Incorporation of viable micro-organisms in a plastic product

Abstract

Disclosed is a method for the manufacture of an article by thermoplastic processing of a thermoplastic composition comprising one or more polymers and viable spores of one or more spore-forming micro-organisms, the method comprising the steps of (1) preparing the thermoplastic composition by mixing one or more polymers with viable spores of one or more micro-organisms; and (2) thermoplastic processing the thermoplastic composition at a temperature of 100 - 350°C and a pressure of 1.5 – 400 bar; wherein the thermoplastic composition further comprises 0.5 – 20 w / w%, based on the polymeric matrix material, of a viscosity modulator selected from the group consisting of an oil, fat, fatty acids and polyalkane glycol, the thermoplastic composition being in liquid form at the conditions of step (2). Further disclosed are a themoplastic composition, an article prepared by the method, and a micro-organism.

Description

[0001] Incorporation of viable micro-organisms in a plastic product

[0002] The invention relates to a method for the manufacture of an article by thermoplastic processing of a thermoplastic composition comprising a thermoplastic polymeric matrix material and viable spores of one or more spore-forming micro-organisms, to such a thermoplastic composition, to an article prepared by the said method, to a micro-organism and to a method for degradation of such an article.

[0003] In the art, there is a desire to incorporate micro-organisms in articles, produced by thermoplastic processing. Micro-organisms confer unique properties to such articles, in particular enzymatic or metabolic activity, resulting in the conversion of one or more substances that are present in the environment of the said article, or within the article itself. Materials comprising micro-organisms therein are also referred herein as bioaugmented materials. However, thermoplastic processing is known to take place at conditions that are prohibitive to the viability of micro-organisms.

[0004] WO2010 / 034776 describes a method for the preparation of such an article, wherein a sporeforming bacterium is incorporated in a thermoplastic composition for the production of a plastic container for a liquid. The spores present in the plastic material would germinate as a result of contact with the liquid in the container and start to metabolize using any oxygen present in the container, so as to avoid oxidation of the contents of the said container. However, the teaching of WO2010 / 034776 is a mere desideratum without any practical utility. For instance, the only examples given in WO2010 / 034776 wherein micro-organisms are incorporated in a plastic material involve providing two plates of polyethylene terephthalate glycol (PETG) and pressing spores of a specific spore-forming bacterium between the said plates. After the plates are pressed together to a single product, the spores are isolated and the viability thereof was evaluated. However, the preparation of a plastic wherein the micro-organisms are evenly mixed is not described, nor any effect evolving from the bacteria after being incorporated in the plastic.

[0005] Indeed, it was found that a mere mixing of microbial spores with a polymeric matrix material does not result in significant incorporation of viable spores in the obtained product. It was found that the spores are vulnerable to the forces, exerted within the polymeric composition during the thermoplastic processing, resulting in deterioration of the spores and loss of viability. Thus, there is a need for a method for the preparation of an article by thermoplastic processing of a thermoplastic composition comprising spores, wherein deterioration of the spores and loss of viability of the spores during processing is reduced.

[0006] The present invention provides for such a method. It has now been found that a viability of 50% or more can be obtained when manufacturing an article by thermoplastic processing of a thermoplastic composition comprising spores of a spore-forming micro-organism and one or more polymers if a viscosity modulator is incorporated in the thermoplastic composition. The inventors found that incorporation of the viscosity modulator in the thermoplastic composition not only resulted in good dispersion of the spores in the polymeric matrix of the article but also leaded to an improved percentage of viable spores in the article. Thus, incorporation of a viscosity modulator in a thermoplastic composition comprising one or more polymers and spores of one or more spore-forming micro-organisms reduces deterioration and loss of viability of the spores during thermoplastic processing to produce an article, thereby leading to improved biodegradability of the obtained article. The method and thermoplastic composition of the invention thus allow the production of articles with improved biodegradability.

[0007] The present invention provides for the manufacture of an article by thermoplastic processing of a thermoplastic composition comprising spores of a spore-forming micro-organism and one or more polymers, said method comprising the following steps:

[0008] (1) preparing the thermoplastic composition by mixing one or more polymers with viable spores of one or more micro-organisms;

[0009] (2) thermoplastic processing the thermoplastic composition at a temperature of 100 - 350°C and a pressure of 1.5 and 400 bar; wherein the thermoplastic composition further comprises a viscosity modulator, capable of lowering the viscosity of the thermoplastic composition at the thermoplastic processing conditions of step (2).

[0010] As will be evident to the skilled person, the one or more polymers in the thermoplastic composition will form a polymeric matrix in the article produced in step (2). The method of the invention thus produces an article comprising thermoplastic polymeric matrix materials and spores of a spore-forming micro-organism, wherein the spores are distributed in the thermoplastic polymeric matrix of the article.

[0011] Without being bound by theory, it is thought that by t incorporating the viscosity modulator, the thermoplastic composition will be less viscous at operating conditions, i.e., the temperature and pressure used during the thermoforming process, resulting in maintenance of the viability of the spores during the process, providing an article wherein at least 50% of the spores as mixed in the thermoplastic composition remain viable in the said article, preferably at least 60% or more.

[0012] The skilled person will be aware of suitable viscosity modulators. The skilled person is aware that the viscosity modulator should be liquid at the thermoplastic processing conditions in order to exert its viscosity modulating activity to arrive at a viscosity reduction of the thermoplastic composition at the said conditions. The skilled person will also be able to find the proper amount of viscosity modulator to be added. Less modulator is needed when the viscosity of the thermoplastic composition is lower as compared with a thermoplastic composition having a higher viscosity. The skilled person will also understand that viscosity modulators that are toxic to a particular micro-organism to be incorporated is not suitable when the said micro-organism is present in the composition. The skilled person can easily test the viability of the spores in the article as e.g. is described in WO2010 / 034776.

