation on surface lysines is a non-invasive chemical reaction which can reduce local conformational entropy of lysines. This chemical modification has been proved to effectively assist crystallization and lattice packaging. These investigators also plan to try different grow conditions, cell hosts or expression vectors to find the suitable condition in order to get the soluble form of the one clone expressing insoluble protein in the original trial.
Molecular engineering of poly(hydroxyalkanoate (PHA) production system: CWU scientists had invented widely adopted PHA-screening PCR methods and a versatile electroporation gene-transfer system that facilitated identification and genetic engineering of PHA-producing organisms. These ARS researchers also had established an impressive collection of native and molecularly (i.e., genetically or metabolically) engineered microorganisms and genes invaluable for the conversion of fats and oils into tailor-made PHAs in good yields (NP306, Component II).
Fermentation and process technologies in poly(hydroxyalkanoate (PHA) 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 (NP306, Component II).
Post-harvest modification of PHA and SL: The 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 (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.
Three provisional patent applications had been filed: 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 and less-than-ideal properties.
Transferred and provided 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.
Foglia, T.A. (Chicago, IL., October 2004) Overview of ERRC fats & oils research. ARS-United Soybean Board Biobased Products Workshop.
Marmer, W.N. (Clemson, SC, October 2004) Overview of ERRC fats & oils research. National Meeting of Fats and Proteins Research Foundation. (rendering industry, allied researchers).Solaiman, D., Catara, V., Greco, S. 2005. Poly(hydroxyalkanoate) synthase genotype and pha production of pseudomonas corrugata and p. mediterranea. Journal of Industrial Microbiology and Biotechnology. 32(2):75-82.
Solaiman, D., Ashby, R.D. 2005. Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications. Biomacromolecules. 6(2):532-537.
Solaiman, D., Ashby, R.D. 2005. Genetic characterization of the poly(hydroxyalkanoate) synthases of various pseudomonas oleovorans strains. Current Microbiology. 50(6):329-333.
Gunther, N.W., Nunez, A., Fett, W.F., Solaiman, D. 2005. Production of rhamnolipids by pseudomonas chlororaphis, a non-pathogenic bacterium. Applied and Environmental Microbiology. 71(5):2288-2293.
Solaiman, D., Ashby, R.D., Foglia, T.A. 2004. Characterization and manipulation of genes in the biosynthesis of sophorolipids and poly(hydroxyalkanoates). Proceedings of the United States-Japan Cooperative Program in Natural Resources, Protein Resources Panel Annual Meeting. p. 215-219.
Ashby, R.D., Nunez, A., Solaiman, D., Foglia, T.A. 2004. Lipidic precursors to control the synthesis and properties of microbial sophorolipids and polyhydroxyalkanoates. Proceedings of the United States-Japan Cooperative Program in Natural Resources, Protein Resources Panel Annual Meeting. p. 225:229.
Solaiman, D., Ashby, R.D., Foglia, T.A. 2005. Production of biosurfactants by fermentation of fats, oils and their coproducts. In:Hou, C.T., editor. Handbook of Industrial Biocatalysis. Boca Raton, FL:Taylor & Francis. p. 14-1-14-9.
Ashby, R.D., Solaiman, D., Foglia, T.A. 2005. Biopolyesters derived from the fermentation of renewable resources. In: Hou, C.T., editor. Handbook of Industrial Biocatalysis. Boca Raton, FL:CRC Press. p. 19-1:19-10.
Solaiman, D., Catara, V., Greco, S., Ashby, R.D., Foglia, T.A. 2004. Rapid genetic characterization of pha synthase and its applications [abstract]. International Symposium on Biological Polyesters. p. 40.
Solaiman, D., Ashby, R.D., Foglia, T.A., Marmer, W.N., Kaplan, D.L. 2004. Biosurfactants from microbial fermentation of renewable substrates [abstract]. Industrial Application of Renewable Resources - A Conference on Sustainable Technologies, American Oil Chemists' Society. p. 14.
Solaiman, D., Ashby, R.D., Zerkowski, J.A., Nunez, A., Foglia, T.A. 2005. Production and modification of sophorolipid biosurfactant [abstract]. Annual Meeting and Expo of the American Oil Chemists' Society. p. 17.
Ashby, R.D., Nunez, A., Solaiman, D., Foglia, T.A. 2004. Production and structural variability of sophorolipids derived from the fermentation of renewable resources [abstract]. American Oil Chemists' Society Meeting. p. 29.
Zerkowski, J.A., Solaiman, D., Ashby, R.D., Foglia, T.A. 2005. Functionalization of hydroxy fatty acids from sophorolipids [abstract]. Annual Meeting and Expo of the American Oil Chemists' Society. p. 64.
Zerkowski, J.A., Solaiman, D., Ashby, R.D., Foglia, T.A. 2005. Mass spectrometric monitoring of the chemical modification of sophorolipids [abstract]. Annual Meeting and Expo of the American Oil Chemists' Society. p. 6.
Zerkowski, J.A., Solaiman, D., Ashby, R.D., Foglia, T.A. 2004. Gemini sophorolipids, a new variety of glycosurfactants [abstract]. American Chemical Society National Meeting. Paper No. 82.
Solaiman, D., Ashby, R.D., Nunez, A., Foglia, T.A. 2004. Production of sophorolipids using soy molasses as substrate. Biotechnology Letters. 26:1241-1245.
Ashby, R.D., Solaiman, D., Foglia, T.A. 2004. Bacterial poly(hydroxyalkanoate) polymer production from the biodiesel co-product stream. Journal of Environment and Polymers. 12(3):105-112.
Ashby, R.D., Solaiman, D., Foglia, T.A. 2005. Synthesis of short-/medium-chain-length poly(hydroxyalkanoate) blends by mixed culture fermentation of glycerol. Biomacromolecules. 6:2106-2112.