scientific findings on full biodegradability in soil...165 of the aliphatic polyesters (sigma/62305)...
TRANSCRIPT
The new European Standard BS EN 17033 for biodegradable mulch films Scientific findings on full biodegradability in soil
London, 21/11/18
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Use of mulch films in agriculture and horticulture: – Functions and benefits: Soil mulching enhances growing conditions and contributes to increased yields and improved crop quality by:
▪ Earlier planting dates
▪ Earlier harvesting dates
▪ Inhibiting the development of weeds
▪ Soil moisture retention
▪ Reduction in leaching of mineral elements and other fertilizer
▪ Protecting leaves and fruits against soil-borne diseases
▪ Protecting the crops from soil
▪ Reduction in soil compaction
► The improvement growing conditions are the same for biodegradable mulch films as those for conventional mulch films
04-Aug-2017 Glauco Battagliarin (BASF SE); Olivier de Beaurepaire (BASF – France); Peter Reuschenbach (BASF SE)
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Disposal of mulch films at the end of the intended service life: – Material recycling or biodegradation?
► Thin films with high soil „attachment rate“ hinder used film collection ► Recommended thickness to enable conventional mulch film collection and
recycling is ≥25 µm
► Reduction of operational costs by using biodegradable mulch films References:
- P. de Lepinau and A. Arbenz. Economic and environmental impact of soil contamination in mulching films. Plasticulture. 39-47. No 135. 2016. - prEN 13655:2016 – Plastics – Thermoplastic mulch films recoverable after use, for use in agriculture and horticulture (2016-09)
Standard for Mulch Film Recycling in the UK - BS EN 13655:2018 Recommended film thickness: 25 µm
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Disposal of mulch films at the end of the intended service life: – Material recycling or biodegradation? ► Disposal of used agricultural plastic in the EU:
► Most agricultural films are not regularly collected and recovered due to the high „attachment rate“ of up to 80%
04-Aug-2017 Glauco Battagliarin (BASF SE); Olivier de Beaurepaire (BASF – France); Peter Reuschenbach (BASF SE)
Source: EPRO
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ecovio® M2351 mulch – Biodegradation in soil according to ISO 17556
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0 50 100 150 200 250 300
Bio
degr
adat
ion
/ %
Time / days
Biodegradation of ecovio® M2351 mulch film relative to cellulose control
à At 181 days an absolute biodegradation of 94.4% (±1.7%) was measured = 89.1% relative to cellulose
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BS EN 17033:2018 “Biodegradable mulch films for use in agriculture and horticulture – requirements and test methods“
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Control of constituents Biodegradability
Dimensional, mechanical and optical properties Ecotoxicity
EN 17033
Transfer into certification schemes
New working item proposal to ISO
21/11/18
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What is ecovio®?
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ecovio® is a compound consisting of:
• Biodegradable and (partly bio based) BASF-Polyester ecoflex®
• Bio based polylactic acid (PLA)
• [Mineral fillers ]
PLA +
Filler
+
sunlight
CO2 plants starch lacticacid PLA
compoundingecovio®
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Let‘s talk about end of life Basics and Standards
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polymer oligomer monomers & other small molecules
CO2
incorporation into biomass
bulk material (e.g. mulch
film)
degradation & transformation
microbial photochemical abiotic
n Mineralization by natural organisms to CO2 and microbial biomass
n CO2 is indicator for biodegradability measurement
n 10% of carbon is estimated to go into biomass, 90% of carbon goes in CO2
1 1 OWSnv (2016) EXPERT STATEMENT (BIO)DEGRADABLE MULCHING FILMS. (European Bioplastics e.V., http://www.european-bioplastics.org/news/publications/).
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Basic understanding and field evaluation are both needed to understand biodegradability
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Elucidating structure- biodegradability relationship
microbialprofiling
enzymecharacterization
cultivation
Polymer characteristics
Microorganisms and enzymes
Abiotic factors
Fundamental understanding Field evaluation Assessing product performance
in field trials
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What is ecoflex®?