[0013] The viscosity modulator is preferably chosen from the group, consisting of fats, fatty-acids, oils, and polyalkane glycols, such as polyethylene glycol. However, oils and fats derived from a natural source, in particular a vegetable source, are preferred. Such vegetable materials are nontoxic, environmental friendly and can also be a carbon source for the micro-organisms after germination of the spores. A particular preferred viscosity modulator comprises a vegetable oil, preferably sunflower oil, more preferably high-oleic sunflower oil, i.e. having an oleic content of about 82 w / w%.

[0014] The thermoplastic composition preferably comprises 0.5 - 20 w / w%, based on the polymeric matrix material, of the viscosity modulator. Above the said range, an excess of oil may tend to form a separate phase. The thermoplastic composition preferably comprises 1 .0 - 20 w / w%, or 5 - 20 w / w%, or 10 - 20 w / w%, even more preferably 15 - 20 w / w% of the viscosity modulator, based on the polymeric matrix material.

[0015] In an attractive embodiment, the thermoplastic composition comprises 106- 1013spores per kg thermoplastic composition, preferably 109- 1012spores per kg thermoplastic composition. This correlates to 0,01 - 10 w / w%, preferably 0,1 - 10 w / w%, more preferably 0,1 - 5,0 w / w / % spores based on the one or more polymers. In a particular embodiment, the thermoplastic composition may comprise 0,1 - 2,0 w / w% spores based on the one or more polymers.

[0016] The viability of the spores can be increased by having the water content in the thermoplastic composition as low as possible. To this end, the constituents of the thermoplastic composition or the thermoplastic composition can be subjected to a drying step, to provide for dry conditions of 5000 ppm water or less.

[0017] The spore-forming micro-organism is preferably a bacterium or a fungus, in particular chosen from one or more of the bacterial genus Bacillus, Brevibacillus, Actinomadura, Amycolatopsis, Alcaligenes, Paenibacillus, Thermopolyspora, and of the fungal genus: Cladosporium, Cryptococcus, Fusarium, Penicillium, Rhizopus, Rhodotorula, Trichoderma. The spore-forming micro-organism preferably is selected from the bacterial genus Bacillus, preferably from the species Bacillus subtilis. Bacillus subtilis is particularly preferred as spore-forming micro-organism because it comprises suitable hydrolase enzymes, capable of hydrolysing the ester bonds of a polyester matrix.

[0018] The micro-organism preferably has a salt tolerance of at least 3.5 w / v%, in another embodiment, the micro-organism has a hydrolase activity, in particular esterase activity, and most preferably the said hydrolase activity is present at such high salt conditions of 3.5 w / v% in the medium. Such micro-organisms are viable at seawater conditions and capable of degrading polyesters present in the sea as pollutants. In a particular suitable micro-organism is Bacillus subtilis PHBS0007 as deposited at the Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584CT Utrecht, the Netherlands, on December 1 , 2023 under access number CBS150835. This particular micro-organism had been bred and selected to be viable at seawater conditions and capable of degrading polyesters at a salt concentration of 3.5 w / v%.

[0019] The skilled person is aware of polymers that can be used to form the suitable thermoplastic polymeric matrix materials. The one or more polymers of the thermoplastic composition can be selected from natural or synthetic polymers. Preferably, the one or more polymer is a synthetic polymer. More preferably, the one or more polymer is selected from the group consisting of the polyesters and polyurethanes and polyvinyl acetate (PVAc) .

[0020] Preferred polyesters are polylactic acid (PLA), polyhydroxyl alkanoates (PHA), polyethylene terephthalate (PET), polybutylene succinate (PBS), polycaprolactone (PCL), polybutylene adipate terephthalate (PBAT), and mixtures thereof.

[0021] Preferred polyurethanes are polyurethane (PU).

[0022] In a particular embodiment, the one or more polymers are selected from the group consisting of PLA, PHA, PET, PBS, PCL, PBAT, PU, and PVAc.

[0023] Polyesters as e.g. described above are suitable materials for the preparation of products that are envisaged to be degraded by the micro-organisms incorporated therein, e.g. by the action of hydrolases originating from the said micro-organism that hydrolyse the ester bonds of the polymeric matrix. Such hydrolases can e.g. be one or more of proteases, lipases, esterases etc. with affinity to the polymeric matrix material. In a preferred embodiment, the polymeric matrix material preferably comprises one or more polyesters, preferably polylactic acid. In case the article needs to be sustained and not degraded, other non-degradable polymers may be added to the thermoplastic composition of the invention. Attractive examples of such non-degradable polymers are polystyrene (PS), polypropylene (PP), polyvinylchloride (PVC, polyvinylidene (PV), polycarbonate (PC), acrylonitrile butadiene styrene (ABS) and polyethylene (PE).

