Polyhydroxyalkanoates:

Biodegradable Polymers & Plastics from

Renewable Resources

M. KOLLER, A. SALERNO, A. MUHR, R. ESSL, H. ANGERER, A. REITERER,

E. RECHBERGER, K. MALLI, G. BRAUNEGG

Graz University of Technology, Austria

Institute of Biotechnology and Biochemical Engineering
martin.koller@tugraz.at

The 20th Jubilee Conference on Materials and Technology

October 17th to 19th, 2012, Portorož, Slovenia

1
 Content of the Presentation
 The „Plastic Situation“ Today
 PHA Biopolyesters - a Sustainable Solution
 Potential Applications of PHAs
 Challenges in PHA Production: Downstream
Processing and Process Design
 Available Feedstocks for PHA Production
 Our Case Studies:
 WHEYPOL Project: Surplus Whey as Feedstock
 ANIMPOL Project: Animal Waste Lipids as Feedstock
 Conclusions and Outlook
2
 Nowadays, We Literally Live in the „Plastic Age“…

250 million 330 million

100 million
tons tons
tons

1,5 million tons

60 years 20 years
2010 2015
ago ago

Figure: Rising amounts of plastics produced globally (Koller et al., 2011)

3
 Quantities of Consumed Plastic Materials in
Different Global Regions
80-120 kg / a 250 Mtons / a
Developed
World production & consumption
Countries of
(average pro capite) Plastic Materials

2-15 kg / a
Developed and
Emerging and
industrialized countries Developing
Emerging and developing Countries
countries
(average pro capite)
TODAYS SITUATION: Polymers Predominately Deriving
from Petro-Industry

1. Highly resistant polymeric materials

2. No natural degradation (landfil crisis!)
3. Insufficient performance of recycling
systems
4. High risk connected to the thermal
conversion of plastics by incineration
(generation of toxines)
5. CO2 generation! Green house gases!
Global warming!

5
 It is Time to Switch.....
1. Fluctuation of petrol price is the
major factor of uncertainty for global
industry.

2. Advanced methods for tracing and

discharging of crude oil exist, but
finally fossil resouces are limited!

3. Since the year 2011: Instability of the

political situation in many oil-
exporting countries!! (Libya, Bahrain
etc.). Future situation in Iran or Saudi
Arabia??
6
 PHAs: a Sustainable Solution!
Polyhydroxyalkanoates (PHAs) are biopolymers produced by a
broad range of prokaryotes from renewable resources.
They are the only family of „bioplastics“ entirely produced AND
degraded by living cells!

The industrial implementation of PHAs

has two major impacts:

•in replacing petrol based plastics

(and reducing problems caused by
them!)

•in solving industrial waste problems

7
 PHAs: a Sustainable Solution!

PHAs can be selected as a sustainable solution for polymer industry:

1. Biobased, Biocompostible and

Biodegradable („green plastics“)

2. Produced by living microorganisms

3. PHAs and their follow-up products

can be processed to create a broad
range of marketable products for a
variety of applications

8
 PHAs: a Sustainable Solution!

PHAs can be selected as a sustainable solution for polymer industry:

1. Biobased, Biocompostible and

Biodegradable („green plastics“)

2. Produced by living microorganisms

3. PHAs and their follow-up products

can be processed to create a broad
range of marketable products for a
variety of applications

9
 Occurence and Composition of
Polyhydroxyalkanoates (PHAs)
• Reserve Compounds for Carbon
and Energy; intracellularily
produced by numerous
procaryotic micro-organisms

• Production under condituions of

Carbon surplus together with a
Limitation of an essential
Growth component (e.g.: N,
PO43-, O2; Mg, S, K)

• Biologically produced and

biologically degradable
Electron microscopic picture of Cupriavidus necator DSM 545;
(kompostierbare) BIOPLASTICS biopolymer content 60 to 70 wt.-%
Picture provided by Dr. E. Ingolić, ZFE-FELMI Graz
Koller et al., Macromolecular Bioscience 7, 218-226, 2007
Inclusions (PHA Granula)

11
 The Cyclic Nature of Polyhydroxyalkanoate Metabolism
Starting from Agricultural Waste Materials

Polysaccharides, Waste Lipids

Oligosaccharides
Change of nutrient conditions to growth (no limitations!)

