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United States Department of Agriculture

Agricultural Research Service

Research Project: Production and Value Enhancement of Biosurfactants and Biopolymers Derived from Agricultural Lipids and Coproducts

Location: Biobased and Other Animal Co-Products

2011 Annual Report

1a.Objectives (from AD-416)
The long term goal of the project is to develop commercially viable integrative bioprocesses encompassing biological and bio/chemical technologies to produce environmentally friendly biobased materials and chemicals using agricultural fats (i.e., tallow, lard, chicken fats, etc.), oils (i.e., soybean oil, cuphea oil, sunflower oil, etc.) and coproducts as feedstocks.

1: Develop biocatalysts and bioprocesses that enable the commercial production of new biobased products from agricultural lipids and related coproducts such as bioglycerol. 1A: Improve the efficiency of feedstocks utilization and enable the production of new varieties of bioproducts in high yields through metabolic engineering of microorganisms. 1B: Systematic optimization of bioprocesses to improve yields and economics of biobased products.

2: Develop new commercially viable applications for sophorolipids, rhamnolipids, cyanophycin and gamma-polyglutamic acid. 2A: Increase the values of product components. 2B: Develop biomaterials for end-use products.

1b.Approach (from AD-416)
Microorganisms capable of producing new variants of rhamnolipids (RLs) and sophorolipids (SLs) will be created by introducing mutated genes that originally encode the synthesis of native RL and SL. Increasing the RL yields in non-pathogenic P. chlororaphis by metabolic engineering will be attempted by increasing the copy-number and transcription level of the responsible genes (i.e., rhlAB), or by ‘knocking out’ the gene(s) responsible for diverting precursors away from RL biosynthesis. We will also enhance the value of P. chlororaphis by introducing another gene, rhlC, to allow the biosynthesis of dirhamnose lipids. The efficiency of RL- and SL-producing microorganisms to utilize soy molasses (SM) will be improved by introducing genes encoding enzymes that breakdown the complex sugars in SM. We will also express in Candida bombicola (an SL-producing yeast) and P. chlororaphis the genes that are used for glycerol metabolism to improve the organisms’ efficiency of using bioglycerol. To enhance colonization and biofilm formation for higher RL yields from P. chlororaphis, we will experiment with attaching a sponge-like adapter to the impellers of a fermenter and then use minimal aeration (slow impeller rotation, reduced air introduction, or both). Separately, process optimization will be carried out for the fermentative synthesis of SL, gamma-polyglutamic acid (PGA) and cyanophycin (Cp) using such low-cost feedstocks as crude glycerine from biodiesel production, soy molasses and meat and bone meal hydrolysates. Fermentation parameters such as carbon source content and concentration, feed rate, aeration, pH etc. will be varied and optimized using Response Surface Methodology (RSM). To increase the values of bio-product subcomponents or for application in end-use products, biological and chemical syntheses will be performed to obtain the followings for subsequent characterization: .
1)novel epoxy and polyhydroxy fatty acids by using functionalized fatty acids (i.e., vernolic acid, ricinoleic acid, lesquerellic acid) as fermentative substrates,.
2)cyanophycin-derived biosurfactants by linking fatty acids to the polar dipeptide unit of Cp,.
3)terminally unsaturated fatty acids by elimination reactions on hydroxy fatty acids,.
4)biopolymer/biosurfactants composites by solution-casting of PHA and SL or RL,.
5)oligoamides by converting the hydroxy fatty acids to amino fatty acids and self-coupling them, and.
6)water-soluble SLs by linking charged amino acids to the sugar headgroups.

