Factors affecting the biodegradability of biodegradable polyester in soil

Chemosphere, Vol.25, No.12, pp 1879-1888, 1992 Printed in Great Britain

0045-6535/92 $5.00 + 0.00 Pergamon Press Ltd.

FACTORS A F F E C T I N G THE B I O D E G R A D A B I L I T Y

Yoshikuni

Yakabe,

Kurume

BIODEGRADABLE

POLYESTER

Kazuo Nohara,

Takaharu Hara

Laboratories, 19-14,

Chemicals

Chuo-machi,

IN

Inspection

Kurume-shi,

OF

SOIL and

Yoshifumi

& Testing

Fukuoka,

Fujino

Institute,

830

Japan

(Receivedin G e r m a n y l 9 August1992;accepted22September1992)

Abstract The were

biodegradabilities

studied

to

biodegradable course

the

residual

of

the

but

did

in

on

induction

period

curve. and

The

the

rate

reached

test sampling

date

dability

among

period

in of

PCL

The

soil.

first-order substance the

the for

and

the

water

same content

were

of

smaller

induction

kind kind

and

used. were

with

of the

soil

the

certain rate

The

The

than

time

in

primarily constant

and

the

residual increased

size

of

the

and

on

the

the

biodegra-

attributed

P(3HB-co-3HV),

was

and

the

soil

the

which

particle

variation

rate

of

constant

test

the

analyzing

P(3HB-co-3HV)

of

of

period

from

the

kind

soil

and

by

early

determined

was

soil.

longer

in

for

in

soil

measured

a

period

soil.

with

The

increasing on

of

was

PCL,

biodegradability

mixed

little after

soil

depended

same

polymer

kinetics and

values

values the

the

the

and

the

were

biodegraded

biodegradation

constant

Both

polymers

of

test

P(3HB-co-3HV)

affecting

The

polymer

the of

substance.

polyester,

factors

biodegradation

constant

to

variation

in

polymer.

dependent

two

the

plastics

time test,

of

examine

to

the

the

induction

respectively.

Introduction Various plastics

in

methods

are

the

development

biodegradability environment

of

plastics

e x p o s e d by

the

reproducible

the

method

using

test p l a s t i c s

employed

result

of is

to new

estimate

the

biodegradability

biodegradable

desirable

wasted plastics

to

be

estimated

such as in soil and

is not e x p e c t e d for this method.

the s p e c i f i c

can e s t i m a t e

enzyme

[4-6]

or b a c t e r i a

the b i o d e g r a d a b i l i t y

1879

of

plastics.[l-4] in

natural

sea-water,

On the other [4,7]

The

that

but hand,

degrade

q u i c k l y and r e p r o d u c i b l y ,

but

1880

it is d i f f i c u l t environment, correlating laboratory

to

factors

plastics.

The

the

used

soil

the

and The

study

waste

of

plastics

[i,5].

the b i o d e g r a d a b i l i t y vary with particle

of p l a s t i c s the basic

of

in

is p r i m a r i l y

order

it step,

which

in this

as

on

we of

the

plastics

occur

content

size of test s u b s t a n c e u s i n g two p o l y e s t e r

that

of

study

on

influencing

assume

reaction

the

depend

factor

is

by

because

the p l a s t i c s

the kind and water

to o[

determined

content

important

at the first

been about

biodegradability

such as water is also

standard

has not

knowledge

plastics soil

in soil b e c a u s e

the

examined test

soil

test

substance;

and

polycapro-

P(3HB-co-SHV),

PCI~.

