Plastics Produced by
Bacteria— Degradable
Polyhydroxyalkanoates
Project,
The development of an industrial
fermentation process for the
production of polyhydroxyalkanoates
Biotechnology and Polymers
? Most Chemically Synthesized Polymers are not
Biodegradable,such as PE,PP,PS and PET
? While most Biologically Synthesized Polymers
are Biodegradable,such as Proteins,Cellulose,
Glycan
? The most Notable Biopolymers with Properties
Similar to Thermoplastics are:
Polyhydroxyalkanoates or PHA
Characteristics of Bioreactions
“Green Chemistry”
? Gentle conditions (30-40oC,1 atm pressure，
aqueous medium)
? High specificity
? Optically pure product
? Cheap substrates（ not toxic,not flammable）
What Biopolymers can be
Microbiologically Synthesized?
？
Biologically Synthesized
Natural Polymers
? DNA,RNA
? Proteins
? Polysaccharides
? Polyhydroxy-
alkanoate-PHA
Genetic Information
Enzymes or Structure
Materials
Structure materials or
Carbon and Energy
reserve materials
Carbon and Energy
Sources and more?
? Mostly hydrophobic
? Chiral monomers
? Great structure variety
? Among PHA,
polyhydroxybutyrate is
the most common
? Molecular weights range
from 10,000 to several
millions
Polyhydroxyalkanoates (PHA)
Applications of PHA
? Biodegradability
? Biocompatibility
? Hydrophobicity
? Gas impermeability
? Piezoelectrocity
? Optical activity
? Special Properties caused by functional
Groups
Biomaterials and
Tissue engineering
Biodegradable Packaging
Electronic Materials?
Smart materials?
PHA Common Structure
PHA molecular structure
—[CH—(CH2)m—C—O]n——
R
O
m=1,2 or 3,R is variable
Commercialized PHA
—[CH—CH2—C—O]n—
O
CH3
R=Methyl,PHA=Polyhydroxy-
butyrate(PHB)
—[CH—CH2—C—O]n—
C2H5
O
R=Ethyl,PHA=Polyhydroxy-
valerate (PHV)
—[CH—CH2—C—O]a——[CH—CH2—C—O]b—
O O
CH3 C2H5
First Generation of PHA
Second Generation of PHA:
PHBV
Third Generation of PHA
—[CH—CH2—C—O]a——[CH—CH2—C—O]b—
O O
CH3 C
3H5
Copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate,
Copolymers of 3-hydroxybutyrate and 3-hydroxyoctanoate
Have been synthesized by Doi,Steinbuchel and P&G.
Comparison of PHA and Plastics
Material Site-Chain C Tg(oC) Tm (oC) Tensile Elongation to
Number Strength(MPa) Break (%)
PHB 1 5 180 40 3
PHBV(10%) 2 150 25 20
PHBV(20%) 2 135 20 100
PHBHHx(10%) 3 -1 127 21 400
PHBHHx(15%) 3 115 23 760
PHBHHx(17%) 3 -2 120 20 850
PP 170 35 400
PET 262 56 7300
HDPE 135 29 -
PS 110 50 -
Novel PHA Monomer Structures
—[CH—CH2—C—O]n—
O
CH2—CH2—CH—CH—(CH2)4—CH3
O
O
CH2—CH—CH—CH—CH2—(CH2)4—CH3
O
—[CH—CH2—C—O]n— CH2—CH2—CH—O—
—[CH—CH2—C—O]n—
O
CN
Novel PHA Monomer Structures
O
—[CH—CH2—C—O]n—
(CH2)2—CF3
—[CH—CH2—C—O]n—
O
(CH2)2—CF2—CF3
—[CH—CH2—C—O]n—
O
(CH2)2—C CH
O O O
O O O
O O O
O
O
O
O
PHA Research in China
Industrialization First Attempt
? From 1986-1990,Chinese 7th Five-Year
Construction Period,Microbiologists begin to
study the possibility for mass production of PHB
for replacement of plastics.
? Alcaligenes,Azotobacter,Bacillus etc,were used
as PHB producers.
? The attempt failed due to high cost of making the
biodegradable PHB.
