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Utilization of Waste in Industrial (White) Biotechnology

Utilization of Waste in Industrial (White) Biotechnology. White Biotechnology . is an emerging field within modern biotechnology that serves industry.

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Utilization of Waste in Industrial (White) Biotechnology

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  1. Utilization of Waste in Industrial (White) Biotechnology

  2. White Biotechnology • is an emerging field within modern biotechnology that serves industry. • It uses living cells like moulds, yeasts or bacteria, as well as enzymes to produce goods and services. Living cells can be used as they are or improved to work as "cell factories" to produce enzymes for industry. • White Biotech can help realize substantial gains for both environment, consumers and industry.

  3. Red Health Care Green Agro-Food Health Unmet Needs White Industrial Sustainability Economy Biotechnology Using nature’s toolset

  4. Industrial (White) Biotechnology Cell factories Biofuels Biomaterials Biochemicals Sugars

  5. The IB Value Chain Bulk Biofuels H2 Ethanol Sugars • Feedstocks • Renewable • - Fossil Biomaterials Polylactic acid 1,3 propane diol PHAs Biochemicals Food Ingredients Pharmaceuticals Fine Chemicals Bioprocesses Fine

  6. Industrial Biotechnology Cleaner and more (cost) efficient ways of making: Faded jeans Detergents Plastics Vitamins Antibiotics Fuel Biosteel Biobatteries DNA computers Reduced environmental foot-print up to 20 – 60 % Added Value of 11-22 billion € per Year Present Future

  7. IB: three P’s go hand in hand People sustainability Planet Profit

  8. The IB Value Chain Bulk Biofuels H2 Ethanol Sugars • Feedstocks • Renewable • - Fossil Biomaterials Polylactic acid 1,3 propane diol PHAs Biochemicals Food Ingredients Pharmaceuticals Fine Chemicals Bioprocesses • Strong points Europe • Enzymes • Biochemicals Fine

  9. Bioenergy Biomass B & B The IB Value Chain Bulk Biofuels H2 Ethanol Sugars • Feedstocks • Renewable • - Fossil Biomaterials Polylactic acid 1,3 propane diol PHAs Bioprocesses Biochemicals Food Ingredients Pharmaceuticals Fine Chemicals Fine

  10. Developing a Strategic Research Agenda and Roadmap (1) Main R&D objectives Strain, biocatalyst & process optimization Novel and/or improved functionalities and products

  11. Developing a Strategic Research Agenda and Roadmap (2) • Research & Technology areas in IB • Novel enzymes and microorganisms – metagenomics • Microbial genomics and bioinformatics • Metabolic engineering and modeling • Performance proteins and nanocomposite materials • Biocatalyst function and optimization • Biocatalytic process design • Innovative fermentation science • Innovative down-stream processing • Integrated biorefineries

  12. Novel biotechnological processes for production of polymers, chemicals, and biofuels from waste

  13. Background • Ecological reasons to promote „White Biotechnology“: • Global Warming, Green house effect • „Rio Declaration on Environment and Development“June 1992: Broad consensus to switch to alternative, sustainable Technologies • Principle 4: • „In order to achieve sustainable development, environmental protection shall constitute an integral part of the development process and cannot be considered in isolation from it.“ • Rising Prices for mineral oil: Economic necessity to promote technologies independent from the availability of fossil feedstocks • Major Drawback for „White Biotechnologie“: Costs for Raw Materials • Solution Strategy: Utilization of Waste Materials for Production of Biopolymers, Biochemicals and Biofuels 13

  14. Need for „White Biotechnology“ for Production of Biopolymers, Biofuels and Biochemicals? 140 130 June 2008: Price surmounted 130 US-$ per barrel July 2008: Price surmounted 140 US-$ per barrel 14 14