Convertible carbon Glycerol Biodiesel Poly-(3-Hydroxybutyrate)

Source: e.g.: Glucose
Depolymerase
Catabolism via e.g. PHA
ß-Oxydation 3-Hydroxybutyrate Synthase
2-Keto-3-Desoxy-6-
C4 - Unit
Phosphogluconate 3HB-Dehydrogenase
Pathway (KDPG)
CO2 Acetoacetate
Pyruvate C4 - Unit 3-Hydroxybutyryl-CoA
C3 - Unit Thiaphorase C4 - Unit
Acetoacetyl-CoA
Limitation Reductase
of growth
component
Acetyl-CoA
plus C2 - Unit 3-Ketothiolase Acetoacetyl-CoA
surplus of C4 - Unit
carbon

„Ideal“ growth conditions:

PHA –
no limitations! Accumulation!!
Growth of Biomass!! 12
 “White Biotechnology”
Upstream
processing
(hydrolysis)
Raw materials Accessible
C-source
fermentation
process

Microorganisms as
„cell factories“
(Archea, Bacteria, Fungi)
Bacillus Xanthomonas Haloferax mediterranei
megaterium campestris Downsstream
(PHA-biopolymer and processing
(PHA-biopolymer (Polysaccharide Polysacharide
production) production) (separation
production)
&
purification)

Bioproducts
13
 Chemical Structure of PHAs
• Building blocks mainly chiral (important exception: 4-
Hydroxybutyrate); chiral building blocks are enantiomerically
pure [always (R) – Konfiguration] !
• Most important representative: Poly-3-Hydroxybutyrate (PHB);
Homopolyester of 3-Hydroxybutyrate → highly crystalline, brittle
material, high melting point hempers most processing steps
• Remedy: Via production strategy change of the polyester
composition by incorporation of different building blocks →
Finetuning of product properties; Possibility to insert
functional groups for post-synthetic chemical and/or enzymatic
modification
Chiral
Center
Microbial Production of PHAs
Depending on the microbial production strain, PHAs can be divided in
2 large groups based on the number of carbon atoms in the monomer
units:

• C3-C5: short-chain-lengh PHA (scl)

• C6-C14: medium-chain-lengh PHA (mcl)

General chemical structure of

PHAs. The chiral center is
indicated by an asterisk (*)

PHAs can be also classified as homo- , co-, or terpolyester s depending

on:
• Substrate and/or co-substrate
• Production strain
Significance: Properties similar to Thermoplasts, Elastomeres or Latexes from
petrol-based industry. High dependence of properties on composition and
production strategy
PHAs: Property Dependance on Composition
Khanna and Srivastava, 2005; Williams and Martin, 2002; Koller et al., 2010

scl-PHAs mcl-PHA

16
 PHAs: a Sustainable Solution!

PHAs can be selected as a sustainable solution for polymer industry:

1. Biobased, Biocompostible and

Biodegradable (green plastic)

2. Produced by living microorganisms

3. PHAs and their follow-up products

can be processed to create a broad
range of marketable products for a
variety of applications

17
Potential Applications of PHAs

•„Smart“ latexes and gels; thermosensitive

adhesives; carriers for active compounds
• Biodiesel obtained by transesterification of PHAs
with longer side chains (mcl-PHA) (sewage water)

18
 Application of PHAs
Surgical Applications: Implants
Ongoing project „BioResorbable Implants for Children“ (BRIC)
[Laura Bassi Center of Expertises; Austrian project]:
Development of BioResorbable Implants for Children surgery
(healing of femoral fractures).
 Coordinated by Medical University Graz, Austria; Prof. A.
Weinberg
 Now: Rat animal experiments; soon: lamb model
 Optimization of experimental in-vitro set-ups for degradation
tests for the implants
Literature: Artificial organs, artifical blood vessels, materials for
wound treatment:

19
 Application of PHAs
Surgical Applications: Implants
Ongoing project „BioResorbable Implants for Children“ (BRIC)
[Laura Bassi Center of Expertises; Austrian project]:
Development of BioResorbable Implants for Children surgery
(healing of femoral fractures).
 Coordinated by Medical University Graz, Austria; Prof. A.
Weinberg
 Now: Rat animal experiments; soon: lamb model
 Optimization of experimental in-vitro set-ups for degradation
tests for the implants
Literature: Artificial organs, artifical blood vessels, materials for
wound treatment:

Chen et al., 2005 Sodian et al., 2000 Rokkanen et al., 2000 20

The Entire Process..

