Combined use of Bacillus strains and Miscanthus for accelerating biodegradation of poly(lactic acid) and poly(ethylene terephthalate)

Synthetic polymer materials

Polymer samples were made from two types of polymer granulate: PLA (Ingeo^TM Biopolymer 2003D, Nature Works LLC, USA) and PET (SKYPET –BL 8050, Korea). Films were extruded from the granulates using a laboratory apparatus consisting of a single-screw Plasti-Corder PLV 151 extruder (Brabender, Germany) with a slit die of working width 170 mm and slit height 0.35 mm, and a water-cooled, three-roller smoothing system of roller diameter 110 mm. A screw of length 25D was used with a mixer head of length 8D and compression level of 3:1. The extruder station was additionally equipped with an apparatus necessary to measure the temperature of the heating zones in the plasticising system and the die. PLA and PET films of thickness 0.087 mm were produced (Janczak et al., 2018).

Microorganisms and bacterial inoculum

In the study, bacteria strains belonging to PGPR were used, Bacillus sp. (ML1-2), whose gene sequence available in GeneBank NCBI was verified using BLAST software. It was established that this sequence is 100% homologous to sequences of B. subtilis. The B. subtilis strain (KM411502.1) was isolated from the mycorrhizosphere of an ectomycorrhizal fungus from degraded soil contaminated with heavy metals. B. cereus (HM989919) was isolated from a fruit body of an ectomycorrhizal fungus Hebeloma mesophaeum associated with Salix caprea from degraded soil contaminated with heavy metals (Hrynkiewicz et al., 2012). Bacteria strains used in the experiments were grown on a solid medium R2A (Oxoid) at 24^∘C for 2 days. Bacterial inoculum containing 5 ×10⁴cfu/mL was prepared in saline solution (0.9% NaCl pH 7.0). The suspensions prepared in this way were used for further analyzes described in individual sections of this manuscript.

According to some researchers, bacteria Bacillus sp. should be used for the biodegradation of polymeric materials, including PLA and PET, and as PGPR bacteria (Bubpachat, Sombatsompop & Prapagdee, 2018; Janczak et al., 2018; Janczak et al., 2020).

Biochemical characteristic of Bacillus strains

Pure cultures of the tested bacterial isolates were checked for their plant growth-promoting properties and for promoting biodegradation of polymeric materials. The following biochemical features were analyzed: production of cellulase on CMC (carboxymethyl-cellulose sodium salt medium) (Hankin & Anagnostakis, 1977), phosphate solubility on PVK substrate (Pikov-skaya’s medium) (Sharma, Kumar & Tripathi, 2011) production of siderophores on CAS (chrome azurol S medium), production of pyoveridin, a fluorescent siderophore in bacteria of the genus Pseudomonas (SM) (succinate medium), lipase production (LP), production of protease (SMA) (skim milk agar) (Ghodsalavi et al., 2013).

All media were prepared according to the literature recommendations, then autoclaved at 121 °C for 20 min, cooled and poured into petri dishes to cool and solidify. After inoculation with bacteria, each medium was incubated at 27 °C for 48 h, except for PVK medium which was incubated for 7 days. After incubation, the test results for each tested bacterial isolate were recorded on a 5-point scale: 0–4, where the numbers: 0, 1, 2, 3, 4 - denote in turn: no reaction, low, medium, high and bar- very high intensity of the observed biochemical reaction. The results are presented in Table 1 (Krawczyk et al., 2016).

The biofilm-forming ability of bacteria

Bacteria isolates of B. subtilis, B. cereus were pre-incubated in flasks containing nutrient broth (50 mL) for 24 h at 25 °C. Next, optical density (value 1) of each culture was determined in relation to OD_600nm. The bacterial suspension (0.1 mL) was transferred to a nutrient broth (10 mL) and put onto the PLA and PET surface. The fragments of films (5 ×5 cm) were incubated for 48 h at 25 °C and then dried at room temperature for 40 min. Next, the films were covered with 1% solution of crystal violet which adhered to the biofilm. After 45 min they were washed with distilled water to remove excess dye and dried for 30 min. Subsequently, they were placed in a beaker and 96% ethanol was added to wash away the absorbed dye. The absorbance of alcohol extract was measured using a BioRad spectrophotometer at a wavelength 595 nm. Biofilm abundance was analyzed as absorbance at 595 nm, and performed in triplicate (Adam & Duncan, 2001; Walczak, Richert & Burkowska-But, 2014; Świontek Brzezinska et al., 2018; Świontek Brzezinska et al., 2020).

