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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 PE is considered as one of the most persistent synthetic polymers ever produced and thus it poses a serious environmental problem. Today, I am going to share with you some evidences on biodegradation of PE by isolated soil bacteria Environmental Engineering,
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Polyethylene for soil mulching
The main source of PE waste is agriculture. The major use is for soil mulching. The annual consumption of PE in the US alone is…….. In the U.S. ~ ca. 1,000,000 Ton/year in agriculture
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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 According to recent estimates the global amount of PE is……
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Polyethylene waste can be recycled to various products such
as fences, side walks, acoustic walls etc. But the cost of some of the recycled products may be higher than the regular non recycled products.. Although to some extent it is predictable, usually the polyethylene waste varies in time and place. Currently, in Israel, only ca. 15% of the polyethylene waste is recycled.
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In order to provide an answer to the polyethylene waste
problem 2 alternative polymers have been developped in the recent years. The first photodegradable polyethylene which contains metal chelators that serve as photosensytizers which initiate a photolytic chain reaction in which the polyethylene breaks eventually to CO2 and water. However, this process still takes relatively long time and requires that the polyethylene will be exposed to sunlight. The second is what is called biodegradable polyethylene which is a copolymer of starch and polyethylene. In soil, only the starch is degraded and the polyethylene deteriorates to small particles or poweder which decreases the volume of the waste.
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We have done a major screening for PE degrading bacteria from various soil samples enriched with PE and amended with nitrogen and phosphorous. As you can see……
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Control Bacteria Isolates were further screened in liquid cultures containing PE as the sole carbon source in the medium
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Few words on the methodology.
Basically we wanted to combine photo and bio degradation. Polyethylene type U.V. partial photodegradation in a Q.U.V accelerated weathering tester. under these conditions after 3 days treatment the photodegradation is equivalent to that obtained after 3 months exposure to sunlight. The polyethylene samples were disinfected and added to the medium.
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Bacterial growth on PE Bacterial C.F.U./ml Time (days) P33- PE P33+PE
10 5 6 7 8 9 P33- PE P33+PE Bacterial C.F.U./ml 10 5 6 7 8 9 P34- PE P34+PE 20 15 10 5 Time (days)
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Effect of pH Weight loss (%) pH 5 7.6 8.5 2 4 6 8 10 12 50 C
2 4 6 8 10 12 pH Weight loss (%) 50 C The optimal pH for polyethylene degradation by these bacteria was 7.6.
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Biofilm on polyethylene
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Hydrophobicity of PE - degrading bacteria measured by contact angle
Medium pH Bacterium Bacillus ± ± ± 3.4 Bacillus ± ± ±0.5 Rhodococcus rhodochrous 49.2 ± ± ± 2.5 ….. A greater contact angle indicates higher hydrophobicity
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In order to increase the hydrophobic interactions between the bacteria and PE we added various non ionic surfactants to the bacterial culture. This slide shows that addition of Tw 20 did not increase the colonization of PE. Similarly other surfactants (Tw 80 or Tw 60) were ineffective. However, the addition of MO improved the biofilm density.
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Here you can see several stages in biofilm formation as affected by MO
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Biofilm + Mineral oil (16hr)
This is a close look at the minearal oil amended biofilm after 16 hr. And you can see the aggregates of the bacterial cells.
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Biofilm + Mineral oil (7 days)
This is a close look at the minearal oil amended biofilm after a week where it’s impossible to see individual bacterial cells.
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Effect of an anionic surfactant and mineral
Mix C30 Effect of an anionic surfactant and mineral oil on biofilm formation on polyethylene In an attempt to improve the attachment of the bacteria to the polyethylene surface and the biofilm formation we tried to add to the medium various unionic surfactatnts like Tween 20, 60, 80 and mineral oil. This slide shows the effect of mineral oil and Tween 60 on biofilm formation of the initial bacterial mixture. The biofilm density was determined using an arbitrary index where 0 = no colnization and 4 = maximal colonization. As you can see the mineral oil enhances the biofilm formation whereas no effect was obtained with Tween 60. Other Tweens also did not improve the colonization of the biofilm. * Biofilm density index: 0 = No biofilm; 4 = Dense biofim
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Improved biodegradation of polyethylene
by mineral oil 10 20 30 40 50 Increased weight loss (%) 0.02 0.05 0.1 Mineral oil concentration (%)
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The activity of the bacterial biofilm could be measured by FDA hydrolysis by extracellular esterases produced by the bacterial biofilm
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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 It is known that UV radiatio initiates some photolysis of the PE. And since the major source of PE waste is from soil mulching where it is exposed to UV we wanted to study the combination of UV radiation with biodegradation
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Biodegradation of polyethylene by strain 707
90 75 60 45 30 15 5 10 20 60 Hr 80 Hr 100 Hr 120 Hr Biodegradation of polyethylene by strain 707 Incubation time (Days) Weight Loss (%) U.V. exposure * 50C
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Low density PE Absorbance Wavenumber (cm-1) 0.5 1 1.5 2 2.5 500 1000
0.5 1 1.5 2 2.5 500 1000 1500 2000 2500 3000 3500 4000 Wavenumber (cm-1) Absorbance 2920 2850 1463 720
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Effect of U.V. on polyethylene
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1300 1400 1500 1600 1700 1800 Wavenumber (cm-1) Absorbance 1718 1463 Control 120 Hr U.V. 60 Hr U.V. 100 Hr U.V.
