Colonization, biofilm formation and biodegradation of polyethylene by soil bacteria

Colonization, biofilm formation and
biodegradation of polyethylene by
soil bacteria
Alex Sivan
The Institute for Applied Biosciences and
The Department of Biotechnology
Engineering, Ben Gurion University, Beer
Sheva, Israel
Environmental Engineering, 17.12.02


Polyethylene for soil mulching

In the U.S. ~ ca. 1,000,000 Ton/year in agriculture


Polyethylene waste
• 25,000,000 Ton/year
• Makes up to 40% of the plastic waste
• Highly flammable; burning releases toxic gases
• Pollutes marine and fresh water habitats
• Highly recalcitrant; > 400 years for degradation


Polyethylene rec

• Applications:
fences, side-walks, aco
• Cost of recycled products.
• Variable quantities in time a
• Currently, only ca. 15% of th

Alternative polyet

• Photodegradable polye

–Contains toxic metals.
–Requires long exposure t

• “Biodegradable” polye

–PE - starch copolymers.
–Only the starch is degrad


PE degradation
40

Soil

30

Loess A
Ein Tama
Loess B
Peat

20

Weight loss (%)

10
0

30

50

Temperature (C°)


Control

Bacteria


Methodology
• Polyethylene:

Linear LDPE
U.V. photosensi
• U.V. pretreatment: Accelerated w
5 U.V. cycles
1 hr water co
between cycles.
• Total irradiation: 60 hr of U.V.
• Disinfestation/sterilization


Biodegradation assay

• ca. 100 mg polyethylene in c
synthetic medium
• Inoculation
• Incubation at 30ºC or 50ºC
• Dry weight of polyethylene


Bacterial growth on PE

Bacterial C.F.U./ml

10
10
10
10
10

10
10
10
10
10

9

P33- PE
P33+PE

8

7
6
5

P34- PE
P34+PE

9
8
7
6
5

0

5

10

15

Time (days)

20


Effect of pH
Weight loss (%)

12

10

50 C

8
6
4
2
0

5

7.6

pH

8.5


Biofilm on polyethylene




Bacterial addhesion to hydr
100

Rhodococcus
rhodochrous

90

Log.
hex
Stat. hex
Log. octa
Stat. octa

80

% of initial O.D.

70
0.00 0.04 0.08 0.12 0.16 0.20

Hydrocarbon added (ml)


Hydrophobicity of PE - degrading bacteria
measured by contact angle
Medium pH
Bacterium

6

7

8

Bacillus 707

3.4

36.7±1.5

60.6±4.0

62.0 ±

Bacillus 712
±0.5

51.6±2.8

58.3±2.8

69.6

Rhodococcus


Effect of mineral oil on biof
on polyethylene
Rhodococcus
rhodochrous
by
5
4

Control
MO

(0.05
Tw 20 (0

3
Colonization index

2
1

00 5 10 15 20 25 30 35 40

Incubation time (days)


Development of bacte
16 hr

7 days

16 hr

16 hr
SM

SM + MO


Biofilm + Mineral oil

(16hr)

Biofilm + Mineral oil (7
days)


Mix C30

Effect of an anionic surfactant and mineral
oil on biofilm formation on polyethylene
Incubation time (days)
Additive

Conc.
(%;v/v)

4

10

17

30

Mineral oil

0.01
0.05
0.1

2*
3
3

3
4
4

3
4
4

3
4
4

Tween 60

0.1
0.5
1.0

0
0
0

0
0
0

0
0
0

0
0
1

0

0

1

1

Control

* Biofilm density index: 0 = No biofilm; 4 = Dense biofim


Increased weight loss (%)

Improved biodegradation of polyethylene
by mineral oil
50
40
30
20
10

0
0.02

0.05

0.1

Mineral oil concentration (%)


FDA hydrolysis by the bacterial biofilm
R2 = 0.983

0.2

3 pieces

0.1

O.D.494

1 piece
R2 = 0.982

0.0

0

100

200

Time (min)

300


Combined photolysis and biodegradation
• Polyethylene:

Linear LDPE MW 100,000
contains UV sensitizer

• UV pretreatment:

Accelerated Weathering
Tester (Q.U.V)
60 Hr of UV 312nm


Biodegradation of polyethylene by strain 707
20

Weight Loss (%)

* 50C
15

U.V. exposure
60

80
100
120

10

5

0
0

15

30

45

60

75

Incubation time (Days)

90

Hr
Hr
Hr
Hr