[0024] It was found that the germination, revival and survival of the micro-organisms is significantly increased when the thermoplastic composition further comprises a hygroscopic agent. The hygroscopic agent will attract water into the polymeric matrix as soon as the article comes in contact with water. Such water contact can be a trigger for germination and growth of the microorganisms. The skilled person is aware of suitable hygroscopic agents. Again, the skilled person is aware not to use a hygroscopic agent that is toxic for the envisaged micro-organism to incorporate in the polymeric matrix. Preferably the hygroscopic agent comprises a powder material originating from a natural origin, preferably selected from flour, starch or cellulose. Other hygroscopic agents could be wood powder, powdered potato peel, dry vegetable leaf powder etc. The thermoplastic composition preferably comprises 5 - 30 w / w%, based on the polymeric matrix material, more preferably 5 - 25 w / w%, even more preferably 10 - 20 w / w% of the hygroscopic agent(s).

[0025] The article is preferably a non-food article, i.e., comprising non-edible thermoplastic polymeric matrix material that can be used in articles for construction, substrate material for the micro-organisms incorporated therein, or activation or cleaning articles to be put in a particular environment where the micro-organisms grow and metabolize and may neutralise undesired substances by enzymatic conversion or metabolization. Incorporation in a food product can however also be envisaged as e.g. a probiotic.

[0026] In a very attractive embodiment, the thermoplastic composition further comprises one or more nutrients, chosen from a carbohydrate source, a phosphate source and a nitrogen source. Another reason for the failure of fruitful incorporation of viable micro-organisms in an article to be produced by thermoplastic processing at a temperature of 100 - 350°C and optionally a pressure of 1 ,5 - 400 bar is the lack of nutrients in the thermoplastic composition. Indeed, the spores may survive the harsh thermoplastic processing conditions, but will eventually fail to germinate / revive for lack of the presence of sufficient nutrients. So, in case the article is to be used under conditions where there are insufficient nutrients in the environment, the nutrients are attractively provided as being incorporated in the matrix material, in terms of quantity, diversity and quality. As explained above, in case an oil or a fat from a natural source is used as viscosity modulator, said oil or fat can function as carbon source for the micro-organisms after germination. Preferably, a phosphate source and a nitrogen source are provided in the matrix as well. The skilled person is aware of suitable compounds and the necessary amounts, e.g. 0.01 - 5 w / w%, or 0.5 - 5 w / w% based on the polymeric matrix material for each source.

[0027] Accordingly, the thermoplastic composition preferably further comprises one or more trace elements such as e.g. magnesium sulphate and the like, preferably in an amount of 0.008 - 2 w / w / %, or 0.01 - 2 w / w%, or 0.1 - 2 w / w% based on the polymeric matrix material.

[0028] Also, the thermoplastic composition preferably further comprises a protein source, in particular a protein hydrolysate, preferably in an amount of 0.01 - 1 w / w% based on the polymeric matrix material. Examples of such protein sources are tryptone and peptone. Tryptone and peptone are also suitable phosphor and nitrogen sources.

[0029] Thermoplastic processing techniques are known to the skilled person, and any thermoplastic process can be applied in the method of the invention. Preferred processes are chosen from extrusion, 3D printing, injection moulding, thermoforming, blow moulding and foaming, as long as the process temperature is within the range of 100 - 350°C, preferably 140 - 220°C, more preferably 150 - 200°C. The pressure during the process is preferably 1.5 - 400 bar, preferably 1.5 - 50 bar.

[0030] In case of extrusion, the extruder preferably has multiple temperature zones, wherein above 120°C, the temperature increase in the subsequent downstream temperature zone preferably does not exceed 20°C.

[0031] The invention also relates to a thermoplastic composition comprising a viscosity modulator as described herein, and to an article, prepared by the method or with the thermoplastic composition as described herein

[0032] The invention also relates to the Bacillus subtilis strain Bacillus subtilis PHBS0007 as deposited at the Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584CT Utrecht, the Netherlands, on December 1 , 2023 under access number CBS150835.

[0033] The invention will now be further exemplified by the following examples. The percentages are in weight percent (w / w%), based on the weight of the polymeric matrix material, unless indicated otherwise.

[0034] LEGEND OF THE FIGURES

[0035] Fig. 1 depicts the germination percentage of different isolates

[0036] Fig. 2 shows a qNMR plot

[0037] Fig. 3 shows a box-plot PLA degradation analysis with qNMR

[0038] Fig. 4 Microbe survival in PLA with different additives, showing the growth curve

[0039] Fig. 5 is a bar graph showing the total growth per treatment (OD600) after 60 hrs.

[0040] Fig. 6 is a flow-diagram of Example 7

[0041] EXAMPLES

[0042] Materials:

[0043] Yeast extract: Yeast Extract - Thermo Scientific Chemicals - China

[0044] MgSO4'7H2O: Sigma - Aldrich - Japan

[0045] Starch: Biological Corn Starch - VehGro - Austria

[0046] Tryptone: Tryptone (vegetable) - Sigma Aldrich I MERCK - India

[0047] High oleic sunflower oil: Wokolie - Ekoplaza - The Netherlands agar: Fisher BioReagents™ Agar, Powder I Flakes - Fisher Bioreagents - Mexico polylactic acid: RENEW 804 - Futerro - China

[0048] Bleach solution: CLEAN Think Bleach - Evora, Renswoude, Netherlands

[0049] Chloroform: Reagent ACS, 99.8+%, Thermo Scientific Chemicals, Switzerland

[0050] Chloroform-d for NMR: 99.8% atom D, containing 1 v / v% TMS, Thermo Scientific Chemicals DCM: Dichloromethane, for HPLC, Stabilised with Amylene, Fisher Scientific Chemical, Germany

[0051] PEG400: CARBOWAX™ PEG400E - Labshop - China

[0052] Betain-ABB: Chempri - United Kingdom

[0053] Acetone 99.5+%: FisherScientific, Alfa Aesar, Germany

[0054] 702 medium: 10 g / l of vegetable tryptone, 2 g / l of yeast extract, 1 g / l MgSO4'7H2O 702+ medium: 702 medium including 35 g / l of NaCI.