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PE degradation with strain 707
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1300 1400 1500 1600 1700 1800 Wavenumber (cm-1) Absorbance U.V. 120 Hr U.V days incubation U.V days incubation 1718 1463
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Carbonyl index of U.V. irradiated PE incubated with strain 707
60 80 100 120 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 15 30 90 U.V. irradiation time (Hr) Carbonyl index A C=O/A CH2 Incubation time (days) Carbonyl index of U.V. irradiated PE incubated with strain 707 Carbonyl index: A 1718/ A 1463 The changes in carbonyl groups can be quantified by using an index called the “arbonyl index” which is the ratio between the absorbance of carbonyl at 1718 and the absorbance of CH2 at There is a significant decrease in carbonyl index following the bacterial treatment in samples that were exposed for 80 hr or longer. Maximal decrease was obtained after exposure of 120hr.
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Degradation of CH2 groups
2.5 2920 60 Hr U.V. 2 Absorbance 1.5 1463 1 0.5 4000 3000 2000 1000 2.5 2920 U.V days incubation 2 In addition to the decrease in carbonyl residues we have noticed in few of the samples a remarkable decrease in CH2 groups especially at 2920 which indicates utilization of the PE backbone. Absorbance 1.5 1463 1 0.5 4000 3000 2000 1000 Wavenumber (cm-1)
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Degradation of CH2 groups
U.V. 60 Hr 2.5 1 2 2500 2700 2900 3100 1.5 Absorbance 0.5 U.V days incubation 2.5 2 This is a close look on the CH2 peaks at 2920 of the control and the bacterial treatment. 1.5 Absorbance 1 0.5 3100 2900 2700 2500 Wavenumber (cm-1)
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Biodegradation of UV irradiated polyethylene
1 2 3 4 20000 40000 60000 80000 100000 120000 140000 Incubation time(days) with bacteria after 120 hr UV Molecular weight No UV No bacteria 15-30 45-60 Biodegradation of UV irradiated polyethylene The combination of UV and biodegradation with strain 707 resulted in a reduction in the molecular weight of the PE. However, this reduction was far greater than that of the gravimetric weight Therefore, we have hypothesized that may be this strain is unable to EFFECTIVELY degrade the oligomers that he produced during degradation of the PE.
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Mineral oil degrading bacteria
Time (hr) 4 8 12 16 20 24 28 32 Utilization of mineral oil (%) 40 60 80 100 st.208 st.489 st.490 st.547 st.555 Since MO resembles to short oligomers of PE we have screened and isolated few MO degrading bacteria which we wanted to test their ability to degrade PE. As you can see these strains can utilize very efficiently MO as a sole carbon source.
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Effect of combinations of bacteria on PE degradation.
Weight loss of polyethylene (%) 5 10 15 20 25 30 35 40 I -707 II-707 II I I II Effect of combinations of bacteria on PE degradation. Reduction of gravimetric weight 1st incubation 2nd incubation 1st+2nd To answer the question of the absence of correlation between the reduction in molecular weight and gravimetric weight we have carried out 2 incubation steps involving the mineral oil degrading bacteria: The PE was first incubated with strain 707 for 30 days, the PE was separated, disinfested and incubated again with 2 MO degrading bacteria. As you can see the degradation during the 2nd incubation with MO degrading bacteria was greater than that of 707. We have tested other combinations which showed that maximal degradation is obtained when the 2 incubation steps are done with the MO degrading bacteria.
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Effect of combinations of bacteria on PE degradation
Reduction in molecular weight I -707 II-707 II I I II Reduction in MW (%) 5 10 15 20 25 30 35 1st incubation 2nd incubation 1st+2nd incubation
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Summary Enrichment cultures gave rise to bacterial strains that could utilize PE as a sole carbon and energy source Maximal biodegradation (up to 30%) was obtained after two-steps incubation with combined cultures PE degrading bacteria are hydrophobic and form a biofilm on the PE surface Mineral oil enhances biofilm formation and PE biodegradation The degrading bacteria utilize carbonyl residues in the PE which are formed during UV irradiation Combination of UV photolysis and biodegradation showed a synergistic effect
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Collaborators Irit Gilan Deborah Bitty Dr. Valentina Pavlov
Dr. Mark Karpassas Prof. Shimona Geresh
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