  15. „Target Areas“: Final Products from Conversion of Waste Materials via „White Biotechnology“ Industrial producers of Waste streams: Final Products: Major substrates for production of biopolymers, biofuels and biochemicals: Monosaccharides: Glucose, Galactose, Fructose, Xylose, Arabinose Dairy Industry: Whey Biopolymers (PHA, PLA) Sugar cane industry: Molasses, Bagasse Biofuels (Bioethanol, Biodiesel) Disaccharides: Sucrose, Lactose, Maltose, Cellobiose Wood processing industry, Paper Industry Polysaccharides: Starch, Cellulose, Lignocellulose Biochemicals (Fine chemicals, Organic acids, Antibiotics, Aromatics, Surfactants, Solvents, Chiral Synthons) Additional agricultural branches (e.g. straw from rice, mais etc., olive oil production, palm oil industry, sugar beet industry) Organic acids Alkohols: Glycerol, Methanol Biodiesel production: raw glycerol phase, low-quality biodiesel fractions Lipids Catalytically active Biomassfor Production of Biopolymers, Biofuels and Biochemicals Slaughterhouses and Rendering Industry: Meat- and Bone Meal, slaughter wastes Proteinaceous materials (Peptides)

  16. Brazil: Integration of Biofuel & Biopolymer Production into Sugar Cane Industry: Actual and Potential Utilization of the waste streams Sugar Cane Fibers potential filler for PHA-based materials?! 561.600 t/a Combustion Bagasse 2,160.000 t/a Bioethanol 52.575 m3/a Steam and electrical power Milling 32,4 GW/h / a 395.000 t steam/ a Hydrolysis Higher Alcohols (Butanol, Pentanols) Extraction Convertible Sugars(Glucose, Xylose, Arabinose) Destillation Raw Juice Extraction solvents! Biofuel Production Fermentative Conversion to Bioethanol 180.000 t/a Downstream Processing:Extraction of PHA from biomass Crystallization Saccharose 1.) Production of catalytically active Biomass PHA Biopolymer Production Selection of production strain! Hydrolysis to Glucose and Fructose 2.) Production of PHA biopolyesters 30.000 t/a Molasses Hydrolysis to peptides and amino acids PHA Biopolymers Residual Biomass 10.000 t/a

  17. PHB INDUSTRIAL S/A – Sao Paulo, Brazil Production of PHB homopolyester and Poly-3-HB-co-3HV copolyesters from sugar cane saccharose; autarkic energy supply! Basic research: TU Graz, Austria View of the PHB Pilot Plant for 50 t/a Production strain: Cupriavidus necator DSM 545 (formerly Wautersia eutropha) Intented industrial scale production of PHA: 10.000 t/a

  18. Whey from Dairy Industry – a versatile Feedstock for Biotechnology • Application of Whey lactose (D-gluco-pyranose-4-ß-D-galactopyranoside) from dairy industry: animal feed, sweets, food processing, baby food, laxatives, pharmaceutical matrices • But: annually 13,500.000 t of Surplus Whey in Europe (620.000 t lactose)! • Ecological problem; polluting whey partly disposed in sea • 2001: EU – project WHEYPOL(G5RD-CT-2001-00591): application of surplus whey from Italian dairy industry as substrate for PHA biopolyester production

  19. From Milk to Whey towards PHA Biopolyesters MILK 2.) Production of PHA biopolyesters 1.) Production of catalytically active Biomass Pasteurization Yield PHA/C-source = 0,33 g/g: ca. 200 000 t/a PHA in EU from surplus whey possible!! Transformation (enzymatic or acidic) Curd cheese Full Fat Whey Skimming (ca. 13 500 000 t/a in EU surplus!) Skimmed Whey Pasterization Storage Concentration Lactose Hydrolysis to Glucose and Galactose ?! (depends on production strain) WHEY CONCENTRATE (ca. 2 700 000 t/a in EU surplus!) Ultrafiltration Whey Retentate Whey Permeate Desalting ?! (necessity depends on production strain) Storage α-Lactoglobulin (2 wt.-%), ß-Lactoglobuline (9 wt.-%); Lactose (15 wt.-%) 20 – 21 wt.-% Lactose (81% of the entire lactose from milk) (ca. 620 000 t/a in EU from surplus whey!)

