bio polyesters

8/6/2019 Bio Polyesters

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Leonardo da Vinci

Human ingenuity may makeHuman ingenuity may make

various inventions, but it will nevervarious inventions, but it will neverdevise any inventions more beautiful,devise any inventions more beautiful,nor more simple, nor more to thenor more simple, nor more to the

purpose than Nature does; because inpurpose than Nature does; because inher inventions nothing is wanting andher inventions nothing is wanting andnothing is superfluous.nothing is superfluous.

Jim Robbins, Second Nature, Smithsonian, July 2002, Vol. 33, No. 4, p. 78-84


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Bio-polyesters Why (Bio) synthesis?

History

1. Interest

b. Biodegradability

a. Applications

Process

a. Nutrientsb. Biochemistry

c. Extractionb. Bio ethics

Future

a. Directions

Overview


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DegradationBiodegradation Environmental

Intracellular Extracellular Thermal Hydrolytic

Depolymeraseenzymes w/ dimer

hydrolase

Excreted DPEs Conditions 0-3380C ester linkages

R--hydroxybutyricacid

acetylacetate

assimilationProduct (Iso)crotonic

acid, dimer,trimer

monomerichydroxy

acid

Griffin G. J. L.


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Factors for degradability? A ? A? A? AEOHCOOHk

tCOOH 2$

xx

Non

enzymatic

COOH groups auto catalyze

Second stage weight loss- porous- bulk ( surface in enzymatic)

Crystallinity reduces reactivity

Scission catalyzed by acids, bases(amines)

Hydroph

obicity reduces th

e catalysis rateHydroph

obicity reduces th

e catalysis rateBlocking end groups(COOH) temporary reduction in rate

Blending ( variable effects) PHBV/CABBlending ( variable effects) PHBV/CABBlending ( variable effects) PHBV/CABEnzymatic

(vivo) Low Crystallinity, low Tg enhances rate, alkylsubstituents retard


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Economics

Bacterial cost Synthetic

PolyestersPHBV$3-4/ lb projected from1994 ($8-10/lb)

Polypropylene$0.30-0.45/lb

1. Substrate ~ 10%2. Separation ~ (50 70)%


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Synthetic strategies(1)O

O

O

O O

O

Sn

OH

OH

O

O

O

O

CH3

CH3

O

O

O

O

CaprolactoneE

Stannous Octoate

Ethylene Glycol

(Catalyst)

(initiator)

D, L - lactide glycolide

rom : Taylor A. E.


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Synthetic strategies(2)

O

O

O

O

O

OH

O

O

O

O

O n

OO

O

O n

+

CaprolactoneSuccinic anhydride

1-methylimidazole

1, 2 dichloroethane

65-70 oC

Carboxylic acid termination

N2, Stannous Octate

65 oC, 3h

115 oC, 15h

Polymer

Glycidyl termination

Storey , R. F., Hickey, T. P. J. Poly Sci.,

Part A 1993, 31, 1825


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Synthetic strategies(3)

C H 2 O

C H2

O

C H2

O

C H 2 O

C H2

O

O

H

O

H

O

C H3

C H2

C

H

P C LO

O

P C LO

O

P C LO

O

P C L OO

P C L OO

P C L OO

P C L O OP C LOO

C

C

C

T r i m e th

y l p r o p a n e i n i t i a t e d P o l y - E - C a p r o l a c t o n e

X y l i t o l - i n i t i a t e d P o l y - E - C a p r o l a c t o n e


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Initiators

% conversion over time

0

20

40

60

80

100120

8 14 2032

.541

.5 57 84 105

129

187

Time (h)

%conversion

% Conversion (AEG-

Init.) (NMR)

% Conversion (AEG-

Init.) (TGA)

% Conversion (Water-

Init.) (NMR)

% Conversion (Water-

Init.) (TGA)

% Conversion (EG-

Init.) (NMR)

% Conversion (EG-

Init.) (TGA)


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Poly hydroxy alkanoates

PHAs

Propionate (H)

Butyrate (CH3)

Valerate (CH2CH3)

Caproate (C3H7)

Heptanoate (C4 H9)