Renewable Raw Material. Here: Sugar cane

The Entire Process..

Selection of powerful microbial production strain

 The Entire Process..

Evaluation of optimimum cultivation conditions

The Entire Process..

Bacterial cell harbouring PHA granules

 The Entire Process..
Biosynthesis under controlled and optimized conditions (bioreactor)
The Entire Process..

PHA granulate
Bacterial cell harbouring PHA granules

Downstream processing for PHA- biopolymer recovery

The Entire Process..

PHA granulate
The Entire Process..

Biodegradation (Composting)
 The Entire Process..

Final products of the oxidative catabolism:

carbon dioxyde and water!
Obstacles for the Market Penetration of PHAs
The production costs of PHA must be in the same range as the
„classical“ petrochemical competitors on the plastic market (PP,
PE, PET etc.)
Hence, they have to be minimized despite the instable market
price for crude mineral oil
This can be accomplished by optimizing:
1. The cost of downstream processing for recovery of PHA
from biomass

2. The process design (discontinuous vs. continuous

fermentation mode)

3. The selection of raw materials 30

Obstacles for the Market Penetration of PHAs
The production costs of PHA must be in the same range as the
„classical“ petrochemical competitors on the plastic market (PP,
PE, PET etc.)
Hence, they have to be minimized despite the instable market
price for crude mineral oil
This can be accomplished by optimizing:

The cost of downstream processing for recovery of PHA from

biomass

31
 @ Downstream Processing

• Extraction of PHA from surrounding cell mass

• Often high throughput of (even toxic!) solvents
and high energy requirements!
• Screening of alternative solvents and efficient
extraction strategies needed
• Mechanical cell disruption, chemical or
enzymatic digestion of non-PHA cell mass

32
Cell Harvest, Separation PHA-rich Biomass
from Aqueous
Supernatant, Drying
Removal of Lipids with
Organic Solvents or sCO2
(Degreasing) Chemical or enzymatic digestion of
non-PHA cell mass
or
Mechanical disruption of cells
or
Disruption of cells with high
intracellular osmotic pressure in
Degreased PHA-rich hypotonic media
Biomass
+ Lipids
Extraction with organic Released PHA
solvent (e.g. Chloroform) Granules
(often still
surrounded by
Crude extracted PHA membrane)
(high purity)

+
PHA-free Cells +
Biomass Digestate
(Cell Debris)
Downstream Processing of Biomass for PHA Isolation:
Extraction

Extraction on laboratory scale: e.g. Industrial scale: e.g.

Soxleth Extractor Podbielniak Extractor 34
 Future of PHA Isolation from Biomass
Alternative: Investigation of „Green Solvents“ that are
easy to recycle (e.g. lactic acid esters)

Avoiding of chlorinated solvents like chloroform!!

PHBISA Process Brazil: PHA extraction via the fusel

alcohol fraction (iso-pentanol) from the destillative
Bioethanol production

Taking profit of the high intracellular pressure of

osmophilic production strains (example: Haloferax
mediterranei) → simple release of intracellular PHA-
granula in hypotonic medium (deionized water); low
purity; membranes of granules remain intact! 35
Novel Strategy for PHA Extraction
Developed at TU Graz
Extraction at temperature above
the solvent´s boiling point in a first
vessel. System under Pressure

Solvent: Classial „non-PHA solvent“

at room temperature!

Pressure release by opening of

valve; residual biomass remains in
filter, PHA dissolved in solvent
passes through to vessel 2

Precipitation of polymer in vessel 2

(cooling)

36
Obstacles for the Market Penetration of PHAs
The production costs of PHA must be in the same range as the
„classical“ petrochemical competitors on the plastic market (PP,
PE, PET etc.)
Hence, they have to be minimized despite the instable market
price for crude mineral oil
This can be accomplished by optimizing:

The process design

(discontinuous vs. continuous fermentation mode)

37
@ Process Design: PHA Production at
Graz University of Technology
Batch vs. Continuous Production Mode