PHA Research in China
Industrialization Second Attempt
? Plastic pollution,“White Pollution”
becomes an increasingly serious problem,
no alternative solution has been found
? Thus PHA industrialization is again listed
as a key research project in the ninth five-
year construction period (1996-2000) after a
forgotten eighth five-year period (1990-
1995)
PHA Research in China
? A multi-disciplinar team consisting of
microbiologists,polymer engineers,
material scientists,chemical engineers was
assembled at Tsinghua University to work
on production strain screening,fermentation
process development and scale up,PHB and
PHA property improvement
PHA Research in China
? Previous works with Alcaligenes,
Azotobacter,recombinant E,coli etc,have
to be abandoned as these are patented and
WTO entrance prohibits the use of these
patented technology in China.
? A new broad screening process has to be
carried out to identify potential industrial
strains for PHB production.
Project PHB Phase 1 Strain selections:
Samples collection from islands,mountains,forests and oil plants
By 吴琼、洪葵、田卫东、林会兰、樊凌等
A rapid,non-invasive PHA detection method,the
FT-IR method was developed
By 洪葵、田卫东、孙素琴
4 0 00, 03 0 002 0 001 5 001 0 004 0 0,0
c m - 1
% T
1 7 2 4
1 2 7 8
1 7 2 3
1 2 7 8
1 7 2 4
1 5 0 51 3 8 0
1 2 7 8
1 1 3 3
1 0 5 8
979
1 7 2 4
1 5 0 51 3 8 0
1 2 7 8
1 0 5 7
979
2 9 8 4
1 7 2 5
1 5 0 41 3 8 3
1 1 7 6
966
2 9 8 4
1 5 0 45
965
PHA Accumulation is a common
phenomenon among many bacteria
by 吴琼、樊凌、林会兰等
0
50
100
150
200
250
300
350
400
BP Ba2 O2 La2 G5 M30
number of strains grown
number of strains producing PHA
Percentage of strains producing PHA
BP,Complex medium,Ba,Butyric acid,O,Octanoate,
La,Lauric acid,G,Glucose,M,Molasses
Project PHB Phase 2,Lab Process Devlopemnt
A Bacillus Strain with Industrial Potential
by 赵锴、李蔓青,吴琼、黄鸿华
Fermentor Results,30 g/l of cell dry weight
with 78% of PHB accumulation after 8 hrs of
growth in a 5 L fermentor
Shake Flask Results,11g/l of cell dry weight
containing 70% of PHB after 48 hrs growth
in molasses only medium
Project PHB Phase 3,Pilot Process Devlopemnt
PHB Pilot Production Process
（ 4 cubic meter fermentor) by 赵锴、李蔓青、吴琼、黄鸿华等
Petri
Disk
Shake
Flask
4 m3
Fermentor
Centrifuge dryer PHB
Extraction
PHB
dryer
PHB powderOrder has been received from Germany
Project PHB Phase 3,Pilot Process Devlopemnt
PHB Pilot Production Process （ 4 cubic meter fermentor)
by 赵锴、李蔓青等、江门生物技术开发中心
? Molasses was treated to remove sediments
? Fermentation was carried out in a 4 m3 vessel
Project PHB Phase 3,Pilot Process Devlopemnt
PHB Pilot Fermentation Process （ 4 cubic meter fermentor)
by 赵锴、李蔓青、陈金春等、江门生物技术开发中心
? 800 L Seed fermentor
? 4000 L Pilot fermentor
Project PHB Phase 3,
Pilot Process Devlopemnt
PHB Pilot Production Process
（ Separation process)
by 赵锴、李蔓青、陈金春
等、江门生物技术开发中心
? The bacterial cells will
be harvested by
continuous
centrifugation
(Westfalia Disc-
Centrifuge,40 tons/h)
? The concentrated cells
will be transferred to a
container for PHB
extraction
Project PHB Phase 3,
Pilot Process Devlopment
PHB Pilot Production Process
（ PHB extraction process)
by 赵锴、李蔓青、陈金春等、
江门生物技术开发中心
? Concentrated cells were
lysed using SDS at 50oC
and pH 10-11
? The PHB granules were
centrifuged and wished.
