Colonization, biofilm formation and Colonization, biofilm formation and biodegradation of polyethylene by biodegradation of polyethylene by

soil bacteriasoil bacteria

Alex SivanAlex SivanThe Institute for Applied Biosciences and The Institute for Applied Biosciences and The Department of Biotechnology The Department of Biotechnology Engineering, Ben Gurion University, Beer Engineering, Ben Gurion University, Beer Sheva, Israel Sheva, Israel

Environmental Engineering, 17.12.02Environmental Engineering, 17.12.02

Polyethylene for soil mulchingPolyethylene for soil mulching

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

Polyethylene wastePolyethylene waste

• 25,000,000 Ton/year25,000,000 Ton/year

• Makes up to 40% of the plastic wasteMakes up to 40% of the plastic waste

• Highly flammable; burning releases toxic gasesHighly flammable; burning releases toxic gases

• Pollutes marine and fresh water habitats Pollutes marine and fresh water habitats

• Highly recalcitrant; > 400 years for degradation Highly recalcitrant; > 400 years for degradation

Control BacteriaControl Bacteria

2020151510105500001010

551010

661010

771010

881010

99 P34- PEP34- PEP34+PEP34+PE

101055

101066

101077

101088

101099

P33- PEP33- PEP33+PEP33+PE

Time (days)

Bac

teri

al C

.F.U

./ml

Bac

teri

al C

.F.U

./ml

Bacterial growth on PEBacterial growth on PE

Effect of pHEffect of pH

55 7.67.6 8.58.500

22

44

66

88

1010

1212

pHpH

Wei

ght

loss

(%

)W

eigh

t lo

ss (

%) 50 C50 C

Biofilm on polyethylene

Hydrophobicity of PE - degrading bacteria Hydrophobicity of PE - degrading bacteria measured by contact anglemeasured by contact angle

Medium pHMedium pH

Bacterium Bacterium 66 77 88

Bacillus 707 Bacillus 707 36.7±1.536.7±1.5 60.6±4.060.6±4.0 62.0 ± 3.462.0 ± 3.4

Bacillus 712Bacillus 712 51.6±2.851.6±2.8 58.3±2.858.3±2.8 69.6 ±0.569.6 ±0.5

Rhodococcus Rhodococcus rhodochrousrhodochrous 49.2 49.2 ±± 2.9 2.9 67.5 67.5 ±± 2.8 2.8 73.7 73.7 ±± 2.5 2.5

SM

7 days

16 hr

Development of bacterial biofilm on PEDevelopment of bacterial biofilm on PE

SM + MO

16 hr

16 hr

Biofilm + Mineral oil (16hr)

Biofilm + Mineral oil (7 days)

Incubation time (days)

Additive Conc.(%;v/v)

4 10 17 30

0.01 2* 3 3 3Mineral oil 0.05 3 4 4 4

0.1 3 4 4 4

0.1 0 0 0 0Tween 60 0.5 0 0 0 0

1.0 0 0 0 1

Control 0 0 1 1

Effect of an anionic surfactant and mineralEffect of an anionic surfactant and mineral oil on biofilm formation on polyethyleneoil on biofilm formation on polyethylene

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

Mix C30Mix C30

0.020.02 0.050.05 0.10.100

1010

2020

3030

4040

5050

Mineral oil concentration (%)Mineral oil concentration (%)

Incr

ease

d w

eigh

t lo

ss (

%)

Incr

ease

d w

eigh

t lo

ss (

%)

Improved biodegradation of polyethylene Improved biodegradation of polyethylene by mineral oilby mineral oil

30020010000.0

0.1

0.2

1 piece

3 pieces

Time (min)

O.D.494

R2 = 0.982

R2 = 0.983

FDA hydrolysis by the bacterial biofilm FDA hydrolysis by the bacterial biofilm

Combined photolysis and biodegradationCombined photolysis and biodegradation

• Polyethylene:Polyethylene: Linear LDPE MW 100,000Linear LDPE MW 100,000contains UV sensitizer contains UV sensitizer

• UV pretreatment:UV pretreatment: Accelerated Weathering Accelerated Weathering Tester (Q.U.V)Tester (Q.U.V)

60 Hr of UV 312nm60 Hr of UV 312nm

907560453015000

5

10

15

20

60 Hr 80 Hr100 Hr120 Hr

Biodegradation of polyethylene by strain 707

Incubation time (Days)

Wei

ght

Los

s (%

)

U.V. exposure

* 50C

Low density PELow density PE

00

0.50.5

11

1.51.5

22

2.52.5

50050010001000150015002000200025002500300030003500350040004000

Wavenumber (cmWavenumber (cm-1-1))

Ab

sorb

ance

Ab

sorb

ance

29202920 28502850

14631463

720720

Effect of U.V. on polyethyleneEffect of U.V. on polyethylene

00

0.20.2

0.40.4

0.60.6

0.80.8

11

1.21.2

1.41.4

1.61.6

130013001400140015001500160016001700170018001800

Wavenumber (cmWavenumber (cm-1-1))

