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Utilization of Waste inUtilization of Waste inIndustrial (White) BiotechnologyIndustrial (White) Biotechnology

White Biotechnology White Biotechnology 2

• 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.

BiotechnologyBiotechnology

GreenGreenAgro-FoodAgro-Food

RedRedHealth CareHealth Care

WhiteIndustrial

HealthHealth Unmet NeedsUnmet Needs

EconomyEconomySustainabilitySustainabilityUsing nature’s toolset

4Industrial (White) Biotechnology Industrial (White) Biotechnology

SugarsBiofuelsBiomaterialsBiochemicals

Cell factories

5The IB Value ChainThe IB Value Chain

BiofuelsH2

Ethanol

SugarsFeedstocks-Renewable- Fossil

BiochemicalsFood IngredientsPharmaceuticalsFine Chemicals

BiomaterialsPolylactic acid

1,3 propane diolPHAs

Bioprocesses

Bulk

Fine

Cleaner and more (cost) efficient ways of making:

Faded jeansDetergentsPlasticsVitaminsAntibioticsFuelBiosteelBiobatteriesDNA computers

Industrial BiotechnologyIndustrial BiotechnologyPr

e se

Pre s

entnt

Fut u

rFu

t ur

ee

Reduced Reduced environmental foot-environmental foot-print up to 20 – 60 %print up to 20 – 60 %

Added Value ofAdded Value of11-22 billion € per 11-22 billion € per

YearYear

IB: three IB: three P’sP’s go hand in go hand in handhand

sustainability

Profit

People

Planet

8The IB Value ChainThe IB Value Chain

BiofuelsH2

Ethanol

SugarsFeedstocks- Renewable- Fossil

BiochemicalsFood IngredientsPharmaceuticalsFine Chemicals

BiomaterialsPolylactic acid

1,3 propane diolPHAs

Bioprocesses

Bulk

Fine

Strong points Europe• Enzymes• Biochemicals

9The IB Value ChainThe IB Value Chain

BiofuelsH2

Ethanol

SugarsFeedstocks-Renewable- Fossil

BiochemicalsFood IngredientsPharmaceuticalsFine Chemicals

BiomaterialsPolylactic acid

1,3 propane diolPHAs

Bioprocesses

Bulk

Fine

Biomass

Bioenergy

B & B

10Developing a Strategic Research Agenda and Roadmap (1)Developing a Strategic Research Agenda and Roadmap (1)

Main R&D objectives

Strain, biocatalyst & process optimization

Novel and/or improved functionalities and products

11Developing a Strategic Research Agenda and Roadmap (2)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

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Novel biotechnological processes for production of polymers,

chemicals, and biofuels from waste

BackgroundBackground 13

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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

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14

140

130

14June 2008: Price surmounted 130 US-$ per barrel

July 2008: Price surmounted 140 US-$ per barrel

Need for Need for „„White BiotechnologyWhite Biotechnology““ for Production of for Production of Biopolymers, Biofuels and Biochemicals? Biopolymers, Biofuels and Biochemicals?

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„Target Areas“: Final Products from Conversion of Waste Materials via „White Biotechnology“

Major substrates for production of biopolymers, biofuels and biochemicals:

Monosaccharides: Glucose, Galactose, Fructose, Xylose,

Arabinose

Disaccharides: Sucrose, Lactose, Maltose, Cellobiose

Polysaccharides: Starch, Cellulose, Lignocellulose

Organic acids

Lipids

Alkohols: Glycerol, Methanol

Industrial producers of Waste streams:

Proteinaceous materials (Peptides)

Biodiesel production: raw glycerol phase, low-quality biodiesel fractions

Dairy Industry: Whey

Wood processing industry,

Paper Industry

Additional agricultural branches (e.g. straw from rice, mais etc., olive oil production, palm oil industry, sugar beet industry)

Slaughterhouses and Rendering Industry: Meat- and Bone Meal, slaughter wastes

Biochemicals(Fine chemicals, Organic acids,

Antibiotics, Aromatics, Surfactants, Solvents,

Chiral Synthons)

Biofuels(Bioethanol, Biodiesel)

Biopolymers(PHA, PLA)

Catalytically active Biomass for Production of Biopolymers, Biofuels

and Biochemicals

Sugar cane industry: Molasses, Bagasse

Final Products:

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Brazil: Integration of Biofuel & Biopolymer Production into Sugar Cane Industry: Actual and Potential Utilization of the waste streams

Sugar Cane

Milling

Extraction

Raw Juice

Crystallization

Molasses

Saccharose

Steam and electrical power

CombustionBagasse

Hydrolysis to Glucose and Fructose

Convertible Sugars (Glucose, Xylose, Arabinose)

PHA Biopolymer Production

Fermentative Conversion to

Bioethanol1.) Production of catalytically active Biomass

2.) Production of PHA biopolyesters

Hydrolysis

Biofuel Production

Destillation

Bioethanol

Higher Alcohols (Butanol, Pentanols)

PHA Biopolymers

Downstream Processing:

Extraction of PHA from biomass

Residual BiomassHydrolysis

to peptides and amino acids

Selection of production strain!

Fibers potential filler for PHA-based materials?!

Extraction solvents!