Octanoate (C5 H11)

MWMWMW (50-1,000) k Da Repeat unitRepeat unitRepeat unit


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Co-polyester properties -PP

P(3HB) P(3HB-3HV) P(3HB-4HB) P(3HO-3HH) PP

Content 20% 16% 11%

Tm (C) 177 145 150 61 176

Tg (C) 2 -1 -7 -36 -10

Crystallinity (%) 70 56 45 30 60

Extension to break (%) 5 50 444 300 400

Polypropylene properties compared with some

Co-polyester content indicated is % with P(3HB).Madison LL, Huisman GW. From Doi, Y. Microbial Polyesters; VCH: New York, 1990.


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PHBV properties

Tensile Strength

020

40

60

0 3 9 14 20 25

%HV

Tensile Strength(M

)

Impact Strength

0

200

400

600

0 3 9 14 20 25

%HV

Notched Izod im pact

strength ( m)

ransition emperatures

10

0

10

20

0 3 9 14 20 25

%HV

Glass Transition (oC)

Griffin G . J.L.

Melting Point (oC )

050

100

150

200

0 3 9 14 20 25

%HV

Meltingpoint

Melting Point (oC )


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ISODIMORPHISM

Melting Point

7

7

7

%HV

M P

P M P

Gr ff G J L


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Crystallinity

Yi Wang, Yasuhide Inagawa, Terumi Saito,Ken-ichi Kasuya,Yoshiharu Doi, and Yoshio Inoue., Enzymatic Hydrolysis of Bacterial Poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by Poly(3-hydroxyalkanoate) Depolymerase fromAcidovoraxSp. TP4, Biomacromolecules, 3 (4), 828-834, 2002

P(3HB-co-3HP), Bacterial P(3HB) & Syn P(3HP)


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Biodegradability

Synthetic polymers w/ester linkages

Hydrolysis wouldinitiate degradation

Structure of

prepolymer couldpropagate

Easy to process

Biosynthetic polymers

Compatible chirality

PHB content in most

living organisms

High reproducibility Quick yield

Renewable


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P(3HB-3HV) in aerobic (20C)

sewage sludge 0, 2, 4, 6, 8, and 10 weeks

Photograph courtesyof Dieter Jendrossek,Georg-August-Universitt, Gttingen,

Germany.

Lara L. Madison and Gjalt W. Huisman

Degradation in soil

0

20

40

60

80

1 2 3 5 7 10 13 17 18Time in wee ks

%

Carbonlost

% labelled

carbon

lost


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Applications

Chiral blocks for syn, chromatography

Various biodegradability products

Toner (paper recyclables)

Insecticide packaging

Drug release matrix in vet MEDICINE

Piezoelectric properties in temp dep

PHBV grades allow var phy properties

Synthetic polymers w/ ester linkages


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History

Maurice Lemoigne (Institut Pasteur) .2525

PHB first MENTIONEDPHB first MENTIONED

Baptist,Werber(W. R. Grace) 60s60sLb of PHB produced for evalLb of PHB produced for eval

Imperial Chemical Industries, Ltd. 77--80s80s

Pruteen developed Ae 70% biomassPruteen developed Ae 70% biomass Biopol, Metabolix Inc. and Monsanto90s90s

Bioidegradable bottle & Wella shampooBioidegradable bottle & Wella shampoo


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Philip Ball, Consulting Editor, Nature

There is no assemblyThere is no assembly

plant so delicate,plant so delicate,versatile and adaptive asversatile and adaptive as

the cell.the cell.



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General enzymatic pathway forPHB and

PHBHx synthesis

Madison LL, Huisman GW.

fatty acid degradation

Athree-step pathway. The three

enzymes are encoded by the genesofthe phbCAB operon. A promoter

upstream ofphbCtranscribes the

complete operon.


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PHBV w/o supplements

Generally, P(3HB-3HV)is synthesized withsupplements ofpropionate, valerate, orother Codd fatty acids.

Some organisms areable to form Propionyl-CoA through themethylmalonyl-CoApathway, from succinyl-CoA in the TCA cycle.



