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United States Department of Agriculture

Agricultural Research Service

Research Project: ENHANCED UTILIZATION OF CARBOHYDRATES AND POLYSACCHARIDES FROM CITRUS PROCESSING WASTE STREAMS

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2008 Annual Report


1a.Objectives (from AD-416)
Characterize the structure and functional properties of the polysaccharides present in the citrus processing waste stream. Develop new and use existing procedures including chemical, physical or enzymatic processes to modify and produce new polysaccharide materials, value added polymers, or resins with unique functional properties. Develop new industrial uses for new products produced either isolated or while still contained in the bulk waste stream residue for use as ion-exchange, building and construction materials, paper products, and other non-food related materials. Develop economically viable method for enzymatic and chemically catalyzed depolymerization of polysaccharides in citrus waste streams to monomeric sugars with subsequent fermentation of hydrolysates to value added products such as ethanol, citric and other organic acids, and similar products.


1b.Approach (from AD-416)
Reactions initiated by acid, base, enzymes, and/or thermal treatments will be performed to modify polysaccharides in bulk citrus processing waste or minimally separated citrus waste stream components. Pectinmethyesterase enzymes from citrus will be characterized for their response to temperature, pH, salt, and modifications made to pectin substrates. The effect of modifications on chemical changes such as degree of esterification, fragmentation size and polymerization will be determined along with how these modifications improve water holding capacity, increase ion-exchange capacity, decrease or increase viscosity and other rheological properties. Polysaccharides in citrus residue products and pectin extracts will also be further modified using nucleophilic reagents such as amines and sulfhydryl compounds to produce substituted polymers with new functionality. New grinding, separation and/or filtration process technologies will also be evaluated for economic and environmental advantages for production of more economically attractive carbohydrate and polysaccharide products from citrus waste streams. Products with desirable properties will be produced on a pilot scale and supplied to industrial partners for testing in specific applications. Modified polysaccharides will be tested in applications such as dry strength additives for paper and building materials, metal chelation and ion exchange applications such as industrial wastewater treatment, and water binding capacity for superabsorbant applications. Commercial enzyme systems for efficient hydrolysis of polysaccharides from citrus waste will be evaluated to optimize enzyme loading, temperature and pH of reactions, ability to recycle enzymes, and degree of mixing necessary in pilot scale-up from lab scale experiments. Efficient separation of hydrolysed citrus waste will then be investigated using centrifugation, rotary and flat bed filters to allow fermentation to produce fuel ethanol and organic acids.


3.Progress Report
Structure–functional analysis of block deesterified pectins was performed on enzyme modified pectin to optimize for several different applications such as chelation and suspension aids. The current challenge has been to release the pectin from the peel in a cost effective manner prior to enzyme or chemical modification. High yields of pectin were observed with microwave heating as compared to resistance heating and by adjusting pH values. The data suggests that by careful selection of pH and temperature, acceptable yields of pectin can be obtained while preserving a significant portion of the molecular properties needed for applications. Initial results indicate acceptable yields can be obtained under relatively mild pH conditions with short reaction times which will aid in developing a cost effective process. Sufficient quantities of a thermally tolerant pectin methylesterase (PME) were purified and used for the structural modification of a model pectin. Characterization of the enzyme-mediated changes to structure has been initiated. Large volumes of fruit tissue extracts have been prepared for purification of remaining PMEs by a newly prepared affinity chromatography matrix. The pretreatment process for preparation of citrus waste to remove limonene for fermentation to produce ethanol was optimized to reduce steam requirements. The stripper system designed to resist fouling, test and optimize removal of ethanol from viscous fermented products was received, set up and preliminary operation testing done for experiments in the coming processing season.

This work relates to National Program 306 Quality and Utilization of Agricultural Products, Component 2 New Processes, New Uses, and Value-Added Foods and Biobased Products, Problem Area 2a New Product Technology.


4.Accomplishments
1. Traditional extraction of pectin from citrus peel is expensive using extended extraction times along with the requirement for high concentrations of strong acids. Our goal was to examine conditions to allow more rapid extraction of pectin from citrus tissue with less acid required. High yields of pectin were observed with microwave heating as compared to resistance heating and by adjusting pH and temperature values. The data indicates that by careful selection of pH and temperature, acceptable yields of pectin can be obtained with a reduced processing time while preserving a significant portion of the properties needed for the selected applications. The new extraction procedure can now be tested in combined enzyme-chemical modification processes to further reduce cost so pectin can be utilized in more cost sensitive applications. This is a major step toward developing a cost effective product-process based on maximum utilization of citrus peel with an emphasis on “green technology”.

This work relates to National Program 306 Quality and Utilization of Agricultural Products, Component 2 New Processes, New Uses, and Value-Added Foods and Biobased Products, Problem Area 2a New Product Technology.

2. A naturally occurring yeast Kluyveromyces marxianus was evaluated for ability to ferment citrus waste into ethanol. This organism can tolerate higher temperatures than Saccharomyces cerevisiae, the traditional yeast used in fermentation and would be better suited for simultaneous fermentation and hydrolysis of the complex carbohydrates in citrus processing waste if capable of producing ethanol in good yield at elevated temperatures which kill S. cerevisiae. Fermentations at an elevated temperature is desirable because the enzymes used in the process perform better at higher temperatures (40-50 ºC or 104-122 ºF) and ability to use higher temperatures would mean shorter processing times. We found K. marxianus could produce ethanol in good yield at 40 ºC and at a lower yield (<70% of theoretical) at 42-45 ºC. S. cerevisiae does not tolerate temperature above 38 ºC. These results indicate K. marxianus might be useful in reducing the process time requirements for production of ethanol from citrus waste.

This work relates to National Program 306 Quality and Utilization of Agricultural Products, Component 2 New Processes, New Uses, and Value-Added Foods and Biobased Products, Problem Area 2a New Product Technology.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of New CRADAS1
Number of New Patent Applications Filed1
Number of Non-Peer Reviewed Presentations and Proceedings8
Number of Newspaper Articles and Other Presentations for Non-Science Audiences1

Review Publications
Luzio, G.A., Cameron, R.G. 2008. Demethylation of a model homogalacturonan with the salt-independent pectin methylesterase from citrus: Part II. Structure-function analysis. Carbohydrate Polymers. 71:300-309.

Cameron, R.G., Luzio, G.A., Goodner, K.L., Williams, M.A. 2008. Demethylation of a model homogalacturonan with a citrus salt-independent pectin methylesterase: Effect of pH on block size and number, enzyme mode of action and resulting functionality. Gums and Stablisers for the Food Industry. 14:141-152.

Luzio, G.A., Cameron, R.G. 2007. Rheological investigation of pectin deesterified using salt-independent pectin methylesterase from citrus. Proceedings of Florida State Horticultural Society. 120:304-309.

Zhou, W., Widmer, W., Grohmann, K. 2007. Economic analysis of ethanol production from citrus peel waste. Proceedings of Florida State Horticultural Society. 120:310-315.

Wilkins, M.R., Widmer, W.W., Grohmann, K. 2007. Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol. Process Biochemistry. 42(12):1614-1619.

Last Modified: 4/18/2014
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