  20. Different Routes from Whey Lactose to Biopolyesters (Koller et al., 2007) Whey Lactose Direct Application of Lactose (sufficient ß-Galactosidase activity of production strain) for production of PHA Hydrolysis towards Glucose and Galactose for Production of PHA Bioconversion 1: via Lactobacilli from Lactose to Lactic Acid Bioconversion 2: from Lactate to PHA Polylactic acid(PLA) (www.igb.frauenhofer.de/WWW/GF/dt/GFDP 21 Molke.dt.html) Conversion to Lactic acid esters→Green Solvents Pyrolysis Lactones Unsaturated compounds (Crotonic acid, 2-Pentenoic acid etc.) Synthons for chemical synthesis

  21. Alternative Biotechnological Products from Whey Lactose • Bioethanol: Golden Cheese Company, California (19.000 m³ Bioethanol/year) (For Europe: Surplus whey would yield 290.000 m³ Bioethanol/year) (www.ethanolfra.org/pr010201.html) • Antibiotics: e.g. Bacteriocin Nisin (polycyclic peptide antibiotic from Lactococcus lactis) against highly pathogenic food-spoiling bacteria Listeria monocytogenes and Clostridium botulinum (Hickmann, Flores, Monte Alegre, 2001) • Sophorolipids: Emulsifiers and Surfactants for pharmaceutical, cosmetic and food industry; chemically: sophorose derivates linked to hydroxy fatty acids • Two –step process: Yeast Cryptococcus curvatus cultivated on whey permeate, accumulates single-cell-oil (SCO) from whey lactose. SCO is converted in a second step to sophorolipids by Candida bombicola (Daniel et al., 1999)) www.lipidlibrary.co.uk/Lipids/rhamno/image006.gif www.profoodinternational.com

  22. The Increasing Amounts of Biodiesel • Legislative Situation by the European Commission: Shares of Biofuels [%]: • 2005: 2% • 2010: 5,75% • possibly up to 20% until 2020 (8 * 1010 Liter/a in Europe) • 2005: Production of 1,925.000 t in Europe (= 192.500 t glycerol) • 2008: 2,649.000 t in Europe (= 264.900 t glycerol) • Austria: 2006 Production of 121.665 t Biodiesel; 2007: 241.381 t (+98%!!!)

  23. Glycerol Liquid Phase: Waste from the Biofuel Production for the Production of Biopolymers WASTE LIPIDS e.g. Waste Cooking Oils, waste animal fats Some lipids: direct application as feedstock! 1.) Production of catalytically active Biomass MeOH (EtOH) OH- 2.) Production of PHA biopolyesters Yield PHA/C-source = 0,33 g/g: ca. 88 000 t/a PHA in EU from surplus GLP possible!! Transesterification Biotechnological Production of PHA Biopolyesters Mixture Biodiesel -Glycerolphase Downstream Processing Demethanolization Separation PHA Biopolyesters Residual Biomass (Proteins, Lipids) Degreasing Washing, Dewatering GLYCEROL LIQUID PHASE (GLP) typically 2-4 wt.-% of biomass Low-quality biodiesel fractions: excellent feedstock for PHA production! BIODIESEL (RME)

  24. Occurence of lignocellulosic waste: wood residues (including sawmill and paper mill discards) municipal paper waste agricultural residues (including corn stover, rice straw and sugarcane bagasse) special energy crops Amounts:non-wood lignocellulosic straw alone is estimated with 2,5*109 t/a Composition of Lignocellulose: Lignocellulosic Feedstocks Carbohydrates Lignin (Methoxyphenylpropane) + Cellulose fraction Hemicellulose fraction Monomer: Glucose (Hexose) Monomers: Xylose, Arabinose (Pentoses)

  25. Biotechnological Utilization of Lignocellulose • Obstacle: Lignocellulose has evolved to resist degradation and to confer hydrolyticstability and structural robustness to the plant cell walls by crosslinking between the carbohydrates and the lignin via ester and ether linkages • Focus of research: UPSTREAM TECHNOLOGY: Enhanced lignocellulose digestion and the development of EFFECTIVE ENZYMES for the degradation of cellulose and hemicellulose into glucose and pentoses are the prerequisite for an efficient production of the desired bio-products

