developing*an*integrated*biorefinery:* potentials ... ·...
TRANSCRIPT
DEVELOPING AN INTEGRATED BIOREFINERY: POTENTIALS OF SWITCHGRASS EXTRACTIVES
Nicole Labbé, Jingming Tao, Lindsey Kline, Alexander Bruce, Bonnie H. Ownley, Kimberly D. Gwinn, Doris D’Souza, Naima Moustaid-‐Moussa, Hasan Jameel, and Carlos Ernesto Aizpurua Gonzalez
University of Tennessee, Texas Tech University and North Carolina State
University
Sun Grant Regional Conference Feb. 2-4, 2015
Auburn, AL
Team
• Multi disciplinary
• Multi Institution
Dr. Hasan JameelChemical Engineer
NCSU
Dr. Kimberly GwinnPlant Pathologist
UT
Dr. Bonnie OwnleyPlant Pathologist
UT
Dr. Doris D’SouzaFood Microbiologist
UT
Dr. Naima Moustaid-MoussaNutritional Biochemist
TTU
Dr. Niki LabbéBiomass Chemist
UT
Project Goal
The main goal of this project: demonstrate that switchgrass extractives can act as bioactive agents against plant and foodborne pathogens, as well as modulate immune and inflammatory responses in humans.
Task 1: Investigate compounds in switchgrass extractives with bioactive properties.
Task 2: Confirm efficiency of extracts against plant pathogens and foodborne bacterial pathogens.
Task 3: Evaluate anti-inflammatory properties of extracts in fat cells.
Task 4: Model techno-economics of the proposed extraction in a bioconversion system.
Cellulose30-40%
Hemicellulose24-30%
Lignin 18-22%
Ash2-6%
Extractives6-20%
Chemical Composition of Switchgrass
Terpenes and Terpenoids
Fatty Acids
Carbohydrates
Phenolics
IntroducCon
IntroducCon: ExtracCves
• Non-‐cell wall components
• Small molecules < C40
• Vary by varie;es, age, loca;on, harvest ;me
• Biomass color, fragrance, durability
• Toxic to fungi, bacteria, and termites
• Inhibitor for saccharifica;on/fermenta;on in bioenergy produc;on
Extractives in Switchgrass
0.0
5.0
10.0
15.0
20.0
25.0
30.0
136-140 176-182 220-234 259-264 301-311 324-348
V2-V3 E3-E5 R0-R3 R2-S1 R5-S4 S5
Ext
ract
ives
(%)
Alamo EG1101 EG1102
11 TN farms, Second growing season
ExtracCves content in switchgrass during growing season
Extractives in Switchgrass
250,000 gallons of ethanol/year3,000 tons switchgrass/year300 tons of extractives/year
Switchgrass free sugars
Pre-‐processed Switchgrass
Crude Extracts in Ethanol
Biomass ExtracCon Ethanol/Water
Organosolv Process MIBK/ethanol/water/acid
IsolaCon of ConsCtuents & Solvents Recovery MIBK/ethanol/water/acid
Cellulose Solid
Hemi-‐cellulose Aq. phase
Lignin Org. phase
EnzymaCc SaccharificaCon Fiber Processing
Carbon Fibers
Mono-‐sugars
Solvent
Extracts FracConaCon & Solvent recovery
FracCon 1
FracCon 2
FracCon n
FracCon Analysis and TesCng
IsolaCon of BioacCve Components
BioacCve Components
Solvent
Process design and analysis
Integrated process
Ethanol
cv NeutralizaCon
SaccharificaCon / FermentaCon
Lignin/Residues
Dilute Acid Pretreatment
DisCllaCon
Chemical ComposiCon Wt%
Cellulose 36.4
Hemicellulose 28.9
Lignin 20.9
Total Ash 1.9
ExtracCves 9.1
Switchgrass chemical composiCon (as received)
Alamo, Harvest Cme: March 2012
10
Switchgrass ExtracCon
Chopped, 1-‐2 inches, fresh switchgrass, recycled solvent 3 Cmes
ExtracCves
ExtracCves-‐free biomass
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Flow-‐through Reactor
Ethanol extracts Water extracts
ExtracCon Cme (min) 60*3
ExtracCon temperature (°C) 100
ExtracCon solvent Ethanol (190 proof , 95%) H2O
Switchgrass wt (g) 532*3
Final volume of crude extracts (L) 3.68 3.83
ExtracCves concentraCon* (mg/mL) 6.45 5.93
ExtracCves removal
*Concentrations of extracts in each fraction determined with 100 mL subsample rotovap and dried at 40˚C
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-‐ Following methods in Qawasmeh et. al (2012) "Influence of fungal endophyte infec;on on phenolic content and an;oxidant ac;vity in grasses: Interac;on between Lolium perenne and different strains of Neotyphodium lollii" J. Agric. Food Chem
-‐ Mazza et al. (1999) “Anthocyamins, phenolics, and color of Cabernet Franc, Merlot, and Pinot Noir Wines from Bri;sh Columbia” J. Agric. Food Chem -‐ Extrac;ves filtered by 0.45 µm filter and diluted (1:5) with ethanol -‐ 50 µL diluted extracts added to 50 µL 95% ethanol containing 0.1% HCl, and 910 µL of 2% HCl -‐ Total phenols measured at 280 nm with gallic acid as standard -‐ Hydroxycinnamic acid deriva;ves measured at 320 nm with caffeic acid as standard -‐ Flavonols measured at 360 nm with querceCn as standard