In order to evaluate the viability of biofilm forming bacteria, microorganisms retained on the filter surface were subjected to viability staining, using a diagnostic LIVE/DEAD set (Invitrogen). Fragments of the film (1 cm² from each film) covered with the aqueous solutions of dyes of LIVE/DEAD BacLight^(TM) Bacterial Viability Kit were incubated for 15 min in the dark at room temperature. After the excess dye was removed, the samples were placed on microscope slides and viewed under oil immersion at ×1000 magnification using ECLIPSE 50i fluorescence microscope (Nikon, Japan) (Kumar & Maiti, 2016; Kumar et al., 2017; Richert & Dąbrowska, 2021).

Plant material and soil parameters

Plant material consisted of giant Miscanthus (M. giganteus) seedlings from a two-year cultivation in a field and 7-weeks-old plants from the in vitro cultures. Seedlings, containing 4–5 shoots, were obtained by mechanically dividing rootstock.

In the pot experiment, two-year old compost soil was used, prepared by mixing plant fragments, sand, clay and peat at equal ratios. The compost soil contained: 8.02% MO, 3.71% C_org, 0.30% Nt, 12.0 C/N, 1.29% CaCO₃, pH_H2O7.5, pH_KCl7.1.

Pot experiments

Plants of Miscanthus were obtained from in vitro cultures. Pot experiment was conducted in three variants of inoculation: control (non-inoculated) plants and inoculated with B. cereus and B. subtilis strain in growth substrate (sterile mixture of sand and vermiculite, 1:1). Plants (n = 30, for each variant) were cultivated in the pot (a volume of each pot 0.3 l) in the temperature: 24 °C ± 2 °C and light/dark: 16/8) for 10 weeks and irrigated with Hoagland medium. The growth parameters such as: fresh and dry biomass of shoots and roots, number of leaves, length of shoots and roots were assessed after 10 weeks.

Studies on the biodegradation of PLA and PET in soil were carried out for 6 months (March–August) under greenhouse conditions in 2200-cm³ pots. In the first week of growing the soil was inoculated with one mL of bacterial suspension.

Pieces of PLA and PET film were placed in the soil. Their dimensions were: 30 ×30 mm, 100 ×15 mm and 130 ×110 mm (3 pieces of each), thickness of film had 0.087 mm. The use of various film sizes was related to their subsequent use in various methods for testing changes in biodegradation on plastics. The following experiment systems were used: soil + film (variants with PLA or PET), soil + film + bacteria strain (variants: PLA + B. subtilis, PLA + B. cereus, PET + B. subtilis, PET + B. cereus), soil + plant + film (PLA + M. giganteus, PET + M. giganteus), soil + plant + film + bacteria strain (PLA + M. giganteus + B. subtilis, PLA + M. giganteus + B. cereus, PET + M. giganteus + B. subtilis, PET + M. giganteus + B. cereus). Three biological replicates were produced for each variant. After 6 months, the film samples were removed from the soil and washed with 70% EtOH and then three times with water. The films were dried on filter paper for 12 h at RT (ISO 846, 1997) used to demonstrate biodegradation changes.

Assessment of film biodegradation

Biochemical characteristics

There are many biochemical features of bacteria that have a beneficial effect on plant growth and accelerate the biodegradation processes of polymeric materials (Robertson et al., 2018; Bandopadhyay, Sintim & DeBruyn, 2020). The study investigated five selected biochemical features, the relationship of which with the increase in shoot length and the increase in the amount of root extracts has been scientifically proven (Ghodsalavi et al., 2013). On the basis of the conducted tests, it was determined which of the tested isolates show biochemical properties potentially favorably influencing plant growth and biodegradation. These results are presented in Table 1.

Among the tested isolates, strong intensities of the biochemical reaction on the CMC medium indicating the presence of the cellulase enzyme and enzymes strongly dissolving inorganic phosphates on the PVK medium were noted. In the case of the CAS medium on which the ability to produce siderophores by bacteria was tested, a positive result was recorded for B. subtilis. In contrast, in the SM medium, no positive reaction was noted for any of the isolates for the presence of pyoveridine, a fluorescent siderophore produced mainly by Pseudomonas. Both B. subtilis and B. cereus have a strong biochemical response to lipases (LP) and proteases (SMA).

There are metabolic differences between strains of the same species in terms of the examined features, which may indicate the ability of bacteria to adapt to the environment they currently occupy. Studying these differences can contribute to a better understanding of the bacteria’s mechanisms of action and help identify genetic markers encoding bacterial biochemical traits that support plant growth.

Biofilm in PLA and PET surfaces

The formation of a biofilm by bacteria is the first step in the biodegradation of polymers ( Richert & Da̧browska, 2021; Janczak et al., 2020). Figure 1 shows the results of biofilm formation by Bacillus strains. The amount of biofilm formed by the B. subtilis bacteria on the individual types of film was significantly greater than in the case of the B. cereus strain. A 17% larger biofilm was noted on the surface of the film made of PLA than on the PET film. The biofilm formed by B. cereus on PLA and PET was practically the same size at 0.022 OD_595nm.