[0055] 802 medium: 702 medium including 15 g / l agar

[0056] 712 medium: 702 medium without yeast extract

[0057] Example 1 - Isolation of spore-forming micro-organisms having hydrolase activity

[0058] A. Sampling

[0059] With the use literature and online sources a virtual map was developed using Google Earth Pro. The locations includes PLA production sites, PLA processing places and PLA application locations. In addition some beaches of Texel (NL) and Lampedusa (IT). In these locations soil and water samples are taken in 50ml sterile centrifuge tubes. Ideally the samples contained fragments of plastic and were no lower than 2 cm below the soil surface

[0060] B. Selection

[0061] From each sample about 10 grams (soil) or 10 ml (water) was added to 20 ml of sterile 702 medium and 20 ml sterilized demi-water, and incubated at 32°C with ample shaking for 7 days. During this period, any microbe present in the sample is allowed to grow, and due to nutrient depletion, spore formation starts.

[0062] Following the incubation the samples were thoroughly shaken (250 rotation / minute). Then a suitable dilution series created. For example: from each sample tube, 500 pl of sample was transferred into a 1.5 ml centrifuge tube, 500 pl sterilized water was admixed. Then 500 pl of this tube was taken and transferred in to a fresh tube with the admixture of 500 pl sterilized water. These steps were repeated to 20 dilutions. Then for each dilution, 5 pl is placed on a 802 medium agar-plate and incubated at 32°C for 14h. After this incubation step, the microorganisms were transferred to new 802 medium agar-plates. These plates were incubated until colonies were sufficiently developed. With the use of microscopy the various microbes could be separated.

[0063] To test the polyester degrading capabilities of the previously obtained isolated microbes, the microbes were further plated on polyester emulsion plates. The plates were produced following the guidance of Fransen et al., (2023), PNAS 120(23), https: / / doi.org / 10.1073 / pnas.2220021120. First, 40 mg virgin polymer of interest were mixed with 0.8 ml dichoromethane and shaken until the polymer was completely dissolved. An agar solution (20 ml demi-water, 0.6 g agar) and a medium solution (0.4 g tryptone, 0.08 g yeast extract, 0.04 g MgSCU, 0.7 g NaCI, 20 ml demi-water) were prepared using the quick-program (134 °C, 2.1 bar, 5.5 min, no drying). The solutions were kept in a water bath at 60 °C. 20 ml agar solution were mixed with 16 ml medium solution in a sterile 50 ml tube. 4 ml medium solution were added to the dissolved polymer and the tube was vigorously shaken before quickly transferring the medium-polymer solution into the warm medium-agar solution. The DCM was allowed to evaporate for 30 s before an ultrasonic probe was placed in the tube. Ultrasonification was performed with a Hielscher LIP100H ultrasonicator (10 min / 30 % amplitude / 15 s pulses) before plates were poured.

[0064] After incubation at 32°C, clear zones formed around degrading colonies, but not around non-degrading colonies. The colonies without a halo do not degrade the polyester, and have insufficient enzyme activity. The micro-organisms with halo were selected and further used to provide a spore-forming micro-organism having enzyme activity, necessary for polyester degradation. Bacterial isolates 7 and 9 were used in the following examples.

[0065] Example 2: Preparation of bioaugmented biodegradable material

[0066] A. Cultivation

[0067] A bacterial isolate, or Bacillus subtilis PHBS0007 was inoculated in sterilized 702 medium [10 g / l Tryptone; 2 g / l yeast extract; 1 g / l MgSOt] to an ODeoo of -0.06 and incubated for 48 hours at 35°C;

[0068] B. Harvest of spores

[0069] After reaching a culture density of an ODeoo of 1 .2 or more, the culture was checked for contaminations and presence of spores under the microscope and the culture medium was centrifuged (Eppendorf 5430R, program 5 (7190 RCF 15 min / 24°C).

[0070] C. Spore solution

[0071] The pelletized spores were re-suspended in 20 ml supernatant using the shaker and 5 grams of starch, mixed until homogenous. For later use, the mixture was poured in sterile Petri dishes, and open Petri dishes were dried in a drying oven for 12 h at 60°C.

[0072] D. Preparation of the thermoplastic composition

[0073] The dried spores of Example 2C were grinded using a manual wheat mill (Hawos, Bad Homburg, Germany) and a dry mixture of 12.5 w / w% tryptone, 1.2 w / w % MgSO4'7H2O and 20 w / w% starch was mixed in a weight ratio of dried spores to dry mixture of 5 : 95. The obtained microbial powder was mixed with the high oleic sunflower oil in a weight ratio powder to high oleic sunflower oil of 5 : 11..

[0074] E. Extrusion preparations

[0075] Before extrusion (filament extruder of 3devo, Utrecht, the Netherlands) fit the machine with a die of 4 mm and the screw with at the end a mixing element; the PLA was dried for at least 4 h (preferable 12h) at 70°C with an airflow of at least 0,5 l / s per kg resin or at least lower than 1 ,0% water content. F. Extrusion Production

[0076] A filament extruder of 3devo, Utrecht, the Netherlands, adjusted to the following PLA manufacture specifications to extrude 400 grams of PLA:

[0077] After the filaments reached a diameter of 2.7 - 3.0 mm, the pump adds the starch-oil mixture as prepared in example 2D in a 15 w / w% ratio to 400 grams of PLA into the hopper.