3.Progress Report
Progress was made toward achieving the project objectives of developing commercially viable biocatalysts, bioprocesses, and new biobased products through genetic improvement, fermentation manipulation, and value-added chemical modification of bio-detergents and bioplastics. These project objectives in turn address NP306 Action Plan Component 3 (Biobased Products) Problems 3B (Develop Biobased Products and Sustainable Technologies/Processes). In this regard, project scientists had developed a fermentation protocol to utilize glycerol (a co-product of the biodiesel industry) and/or levulinic acid (a byproduct of the pulp and paper industry) to produce polyhydroxyalkanoates (PHA) with varying monomer ratios; demonstrated that the amount of residual methanol in the crude glycerol from biodiesel production could decrease the molecular weights of PHAs biosynthesized by bacteria; demonstrated in collaboration with other Center scientists the biodegradability of isostearic acid which was made for potential application as a lubricant substitute; identified chemical methods for appending a wide range of chemical functional groups to a fatty acid that is derived from fermentation of agricultural products such as fats and oils; elucidated the enzymatic function of a glucosyltransferase gene cloned from sophorolipid-producing organism; verified transcriptional expression of a fusion protein of class 3 PHA synthase; constructed genetically modified nonpathogenic organism capable of synthesizing non-native rhamnolipids; and completed the cloning of phaG (hydroxyacyl-ACP transacylase) gene of Pseudomonas chlororaphis and lipAB (lipase and its modulator protein) genes of P. resinovorans.

1. Low cost tailor-made biodegradable plastics. Biodegradable plastics must possess the desired property for intended applications and be cost competitive in order to be commercially successful. ARS researchers at Wyndmoor, PA, developed a fermentation protocol to utilize the surplus industrial byproducts glycerol (a co-product of the biodiesel industry) and levulinic acid (a byproduct of the pulp and paper industry) either alone or in combination to produce bacterial polyesters (polyhydroxyalkanoates, PHA) with varying monomer ratios. This allowed the formation of polymer films whose properties (e.g., tensile strength, elongation-to-break, modulus, toughness, breakability, malleability, and flexibility) are controlled at the fermentation level simply by varying the ratio of glycerol to levulinic acid in the media. The impact is that a wider range of practical consumer products can be made from “environmentally friendly” plastics at reduced production costs due to the inexpensive starting feedstocks. The impact to biodiesel and the pulp and paper industries is that this accomplishment imparts additional value to both glycerol and levulinic acid as new outlets become available.

2. New building blocks for oil-based polymers. While fats and oils have long been used as components for industrial materials, the shapes that these molecules have in their natural state can impose limitations on the properties of the final materials. ARS researchers at Wyndmoor, PA, have found chemical methods to alter the shapes and reactivities of agriculture-derived fats and oils so that these substances will have greater value as building blocks for advanced polymers, coatings, lubricants, and nanomaterials. One beneficial aspect is that the modified fats and oils can be produced from low-value agricultural byproducts such as soy molasses. The newly prepared compounds could be used to impart special new properties to materials, such as by making stronger/tougher plastics or UV-resistant ones or thin films that stick to metal surfaces. The critical chemical intermediate required for such polymers is available from ARS scientists for use by partners. The expected impact is that agricultural producers may experience enhanced demand or broader market options because their products are needed to make these building blocks.

Review Publications
Ashby, R.D., Zerkowski, J.A., Solaiman, D., Liu, L.S. 2011. Biopolymer scaffolds for use in delivering antimicrobial Sophorolipids to the acne-causing bacterium propionibacterium acnes. New Biotechnology. 28(1):24-30.

Ashby, R.D., Solaiman, D., Strahan, G.D. 2011. Efficient utilization of crude glycerol as fermentation substrate in the synthesis of poly(3-hydroxybutyrate) biopolymers. Journal of the American Oil Chemists' Society. 88:949-959.

Solaiman, D., Garcia, R.A., Ashby, R.D., Piazza, G.J., Steinbuchel, A. 2011. Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer. New Biotechnology. 28(6):552-558.

Zerkowski, J.A., Nunez, A., Solaiman, D. 2011. Selectively functionalized glycerol/diacid dendrimers via click chemistry of azido fatty acids. Journal of the American Oil Chemists' Society. 88(3):403-413.

Last Modified: 4/21/2014
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