Materials

and

methods

substances 1) ( :31{t3- c o - 3IIV)

molecular Inc..

weight PCI.

fractionated to

325

of

P(3IIB-eo

100

g of

]25

P(311B-co-3ttV)

glass

evaporated 105

coated

SHV)

um t o on

film

bead

with

a

and

750,000 DAISE1

four

of

um a n d

coated

on

el iminate bead

purchased

CENICALS

250

glass

in

average

was

fractions

250

dissolved

rotary

gl ass

chlotoform

in to

was

to

from

sieving

urn,

content

3-hydroxyvalerate

400,000

supplied

by

74

having of

was

urn,

Test

the

for the

po]y(S-hydroxybutyrate-co-S-hydroxyvalerate),

or

to

biodegradable

that b o d y g u a r d

[8-9]

how

Test

required

the

From these d i s c u s s i o n

and

lactone,

is

but

is i n i t i a t e d by the c l e a v a g e

depolymerase

of p l a s t i c s

natural

for

biodegradability

area of test s u b s t a n c e

of' p l a s t i c s

in

biodegradability

plastics,

is to o b t a i n

test m e t h o d

a c t i v i t y of the b a c t e r i a

extrace]]u]ar

surface

of

b i o d e g r a d a b i l i t y of p l a s t i c s

biodegradability

the

environment

important p r o p e r t y

in test and the p r o p e r t y

surface

biodegradation by

soil

laboratory

soil

in n a t u r a l

of

soil b i o d e g r a d a b i l i t y

of this

affecting the

is the

landfill

for the

The p u r p o s e

the b i o d e g r a d a b i l i t y

estimation

test method.

to

test m e t h o d

standardize

them.

the b i o d e g r a d a b i l i t y

relation

established.

the result w i t h

reproducible

biodegradability in

laboratory

number

and

biodegradability

Soi] plastics

the

to c o r r e l a t e Quick

about

diameter

of to

bead

of

the

substance.

were

determined

with

high

by

(PLACCEL

prepared

as

They

of

',37

follows:

chloroform and

74

siever

The

amounts

extracting liquid

and

urn.

with

performance

H4)

size

a\erage Che.micaJs were to

74

respectively.

of 50

sieved

and

Aldrich

particle

urn,

ml

40,

and

quantifying

the

was

100

evaporator lumped

Co.,.

420

22%

of from

The

of

solvent

was of'

P(StIB

g

with

aperture

of the

0.75

mixed

74

co-3tlV)

substance

w i th

chromatography.

soil Two

\'olcarllc Kumnmoto

ash

kinds soil

pref'(;(:ture

of'

sot], was

]n

volcanic

collected

,Japan

three

ash from t~mes.

soJ] the

and

sand

surface

Sand

so;i]

soJ],

wet'(?

of'

cultivated

was

col ]ccted

used.

The

field from

of'

native

1881

field

of

0ita

prefecture

refrigerator carbon

at

4 "C

content

and

in

Japan.

sealed

total

in

These

plastic

number

of

soils

were

container.

bacteria

sieved The

of

<2mm

water

these

and

stored

content,

soils

are

in

organic

shown

in

Table

i.

Table

i.

Selected

soil

Sampling

Soil

properties

water

date

Organic

content (%)

number

(%)

(CFU/g

dry)

Volcanic

ash

i

0ct,1990

61.2

23.4

1.4x108

Volcanic

ash

2

Jan,1991

66.7

23.4

1.7x108

Volcanic

ash

3

Sep,1991

50.8

22.9

2.3xi08

Jun,1991

17.6

Sand

Biodegradability 15 in

100ml

capped was

test of

of

Erlenmeyer

At

once was

solution

twice.

of

(Woelm)

in three

and

was

order

biodegradability

dry

weight

of

to

be

by room

The

in

the

the

ash

was of

25

number

were

cultivating

3

weeks

at

25

of

bacteria

residual percent

a

by and

put

was

in

stoppered of

residual

v/v)

acid

with

10ml

for

with

was 15

added

minutes

tightly. of

was

thermostated

test

added

room

weight was

mixed aluminum

determined

"C

content water

reduced

distilled

P(3HB-co-3HV) 120

"C

vessel

the

dissolved were

at

water

amount

of

soil 25

test

(1/4 3g

control,

of

measured

dilution

The

out

substance

autoclave

of at

supplemented taken

residue

P(3HB-co-3HV)

by

Analysis

weight room

of

added

at

soil 20

g

25

"C

for

the

and

on

to

soil

as a

was

incubated

in

"C.