38
 5-Stage Continuous Process
• Microbial growth in first vessel („ideal“ cultivation
conditions; autocatalytic process)
• Vessel 2 to 5: PHA-production (Carbon supply;
limitation of essential growth component)
• Vessel 2 to 5 are process engineering substitute for a
tubular reactor! According to PHA production
kinetics (linear increase of PHA concentration)!
• High productivities!
• Constant product quality (Molecular mass,
Thermoanalytical data) during steady state
39
40
Atlić, A. et al. (2011). Continuous Production of Poly([(R]-3-hydroxybutyrate) by Cupriavidus
necator in a Multistage Bioreactor Cascade, Applied Microbiology and Biotechnology 91: 295-304

41
Obstacles for the Market Penetration of PHAs
The production costs of PHA must be in the same range as the
„classical“ petrochemical competitors on the plastic market (PP,
PE, PET etc.)
Hence, they have to be minimized despite the instable market
price for crude mineral oil
This can be accomplished by optimizing:

The selection of raw materials

42
Carbon-Rich Waste Streams Selection

No interference
with food- or feed
applications!!!

43
Our Selected Alternative Carbon Sources:

1. Whey from dairy industry (Lactose): EU-FP5 PROJECT

WHEYPOL (Dec. 2001 to Dec. 2004; coordinated by Graz
University of Technology)

2. Crude glycerol phase from the biodiesel production

(Glycerol) EU-FP5 PROJECT BIODIEPRO (Jan. 2003 to Dec.
2005; coordinated by ARGENT Energy; Graz University of
Technology as partner)

3. Molasses from the sugar industry (Sucrose) (Bilateral

project with Brazilian company PHBISA/Copersugar)

4. Animal Derived Waste Lipids (EU-FP7 PROJECT ANIMPOL):

ongoing since 2010; coordinated by Graz University of
Technology)
44
Our Case Study 1: FP5 Project WHEYPOL

The WHEYPOL project developed a

sustainable and sound process for the conversion of

surplus whey from dairy industry to PHA biopolyesters

in order to create a viable strategy that enables the

production of PHAs in Europe in future

45
 Significance of the WHEYPOL Project
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 13,462.000 t of surplus whey in Europe (620.000 t lactose)!

 Global amounts: up to 1.60*108 t (data for 2008); annual increase about 2%!!

 Ecological problem; polluting whey (high COD and BOD!) partly disposed in rivers or
sea

 2001: EU – project WHEYPOL (G5RD-CT-2001-00591): application of surplus whey from

Italian dairy industry as substrate for PHA biopolyester production; Coordinated by
Graz University of Technology
46
 Composition of Different Types of Whey
(Braunegg et al., 2007)

Compound Sweet Fermented Whey Permeate Whey

[% (w/w)] Whey Whey (Substrate for Retentate
Biotechnology!) (Marketable
Proteins)
Lactose 4.7–4.9 4.5–4.9 23 14
Lactic acid traces 0.5 - -
Proteins 0.75–1.1 0.45 0.75 13
Lipids 0.15–0.2 traces - 3-4
Inorganic ca. 7 6-7 ca. 27 ca. 7
compounds
(minerals like
e.g. calcium) 47
 WHEYPOL: PHA Production from Surplus Whey

Dairy industry waste is a potential

source of biologically-produced
polymers with commercial
applications in packaging.
WHEYPOL developed a cost-effective
method to tap this abundant
and sustainable resource.

http://news.cec.eu.int/comm/research/industrial_technologies/
articles/article_805_en.html

Whey production in Europe: 40,420.800 tons/y

Surplus WHEY: 13,462.000 tons/y

Lactose: 619.250 tons /a 205.000 t PHA/a

48
 The Consortium of WHEYPOL
The research was performed by a consortium from 6 European countries: close cooperation
of 6 academic and 3 industrial partners from 5 countries!
Academic Partners:
Partner Partner Logo Key Researcher Main Roles Country

Graz University of Prof. Gerhart Braunegg, Coordination; Biotechnological production of Austria

Technology Prof. Michael PHA biopolyesters (Institute of Biotechnology
Narodoslawsky, and Biochemical Engineering);
Prof. Rolf Marr Downstream Processing; Life Cycle Assessment,
Cleaner production studies; (Institute of Process
and Particle Engineering)

Università di Prof. Sergio Casella Microbiology, Genetics Italy

Padova
Slovak Academy of Prof. Ivan Chodak Characterization of PHAs and follw-up products Slovakia
Science

Università di Pisa Prof. Emo Chiellini Characterization of PHAs; formulation of PHA- Italy
based composites and blends
Polish Academy of Prof. Marek Kowalczuk Characterization of PHA and derived composites Poland
Science and blends
National Institute Dr. Andrej Kržan Characterization of PHA and derived composites Slovenia
of Chemistry and blends

49
 Industrial Waste-Streams from…

Biotechnological conversion of waste streams from dairy industry

(surplus whey) towards PHA biopolyesters.

