Presentation on theme: "Colonization, biofilm formation and biodegradation of polyethylene by soil bacteria Alex Sivan The Institute for Applied Biosciences and The Department."— Presentation transcript:
1 Colonization, biofilm formation and biodegradation of polyethylene by soil bacteria Alex SivanThe Institute for Applied Biosciences and The Department of Biotechnology Engineering, Ben Gurion University, Beer Sheva, IsraelPE 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 bacteriaEnvironmental Engineering,
2 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
3 Polyethylene waste 25,000,000 Ton/year Makes up to 40% of the plastic wasteHighly flammable; burning releases toxic gasesPollutes marine and fresh water habitatsHighly recalcitrant; > 400 years for degradationAccording to recent estimates the global amount of PE is……
4 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 wasteis recycled.
5 In order to provide an answer to the polyethylene waste problem 2 alternative polymers have been developped in therecent years.The first photodegradable polyethylene which contains metalchelators that serve as photosensytizers which initiate aphotolytic chain reaction in which the polyethylene breakseventually to CO2 and water.However, this process still takes relatively long time andrequires that the polyethylene will be exposed to sunlight.The second is what is called biodegradable polyethylenewhich is a copolymer of starch and polyethylene. In soil, onlythe starch is degraded and the polyethylene deteriorates tosmall particles or poweder which decreases the volume ofthe waste.
6 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……
7 Control BacteriaIsolates were further screened in liquid cultures containing PE as the sole carbon source in the medium
8 Few words on the methodology. Basically we wanted to combine photo and bio degradation.Polyethylene typeU.V. partial photodegradation in a Q.U.V acceleratedweathering 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.
16 Hydrophobicity of PE - degrading bacteria measured by contact angle Medium pHBacteriumBacillus ± ± ± 3.4Bacillus ± ± ±0.5Rhodococcus rhodochrous 49.2 ± ± ± 2.5…..A greater contact angle indicates higher hydrophobicity
17 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.
18 Here you can see several stages in biofilm formation as affected by MO
19 Biofilm + Mineral oil (16hr) This is a close look at the minearal oil amended biofilmafter 16 hr. And you can see the aggregates of the bacterial cells.
20 Biofilm + Mineral oil (7 days) This is a close look at the minearal oil amended biofilmafter a week where it’s impossible to see individualbacterial cells.
21 Effect of an anionic surfactant and mineral Mix C30Effect of an anionic surfactant and mineraloil on biofilm formation on polyethyleneIn an attempt to improve the attachment of the bacteria tothe polyethylene surface and the biofilm formation we triedto add to the medium various unionic surfactatnts like Tween20, 60, 80 and mineral oil.This slide shows the effect of mineral oil and Tween 60 onbiofilm formation of the initial bacterial mixture. The biofilmdensity was determined using an arbitrary index where 0 = nocolnization and 4 = maximal colonization.As you can see the mineral oil enhances the biofilm formationwhereas no effect was obtained with Tween 60. Other Tweensalso did not improve the colonization of the biofilm.* Biofilm density index: 0 = No biofilm; 4 = Dense biofim
22 Improved biodegradation of polyethylene by mineral oil1020304050Increased weight loss (%)0.020.050.1Mineral oil concentration (%)
23 The activity of the bacterial biofilm could be measured by FDA hydrolysis by extracellular esterases produced by the bacterial biofilm
24 Combined photolysis and biodegradation Polyethylene: Linear LDPE MW 100,000contains UV sensitizerUV pretreatment: Accelerated Weathering Tester (Q.U.V)60 Hr of UV 312nmIt 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
25 Biodegradation of polyethylene by strain 707 9075604530155102060 Hr80 Hr100 Hr120 HrBiodegradation of polyethylene by strain 707Incubation time (Days)Weight Loss (%)U.V. exposure* 50C
26 Low density PE Absorbance Wavenumber (cm-1) 0.5 1 1.5 2 2.5 500 1000 0.511.522.55001000150020002500300035004000Wavenumber (cm-1)Absorbance292028501463720
27 Effect of U.V. on polyethylene 0.20.40.60.8220.127.116.1130014001500160017001800Wavenumber (cm-1)Absorbance17181463Control120 Hr U.V.60 Hr U.V.100 Hr U.V.
28 PE degradation with strain 707 0.10.20.30.18.104.22.168.80.9130014001500160017001800Wavenumber (cm-1)AbsorbanceU.V. 120 HrU.V days incubationU.V days incubation17181463
29 Carbonyl index of U.V. irradiated PE incubated with strain 707 60801001200.00.10.20.22.214.171.124.70.8153090U.V. irradiation time (Hr)Carbonyl index A C=O/A CH2Incubation time (days)Carbonyl index of U.V. irradiated PEincubated with strain 707Carbonyl index: A 1718/ A 1463The changes in carbonyl groups can be quantified by using an index called the “arbonyl index” which is the ratiobetween the absorbance of carbonyl at 1718 and the absorbance of CH2 atThere is a significant decrease in carbonyl index followingthe bacterial treatment in samples that were exposed for80 hr or longer. Maximal decrease was obtained afterexposure of 120hr.
30 Degradation of CH2 groups 2.5292060 Hr U.V.2Absorbance1.5146310.540003000200010002.52920U.V days incubation2In addition to the decrease in carbonyl residues we have noticed in few of the samples a remarkable decrease in CH2 groups especiallyat 2920 which indicates utilization of the PE backbone.Absorbance1.5146310.54000300020001000Wavenumber (cm-1)
31 Degradation of CH2 groups U.V. 60 Hr2.51225002700290031001.5Absorbance0.5U.V days incubation2.52This is a close look on the CH2 peaks at 2920 of the control and the bacterial treatment.1.5Absorbance10.53100290027002500Wavenumber (cm-1)
32 Biodegradation of UV irradiated polyethylene 123420000400006000080000100000120000140000Incubation time(days) with bacteria after 120 hr UVMolecular weightNo UVNo bacteria15-3045-60Biodegradation of UV irradiated polyethyleneThe 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 weightTherefore, we have hypothesized that may be this strain is unable to EFFECTIVELY degrade the oligomers that he produced during degradation of the PE.
33 Mineral oil degrading bacteria Time (hr)48121620242832Utilization of mineral oil (%)406080100st.208st.489st.490st.547st.555Since 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.
34 Effect of combinations of bacteria on PE degradation. Weight loss of polyethylene (%)510152025303540I -707II-707IIIIIIEffect of combinations of bacteria on PE degradation.Reduction of gravimetric weight1st incubation2nd incubation1st+2ndTo 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.
35 Effect of combinations of bacteria on PE degradation Reduction in molecular weightI -707II-707IIIIIIReduction in MW (%)51015202530351st incubation2nd incubation1st+2nd incubation
36 SummaryEnrichment cultures gave rise to bacterial strains that could utilize PE as a sole carbon and energy sourceMaximal biodegradation (up to 30%) was obtained after two-steps incubation with combined culturesPE degrading bacteria are hydrophobic and form a biofilm on the PE surfaceMineral oil enhances biofilm formation and PE biodegradationThe degrading bacteria utilize carbonyl residues in the PE which are formed during UV irradiationCombination of UV photolysis and biodegradation showed a synergistic effect
37 Collaborators Irit Gilan Deborah Bitty Dr. Valentina Pavlov Dr. Mark KarpassasProf. Shimona Geresh