soils

the

dry

including

through

room

the

blank

of

As

3 and

water,

was

test

influence

and

activity

the the

with

After

as

soil was

passed

fixed

soil

20g

Acetone/Dichloroemthan

dryness,

plastics,

distilled

bacteria

of

test

soil

thermostated

examine of

at

1.6x108

thermostated

weight

test

sterilized

equilibrated.

thermostated test

100ml to

with in

of

chromatography.

put to

the

amount

soil

volcanic

supplemented

fixed

reduced

extract

residual

liquid

the

the

with

ash

and

In

weight

evaporated

The

mixed

incubated

Total

and

the

was

and

intervals, After

volcanic time

week

foil.

a week

performance the

substance flask

extracted

chloroform.

high

test

appropriate

substance oxide

the

aluminum

measured

3.49

method

mg

with

water.

and

Bacteria

carbon

in

soil

at

appropriate

plate

method

in

order

to

during

the

test.

'C

using

examine

intervals albumin the

with agar

change

the medium

of

data residue

decreased

according

to

the

following

first-order

the

1882

kinetic

model

after

a certain

time,

which

depends

on t e s t

substance

and

soil:

-kt R = R0 e where

R,

R 0,

k and

a first-order to

the

rate

data

induction

t are

the p e r c e n t

constant

and

and

ti,

residue

calculated

equation

2.

was

from

of

as

fitted

test

respectively.

determined

defined

the

residue

time,

R 0 and k were

period,

ti

equation

the

by

time

curve

substance,

This

when

become

square

the

100%

constant, was

and was

applied

method.

theoretical

in(RO/lOO)/k

=

a

equation

least

1

The

percent

calculated

equation

by

2

R e s u l t s and d i s c u s s i o n B a c t e r i a n u m b e r of test soil Figure blank

soils

1 shows

with

the v a r i a t i o n

time.

value

bacteria

in the

soil w a s

kept

increased

slightly

after

increment 3HV)

of

the

end of

the b a c t e r i a

degrading

in the b a c t e r i a

bacteria

constant soil

until

The

the

number

test

during

number

indicating

whole

28 days

test

and

number

of the b l a n k period,

reached

is a t t r i b u t e d

that

of

the

soil the

while

the

at

of

the

the

test

number.

The

one

induction

and

kept

activity

to a c o n s t a n t

to

test

was of

of

P(3HB-co-

bacteria.

D

2.o

b

1.5"

2.5

o

Blank

1.0" o rn

0.5

0.0

i

i

20

i

40

60

80

Days

Figure

i.

volcanic

Residual

Variations

ash

in

2 with

the

time.

bacteria

number

Particle

size

of of

test

and

P(3HB-co-3HV):

blank

in

soils

37/75um.

curve

Figure for

soil

P(3HB-co-3HV)

2

shows in

the volcanic

percent ash

residue soil

2.

and The

its polymer

logarithms diminished

vs.

time below

plots 10%

of

1883

the

initial

in

the

value

control

P(3HB-co-3HV) soil.

in

The

but

at at

it

the

were

R 0 were

the

regression

curves

to

was

enough

number

bacteria

that

but

after

the

the

the residue

that

by

calculated rate

compared

among

various

was

observed. i.

results to

soil

are

equation

the

to

in

by

the

18

days

degraded

induction

in

period

when

the

1

become

equation

calculated

of are

follows:

during

time

the 2

as

was

the

as

and

Figure

interprets induced

Hence

defined

period

in

P(3HB-co-3HV)

bacteria. was

linearly

k

P(3HB-co-3HV)

was

days,

coefficient

lines

biodegrade

and

regression

test

solid

in

18

for

High

of

bacteria

until

constant

method.

The

the

data

rate

P(3HB-co-3HV)

induction

by The

the

squares

These

induced

the

1 and

P(3HB-co-3HV)

disappearance

little

afterward.

bacteria

of

the

decreased

least

plastics

and

residues that

biodegradation

the

with

the

from

constant

time

degrade

degrade

the

residue

equation

of

mixed

percent

indicating

to

equation

was

kinetics

bacteria

The

r 2 = 0.991)

polymer

first-order

to by

determination(

soil,

due with

fitted

determined

not

average

93.9%

percent

linearly

part

the there

The

was

2 was

of

decreased

constant

days.

days

Figure

logarithm

decreased

66

66

this

of

percent 100%.

method

were

conditions.