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The two ketothiolases forPHBV synthesis

Steven Slateret al


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Tapping the fatty acid synthesis

Monomers from (R)-3-hydroxyacyl-ACP

intermediates areconverted to (R)-3-hydroxyacyl-CoAthrough an acyl-

ACP:CoAtransacylaseencoded by the

phaG gene.



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PhaG mediated synthesis

Silke Fiedler, Alexander Steinbch

el, and Bernd H. A. Reh

m,


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MCL PHA syn in 'thioesterase

I- E. coli JMU193 onGluconate1. B-ketoacyl-ACP synthase

2. B-ketoacyl-ACP reductase

3. hydroxyacyl-ACP

dehydrase4. enoyl-ACP reductase

5. 'thioesterase I

6. acyl-CoA synthase

7. acyl-CoA dehydrogenase

8. Enoyl-CoA hydratase9. Isomerase

10. specific hydratase

11. PHA polymerase

Klinke, S.; Ren, Q.;Witholt, B.; Kessler B


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MCL PHA syn in Transgenic

Plants The oxidation of

unsaturated fatty

acids withcis doublebonds at an evencarbon are indicatedby dashed lines.

VolkerMittendorf et al


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PHBHx Synthesis

Fukui T., Abe H., and Doi Y

Aeromonas caviae

PhaC, PhaJ

Ralstonia eutropha

PhbA, PhbB, BktB, (S)-3HB-CoAdehydrogenase

Streptomycescinnamonensis

Crotonyl-CoA reductase


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Plasmid forPHBHx synthesis in R. eutrophus

Fukui T., Abe H., and Doi Y

The direction

Of the codons isImportant as

well as the

promoters

which are

inducedusing external

Control agents..


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Gene constructs for Transgenic

plants

Yves Poirier, Giovanni Ventre, and Daniela Caldelari

PTS,Peroxisomaltargeting

sequencePhaC1

synthaseFatB3

thioesteraseOCS, octopine

synthase


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Amount of PHA relative to

growth stage of transgenic plants7-day-old seedlings grown

in Murashige and Skoog

media, green & senesced

leaves from soil-grown

plants. Average of2 ind.

measurements.

http://www.pnas.org/cgi/content/full/95/23/13397

The beta-oxidation cycle is induced upon seedgermination, involved in the mobilization of reservelipids, so the highest amount of PHA is expected to besynthesized at this stage. VolkerMittendorf et al


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Some Organisms and their copolyesters or enzymes and genesSome Organisms and their copolyesters or enzymes and genes

Rhodosporiillu

m rubrum

C3- C6

(of specialinterest)

Pseudomonas

oleo

PHA

depolymerasePHA synthase

phaC2

Rhodocyclusgelatinosus

PHA synthase phaC1

Rhodococcusruber

Alcaligeneseutrophus

PHA synthase

CoA reductase

phbC-A-B

Pseudomonad C4- C12 Streptomycescinnamonensis

Crotonyl-CoA

reductase

ccrSc

AeromonasCaviae

P(3HB-CO-3HHx)

even C-

alkanoates or

plant oils

PHA synthase

enoyl-CoA

hydratase

phaC-JAc

Escherichiacoli

P(3HB-CO-4HB)

glucose or

glutamate

PHA gluconate

Ralstonia

eutropha

3HB, 3HA glucose


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A. CompletephbCAB

B. Intrptd phblociC. Incomp phbloci

D. From 2plmrsasE. P(3HB-3HH)F. msc

Operons



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Formation of a PHA granule

C and Z are similar to Lacting on the surface ofthe granule

Proposed mechanism:- Econverts monomerCoA tooligomers which cleave atcritical length or conc. E-oligomrers formcompartments orPHAgranules which coalesce toform larger bodies.

C= PHA polymerase

Z= PHA depolymerase

L= lipase ( cleaves ester bonds)

E= soluble enzyme

TG=Triglyceride Madison LL, Huisman GW.