? The washed PHB
granules suspensions were
treated with protease and
Na-hyperchloride to
increase PHB purity.
Project PHB Phase 3,
Pilot Process Devlopment
（ PHB extraction process)
by 赵锴、李蔓青、陈金春等、江
门生物技术开发中心
? The PHB granules were
be further purified with
protease and Na-NaOCl
to whiten the product.
Project PHB Phase 3,
Pilot Process Devlopment
（ PHB drying process)
by 赵锴、李蔓青、陈金春等、江门
生物技术开发中心
? Spread dry the PHB granules
suspension to obtain the PHB
in final powder form,
Dried cell mass Dried PHB
Project PHA
Production of Copolymers of 3-hydroxybutyrate
and 3-hydroxyhexanoate (PHBHHx)
by 洪葵、田卫东、张广、姚俊、林会兰、陈金春等、
江门生物技术开发中心
Project PHA phase 1,Strain selection
An unidentified strain QH was isolated from
oil-contaminated soil,Strain QH was found to
synthesize a copolymer consisting of
3-hydroxybutyrate and 3-hydroxyhexanoate
Project PHA phase 2,Lab process development
Pilot Production of PHA,PHBHHx
polyacrylamide
Rolling Press FilterStrain QH
A flocculation process was used to replace the centrifugation process,
The rest of the process development is similar to PHB project
PHB Genetic Engineering
* phbA phbB phbC
Plasmid
PHB genes
Insert PHB genes into a vector
E,coli cell
Transform a vector into E,coli
PHA Produced by Genetic Engineering
PHB (C4)
PHBV (C4—C5）
PHBHHx（ C4—C6）
PHOD（ C8-C10）
PHB genes have been expressed in Plant
Future Plastic
Acknowledgement
? China Biotechnology Center
? Procter & Gamble Co,USA
? Jiangmen Center for Biotech Development
? Dagang Oil Field,Tianjin,China
? Shengli Oil Field,Shangdong,China
? Daqing Oil Field,Helongjiang,China
? Tsinghua University Analysis Center
谢谢大家！
*
Bacteria— Degradable
Polyhydroxyalkanoates
Project,
The development of an industrial
fermentation process for the
production of polyhydroxyalkanoates
Biotechnology and Polymers
? Most Chemically Synthesized Polymers are not
Biodegradable,such as PE,PP,PS and PET
? While most Biologically Synthesized Polymers
are Biodegradable,such as Proteins,Cellulose,
Glycan
? The most Notable Biopolymers with Properties
Similar to Thermoplastics are:
Polyhydroxyalkanoates or PHA
Characteristics of Bioreactions
“Green Chemistry”
? Gentle conditions (30-40oC,1 atm pressure，
aqueous medium)
? High specificity
? Optically pure product
? Cheap substrates（ not toxic,not flammable）
What Biopolymers can be
Microbiologically Synthesized?
？
Biologically Synthesized
Natural Polymers
? DNA,RNA
? Proteins
? Polysaccharides
? Polyhydroxy-
alkanoate-PHA
Genetic Information
Enzymes or Structure
Materials
Structure materials or
Carbon and Energy
reserve materials
Carbon and Energy
Sources and more?
? Mostly hydrophobic
? Chiral monomers
? Great structure variety
? Among PHA,
polyhydroxybutyrate is
the most common
? Molecular weights range
from 10,000 to several
millions
Polyhydroxyalkanoates (PHA)
Applications of PHA
? Biodegradability
? Biocompatibility
? Hydrophobicity
? Gas impermeability
? Piezoelectrocity
? Optical activity
? Special Properties caused by functional
Groups
Biomaterials and
Tissue engineering
Biodegradable Packaging
Electronic Materials?
Smart materials?
