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 30 50 Temperature (C°) Control Bacteria Methodology • Polyethylene: Linear LDPE U.V photosensi • U.V pretreatment: Accelerated w U.V cycles 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 P33- PE P33+PE P34- PE P34+PE 5 10 15 Time (days) 20 FDA hydrolysis by the bacterial biofilm R2 = 0.983 0.2 pieces 0.1 O.D.494 piece R2 = 0.982 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 0 15 30 45 60 75 Incubation time (Days) 90 Hr Hr Hr Hr Low density PE 2.5 2920 2850 1463 1.5 720 0.5 4000 3500 3000 2500 2000 1500 Wavenumber (cm-1) 1000 500 Absorbance Effect of U.V on polyethylene 1.6 1.4 1.2 0.8 1718 0.6 0.4 0.2 1800 1700 1600 1500 1400 Wavenumber (cm-1) 1300 Absorbance 1463 Control 60 Hr U.V 100 Hr U.V 120 Hr U.V PE degradation with strain 707 0.9 U.V 120 Hr U.V + 60 days incubation U.V + 90 days incubation 1463 0.8 1718 0.6 0.5 0.4 0.3 0.2 0.1 1800 1700 1600 1500 Wavenumber (cm-1) 1400 1300 Absorbance 0.7 Carbonyl index A C=O/ A CH Carbonyl index of U.V irradiated PE incubated with strain 707 Incubati on time (days) 0.8 0.7 0.6 15 30 60 0.5 0.4 90 0.3 0.2 0.1 0.0 60 80 100 120 U.V irradiation time (Hr) Carbony l index: A 1718/ A 1463 60 Hr U.V 2920 2.5 1.5 1463 0.5 Absorbance Degradation of CH groups 3000 2000 1000 2.5 2920U.V + 30 days incubation 1.5 1463 0.5 4000 3000 2000 1000 Wavenumber (cm-1) Absorbance 4000 Degradation of CH groups U.V 60 Hr 1.5 0.5 2900 2700 U.V + 30 days incubation 2500 2.5 1.5 0.5 3100 2900 2700 Wavenumber (cm-1) 2500 Absorbance 3100 Absorbance 2.5 Biodegradation of UV irradiated polyethylene Molecular weight 140000 120000 100000 80000 60000 40000 20000 No UV No bacteria 22 15-30 45-60 Incubation time(days) with bacteria after 120 hr UV Utilization of mineral oil (%) Mineral oil degrading bacteria 100 80 60 40 st.208 st.489 st.490 st.547 st.555 20 0 12 16 20 Time (hr) 24 28 32 Weight loss of polyethylene (%) Effect of combinations of bacteria on PE degradation Reduction of gravimetric weight 40 35 30 1st incubation 2nd incubation 1st+2nd 25 20 15 10 I -707 II-707 I -707 II-490+555 I -490+555 II-490+555 I -707+490+555 II-707+490+555 Effect of combinations of bacteria on PE degradation Reduction in molecular weight Reduction in MW (%) 35 30 1st incubation 2nd incubation 1st+2nd incubation 25 20 15 10 I -707 II-707 I -707 II-490+555 I -490+555 I -707+490+555 II-490+555 II-707+490+555 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 Collaborators Irit Gilan Deborah Bitty Dr Valentina Pavlov Dr Mark Karpassas Prof Shimona Geresh [...]... 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... 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... • 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... degrading bacteria 100 80 60 40 st.208 st.489 st.490 st.547 st.555 20 0 0 4 8 12 16 20 Time (hr) 24 28 32 Weight loss of polyethylene (%) Effect of combinations of bacteria on PE degradation Reduction of gravimetric weight 40 35 30 1st incubation 2nd incubation 1st+2nd 25 20 15 10 5 0 I -707 II-707 I -707 II-490+555 I -490+555 II-490+555 I -707+490+555 II-707+490+555 Effect of combinations of bacteria. ..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... 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 Low density PE 2.5 2920 2850 1463 1.5 720 1 0.5 4000 3500 3000 2500 2000 1500 Wavenumber (cm-1) 1000 0 500 Absorbance 2 Effect of U.V on polyethylene 1.6 1.4 1.2 1 0.8 1718 0.6 0.4 0.2... Degradation of CH 2 groups U.V 60 Hr 2 1.5 1 0.5 2900 2700 U.V + 30 days incubation 0 2500 2.5 2 1.5 1 0.5 3100 2900 2700 Wavenumber (cm-1) 0 2500 Absorbance 3100 Absorbance 2.5 Biodegradation of UV irradiated polyethylene Molecular weight 140000 120000 100000 80000 60000 40000 20000 0 No UV No bacteria 1 22 0 15-30 3 45-60 4 Incubation time(days) with bacteria after 120 hr UV Utilization of mineral... 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 ... (cm-1) 1400 0 1300 Absorbance 0.7 Carbonyl index A C=O/ A CH 2 Carbonyl index of U.V irradiated PE incubated with strain 707 Incubati on time (days) 0 0.8 0.7 0.6 15 30 60 0.5 0.4 90 0.3 0.2 0.1 0.0 0 60 80 100 120 U.V irradiation time (Hr) Carbony l index: A 1718/ A 1463 60 Hr U.V 2920 2.5 2 1.5 1 1463 0.5 Absorbance Degradation of CH 2 groups 0 3000 2000 1000 0 2.5 2920U.V + 30 days incubation 2 1.5 1