120 100 '

0

0

4'

8O O0

"o o

I

3.

6O _J

o

~_

40

20

l

i

i

20

40

60

20

80

40

Days

Figure the

2.

Percent

residue

biodegradation

of

P(3HB-co-3HV): order

Particle

size

on

and

Solid

its in

lines

are

of

test

the the

of

the

biodegradability

of p l a s t i c s

in

soil

the

in the

plastics

surface

frequency of

used

constant

and

for

powder

respectively. the

particle

the

(right) ash

soil

vs. 2.

regression

time

Particle

curves

to

plot

for

size the

of

first-

substance contact

area

the

logarithm

volcanic

model.

Since depend

left)

P(3HB-co-3HV)

125/250um.

kinetic

80

60

Days

the

The size

test.

Figure

induction

period

and

film

the

rate for

constant

the

is

3 and

with

and

the

in

it

is

influenced 4 show

the

coated

P(3HB-co-3HV)

bacteria

plastics,

the

glass

induction

powder

by

particle

on in

the

soil

with

anticipated the

size

period range

particle

variations

bead of

of of were 74

plastics that size in the

test

the of rate

substance

P(3HB-co-3HV), independent to

420um

as

on the

1884

average

particle

substance

of

size

except

37/74um,

while

for

P(3HB-co-3HV)

film

to

76um.

results

These

constant but

of

it

about

reach 70

with

P(3HB-co-3HV) to

um

the

for

small

induction

values

increased

and

increasing

average

o

diameter

of

glass

induction

increase

with

the

particle

size

certain

particle

size,

important

to

values

above

soil

i.

a

This

0.10

20

0.08 '

15 ~

result

period

is

test

80 ~

~0

~_

50 rate

substance, which

was

select

test

5

~ sO

e

37/74#m

o []

74/125#m 125/250#m

60

~d

~d

o.o2

L

0.00 100

,

200

•

g-

c

20-

0

300

0

400

I

0

I

20

KJgure

3.

Right:

varied

regression between

Comparison

particle curves

the

powder

to

the

the

in

the

first-order and

of

size

rate

constant

and

particle

size

S

0.04-

P(3flB-co-3HV)

2.

Solid

the

model.

the

test

period

80

powders

lines

Left:

induction

of

are

the

Relationship for

P(SHB-co-

substance.

120 lOO

-~8 "Z3 "~

~

~-

~u

t<

o

40p.m

\

•

50p.m

80

K 60

2_ 40

r5

0,02 "

2O

~

0.00 30

coated

for

soil

20

10

Figure

60

curve

kinetic

average

0.08 -

0.06 -

ash

first-order

0.10-

cd

residual

volcanic

I

40 Days

Average particle size (Ltm)

are

the

0.04-

cr

3HV)

from

and

of

the values

100

£

of'

the bead

the

ash

with

to

that

0.06 -

c

period reached

indicate

constant

volcanic

slightly both

4. on

,

,

,

0

40 50 60 70 Average diameter (#.m)

Right: glass

the

Relationship

Comparison

bead

of

regression between

f'or P ( S H B - e o - 3 H V )

film

of

varied curves

the on

20

0

the

to

the

first-order glass

residual

diameters

bead

curve

in v o l c a n i c first-order

rate and

constant

the

average

for ash

40 Days

P(3HB-co-3HV) soil

kinetic and

60

the

2.

Solid

80

film lines

model.

Left:

induction

period

diameter

of

glass

bead.