Ab

sorb

ance

Ab

sorb

ance

17181718

14631463 ControlControl

120 Hr U.V.120 Hr U.V.

60 Hr U.V.60 Hr U.V. 100 Hr U.V.100 Hr U.V.

PE degradation withPE degradation with strainstrain 707707

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

130014001500160017001800

Wavenumber (cmWavenumber (cm-1-1))

Ab

sorb

ance

Ab

sorb

ance

U.V. 120 Hr U.V. + 60 days incubation U.V. + 90 days incubation

1718

1463

00 6060 8080 100100 1201200.00.0

0.10.1

0.20.2

0.30.3

0.40.4

0.50.5

0.60.6

0.70.7

0.80.8

0 0

15 15

30 30

60 60

90 90

U.V. irradiation time (Hr)U.V. irradiation time (Hr)

Car

bon

yl in

dex

C

arb

onyl

ind

ex AA

C=

O/

C=

O/ AA

CH

CH

22 IncubatioIncubation time n time (days)(days)

Carbonyl index of U.V. irradiated PE Carbonyl index of U.V. irradiated PE incubated with strain 707 incubated with strain 707

Carbonyl index: A 1718/ A 1463Carbonyl index: A 1718/ A 1463

Degradation of CHDegradation of CH22 groups groups

U.V. + 30 days incubationU.V. + 30 days incubation

00

0.50.5

11

1.51.5

22

2.52.5

0010001000200020003000300040004000

Wavenumber (cmWavenumber (cm-1-1))

Ab

sorb

ance

Ab

sorb

ance29202920

14631463

60 Hr U.V.60 Hr U.V.

00

0.50.5

11

1.51.5

22

2.52.5

0010001000200020003000300040004000

Ab

sorb

ance

Ab

sorb

ance29202920

14631463

U.V. 60 HrU.V. 60 Hr

0.50.5

1.51.5

2.52.5

00

11

22

25002500270027002900290031003100

Ab

sorb

ance

Ab

sorb

ance

U.V. + 30 days incubationU.V. + 30 days incubation

00

0.50.5

11

1.51.5

22

2.52.5

25002500270027002900290031003100

Wavenumber (cmWavenumber (cm-1-1))

Ab

sorb

ance

Ab

sorb

ance

Degradation of CHDegradation of CH22 groups groups

11 22 33 44

00

2000020000

4000040000

6000060000

8000080000

100000100000

120000120000

140000140000

Incubation time(days) with Incubation time(days) with bacteria after 120 hr UV bacteria after 120 hr UV

Mol

ecu

lar

wei

ght

Mol

ecu

lar

wei

ght

No UVNo UVNo bacteriaNo bacteria

00 15-3015-30 45-6045-60

Biodegradation of UV irradiated polyethyleneBiodegradation of UV irradiated polyethylene

Mineral oil degrading bacteriaMineral oil degrading bacteria

Time (hr)Time (hr)00 44 88 1212 1616 2020 2424 2828 3232

Uti

liza

tion

of

min

eral

oil

(%

)U

tili

zati

on o

f m

iner

al o

il (

%)

00

2020

4040

6060

8080

100100

st.208 st.208 st.489 st.489 st.490 st.490 st.547 st.547 st.555 st.555

Wei

g ht

loss

of

po l

y eth

ylen

e (%

)

0

5

10

15

20

25

30

35

40

I -707II-707

I -707II-490+555

I -490+555II-490+555

I -707+490+555II-707+490+555

Effect of combinations of bacteria on PE degradation.

Reduction of gravimetric weight

1st incubation2nd incubation1st+2nd

Effect of combinations of bacteria on PE Effect of combinations of bacteria on PE degradationdegradationReduction in molecular weightReduction in molecular weight

I -707I -707

II-707II-707I -707I -707

II-490+555II-490+555I -490+555I -490+555

II-490+555II-490+555I -707+490+555I -707+490+555

II-707+490+555II-707+490+555

Red

uct

ion

in M

W (

%)

Red

uct

ion

in M

W (

%)

0

5

10

15

20

25

30

351st incubation2nd incubation

1st+2nd incubation

SummarySummary

• Enrichment cultures gave rise to bacterial strains that Enrichment cultures gave rise to bacterial strains that could utilize PE as a sole carbon and energy sourcecould utilize PE as a sole carbon and energy source

• Maximal biodegradation (up to 30%) was obtained after Maximal biodegradation (up to 30%) was obtained after two-steps incubation with combined culturestwo-steps incubation with combined cultures

• PE degrading bacteria are hydrophobic and form a PE degrading bacteria are hydrophobic and form a biofilm on the PE surface biofilm on the PE surface

• Mineral oil enhances biofilm formation and PE Mineral oil enhances biofilm formation and PE biodegradationbiodegradation

• The degrading bacteria utilize carbonyl residues in the The degrading bacteria utilize carbonyl residues in the PE which are formed during UV irradiationPE which are formed during UV irradiation

• Combination of UV photolysis and biodegradation Combination of UV photolysis and biodegradation showed a synergistic effectshowed a synergistic effect

Irit GilanIrit Gilan

Deborah BittyDeborah Bitty

Dr. Valentina PavlovDr. Valentina Pavlov

Dr. Mark KarpassasDr. Mark Karpassas

Prof. Shimona GereshProf. Shimona Geresh

CollaboratorsCollaborators