Latterie Vicentine Soc.Coop. A R.L.

Large Italian dairy company.
Key representative: Mr. Luigi Sibilin

50
Additional Industrial Partners:
How industry can support and optimize academic research!

BDI - BioEnergy International AG

Large Austrian company specialized in
construction of technical plants (biodiesel). Role
in WHEYPOL: Process design & Engineering
Key representative: Dr. Edgar Ahn

Idroplax srl, Italy.

Representative of Polymer Industry! Interested
in switching to bioplastics.
Role in WHEYPOL: Processing of biopolymers on
large scale
Key representatives: Mr. Luca Landini; Mr. Bayan
Giltsoff 51
 Biotechnological Example:
Fermentation Pattern for PHA Production from Hydrolyzed Whey
Lactose on a Highly Saline Nutrient Medium by the Archaeon
Haloferax mediterranei

14 Limitation of
Glucose Galactose growth
12
3-PHA Protein component
(nitrogen,
10 phosphate):
residual
[g/L]

8
biomass
(expressed as
6
protein)
4 concentration
remains
2 constant,
carbon flux
0 towards PHA
0 50 100 accumulation)
Time [h]

Koller et al., Biomacromolecules 6, 561-565, 2005 52

 Main Results:

Process Parameters Values

Cell Dry Mass 11.0 [g/L]

PHA 5.5 [g/L]

Residual Biomass 5.5 [g/L]

PHA / CDM 49.6 [%]

µ max. 0.11 [1/h]

Volumetric Productivity 0.05 [g/Lh]

Yield PHA / Whey sugars 0.33 [g/g]

Koller et al., Biomacromolecules 6, 561-565, 2005

53
 The WHEYPOL Process:
Economic Assessment
Beneficial Combined Effects
Waste stream (Whey) as
Raw Material
High-value Copolyester
from „simple“ carbon-source
Lactose (no addition of
precursor)
Insterile „septic“ Process
possible; safes energy for
sterilization (extreme
halophilic!)
Product isolation: simple
release of PHA granula in
Choi and Lee Choi and Lee Reddy et al., Haloferax Hydrogenophaga Pseudomonas
1997 1999 1999 mediterranei, pseudoflava, hydrogenovora, deionized water (high
Koller et al. Koller et al. 2007 Koller et al. 2007
2007 intracellular osmotic pressure)
Koller et al., Macromolecular Bioscience 7, 218-226, 2007 54
Example 2: FP7 Project ANIMPOL
„Biotechnological conversion of carbon containing wastes
for eco-efficient production of high added value
products”

Project Start: January 1st, 2010

Entire Project Volume: € 3,7 Mio.; EU contribution: € 2,9 Mio

Coordinated by Graz University of Technology, Austria

55
 FP7 Project ANIMPOL

The Animpol project aims at the

sustainable and value added conversion of waste-lipids from
animal processing industry (waste streams from
slaughterhouses, the animal rendering industry and
waste fractions from conventional biodiesel
production)
in order to create a viable strategy that enables the
production of PHAs in Europe in future.
Bring together waste producers from animal processing
industry and biofuel industry with the polymer industry.
Development of an integrated, sound industrial process!
56
 WASTE LIPIDS
Transesterification

MIX BIODIESEL-GLYCEROL
Separation

BIOFUEL (RME) GLYCEROL LIQUID PHASE (GLP)

MICROBIAL PHA PRODUCTION

(group 1 and group 2 production strains)