PHA Common Structure
PHA molecular structure
—[CH—(CH2)m—C—O]n——
R
O
m=1,2 or 3,R is variable
Commercialized PHA
—[CH—CH2—C—O]n—
O
CH3
R=Methyl,PHA=Polyhydroxy-
butyrate(PHB)
—[CH—CH2—C—O]n—
C2H5
O
R=Ethyl,PHA=Polyhydroxy-
valerate (PHV)
—[CH—CH2—C—O]a——[CH—CH2—C—O]b—
O O
CH3 C2H5
First Generation of PHA
Second Generation of PHA:
PHBV
Third Generation of PHA
—[CH—CH2—C—O]a——[CH—CH2—C—O]b—
O O
CH3 C
3H5
Copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate,
Copolymers of 3-hydroxybutyrate and 3-hydroxyoctanoate
Have been synthesized by Doi,Steinbuchel and P&G.
Comparison of PHA and Plastics
Material Site-Chain C Tg(oC) Tm (oC) Tensile Elongation to
Number Strength(MPa) Break (%)
PHB 1 5 180 40 3
PHBV(10%) 2 150 25 20
PHBV(20%) 2 135 20 100
PHBHHx(10%) 3 -1 127 21 400
PHBHHx(15%) 3 115 23 760
PHBHHx(17%) 3 -2 120 20 850
PP 170 35 400
PET 262 56 7300
HDPE 135 29 -
PS 110 50 -
Novel PHA Monomer Structures
—[CH—CH2—C—O]n—
O
CH2—CH2—CH—CH—(CH2)4—CH3
O
O
CH2—CH—CH—CH—CH2—(CH2)4—CH3
O
—[CH—CH2—C—O]n— CH2—CH2—CH—O—
—[CH—CH2—C—O]n—
O
CN
Novel PHA Monomer Structures
O
—[CH—CH2—C—O]n—
(CH2)2—CF3
—[CH—CH2—C—O]n—
O
(CH2)2—CF2—CF3
—[CH—CH2—C—O]n—
O
(CH2)2—C CH
O O O
O O O
O O O
O
O
O
O
PHA Research in China
Industrialization First Attempt
? From 1986-1990,Chinese 7th Five-Year
Construction Period,Microbiologists begin to
study the possibility for mass production of PHB
for replacement of plastics.
? Alcaligenes,Azotobacter,Bacillus etc,were used
as PHB producers.
? The attempt failed due to high cost of making the
biodegradable PHB.
PHA Research in China
Industrialization Second Attempt
? Plastic pollution,“White Pollution”
becomes an increasingly serious problem,
no alternative solution has been found
? Thus PHA industrialization is again listed
as a key research project in the ninth five-
year construction period (1996-2000) after a
forgotten eighth five-year period (1990-
1995)
PHA Research in China
? A multi-disciplinar team consisting of
microbiologists,polymer engineers,
material scientists,chemical engineers was
assembled at Tsinghua University to work
on production strain screening,fermentation
process development and scale up,PHB and
PHA property improvement
PHA Research in China
? Previous works with Alcaligenes,
Azotobacter,recombinant E,coli etc,have
to be abandoned as these are patented and
WTO entrance prohibits the use of these
patented technology in China.
? A new broad screening process has to be
carried out to identify potential industrial
strains for PHB production.
Project PHB Phase 1 Strain selections:
Samples collection from islands,mountains,forests and oil plants
By 吴琼、洪葵、田卫东、林会兰、樊凌等
A rapid,non-invasive PHA detection method,the
FT-IR method was developed
By 洪葵、田卫东、孙素琴
4 0 00, 03 0 002 0 001 5 001 0 004 0 0,0
c m - 1
% T
1 7 2 4
1 2 7 8
1 7 2 3
1 2 7 8
1 7 2 4
1 5 0 51 3 8 0
1 2 7 8
1 1 3 3
1 0 5 8
979
1 7 2 4
1 5 0 51 3 8 0
1 2 7 8
1 0 5 7
979
2 9 8 4
1 7 2 5
1 5 0 41 3 8 3
1 1 7 6
966
2 9 8 4
1 5 0 45
965
PHA Accumulation is a common
phenomenon among many bacteria
by 吴琼、樊凌、林会兰等
0
50
100
150
200
250
300
350
400
BP Ba2 O2 La2 G5 M30
number of strains grown
number of strains producing PHA
Percentage of strains producing PHA