At the same time, the bacteria forming the biofilm on the PLA surface had a high viability, and therefore the number of microorganisms was greater than on the PET surface, which was clearly confirmed by the LIVE/DEAD analysis (Fig. 2). There is no doubt that a biofilm has formed on the surface of the polymers degrades these materials. However, the rate of biodegradation depends on activity of microorganisms and type of polymers. Polymers withwith high molecular weight have a slower rate of degradation than those with they have a low molecular weight and take many years to degrade long-chain polymers for simple hydrocarbons (Kim & Park, 2010; Lee, Kim & Song, 2014; Wang et al., 2020).

The growth of miscanthus in presence of Bacillus sp. strains

The effect of B. cereus and B. subtilis bacterial strains on the growth of miscanthus was tested (Table 2). The plant inoculation with bacteria stimulates the growth of plant roots and shoots. There was also an increase in the biomass of roots and shoots, but the result was not statistically significant. The presence of bacteria increased the survival rate of plants.

Janczak et al. (2020) have conducted similar research work using other bacterial strains (Janczak et al., 2020). B. cereus has been shown increase biomass production of Trifolium repens (Azcon et al., 2010) and Salix viminalis (Hrynkiewicz et al., 2012).

Assessment of film surface

Photographic documentation of film samples was prepared after six months of incubation in soils inoculated with bacteria and in the control, not containing inoculum (Fig. 3). Analyses of both films, PLA and PET, using SEM, showed presence of microorganisms on the surface of films in variants inoculated with bacteria from the Bacillus genus. More changes were found on PLA film, versus PET.

Following incubation in soil, the samples of PLA film were changed; cracks and chips were observed in the film. These changes were more pronounced in variants with the plant. In variants without the plant, only films incubated in soil in the presence of B. cereus were cracked, and in SEM images, numerous cavities were additionally seen in the film. These bacteria also caused degradation changes in the PLA surface in variants with the plant, which were visible as worn areas with irregular fragments of non-degraded polymer. The presence of M. giganteus contributed to formation of cavities in the PLA film, possibly due to its contact with roots and their exudates (Figs. 3–4).

Following PET film incubation in soil inoculated with bacteria, no film deformation was observed. Similar changes were present in variants with the plant. This may be an effect of root exudates or autochthonous microorganisms (Fig. 3). Similar results have been reported by others (Ziembinska & Gatlik, 2012; Lee, Kim & Song, 2014; Hermanova et al., 2015).

Loss of film weight

Mechanism and process of biodegradation depends on the molecular weight of the plastic and/or its composition (e.g., addition of plasticizers, dyes, flame retardants), on the presence of microorganisms and on environmental conditions (e.g., soil reaction, temperature) (Bano et al., 2017). Most polymers are too large to pass through cell membranes. The biological degradation of polymers actively involves at least two categories of enzymes: extracellular depolymerases and intracellular depolymerases (Muthukumar & Veerappapillai, 2015). During degradation, exoenzymes from microorganisms break down complex polymers, resulting in smaller short-chain molecules, e.g., oligomers, dimers and monomers, that are small enough to pass through semi-permeable outer bacterial membranes and then be used as a carbon and energy source. The process is called “depolymerization”. The microorganisms use the plastics as a source of carbon when nutrients are in poor supply or inaccessible.

Therefore, there is a general opinion that PLA may contribute to environmental pollution (Karamanlioglu & Robson, 2013). In films from variants with soil inoculated with Bacillus, a significant loss of film weight was found for PLA film versus the control (film from non-inoculated soil), excluding PLA from soil inoculated with B. subtilis. A ca. 70% loss of weight was demonstrated versus the control film for all the remaining variants inoculated with bacteria. Presence of the plant resulted in ca. 55% loss of weight (Table 3).

Analyses of film properties (EDX, FTIR, Film tensile strength test)

To demonstrate biodegradation changes in PLA and PET films, oxygen and carbon content (%_w) on the surface of analysed samples was verified using the EDX analysis. The oxygen-to-carbon ratio O/C (%) was calculated, and in all samples of PLA film it was higher than in the control film (Table 4), and this indicates biodegradation changes (Vieira, Guedes & Tita, 2013; Lv et al., 2017).

The most significant increase in this value was observed for samples inoculated with B. cereus in the presence of the plants, for PLA and PET a like. For the PLA film, an increase in the O/C ratio was additionally observed in variants inoculated with one of the Bacillus strains in the presence of the plants, and with B. subtilis strain in soil without plants (Table 4). For the PET film, a slight increase in the O/C ratio was noticed following inoculation with each of the bacteria strains (Table 4). In our six-month study (180 days), changes of 0.3 and 0.6 in the O/C ratio were noted in the presence of B. subtilis and B. cereus, respectively.