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Terephtalic acid Butanediol Adipic acid
3 Monomers together build a long chain polymer
ester bond
Terephtalic acid
Butanediol Adipic acid Butanediol
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Process of biodegradation of PBAT
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watersoluble fragments
biodegradable polymer (e.g. ecoflex®) microbes
1. Microbial colonization of the surface and excretion of enzymes (e.g. cutinases)
2. Enzymatic hydrolysis of ester bonds
3. Release of water soluble fragments
4. Uptake and metabolization by microbes
CO2 H2O
ester bond
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extracellular enzymes
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1. Microbial colonization Polyester (PBAT) in agricultural soil
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negative control
unicellular organisms
fungal hyphae
surface & deptherosion
Laboratory experimentsIncubations in agricultural soilà 6 weeks @ 25°CO
O
O
O
O
O
OO
m n
2 µm
intact film surface
Scanning electron microscopy images
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Process of biodegradation of PBAT
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watersoluble fragments
biodegradable polymer (e.g. ecoflex®) microbes
1. Microbial colonization of the surface and excretion of enzymes (e.g. cutinases)
2. Enzymatic hydrolysis of ester bonds
3. Release of water soluble fragments
4. Uptake and metabolization by microbes
CO2 H2O
ester bond
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extracellular enzymes
Where does the carbon end up? Does ecovio® M fully biodegrade?
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4. Microbial metabolism How to show the biomass formation?
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https://youtu.be/_ii1uyRHXOE
è By tracing marked carbon in the polymer!
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4. Microbial metabolism How to show the biomass formation?
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Labelled carbon
Polymer with labelled carbon
Fungal hypha and bacteria
Enzyme
Water soluble fragment with labelled carbon
Fungal hypha and bacteria with labelled biomass
1. Microbial colonization of the surface and excretion of enzymes (e.g. cutinases)
2. + 3 Enzymatic hydrolysis of ester bonds and release of watersoluble fragments
4. Uptake and metabolization by microbes èFormation of biomass from labelled carbon
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4. Microbial metabolism Conversion into microbial biomass
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negative control poly(butylene adipate-co-terephthalate)PBAT: labeled in adipate
Primary ion beam (Cs+)
Secondary ions
Mass spectrometer(12C14N; 12C13C)
Nanoscale secondary ion mass spectrometry (NanoSIMS)
O
O
O
O
O
O
OO
m n* *
Seco
ndar
y el
ectro
ns12
C14
N -
ions
Zumstein et al., Science Advances, Biodegradation of synthetic polymers in soils: Tracking carbon into CO2 and microbial biomass, 2018
13C / (12C + 13C) (%)13C atom percent
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4. Microbial characterization Microflora is a dominating factor
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ecovio
isolation from soil
direct isolation from partially
degraded polymer
characterization
DNA isolation & community
analysis
cultivation
1. 2.
3.
Exemplary soil organisms (not necessarily ecovio degraders)
èWho is eating our product?
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4. Microbial characterization Tracing the label in DNA
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12C DNA
13C DNA
fully labeled control
unlabeled control
13C-labeled polymer
12C-DNA 13C-DNA
0%
20%
40%
60%
80%
100%
0 50 100 150
Bio
degr
adat
ion
[%]
Incubation time [d]
DNA - Stable Isotope Probing (13C DNA-SIP) 13C polymer + soil
Incubation, extraction of total DNA
(12C & 13C strands) Separation via
ultracentrifugation
light (12C)
heavy (13C) Fractionation Labeled
metagenome
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Cooperation ETH Zürich and BASF on biodegradation in soil
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Biodegradation of Polyesters in Agricultural SoilsRebekka Baumgartner1, Michael T. Zumstein1, Taylor Nelson1, Melissa Maurer-
Jones1, Hans-Peter Kohler2, Kristopher McNeill1, Michael Sander1
Glauco Battagliarin3, Katharina Schlegel3, Carsten Sinkel3, Ulf Küper3
1. Swiss Federal Institute of Technology (ETH), Department of Environmental Systems Science, Environmental Chemistry, Zurich, Switzerland
2. Swiss Federal Institute of Aquatic Science and Technology (Eawag), Environmental Biochemistry, Dübendorf, Switzerland
3. Advanced Materials & Systems Research – Biopolymers, BASF, Ludwigshafen, Germany
à Landmarking cooperation for sustainable chemistry