  26. Composition of Different Lignocellulosic Materials

  27. Conversion of Lignocellulose to Value-added Bioproducts Development of efficient hydrolysis methods required!! Plant Biomass Hemicellulose Hydrolysis (enzymatic or chemical) High energy input needed! Alternatives have to be developed! e.g.: Solid State Fermentation! Steam Explosion Pentoses (Xylose, Arabinose) Fermentation to Bioethanol Extraction with water Lignin Alkaline extraction Biotechnological Production of Biopolyesters Adhesives Energy Hydrolysis (enzymatic or chemical) Glucose Cellulose Petschacher Barbara, Diploma Thesis, Graz University of Technology, 2001

  28. Follow-up Products of PHAs: Chiral Synthons for Organic Synthesis Chiral synthons: Stereoregular compounds acting as chiral precursors, e.g. production of pharmaceuticals, pheromons, vitamins, antibiotics, aromatics, perfumes PHAs: Biobased Polyesters consisting mainly of optically pure monomers Chiral center Hydrolysis leads to a rich source of bifunctionel, R(-)-configurated hydroxy acids. Market value higher than for the polymer itself! In-vivo degradation of PHA by adjusting the enzymatic systems involved in intracellular PHA metabolism via the cultivation conditions (C-source, pH, T); excretion of metabolites Highly efficient process! App. 130 PHA buliding blocks reported- broad range of possible chiral synthons Classical Hydrolysis: In-vivo degradation Isolation of PHA (Seebach et al., 1992; Seebach and Züger, 1982) PHA Optically pure monomers acidic alcoholysis of the isolated PHA Process rather complex and highly Solvent-demanding! (Lee et al., 1999)

  29. Meat- and Bone Meal (MBM) from Slaughterhause Waste & Rendering Industry – a Precious Nitrogen Source for Biotechnological Purposes • Classical Utilization of MBM: Animal Feed • Problem: The emerge of Bovine Spongiform Encephalopathy (BSE, „Mad Cow Desease“) • Peak: Infection of 3500 head of caddle weekly in Great Britain • Alternative Utilization: Energy production by Combustion → low value-creation • 2001: Task Force Graz University of Technology for Safe Utilization of MBM to produce value-added products!

  30. Structure of a Prion Causing BSE Hydrolysis of Meat- and Bone Meal • Precondition of Safe Utilization of MBM: Hydrolysis of MBM to destroy prions Hydrolysis time [h] SDS-Gel-Electrophoresis of alkaline Hydrolysis of MBM (PhD thesis José Neto, Graz University of Technology, 2006)

  31. Production of Meat- and Bone Meal Possible: Removal of Lipids prior to hydrolysis („Degreasing step“) Application of lipids for Biodiesel Production or as carbon source for fermentative Production of e.g. Biopolymers Application of hydrolyzed MBM for Biomass production

  32. Concluding Remarks • A broad range of waste materials from different origins exists to be potentially utilized for biotechnological production of biopolymers, biofuels and biochemicals • Selection of the appropriate waste stream for biotechnological purposes depends on the global region where the production is intended. Facilities for production should be integrated into existing production lines, where the waste streams directly accrue (Prime example: Integration of sugar-, bioethanol and biopolymer production in Brazil) • Improvement of upstream technologies, selection of optimized biocatalysts, enhanced downstream processing and autarkic energy supply are required to achieve cost efficiency in the production of biopolymers, biofuels and biochemicals from waste.

  33. Content : • Limitation and rising prices of fossil feedstocks and the increasing need for „White Biotechnology“: Ecological and Economic needs • „Target Areas“: Final products from conversion of waste materials via „White Biotechnology“ • What waste materials are available for biotechnological purposes (occurence and the challenges of their utilization) • Meat and Bone Meal (Slaughterhouses and Rendering industry) • Sugar Cane industry – Integration of Biofuel and Biopolymer Production • Whey (Dairy Industry) • Raw Glycerol Liquid Phase (from Biodiesel Production) • Waste Lipids • Cellulosic and Lignocellulosic Feedstocks • Follow-up Products of PHAs: Chiral Synthons for Organic Synthesis • Summary

  34. THANK YOU

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