180.000 t/a

30.000 t/a

10.000 t/a

561.600 t/a

32,4 GW/h / a

395.000 t steam/ a

52.575 m3/a2,160.000 t/a

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PHB INDUSTRIAL S/A – Sao Paulo, Brazil

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

Production of PHB homopolyester and Poly-3-HB-co-3HV copolyesters from sugar cane saccharose; autarkic energy supply!

Basic research: TU Graz, Austria

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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

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From Milk to Whey towards PHA Biopolyesters

Pasteurization

Transformation (enzymatic or acidic)

Skimming

Full Fat Whey

Pasterization

Concentration

WHEY CONCENTRATE

Ultrafiltration

Whey Whey PermeatePermeate

Whey Retentate

Curd cheese

Desalting ?!

(necessity depends on production strain)

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!)

Skimmed Whey (ca. 13 500 000 t/a in EU surplus!)

(ca. 2 700 000 t/a in EU surplus!)

Storage

Storage

MILK

Lactose Hydrolysis to Glucose and Galactose ?!

(depends on production strain)

1.) Production of catalytically active Biomass

2.) Production of PHA biopolyesters

Yield PHA/C-source = 0,33 g/g: ca. 200 000 t/a PHA200 000 t/a PHA in EU from surplus whey possible!!

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Different Routes from Whey Lactose to Biopolyesters (Koller et al., 2007)

Whey Lactose

Hydrolysis towards Glucose and Galactose for Production of PHA

Direct Application of Lactose (sufficient ß-Galactosidase activity of production strain) for production of PHA

Bioconversion 1: via Lactobacilli from Lactose to Lactic Acid

Bioconversion 2: from Lactate to PHAPolylactic acid

(PLA)(www.igb.frauenhofer.de/WWW/GF/dt/GFDP 21 Molke.dt.html)

Conversion to Lactic acid esters → Green Solvents

Pyrolysis

Unsaturated compounds (Crotonic acid, 2-Pentenoic acid etc.) Synthons for chemical synthesis

Lactones

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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

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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%!!!)

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Glycerol Liquid Phase: Waste from the Biofuel Production for the Production of Biopolymers

WASTE LIPIDS

MeOH (EtOH) OH-

Transesterification

Mixture Biodiesel -Glycerolphase

Separation

Washing, Dewatering

BIODIESEL (RME)

GLYCEROL LIQUID PHASE (GLP)

Degreasing

Demethanolization

1.) Production of catalytically active Biomass

2.) Production of PHA biopolyesters

Yield PHA/C-source = 0,33 g/g: ca. 88 000 t/a PHA88 000 t/a PHA in EU from surplus GLP possible!!

Biotechnological Production of PHA Biopolyesters

Downstream Processing

PHA Biopolyesters

Residual Biomass (Proteins, Lipids)

e.g. Waste Cooking Oils, waste animal fats

typically 2-4 wt.-% of biomass

Some lipids: direct application as feedstock!

Low-quality biodiesel fractions: excellent feedstock for PHA

production!

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Lignocellulosic Feedstocks

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:

Carbohydrates Lignin (Methoxyphenylpropane)

Cellulose fraction Hemicellulose fraction

Monomer: Glucose (Hexose) Monomers: Xylose, Arabinose (Pentoses)

+

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Biotechnological Utilization of Lignocellulose Obstacle: Lignocellulose has evolved to resist

degradation and to confer hydrolytic stability 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

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Composition of Different Lignocellulosic Materials

Cellulose [wt.-%]

Hemicellulose [wt.-%]

Lignin[wt.-%]

Corn cobs 42 – 45 33 – 35 10 - 15

Corn stover 35 25 - 38 35

Wheat straw 33 - 47 22 – 30 13 - 19

Hemp straw 44 - 45 19 - 21 20 - 22

Rice straw 39 36 10

Bagasse 40 29 13

Beech wood 46 31 23

Fir wood 43 27 29

Poplar wood 50 31 17

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Conversion of Lignocellulose to Value-added Bioproducts

Plant Biomass

Steam Explosion

Extraction with water

Alkaline extraction

Cellulose

Hydrolysis (enzymatic or chemical)

Glucose

EnergyAdhesives

Lignin

Hemicellulose

Pentoses (Xylose, Arabinose)

High energy input needed!

Alternatives have to be developed!

e.g.: Solid State

Fermentation!

Hydrolysis (enzymatic or chemical)

Biotechnological Production

of Biopolyesters

Development of efficient hydrolysis methods required!!

Fermentation to Bioethanol

Petschacher Barbara, Diploma Thesis, Graz University of Technology, 2001

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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!

Classical Hydrolysis:

Isolation of PHA

PHA

acidic alcoholysis of the isolated PHA

Optically pure monomers

(Seebach et al., 1992; Seebach and Züger, 1982)

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

(Lee et al., 1999)Process rather complex and highly Solvent-demanding!

In-vivo degradation

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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!

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Hydrolysis of Meat- and Bone Meal

Precondition of Safe Utilization of MBM: Hydrolysis of MBM to destroy prions

Structure of a Prion

Causing BSE SDS-Gel-Electrophoresis of alkaline Hydrolysis of MBMSDS-Gel-Electrophoresis of alkaline Hydrolysis of MBM

(PhD thesis José Neto, Graz University of Technology, 2006)(PhD thesis José Neto, Graz University of Technology, 2006)

Hydrolysis time [h]

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Production of Meat- and Bone Meal

Application of hydrolyzed MBM for Biomass production

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

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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.

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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

THANK YOU

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