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Recommendation for Policy Makers
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Biodegradation of Polyesters in Agricultural Soils
à All agricultural films marketed as “degradable” or “biodegradable” to meet British standard BS EN 17033:2018
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4. Microbial characterization Tracing the label in DNA
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Bacteria Fungi (ITS region)
PBS = poly(butylene succinate)
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Process of biodegradation of PBAT
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watersoluble fragments
biodegradable polymer (e.g. ecoflex®)
extracellular enzymes
microbes
1. Microbial colonization of the surface and excretion of enzymes (e.g. cutinases)
2. Enzymatic hydrolysis of ester bonds
3. Release of water soluble fragments
4. Uptake and metabolization by microbes
CO2 H2O
ester bond
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2. Enzymatic hydrolysis Polyester (PBAT) hydrolysis
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negative controlPBATx with x= T/(T+A)
PBAT0 B-A-B-A
PBAT50 B-A-B-T
PBAT10 PBAT20 PBAT29 PBAT42
Quartz Crystal microbalance with dissipation monitoring (QCM-D)
80 n
m
Zumstein et al., Environ. Sci. Technol., 2017, 51, 7476-7485
161 formation). The hydrolysis of the aliphatic-aromatic copolyest-
f2f3 162 ers (data in Figures 2 and 3) by FsC and RoL was investigated
163 at an activity of 16.2 × 10−9 kat/mL for both enzymes. We note
164 that we employed two different RoL batches for the hydrolysis
165 of the aliphatic polyesters (Sigma/62305) and aliphatic-
166 aromatic copolyesters (Sigma/80612). The use of two RoL
167 batches and different enzyme concentrations impairs a direct168 comparison of the hydrolysis data in Figures 1, 2, and 3.
169Polyester Hydrolysis. We used a QCM-D E4 system (Q-170Sense, Sweden) with four flow cells to follow the enzymatic171hydrolysis of polyester thin films coated onto QCM-D172sensors.32 QCM-D is a piezoacoustic resonator technique that173monitors the changes in the resonance frequencies, Δf i (Hz),174and the energy dissipations, ΔDi, of a piezoquartz crystal175embedded into the QCM-D sensor. During the hydrolysis176experiment, the fundamental tone (i = 1) as well as six177oscillation overtones (i = odd numbers between 3 and 13) were
Figure 2. Hydrolysis of poly(butylene adipate coterephthalate) (PBATx) thin films by Fusarium solani cutinase (FsC) and Rhizopus oryzae lipase(RoL) at pH 6, as measured with a quartz-crystal microbalance with dissipation monitoring (QCM-D). The different PBATx polyesters varied in themolar fraction of the aromatic diacid terephthalate (T) to the aliphatic diacid adipate (A) (i.e., x = T/(A + T) × 100). (a, b) Changes in the adlayermass during the hydrolysis of PBATx thin films by FsC (a) and RoL (b) at 30 °C. Enzymes were added at time t = 0 h, as indicated by the verticaldashed lines. The insets show the adlayer mass progress curves within the first hour of carboxylesterase addition. (c) Fraction (%) of initially coateddry polyester mass that was removed during the hydrolysis experiments. (d) Estimated rates for the hydrolysis of PBATx films by FsC at threedifferent experimental temperatures. Error bars in panels c and d represent deviations of duplicate measurements from their mean.
Figure 3. Hydrolysis of films of poly(butylene adipate) (PBAT0 = PBC6) (a) and poly(butylene adipate coterephthalate) with a terephthalatecontent of 50% relative to the total diacid content (i.e., PBAT50, b) by a set of carboxylesterases (abbreviations of enzymes shown next to curves; seeTable 1 for full names and details) at pH 6 and 30 °C as investigated by quartz-crystal microbalance with dissipation monitoring (QCM-D)measurements. For clarity, we show one representative measurement of duplicates for each enzyme−polyester combination.
Environmental Science & Technology Article
DOI: 10.1021/acs.est.7b01330Environ. Sci. Technol. XXXX, XXX, XXX−XXX
D
faster
Fusarium solani cutinase
slower
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4. Microbial characterization Phylogeny of primary disintegrating microbes
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n Isolation of microorganisms directly from partial degraded polymer films (à more than 400 isolates, esp. fungi)
è Fungi have been identified to be the most potent but not exclusively degrading microorganisms in soil
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EN 17033: „Biodegradable mulch films for use in agriculture and horticulture – requirements and test methods“
26
Control of constituents Biodegradability
Dimensional, mechanical and optical properties Ecotoxicity
EN 17033
Transfer into certification schemes
New working item proposal to ISO
21/11/18