[0078] After leaving the die, the extruded product in the form of a filament having the above diameter was directly cooled down to room temperature of 20°C.

[0079] Likewise, a second and third batch of filaments was made by adding the starch-oil mixture as prepared in Example 2D in a 1 and 2 w / w% ratio, respectively, to 400 grams of PLA into the hopper, and cooling the extruded filaments down to room temperature of 20 °C.

[0080] Example 3: Salt germination test

[0081] OD measurements at 600 nm were performed with a Genway Genova Bio spectrophotometer. 702 medium was used as blank.

[0082] To 20 ml of 702 medium or 702+ medium, 2 ml of the endospore solution of Example 2C was mixed, resulting in a mixture having an ODeoo of between 0.3 and 0.9.

[0083] The mixtures were incubated at 32°C and every 30 min the ODeoo was measured. A drop in ODeoo indicates germination of the spores, whereas an increase in ODeoo would indicate microbial growth. If the drop in ODeoo is more pronounced in 702+ medium as compared to that observed for the 702 medium, the degree of germination in the 702+ medium is stronger (not growth rate nor growth level) compared to the germination in 702 medium. The difference in ODeoo value for both ambient conditions and non-ambient activation conditions can be expressed as the AODeoo value. Reliability of the measurements are improved by performing multiple measurements and calculating the averaged AODeoo, expressed as RAOD value. Based on the measured ODeoo values, The degree of changes in optical density (AODeoo) can be calculated with the lowest OD values (ODTL) and the first OD values (ODTO) used with the formula AODeoo = 100 - ODTL / ODTO X 100. The RAOD is then calculated as RAOD=AODeoo 702+ medium I AODeoo 702 medium x 100.

[0084] Table 1 shows the ODeoo values for a bacterial isolate with lower preference for high salt germination (isolate 7) and a microbial isolate with higher preference for high salt germination (isolate 9, eventually providing Bacillus subtilis PHBS0007). Figure 1 depicts the percentage of ODeoo decrease in 702+ medium as compared to that in 702 medium. Table 1 : Germination at normal and high salt conditions

[0085] Example 4: PLA hydrolysis tests

[0086] A. Preparation of microbes

[0087] Colonies of isolate 7, isolate 9 and Bacillus subtilis PHBS0007 are grown on 802 medium agar plates at 34°C for about 12 hours until colonies form. The selected colonies are inoculated in 702+ medium until the stationary growth phase is reached.

[0088] B. Compounding + 3D-printing

[0089] Filaments of Example 2F made from PLA and 15 w / w% spore-starch-oil mixture were taken and fed to an Ultimater 3 3D printer Yandex, Geldermalsen, Netherlands) to produce printed sheets of 25 x 92 x 0.1 mm through an AA 0.4 mm nozzle.

[0090] C. Incubation of the microbes with PLA

[0091] Different samples as prepared according to example 4A were adjusted to the same optical density between 1.0 and 1.5 to assure a similar number of microbes for each sample.

[0092] The printed PLA sheets were sterilised by washing with 10% bleach, followed by a 70% ethanol rinse, and placed in 50-ml tubes comprising 25 ml 712 medium, that were inoculated with 200 l of a sample. The tubes were incubated for 14 days at 34°C in (an)aerobic conditions with abundant shaking.

[0093] D. qNMR preparations

[0094] After the incubation of Example 4C, 0.025 - 0.030 gram of film material was put in 1 ,5 ml centrifuge tubes. To each tube, 1.00 ml chloroform-D was added. The tubes were shaken until the sample was fully dissolved. The tubes were centrifuged in an Eppendorf 5430R centrifuge for 5 minutes at 22°C at 20817 ref to separate any undissolved substances. From the supernatant, 600 pl was taken using a pipet (P1000, pipetman, Gilson) and added into a, previously cleaned with chloroform, NMR tube (Bel-Art™ SP Scienceware™ Wilmad™ Disposable NMR Tube Caps, 5.0mm Dia., Bel-Art™, USA). The samples were stored at -20°C in a labelled and sealed bag.

[0095] E. qNMR measurements

[0096] The temperature in the room was measure and logged by Raspberry pi Model 2B+ (Rasperry Pi Foundation, Cambridge, UK) provided with a DHT22 AM2302 temperature and humidity sensor (AZ-delivery, Deggendorf, Germany).

[0097] The machine preparation starts with ‘full autoshimming’ ("calibration") process on a Nanalysis 60Hz NMR (Nanalysis Scientific Corp., Calgary, Canada) with the blue reference standard, NMR tube, as provided by the manufacturer. Previous to analysis a Medium shim: recommended for qNMR 1 D analysis was run. The magnet temperature was monitored via ‘Status’. The sample warmer temperature was set to the same temperature as the magnet temperature of 34.0 °C with a hysteresis of 0.3 °C at interval of 2 minutes.

[0098] The samples were placed in the sample warmer for 5 minutes or more. The environmental conditions were monitored to be for 18 - 26°C for current temperature, 15-80% for humidity and a temperature variation of less than 1.0°C per hour.

[0099] Samples with the most degradation, as observed by the eye, were selected and a 1 D test was performed with 4 scans, while noting the Gain value in the T1 spreadsheet. The setting was changed from Auto gain to manual with half the gain value of the first automatic. This was performed 3 to 4 times by setting gain values that were actually too low for one scan and then auto for the following scan to evaluate the optimum gain. From the obtained Gain values, the lowest value was selected to proceed for the remainder of the experiment.