1885

substance,

but

between

particle

Test

the

studies

size

and

need

to

derive

the

the b i o d e g r a d a b i l i t y

general

soil.

soil

Figure

PCL

PCL,

P(3HB-co-3HV)

and

And

rate

until

151 for

Jt

the

0.0157

with

than

100 8O

"o

soil

of

ash

3.8

72.0

and

two

rate

than soil

the

soils

except

ash

ash

soils when it

is

water soil

the w a t e r

concluded

biodegradability

to

same

not that

constant and

shown

not

of

other

below,

the

to ttlose

adjusted

was

and

longer, number,

those

identical

was

the period

bacteria to

As

rate

0.0824,

Longer

rate

same

nearly

volcanic

induction

identical

and

6) T h e

smaller

the

change

site

0.0637,

The

much

to the

on v o l c a n i c

the

of same

seasonal ash

soil.

120 , PCL

3HB-co-3HV) 100 " •

•

•

•

•Sand

soil

0

•

80" 60-

40

40-

2O

20"

0

0 20

30

40

50

I

0

60

50

Days

5.

amount was

on t h r e e

were

of P ( 3 H B - c o - 3 H V )

~_

for

the

The

there

largely

bacteria

respectively.

content

that

was

date.(Figure

content.

3 became

both period

initial

expected

examined

was.

nearly

in

kinetics

from

soils

was

approximately

60

Figure

PCL of

with

period. is

constant.

3

K

10

of

test

days,

rate

soil

period

degrading

were

three

Compared

induction

23.4%

bacterium

despite

3 were

PCL

constant

the

ash

only

at d i f f e r e n t for

soil

in v o l c a n i c

in i n h e r e n t

site

the

same

in

P(3HB-co-3HV)

soil.

induction the

is c o l l e c t e d

periods

volcanic

other

volcanic

~

larger

date m o r e

in

Consequently,

120

the

16.4,

properties

different

during

of soil

if the

induction

sampling

volcanic

value.

even

was,

period

two

enough

of p l a s t i c s of

by the f i r s t - o r d e r

indicating

activity

from

and

biodegradability other

induced

the

period

respectively,

two

not

short

degraded

same

(day -]. )

the

soil

sand

biodegradability

P C L was

of P ( 3 H B - c o - 3 H V )

induction

but

sand

curves

and

nearly

collected and

induction

the

with

biodegradabilities

constants

varied

in

soil

fast with

the b i o d e g r a d a t i o n

and a n n u a l l y

soils

while

residual

ash

degraded

soils.

biodegradable

seasonally hence

was

the b i o d e g r a d a b i l i t y

of the

volcanic

biodegraded

soils,

and

determining

comparison

soil,

both

PCL was

The

of

two

days

biodegrade

the

was

between

observed

factor

P(3HB-co-3HV) in

different

ash

is k e y 5 shows

in two k i n d s

soils.

soJ]

relationship

of p l a s t i c s .

soil Test

and

further

Comparison

I

I

100

150

200

Days

of r e s i d u a l

P(3HB-co-3HV)(left)

0

curves

between

and PCL(right).

volcanic

ash

soil

2 and

sand

1886

120 Q 1 O0 •

0

0

0

80-

60

e,(..

Soil 2

Soil 3

40

20

0 0

50

100

150

200

Days Figure ash

6. soils

Comparison

of

collected

from

P(3HB-co-3HV):

125/250

first-order

Water

kinetic

content The

was

the

ash

soil

soil.

3

a

in

constant

Both the

values

in

volcanic

presumed

of of

at for

66

to

in date.

the

three

volcanic

Particle

regression

about % of 3

the

the

size

of

to

the

3

was

curves

the plastics

the

cause

20 the

days

and

water

ash

soil

can

be

the

of

low

content 1 and

were 2.

attributed

induction

of

the

it

required

rate

the

rate

bacteria

content of

the

and

the

volcanic

ash

decreased

to

increased.

consistent low

of

volcanic

constant

approximately the

water

constant in

at

groups

using

period

Consequently to

the

site

biodegradability

content the

both

of

induction

reversely

same

examined

the

the

soil

the

between

was

water

content,

ash

from

influence

between

and

water

volcanic

property

of

relationships

the

in collected

accordingly

P(3HB-co-3HV)

soil

retard

in

elucidate

the

volcanic ash

and

to

shows

increasing

values

are

soils

biodegradability

order

value

two

difference

content

7

With

lines

P(3HB-co-3HV) different

P(3HB-co-3HV)

other

main

the

period

3.