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S. Cerevisiae

Poirier, Y., Erard, N., Petetot, J.M.-C. (2001). Synthesis of Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae by UsingIntermediates ofFatty Acid beta -Oxidation.Appl. Environ. Microbiol. 67

Grown on 0.1% glucose 0.1% Oleic acid, 2% Pluronic 127Awt, B recombinant w/ PHA synthase, 1 um bar, arrow to PHAgranules.


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Cell medium effects (phaG)

1. PHA accumulation andcomposition ofP. fragi

2. NH4Cl concentrations.

3. 50 ml of MM with 1.5%(wt/vol) sodiumgluconate

4. At 30C for 48 h.

5. 3HA, 3-hydroxyalkanoate;3HDD:1, 3-hydroxydodecanoate;3HDD, 3-hydroxydodecanoate;3HD, 3-hydroxydecanoate; 3HO,3-hydroxyoctanoate; 3HHx, 3-hydroxyhexanoate. Silke Fiedler, Alexander Steinbchel, and Bernd H.Silke Fiedler, Alexander Steinbchel, and Bernd H. A. RehmA. RehmApplied and Environmental Microbiology,Applied and Environmental Microbiology, May 2000, Vol. 66, No. 5, p. 2117May 2000, Vol. 66, No. 5, p. 2117--2124.2124.


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Promoter induction (thioesterase-I [bad])

1. PHA accumulation and

'thioesterase I activity inE. coli

2. % Arabinose as indicated.

3. 25 h after stat phase

4. GC (open circles).

5. Spectrophotometric (solidsquares)

6. Control lacked the'thioesterase I-encodinggene.

Klinke, S.; Ren, Q.; Witholt, B., Kessler B. ;Appl. Environ. Microbiol. 1999, 65, 540-8.


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Promoter induction (polymerase [alk])

1. PHA accumulationby E. coli

2. DCPK(dicyclopropylketone) conc.

3. 25 h (shaded bars)

4. 44h

(open bars)5. Analyzed by GC.

Klinke, S.; Ren, Q.; Witholt, B., Kessler B. ;Appl. Environ. Microbiol. 1999, 65, 540-8.


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Enzymatic effects (PhbA & BktB)

1. Comparison ofPhbA and

BktB in synthesis of

PHBV inE. coli.

2. Plasmidfrom R. eutropha

phb Bplus a -

ketothiolase gene.

3. bktB (squares) EE245

4. phbA(triangles) EE247

5. Propionate in the

medium.Steven Slater, Kathryn L. Houmiel, Minhtien Tran, Timothy A. Mitsky,Nancy B. Taylor, Stephen R. Padgette, and Kenneth J. Gruys Steven S


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Phasin effects (PhaR & PhaP)

1. PhaP inR. eutropha

2. wt (open squares)

3. phaR strains (solid)4. 72 h.

5. Average value for2cultures

6. PhaP regulates PHBsyn, PhaR regulatesPhaP syn.

Gregory M. York,1 JoAnne Stubbe,1,2

and Anthony J. Sinskey1


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Ca+ Pump in P(3HB) helix

imbedded in a membrane

Madison LL, Huisman GW.,

E coli is proposed totransport Ca+ out and

DNA

in. The Ca+

(green) is liganded tocarbonyl oxygen andthe polyphosphate

molecule within thehelix, transport isfacilitated byenzymatic action on

the molecule.


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Transgenic

Aeromonas.caviae

Alcaligeneseutrophus

96% yield

P. putida

T. pfennigii P. putida 3HB-co-3HHx-co-3HOctanoate

Alcaligenes

eutrophus

E coli ketothiolase

50%-80%

Cosmid

pVK102

Plants P(3HB-co-3HV)

PCR PHA synthase


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Transgenic w/ modifications

Plants --- Zea Mays Corn

G hirsutum (cotton).

Alfalfa Arabidopsis thaliana w/

Synthase modification

Yeast --- S. Cervisiae w/ Oleic

acid

Insect cells w/ Fattyacid synthase

Microbial mats

Results---- Unsuccessful

(0.34% fiber weight)

0.025to 1.8 g kg-1 dry weight

0.2 to 4.0 g kg-1 dry weight

0.5% of cell dry weight

250 to 500 g of PHA/gof dry mat


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Mats NESpain & Mass.