[0100] The T1 test was started with Scan Delay of 70 seconds and a Tao stop of 4000.00 milliseconds and a Tao start of 10.00 milliseconds.

[0101] In the T1 spreadsheet, the values for the next ‘scan delay being the T 1 times 6’ and the Tao stop being T1 times 4000 are automatically calculated. Scan delay values are continue to be collected until the value start to stabilize over the number of measurements. The highest value where the trend seems to stabilize was selected. Table 2: qNMR settings

[0102] Before scanning was started, the 'fixed Gain' value was selected and the found Gain value was entered for all other samples. The 1 D test was stared with a randomly selected sample with the above stated settings. Without saving the first run, all the samples were scanned until all in random order were scanned and data saved. A medium shim was performed after each 10 scans.

[0103] F. Data analysis

[0104] The NMR output-files were safely stored and the generated .dx files were imported into Mnova (Mestrelab).

[0105] The peak at 5 ppm was zoomed in, see figure 2. The noise was cleared by using Apodization and the exponential function (around 2.0000Hz). The baseline was corrected manually to be straight on both sides of the peaks.

[0106] Peaks at 5 ppm were integrated manually, and the integral numbers were put in a spreadsheet using the NMR integrals table. A bar-graph was made for data interpretation. The surface area under de peak (the intergral) is proportional to concentration of functional groups. The more functional groups the more hydrolysis occurred. If there is no statistical difference among samples then there is no degradation. If there is difference in the integral numbers there is degradation.

[0107] Table 3 below shows the 'arbitrary integral values' for 3 example isolates. Every value represents a piece PLA in the form of a film. Each piece of film had been incubated with the three isolates (start, middle, end) at 32°C for 14 days. Then, using the qNMR method, described in example 4, the hydrolysis is evaluated. Table 3: qNMR integral values at marine and aquatic conditions

[0108] Figure 3 shows a box-plot PLA degradation analysis with qNMR, summarizing the data shown in table 3 by grouping the three isolates (start, middle and end) either incubated in aquatic or marine conditions. The dots are the outliers. As selection continuous the difference in concentration of functional groups between marine and aquatic increases; which is proportional to the degradation. Also the uncertainty regarding the differences decreases, as noted with the respective p-values 0.33, 0.032 and 2.8e-05.

[0109] Example 5 - Extrusion survival test

[0110] Filaments having a diameter of 2.7 - 3.0 mm and 15 w / w% spore-starch-oil mixture were produced according to example 2F. The filaments were made with spores of B. subtilis PHBS0007, bacterial isolates 7 and bacterial isolate 9. As a control, filaments were produced similarly without the presence of micro-organisms.

[0111] The filaments were cut in a sterile fashion in fragments having a length of 5mm and subsequently into two longitudinal halves. The samples were placed on 802 medium agar plates and incubated at 34°C for 48 hours. Microbial growth was assessed by counting the colony numbers around the filament piece.

[0112] The results are depicted in table 4 below. Table 4 Extrusion survival

[0113] Example 6: Degradation test

[0114] The overall degradation test of the extruded PLA with 15 w / w% spore-starch-oil mixture of example 2F was compared with the reference materials coton powder and virgin PLA in accordance with the ASTM D6691-17 test. According to this test, each powdered sample (cryogenic milling for 120 seconds SPEX 6775 Freezer / Mill®) was put in a small bottles and over the course of 25 days the CO2 concentration in the seawater and head-space is measured. The more degradation occurs, the more CO2 is produced. The results are shown in table 5.

[0115] Table 5. Degradtaion results

[0116] Example 7: Survival test of spores with various viscosity modulators

[0117] A. Cultivation

[0118] A bacterial isolate, or Bacillus subtilis PHBS0007 was inoculated in sterilized 702 medium [10 g / l Tryptone; 2 g / l yeast extract; 1 g / l MgSO4] to an OD600 of -0.06 and incubated for 48 hours at 35°C;

[0119] B. Harvest of spores

[0120] After reaching a culture density of an OD600 of 1 .2 or more, the culture was checked for contaminations and presence of spores under a phase-contrast microscope and the culture medium was centrifuged (Eppendorf 5430R, program 5 (7190 RCF / 5 min / 24°C).

[0121] C. Spore mixture

[0122] The pelletized spores were re-suspended in 20 ml supernatant using the shaker and 5 grams of starch, mixed until homogenous. For later use, the mixture was poured in sterile Petri dishes, and open Petri dishes were dried in a drying oven for 12 h at 70°C. The microbial powder can be used for mixing with the PLA at a later stage.

[0123] D. PLA with various viscosity modulators The viscosity modulator was mixed in a 3 w / w% ratio with PLA into a 10L LDPE bag and shaken and massaged until liquid was homogeneously distributed over the PLA. Three samples with each a different viscosity modulator - sunflower oil, Betain-ABB and PEG400 - were made; an contro sample with sunflower oil and PLA in a 10L LDPE bag was made as negative control (Neg. Ctrl).

[0124] E. Extrusion procedure - pre-compounding

[0125] For each sample of 7D, the contents of the LDPE bag was fed into to a double screw extrude with a rotation speed of 40 rpm, with the set temperatures in table 6 to produce a filament with a diameter of approximately 2 - 4 mm. Pure PLA (i.e. no viscosity modulator) was fed into to a double screw extrude with a rotation speed of 40 rpm, with the set temperatures in table 6 to produce a filament with a diameter of approximately 2 - 4 mm.