CharacterisCcs of extracCves
Ethanol extracts
Water extracts
Total Phenols (at 280 nm, mg/mL) 2.51 (0.05) 1.52 (0.02)
Hydroxycinnamic acid deriva;ves
(at 320 nm, mg/mL) 0.49 (0.01) 0.29 (0.00)
Flavonols (at 360 nm, mg/mL) 0.22 (0.01) 0.20 (0.00)
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CharacterisCcs of extracCves Free sugars
-‐ Crude extracts rotary evaporated at 40˚C and dried in oven -‐ Extracts in water (10 mg/mL) boiled at 100˚C for 10 minutes, filtered with 0.45µm nylon syringe filter -‐ Analyzed by HPLC with Bio-‐rad HPX-‐87P carbohydrate column and guard at 85˚C, 20µL injec;on volume, water as
eluent, 0.25mL/min, RI detec;on at 50˚C, 60 min run ;me
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0 10 20 30 40 50 60
Respon
se (u
RIU)
Time (min)
3x EtOH
3x H2O
Sucrose
Glucose
Ribo
se
Rham
nose
Fructose
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Py-‐GC/MS of dried crude extracCves
Water extracts
Ethanol extracts
Water extracts & ethanol extracts
Furfural4-Vinyl Guaiacol
2,3-dihydro-Benzofuran,
Guaiacol
Syringol
4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenolLevoglucosanAcetic Acid
Vanillin
• Foodborne Pathogens: An;-‐bacterial
• Plant Pathogens: An;-‐bacterial, An;-‐fungal and an;oxidant
• An;-‐inflammatory
ExtracCves applicaCons
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• Foodborne pathogen infecCons affect ~48 million people/year in the U.S. (CDC, 2014).
• A growing increase in the number of mulCdrug resistant bacterial related foodborne outbreaks including Salmonella outbreaks. • Salmonella are gram-‐nega;ve bacteria associated with gastroenteri;s in humans.
• Symptoms last for 4 to 7 days aler an incuba;on period of 12 to 72 hours. • Salmonella enterica serovars Typhimurium (ST) and Enteri;dis (SE) are the most common serovars associated with foodborne human infec;ons.
Foodborne Pathogens
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Persons infected with the outbreak strain of Salmonella Typhimurium by State on October 28th, 2013 (CDC, 2014)
Salmonella outbreaks
18 Increased interest in determining the suitability of natural anCbacterial compounds as alternaCve therapeuCc and prevenCve opCons.
• Bacterial plant pathogens can cause total crop loss, par;cularly in warm, humid environments.
• Infec;on can cause severe damage on foliage and fruit. Foliar lesions lead to defolia;on and sunscald of fruit. Lesions on fruit further reduce their quality and marketability.
• Current management strategies are focused on applica;on of preventa;ve foliar sprays of copper compounds, which are approved for organic produc;on, but are marginally effec;ve.
Bacterial spot on tomato leaves. Photo -‐ J. Mixon.
Bacterial spot on fruit. Photo – Ritchie, D.F. 2000. The Plant Health Instructor. DOI: 10.1094/PHI-‐I-‐2000-‐1027-‐01. Updated 2007.
Bacterial Plant Pathogens
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• Excessive reliance on copper creates selec;on pressure for copper resistance, with worldwide reports of copper-‐resistant bacterial pathogens.
• Movement of infected seeds and transplants has facilitated distribu;on of copper-‐resistant bacterial strains.
Damage caused by bacterial spot. Photo -‐ Mohammad Babadoost, hqp://agronomyday.cropsci.illinois.edu/2009/tours/b4diagnos/
• Alterna;ve control measures, such as the natural an;microbial compounds found in switchgrass extrac;ves, are needed to protect against foliar bacterial pathogens and reduce the use of heavy metal sprays.
Bacterial Plant Pathogens
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ü Swithgrass extrac;ves ü Extrac;ves against foodborne and plant pathogens ü An;-‐inflammatory
• Future work – Saccharifica;on/Fermenta;on of extracted switchgrass – Techno-‐economic analysis
Summary
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Grant No. 2010-‐38502-‐21854
Agriculture and Food Research Ini;a;ve Grant No. 013-‐67021-‐21158
THANK YOU!
Acknowledgement
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