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Concentrated pasteConcentrated pasteConcentrated paste

cellcell lysislysis

purification (purification (depolymerizationdepolymerizationdepolymerization))

centrifugationcentrifugation

Extraction(Phy, Chem, Biochem)(Phy, Chem, Biochem)

Griffin G. J. L.


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solvent extraction

PhysicalPhysical

Pretreatment with methanol or acetone toincrease permeability, removes lipids and

denatures proteinso chloroform

o methylene chloride

o di and tri chloroethane

o propylene carbonate Purification expensive , high volumes of

solvents, crystalline precipitates with nonsolvents methanol, diethyl ether orhexane.

Griffin G. J. L.


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Sodium hypochlorite digestion

ChemicalChemical

Degrades and dissolves cell wall leavingpolymer granules intact

Depolymerization due to alkalinity reduced bytreating with phenyl acetic acid and freezedrying before procedure. surfactants

Difficult to remove sodium hypochlorite.Careful control of pH and digestion timeimproved purity (95%)

Griffin G. J. L.


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Enzymatic digestion

BiochemicalBiochemicalBiochemical

Lysozymes,& deoxy ribonuclease treatmentto solubilize peptidoglycans and proteins.

Weakened cell walls ruptured ultrasonically.90% PHA and some peptidoglycans andproteins. Closest to vivo state.

May be further purified with solvents. MWmay be controlled by heat treatment andspray drying.

Griffin G. J. L.


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Some Stats and Concerns

E coli O157:H7.virulent strain(60)

rBGH..(14 days)

Processed foods(60%)

Salmonella DT104(resists 5 antibiot)

Bacteria in a colon = 200 x all humans US..76mill...325k.5k

$ 20 milliongoatsJennifer Ackerman, Food How safe? How altered?, National Geographic, May 2002, Vol. 201, No. 5, p2-50.

Jim Robbins, Second Nature, Smithsonian, July 2002, Vol. 33, No. 4, p78-84.


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How Altered? 50-fold over 6 yrs

Ge et ca y E eere Foo ro s

0

20

40

60

80

100

1996 1997 1998 1999 2000 2001

Year

o

s

of

acres

o abean

orn

Jennifer Ackerman, F d How afe? How altered?, National Geographic, May 2002, Vol. 201, No. 5, p2-50.


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Pros and Cons

Higher Yields- Tocombat world hunger.

FewerPesticides- Lessspraying soilconservation withherbicide resistant plants.

BetterNutrition-

Enhanced foods rich innutrients.

Docile farm animals

Gene Flow-mutantshard to manage

Toxin build up- BtCrops add it to soil.

Allergens-Foods

may contain chemicalby products of

alteration.

Jennifer Ackerman, Food How safe? How altered?, National Geographic, May 2002, Vol. 201, No. 5, p2-50.Jim Robbins, Second Nature, Smithsonian, July 2002, Vol. 33, No. 4, p78-84.


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Preventive measure

Bt Cropresistantinsects

moatrefuge

insects

Susceptible offspring

Presumably by growingregular crops near the

Bt crops , the cross-breeding of insects willdelay development ofresistant strains.

Bt = Bacillus thuringiensis(source of genes capable of producing insecticide)

Bt Corn toxic to monarch caterpillarsJennifer Ackerman, Food How safe? How altered?, National Geographic, May 2002, Vol. 201, No. 5, p2-50.


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Biomimicry

Larvae mandiblesChain saw

Cocklebur.Velcro Snake skin . Entropy

GeckoAdhesive(re)

SpiderDragline(tendons)

GiraffeLubricant



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Proposed directions

Nutrients Inexpensive and abundant substrate

Organism Bio Stress, balance between vital cell activities and mfgSide effects of long term use,

genetic and/ or other changes in cell chemistry

Regulation triggers inducing enzyme production & releasechemically induced promoters to turn genes on and off.

Process Natural excretion or secretion desired, keeping organism viableMonitoring desired, possibly with controllable viral infections,

Purification ease of extraction, storage in specific areas, natural excretion

Bio Ethics Sanctity of life and other issues

bio polyesters

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