[0126] The filament is passed through a water bath at room temperature (20°C) approximately to solidify the filament. The filament is then fed into a cutter that cuts the filament into approximately 4 mm length pieces and received into five separate labelled bags; respectively Neg. control, Pure PLA, oil PLA, CAPB PLA and PEG400 PLA.

[0127] Table 6: Extruder temperature profile

[0128] F. Preparation of thermoplastic composition

[0129] Except fror the PLA mixture of the negative control, the respective PLA mixtures Pure PLA, oil PLA, CAPB PLA and PEG400 PLA of 7D were mixed with the dry microbial powder obtained in example 7C in a 0.3% ratio using a 10L LDPE bag and shaken and massaged until all the granules are covered with microbial powder.

[0130] G. Extrusion procedure - compounding

[0131] The content of each bag of Neg. control, Pure PLA, oil PLA, CAPB PLA and PEG400 PLA were then fed one by one into to a double screw extrude with a rotation speed of 40 rpm, with the set temperatures in table 6 to produce a filament of approximately 2 - 4 mm in diameter. To ensure no cross contamination the between the processing of each compound, the barrel of the extruder should be fed with virgin PLA until all the filament contains no trace of the microbial powder mixture before continuing to the next sample; pure PLA is completely translucent.

[0132] The filament is passed through a water bath at room temperature (20°C) approximately to solidify the filament. The filament is then fed into a cutter that cuts the filament into approximately 4 mm length pieces and received into the five separate labelled bags.

[0133] H. Surface sterilization For each of the 5 treatments three times 16 granulates in sterile 50 ml tubes are surface sterilized in 10% bleach for 20 min in a sterile 15 ml glass test tube. Following the sterilization, the granules washed three times with sterile demineralized water.

[0134] I. Dissolving

[0135] The granules treated in 7H are partially dissolved in 2 ml acetone by incubation for 60 min at 60°C with vigorous shaking every 5 min. A sixth tubes with acetone is introduced, however, without the addition of granules. This treatment will function as medium control later in the process.

[0136] J. Incubation

[0137] Then 10 ml sterile growth medium (10 g / l Tryptone premium, 2 g / l yeast extract, 1 g / l MgSOt) was added to each of the six tube and incubated at 37°C for 5 h with shaking every hour.

[0138] K. Growth-curve registration

[0139] Following this initial incubation of 7J three times 200 ul from each tube are added to individual wells in a sterile 96-well plate (Assay Plate with Low Evaporation Lid, 96 Well Flat Bottom, Nontreated, Sterile, Product No.: 3370, LOT: 22923045) and the OD600 is measured every 5 min at 32°C with orbital shaking in between reads. (SpectraMax i3 Platform, Molecular Devices, Austria)

[0140] L. Analysis

[0141] For analysis, the initial measured OD value was subtracted from all values to ensure same starting point of all curves. The mean and standard deviation for each sample was calculated from the three individual measured wells and visualized as line with shaded area.

[0142] Bar graphs in FIG 5 show this mean corrected OD600 value for each replicate as dots and the mean and standard deviation per treatment as a bar.

[0143] M. Results

[0144] In Fig. 4, only small fluctuations in the measured OD600 were observed the negative controls, which never exceed a value of 0.1 , indicating that no growth was detected in these conditions. In the pure PLA condition, the GD600 of two of the three replicates is increasing in the first hours of the experiment towards OD=0.2 and continue fluctuating around this value during the remainder of the measurement. In contrast the values of the third replicate continues to fluctuate around 0 the whole time. The same can be observed for the third replicate of the oil PLA, but here the GD600 of the second replicate only starts increasing after 30 h, while the GD600 of the first replicate starts to continually increase after 5 h and reach a value of 0.5 at 60 h. For CAPB PLA (PLA with Betain-AAB) the GD600 of the first replicate starts increasing from the beginning until reaching an OD of around 0.18 at 10 h, then drops a little before increasing further for the rest of the measurements up to an OD of 0.35. In the PEG400 PLA condition the measurements of the first replicate continue to fluctuate strongly around 0 during the whole measurement, while in the second replicate there is an initial increase up to 0.1 then a long plateau phase until 40 h before it further increases up to 0.2. For the third replicate a continuous strong increase can be observed starting within the first few hours and reaching nearly 0.5 at 50 h before starting to slightly decrease again.

[0145] FIG 5 shows the relative total growth for both controls. The average of CAPB PLA is somewhat similar to growth control group pure PLA. However, the average of the PEG400 PLA and oil PLA are higher than the pure PLA group. This is especially the case for the replicates that had growth.

[0146] N. Discussion & Conclusion

[0147] The results suggest that both the medium control and the negative control have little to no growth suggesting a relatively sterile work environment was maintained for the experiment. Thus, affirming the validity of the test. The large standard deviation could be explained by relative low survival rates among the replicates combined with a small sample size. As can be seen from Fig.

[0148] 4, the oil PLA group and the PEG400 PLA group have more growth than the pure PLA which does not have viscosity modulator. The relatively equal growth of the Pure PLA group and the CAPB PLA group could potentially be explained by the water content of the CAPB which contains 67-72 28-33 w / w% water. Thereby, potentially mitigating any advantage the active compound could provide as a viscosity modulator. However, it can be concluded that the addition of viscosity modulator increases the survival rates of microbes following extrusion PEG400 PLA and oil PLA.