of than

The

on

Figure

induction soil

Solid

at

soil

water

soil

of

site

model.

of

date.

test

curves

same

um.

lower

different soil

the

biodegradability

considerably

of

residual

with

biodegradability

low

water

content,

and

lower

the

which

is

biodegradable

activity. From soil could constant

in

these

results,

standardized

not

be had

the

is

biodegradability

determined tendency

from to

the

increase

to

control

test, result with

but of

this

water

the the

water

content

optimum study

content

of

water because

even

at

the 65

test

content %.

rate

1887

80

0.10

1oo

°

~I\

~, o.o8• 6o

~,

~

80-

m

60

° o ~ L~

Water content o 52%

o k

0.06• 40

o . m 0.04 n-

•

~"

20

o

~ 40

~

r,

0.02 '

20-

0.00

0

i

i

i

55

60

65

0

0

0

I

I

I

50

100

150

Water content(%)

Figure ash

7. soils

adjusted

Left:

Comparison

having

varied

by

Right:

adding

of water

appropriate

Variations

P(3HB-co-3HV)

of

in

200

Days

the

residual

curves

contents.

The

amount

of

water

biodegradation

volcanic

ash

of

P(3HB-co-3HV)

water in

rate

in

contents volcanic

and

of ash

volcanic

the

soil

soil

3.

half-degradation

were

time

for

soil.

References

1

Holmes,

P.A.

2.

Kunioka,

(1985).

Biodegradable

Thermoplastic. M.,

Kawaguchi,

Biodegradable Doi,

eutrophus.

Y.,

zation

Segawa, of

Int.

Hosokawa,

J.,

dable 5.

Res.

Doi, of

Y.,

Appl. and

-A

Microbially

Technol.

Doi,

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16,

(1989).

32-36. Production

3-hydroxybutyrate Microbiol.

Kunioka,

and

(1990).

J.

Biol.

Macromol.,

Derived

M., from

12,

Yoshihara,

Chitosan

of

4-hydroxybutyrate

Biotechnol.

M.

Produced

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by

569-573.

Biosynthesis

and in

characteri-

Alcaligenes

106-111. K.

and

Kubo,

and

Homogenized

and

Saito,

T.

(1990).

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Cellulose.

Ind.

Eng.

29,800-805. Kanesawa,

Microbial

and

of

Nishiyama,

Film

Chem.

and

PHB

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

eutrophus. 4.

A.

of

Phys. Y.

Copolyesters

Alcaligenes 3.

Applications

Y.,

Kunioka,

Copolyester:

M.

T.

(1990).

Biodegradation

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate

Poly(3-hydroxybutyrate-co-4-hydroxyvalerate).

Macromolecules,

23,

26-31. 6.

Tokiwa,

Y.

Nature,270, 7.

Tokiwa, tone

8.

Tanio, and

Ando,

a Fungus. T.,

Masamune,

merase

Suzuki,

T.

(1977).

Hydrolysis

of

Polyesters

by

Lipases

76-78. Y.,

by

and

from

Fukui, S.

T,

and

J.

Ferment.

T.,

T.

An

(1976).

Technol.,

Shirakura,

(1982).

Alcaligenes

Suzuki,

Y.,

Saito,

Extracellular

faecalis.

Eur.

Degradation

54,

of

Polycaprolac-

603-608. T.,

Tomita,

K.,

Kaiho,

Poly(3-hydroxybutyrate) J.

Biochem.,

124,

71-77.

T. Depoly-

1888

9.

Nakayama, K., S a i t o , T . , F u k u i , T . , S h i r a k u r a , Y. and T o m i t a , K. ( 1 9 8 5 ) Purification and P r o p e r t i e s of Extracellular Poly(3-hydroxybutyrate) Depolymerases 63-72.

from Pseudomonas

lemoignei.

Biochim.

Biophys.

Acta,

827,