Downstream Processing
RECOVERY OF PHA FROM
BIOMASS
Proteins Lipids

Hydrolysis
Purification/Refining RESIDUAL BIOMASS
PHA Waste Fraction

57
Amounts of Waste in EU Relevant for ANIMPOL

ANIMAL WASTE LIPIDS

500.000 t/y

CRUDE GLYCEROL
265.000 BIODIESEL
metric tons/year

SATURATED UNSATURATED
FRACTION FRACTION
CATALLYTICALLY PHA 50.000 t/year
ACTIVE 120.000 t
BIOMASS (0.3 g/g)
(0.4-0.5 g/g) Excellent
PHA 2nd generation Biofuel!
35.000 t
(0.7 g/g)
58
Biodiesel
 The Consortium of ANIMPOL
The research is performed by a consortium from 6 European countries: close cooperation
of 7 academic and 4 industrial partners from 7 countries
Academic Partners:
Partner Partner Key Researcher Main Roles Country
Logo
Graz University of Dr. Martin Koller, Coordination; Biotechnological production of Austria
Technology Prof. Michael PHA biopolyesters (Institute of Biotechnology
Narodoslawsky, and Biochemical Engineering);
Prof. Hans Schnitzer Life Cycle Assessment, Cleaner production
studies; Process engineering (Institute of
Process and Particle Engineering)

Università di Padova Prof. Sergio Casella Microbiology, Genetics Italy

University of Zagreb Prof. Predrag Horvat Mathematical modeling of bioprocesses Croatia

University of Graz Prof. Martin Mittelbach Enhanced transesterification of waset animal Austria
lipids; assessment of composition and quality of
raw materials

Università di Pisa Prof. Emo Chiellini Characterization of PHAs; formulation of PHA- Italy
based composites and blends
Polish Academy of Prof. Marek Kowalczuk Characterization of PHA and derived composites Poland
Science and blends
National Institute of Dr. Andrej Kržan Characterization of PHA and derived composites Slovenia
Chemistry and blends 60
 Industrial Waste-Streams from…
Biotechnological conversion of waste streams from two industrial
branches towards PHA biopolyesters.
U. Reistenhofer GesmbH, Austria.
Slaughtering industry: lipid rich animal residues.
Key representative: Mr. Thomas Reistenhofer

Argent Energy, Great Britain.

Large biodiesel producer from tallow (highly
saturated biodiesel fractions) and waste cooking
oil; delivers saturated biodiesel fraction and crude
glycerol phase.
Key representative: Dr. Mike Scott

61
 Additional Industrial Partners:
How industry can support and optimize academic research!

Argus Umweltbiotechnologie GmbH, Germany.

Scale-up of industrial process from lab scale
(from 1L to industrial scale 70000 L).
Role in ANIMPOL: development of sustainable
Downstream Processing
Key representative: Dr. Horst Niebelschütz

TERMOPLAST srl, Italy.

Representative of Polymer Industry! Interested
in switching to bioplastics.
Key representative: Dr. Maurizio Malossi
62
Advisory Board of Companies Acting as an „Enduser
Group“

• Advisory Board members are no beneficiaries

of the project; they give advice in how to
proceed with the activities
1. Novamont, Italy: biodegradables

2. ChemTex Italia (gruppo Mossi &

Ghissolfi; Italy): biobased products
3. KRKA, Slovenia: large scale
fermentations
4. Eksportera UAB, Lithuania: long
expertise in by-product business
63
The Holistic Nature of Animpol

64
Biotechnological Example: Fermentation Pattern for scl-PHA
Production from Animal-Derived, Saturated Biodiesel. Production
strain Cupriavidus nector.
Additional: 3HV Precursor Valeric Acid
50,00
Nitrogen limitation at t = 65 h
45,00

40,00
concentration [g/L]

35,00

30,00

25,00

20,00 CDM

15,00 PHA

10,00

5,00

0,00
0 10 20 30 40 50 60 70 80 90 100
time [h]

65
Biotechnological Example: Fermentation Pattern for scl-PHA
Production from Animal-Derived, Saturated Biodiesel.
Production strain Cupriavidus necator.
Additional: 3HV Precursor Valeric Acid
70

Nitrogen limitation at t = 65 h
60

50
Addition of 3HV precursor
valeric acid from t = 69 h
[wt.-%]

40

PHA/CDM
30
3HV/PHA

20

10

0
16 19 24 40 44 46 49 64 66 69 72 75 88 90 91
time [h]

66
 Main Results:
Process Parameters Values
Cell Dry Mass 47.2 [g/L]

PHA 30.0 [g/L]

Residual Biomass (non-PHA Biomass) 21.1 [g/L]

PHA / CDM 63.6 [%]

µ max. 0.04 [1/h]

3HV / PHA 4.3 [mol-%]

Volumetric Productivity (Accumulation Phase) 1.08 [g/Lh]

Specific Volumetric Productivity (Accumulation Phase) 0.37 [g/gh]