BP,Complex medium,Ba,Butyric acid,O,Octanoate,
La,Lauric acid,G,Glucose,M,Molasses
Project PHB Phase 2,Lab Process Devlopemnt
A Bacillus Strain with Industrial Potential
by 赵锴、李蔓青,吴琼、黄鸿华
Fermentor Results,30 g/l of cell dry weight
with 78% of PHB accumulation after 8 hrs of
growth in a 5 L fermentor
Shake Flask Results,11g/l of cell dry weight
containing 70% of PHB after 48 hrs growth
in molasses only medium
Project PHB Phase 3,Pilot Process Devlopemnt
PHB Pilot Production Process
（ 4 cubic meter fermentor) by 赵锴、李蔓青、吴琼、黄鸿华等
Petri
Disk
Shake
Flask
4 m3
Fermentor
Centrifuge dryer PHB
Extraction
PHB
dryer
PHB powderOrder has been received from Germany
Project PHB Phase 3,Pilot Process Devlopemnt
PHB Pilot Production Process （ 4 cubic meter fermentor)
by 赵锴、李蔓青等、江门生物技术开发中心
? Molasses was treated to remove sediments
? Fermentation was carried out in a 4 m3 vessel
Project PHB Phase 3,Pilot Process Devlopemnt
PHB Pilot Fermentation Process （ 4 cubic meter fermentor)
by 赵锴、李蔓青、陈金春等、江门生物技术开发中心
? 800 L Seed fermentor
? 4000 L Pilot fermentor
Project PHB Phase 3,
Pilot Process Devlopemnt
PHB Pilot Production Process
（ Separation process)
by 赵锴、李蔓青、陈金春
等、江门生物技术开发中心
? The bacterial cells will
be harvested by
continuous
centrifugation
(Westfalia Disc-
Centrifuge,40 tons/h)
? The concentrated cells
will be transferred to a
container for PHB
extraction
Project PHB Phase 3,
Pilot Process Devlopment
PHB Pilot Production Process
（ PHB extraction process)
by 赵锴、李蔓青、陈金春等、
江门生物技术开发中心
? Concentrated cells were
lysed using SDS at 50oC
and pH 10-11
? The PHB granules were
centrifuged and wished.
? The washed PHB
granules suspensions were
treated with protease and
Na-hyperchloride to
increase PHB purity.
Project PHB Phase 3,
Pilot Process Devlopment
（ PHB extraction process)
by 赵锴、李蔓青、陈金春等、江
门生物技术开发中心
? The PHB granules were
be further purified with
protease and Na-NaOCl
to whiten the product.
Project PHB Phase 3,
Pilot Process Devlopment
（ PHB drying process)
by 赵锴、李蔓青、陈金春等、江门
生物技术开发中心
? Spread dry the PHB granules
suspension to obtain the PHB
in final powder form,
Dried cell mass Dried PHB
Project PHA
Production of Copolymers of 3-hydroxybutyrate
and 3-hydroxyhexanoate (PHBHHx)
by 洪葵、田卫东、张广、姚俊、林会兰、陈金春等、
江门生物技术开发中心
Project PHA phase 1,Strain selection
An unidentified strain QH was isolated from
oil-contaminated soil,Strain QH was found to
synthesize a copolymer consisting of
3-hydroxybutyrate and 3-hydroxyhexanoate
Project PHA phase 2,Lab process development
Pilot Production of PHA,PHBHHx
polyacrylamide
Rolling Press FilterStrain QH
A flocculation process was used to replace the centrifugation process,
The rest of the process development is similar to PHB project
PHB Genetic Engineering
* phbA phbB phbC
Plasmid
PHB genes
Insert PHB genes into a vector
E,coli cell
Transform a vector into E,coli
PHA Produced by Genetic Engineering
PHB (C4)
PHBV (C4—C5）
PHBHHx（ C4—C6）
PHOD（ C8-C10）
PHB genes have been expressed in Plant
Future Plastic
Acknowledgement
? China Biotechnology Center
? Procter & Gamble Co,USA
? Jiangmen Center for Biotech Development
? Dagang Oil Field,Tianjin,China
? Shengli Oil Field,Shangdong,China
? Daqing Oil Field,Helongjiang,China
? Tsinghua University Analysis Center
谢谢大家！
*