In the ATR spectrum for the PLA film obtained in FTIR analysis the following bands were identified: 1746 cm⁻¹ –absorption of vibrations of the C=O group; 1450 cm⁻¹ –absorption of asymmetric vibrations of the CH₃ group; 1265 cm⁻¹, 1179 cm¹, 1126 cm⁻¹, 1078 cm⁻¹, 1041 cm⁻¹ –absorption of stretching vibrations of O-C-C; 866 cm⁻¹ –absorption of vibrations of the O-CH₃-CH₃ group; 753 cm⁻¹, 701 cm⁻¹ –absorption of deformation vibrations of αCH₃ (Cai, Lv & Feng, 2013). The area at the wave number of 1746 cm⁻¹ was more closely analysed, as it is characteristic for the carbonyl group, which is susceptible to biodegradation processes. The carbonyl index was calculated in relation to absorption from asymmetric vibrations of the CH₃ group at the wave number of 1450 cm⁻¹. A drop in the value of the carbonyl index was observed in all the remaining variants versus the control, at a comparable level.

In the ATR spectrum for the PET film the following bands were identified: 2960 cm⁻¹ –absorption of asymmetric, stretching vibrations of the C-H group; 1712 cm⁻¹ –absorption of vibrations of the C=O group; 1450 cm⁻¹ –absorption of vibrations of the C-H group; 1407 cm⁻¹ –absorption of deformation vibrations of the C-H group; 1235 cm⁻¹ –absorption of asymmetric, stretching vibrations in the C-C-O group with a carbon atom in an aromatic ring; 1087 cm⁻¹ –absorption of asymmetric, stretching vibrations of the C-C-O group; 871 cm⁻¹ –absorption of vibrations of the C-H group in an out-of-plane aromatic ring; 722 cm⁻¹ corresponding to absorption of rotational vibrations of the C-H group in the aromatic ring (Cai, Lv & Feng, 2013). The area at the wave number of 1712 cm⁻¹ was more closely analysed, as it is characteristic for the C-H group in the aromatic ring at the wave number of 871 cm⁻¹. The most significant decrease in the value, indicating biodegradation changes, was observed for the PET film from soil inoculated with B. cereus (14% versus the film from non-inoculated soil) and containing plants. However, the highest value was noted for the film in non-inoculated soil with the plant.

The tensile strength analysis of the film based on measurements of elongation at break –AB (%) and the break force –FB (N) demonstrated biodegradation changes in both films. Changes in strength were visible as a reduction in the break force (FB) in the PLA film, and as shortening of elongation at break (AB) for PET (Table 4). For PLA, lower AB was noted for all analysed variants, excluding the film incubated in soil with B. subtilis without plants. A greater reduction in strength was observed for FB values, where the most significant result was obtained in the variant inoculated with B. cereus with the plant (Table 4). For PET, the most significant reduction in AB was noted for variants with plants in soil inoculated with one of the strains, and in the variant without the plant in soil inoculated with B. cereus. The presence of the plant had a stronger effect on loss in PET tensile strength than inoculation with B. subtilis (Table 4). Similar results have been reported by others (Kim & Park, 2010; Iwanczuk et al., 2013).

Analysis of film permeability to H₂O↑, O₂ and CO₂ gases

A parameter frequently analysed as an indicator for plastic degradation is film permeability to gases such as water vapour, oxygen or carbon dioxide (Hulsmann & Wallner, 2017). Film permeability to H₂O↑, O₂ and CO₂ gases was measured for the PET film only. The PLA film was too defragmented to prepare samples of 130 ×110 mm required for this analysis. During the biodegradation process permeability to gases increases. An elevated permeability to H₂O↑ (g/m² day) was noted only for variants inoculated with bacteria in presence of the plant. On the other hand, the greatest increase in permeability to O₂ (g/m² day) was noted after inoculation with B. cereus, regardless of whether the plants were present or not. A significant effect of the plants was also noted, both for the non-inoculated variant and in the presence of B. subtilis. An increase in permeability to CO₂ (mL/m²day) was noted after inoculation with B. cereus, regardless of whether the plants were present or not, and following soil inoculation with B. subtilis without plants. In our studies, an increase in permeability to water vapor by ca. 1 g/m²day was demonstrated for samples from soil inoculated with Bacillus strains. An increase in permeability to oxygen and carbon dioxide of 15 mL/m² day and 17 mL/m² day, respectively, was also found for film incubated in the presence of B. cereus, and by ca. 6% and 9%, respectively, in the presence of B. subtilis. This data implies that the strains used contribute to degradation changes in PET film during its incubation in soil (Table 5).