Yield (Biomass / Biodiesel) 0.6 – 0.7 [g/g]

67
 The Crude Product:
Poly-(3HB-co-4.3%-3HV)

68
 Biodegradable Latexes from Animal-Derived Waste:
Biosynthesis and Characterization of mcl-PHA
Accumulated by Ps. citronellolis
Submitted to be published in Reactive and Functional Polymers

69
 mcl-PHA from Saturated Biodiesel by
Ps. citronellolis : Polymer Composition
• According to GC-FID analysis, the obtained biopolyester
predominantly consists of
• 3-hydroxyoctanoate (C8) and 3-hydroxdecanoate
(C10), and,
• to a minor extent, 3-hydroxydodecanoate (C12), 3-
hydroxynonanoate (C9), 3-hydroxyhexanoate (C6), and
3-hydroxyheptanoate (C7) monomers.
• This was confirmed by 1H-NMR, also evidencing the
occurrence of low quantities of unsaturated and 3-
hydroxyvalerate (C5) building blocks.
70
mcl-PHA from Animal-Derived Biodiesel by Ps. citronellolis:
Concentrations of Building Blocks during the Cultivation
(GC-FID)

Nitrogen limitation

71
mcl-PHA from Animal-Derived Biodiesel by Ps. citronellolis:
Shares of Building Blocks in Polymer during the Cultivation
(GC-FID)

Nitrogen limitation

72
 Main Results:
Process Parameters Values
PHA 3.0 [g/L]

Catalytically Active Biomass (Protein) 11.2 [g/L]

Sum Protein + PHA 14.2 [g/L]

PHA / CDM 20.1 [%]

µ max. 0.08 [1/h]

Volumetric Productivity (Accumulation phase) 0.07 [g/Lh]

Specific Volumetric Productivity (Accumulation Phase) 0.02 [g/gL]

Yield (Biomass / Biodiesel) 0.5 – 0.6 [g/g]

73
 Comparison of Material Properties
scl-PHA mcl-PHA
Production strain Cupriavidus necator Ps. citronellolis
Composition P(3HB-co-4.3%-3HV) Mainly 3HO and 3HD;
to minor extend: 3HDD, 3HN, 3Hx, 3Hp
Tm [°C] 163.0 48.6
δHm [J/g] 78.9 7.1
Xc [%] 54.0 12.3
Tg [°C] -2.8 -46.9
Td [°C] 282 296
Mw [kDa] 318 66
Mn [kDa] 233 35
Pi (Polydispersity) 1.4 1.9
Mw via SEC-MALS [kDa] 18.7 n.d.
Mn via SEC-MALS [kDa] 27.0 n.d.

SEC-MALS for scl-PHA: group Dr. Andrej Kržan, National Institute of Chemistry, Ljubljana, SLO
Measurements scl-PHA: group Prof. Emo Chiellini, University of Pisa, I
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Measurements mcl-PHA: group Prof. Marek Kowalczuk, Polish Academy of Science, PL
Potential Impact of the Projects
1. General Impact:
• solutions for waste problems arising on local
scales that can be applied for all Europe.

2. Transitional Impact:
• creation of ecological and economic benefits by
converting waste into value-added materials

3. Socioeconomic Impact:
• new jobs directly in the industrial branches
and high-qualified scientific jobs in academia.
 PHA-Biopolymer Production can Become
Economically Competitive by:

• Utilization of locally available waste

materials

• Integration of PHA production into existing

production line

• Alternative extraction methods

Especially for WHEYPOL (and soon for ANIMPOL):

Data for designing a pilot plant to be integrated in

large dairies are available!

Willingness of responsible policy-makers from

waste-generating industrial branches and from
polymer industry to break new ground in
sustainable production is needed!!!

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 Is there a Need for „White Biotechnology“ for Production of
Biopolymers, Biofuels and Biochemicals??

Exponential increase

Linear increase

January 2007 to June 2008: Price jumped by more than 100% and surmounted 130 US-$ per barrel
January 2009: Back to less than 40 US-$ per barrel
June 2010: Again 77 US-$ per barrel!!
April 2011: 127 US-$ per barrel!!
October 15th, 2012: 116 US-$ per barrel!! 78
TOMORROW: WHO KNOWS????? Increasing uncertainties in global political situation!
 Acknowledgements
The audience and the organizers of The 20th Jubilee
Conference on Materials and Technology !

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