Claims

CLAIMS1. Method for the manufacture of an article by thermoplastic processing of a thermoplastic composition comprising a one or more polymers and viable spores of one or more sporeforming micro-organisms, the method comprising the steps of:(1) preparing the thermoplastic composition by mixing one or more polymers with viable spores of one or more micro-organisms;(2) thermoplastic processing the thermoplastic composition at a temperature of 100 - 350°C and optionally a pressure of 1.5 - 400 bar; wherein the thermoplastic composition further comprises a viscosity modulator, capable of lowering the viscosity of the thermoplastic composition at the thermoplastic processing conditions of step (2).

2. Method of claim 1, wherein the viscosity modulator is selected from the group consisting of fats, oils and polyalkane glycols, preferably oils and fats derived from a natural source, in particular vegetable oils and fats, more in particular the viscosity modulator is a vegetable oil.

3. Method of claim 1 or 2 wherein the thermoplastic composition comprises 0.5 - 20 w / w%, based on the one or more polymers, of the viscosity modulator.

4. Method of any of the preceding claims, wherein the thermoplastic composition comprises 0.01 - 10 w / w% spores, preferably 0.1 - 10 w / w%, more preferably 0.1 - 5 w / w%, even more preferably 0.1 - 2.0 w / w%, based on the total weight of the one or more polymers.

5. Method of any of the preceding claims, wherein the thermoplastic composition further comprises a hygroscopic agent, preferably 5 - 30 w / w%, based on the polymeric matrix material.

6. Method of claim 4, wherein the hygroscopic agent comprises a powder material originating from a natural origin, preferably chosen from flour, starch, cellulose.

7. Method of any of the preceding claims, wherein the article is a non-food article.

8. Method of any of the preceding claims, wherein the one or more polymers is a synthetic polymer, preferably a synthetic polymer selected from the group consisting of polyesters, polyurethanes and polyvinyl acetate (PVAc), more preferably a polyester.

9. Method of any of the preceding claims, wherein the one or more polymers is selected from polylactic acid (PLA), polyhydroxyl alkanoates (PHA), polyethylene terephthalate (PET), polyurethane (Pll), polybutylene succinate (PBS), polycaprolactone (PCL), polybutylene adipate terephthalate (PBAT), polyvinyl acetate (PVAc), and of polystyrene (PS), polypropylene (PP), polyvinylchloride (PVC, polyvinylidene (PV), polycarbonate (PC), acrylonitrile butadiene styrene (ABS), polyethylene (PE), the one or more polymers preferably comprising one or more polyesters, more preferably polylactic acid (PLA).

10. Method of any of the preceding claims, wherein the thermoplastic composition further comprises one or more nutrients, preferably nutrients selected from a carbohydrate source, a phosphate source and / or a nitrogen source, preferably in an amount of 0.01 - 5 w / w% based on the one or more polymers.

11. Method of any of the preceding claims, wherein the thermoplastic composition further comprises one or more trace elements, preferably in an amount of 0.001 - 2 w / w% based on the one or more polymers.

12. Method of any of the preceding claims, wherein the thermoplastic composition further comprises a protein source, preferably a protein hydrolysate, more preferably in an amount of 0.01 - 1 w / w% based on the one or more polymers.

13. Method of any of the preceding claims, wherein the spore-forming micro-organism is one or more selected from the group consisting of the bacterial genus Bacillus, Brevibacillus, Actinomadura, Amycolatopsis, Alcaligenes, Paenibacillus, Thermopolyspora, and the fungal genus Cladosporium, Cryptococcus, Fusarium, Penicillium, Rhizopus, Rhodotorula, Trichoderma, the spore-forming micro-organism preferably being selected from the bacterial genus Bacillus, more preferably the species Bacillus subtilis.

14. Method of claim 12, wherein the micro-organism has a salt tolerance of at least 3.5 w / v%.

15. Method of claim 12 or 13, wherein the micro-organism has a hydrolase activity, in particular esterase activity, the hydrolase activity preferably being present at a salt concentration of 3.5 w / v%.

16. Method of claim 14, wherein the micro-organism is Bacillus subtilis PHBS0007 as deposited at the Westerdijk Fungal Biodiversity Institute on December 1 , 2023 under access number CBS150835.

17. Method of any of the preceding claims, wherein the thermoplastic processing is selected from extrusion, 3D printing, injection moulding, thermoforming, blow moulding orfoaming.

18. Thermoplastic composition comprising one or more polymers, viable spores of one or more spore-forming micro-organisms, and a viscosity modulator as defined in any of the preceding claims.

19. Article comprising a thermoplastic polymeric matrix, viable spores of one or more sporeforming micro-organisms dispersed in the thermoplastic polymeric matrix, and a viscosity modulator, prepared with the method of any of claims 1 - 17 or with the thermoplastic composition of claim 18.

20. Bacillus subtilis PHBS0007 as deposited at the Westerdijk Fungal Biodiversity Institute on December 1 , 2023 under access number CBS150835.21 . Use of a viscosity modulator selected from the group consisting of fats, oils, fatty acids, and polyalkane glycols in a method for the production of an article by thermoplastic processing of a thermoplastic composition comprising one or more polymers and viable spores of one or more spore-forming micro-organisms.

22. Use according to claim 21 , wherein the viscosity modulator is an oil or fat derived from a natural source, preferably a vegetable oil or fat, more preferably a vegetable oil.

23. Use of a thermoplastic composition comprising one or more polymers, viable spores of one or more spore-forming micro-organisms, and a viscosity modulator according to claim 18 in a method for the production of an article comprising a thermoplastic polymeric matrix materials and spores of a spore-forming micro-organisms dispersed in the polymeric matrix.

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