Plastic degradation
The findings of this study provide important insights into the biodegradation dynamics of various mulch films at different temperatures. The considerable degradation of biodegradable plastic mulch films (MB and TMB) during six months at RT demonstrates their promise as long-term alternatives to non-biodegradable options (LDPE). The biodegradation of TMB by 51.36% and MB mulch by 69.15% indicates their ability to decompose over time, concerning LDPE mulch recalcitrant to biological decomposition during the six-month trial, according to previous establishment [13]. Further, the impact of temperature on degradation rates is an important inquiry. The rapid breakdown seen at 30°C, particularly with MB mulch (88.90%) as well as no degradation at 45°C, indicates that temperature impact on biodegradation efficiency is linked to microbial catabolic activity. Sintim et al. [40], observed that mulch films' biodegradability in soil field conditions varied with temperature, and higher temperatures considerably increased the biodegradability of polymers. Nevertheless, our study demonstrated that the increasing of temperatures up to 45°C affects survival rate of mesophilic bacterial population, corresponding to the non-degradation of mulch residue [41]. This evidence concerning the impact of temperatures emphasizes the need to consider this environmental variable to optimize the degradation rates based on local climate. Nevertheless, it must be considered that in a real scenario, the temperature along with other biotic (variety of microorganisms such as bacteria, fungi, and Archaea) and abiotic factor (e.g. light, oxygen concentrations, humidity, and acidity) affects polymer biodegradability [42, 43].
Microbial community composition and diversity
This work elucidated the impact of biodegradable mulch films tilled in soil ecosystems comparing the effects of biodegradable and non- biodegradable mulch film residues on soil microbial community diversity and functionality.
Within the presented study, MB and TMB amended soils showed similar trends with general decreases of Cyanobacteria and Planctomycetes after 3 months of incubation, whereas bacterial composition was dominated by Actinobacteria, Proteobacteria, Chloroflexi, Firmicutes, Bacteroidetes, and Acidobacteria, confirming the observations of other research on plastics in different environments [44–46]. Previous research showed that biodegradable plastic mulches enrich Proteobacteria [19, 23]. This phylum has a strong correlation with the total N and organic C levels of soil, indicating its involvement in biogeochemical cycles [47]. The phylum Bacteroidetes is widely dispersed throughout various ecosystems and serves a functional role in the degradation of complex organic materials [48]. Members of the Bacteroidetes family, including Flavobacterium, can facilitate the breakdown and use of complex polysaccharides [49], hence influencing the process of denitrification [50].
Evidence that biodegradable plastics tilled in agricultural soils contribute a little amount of carbon is another factor supporting these shifts. Soil bacteria in agricultural settings are often limited by carbon availability, therefore these inputs cause a response [19]. Furthermore, main variations were at RT and 30°C supporting the results of plastic breakdown and highlighting that burial of biodegradable plastics can lead to changes in soil microbial community structures [20, 22, 23].
LDPE treated soil showed slight changes in its bacterial community composition, as well as its profile was comparable to the control after six months. Otherwise, according to the phyla found, the literature suggests that microplastic cultures were enriched in polyethylene (or other polymers) degrading taxa, such as Bacteroidetes and Proteobacteria [51, 52].
All samples incubated at 45°C showed a stable bacterial composition up to 6 months, probably due to bacterial decrease in activity with higher temperature occurrences [41], aligning with the absence of plastic degradation
No fluctuations in the abundance of predominant fungal phyla Ascomycota, Basidiomycota, and Mortierellomycota were observed at RT and 30°C. Previous research showed that the phyla Ascomycota and Basidiomycota were responsible for the breakdown of oil-based polymeric polymers [19, 23, 53]. Therefore, these findings suggest that, at least throughout the 6-month trial, no mesophilic fungal communities linked to plastic degradation emerged. Otherwise, the shifts observed at 45° C in fungal community composition highlight the influence of both plastic and temperature on fungal dynamics, in absence of plastic degradation. In all samples, the Ascomycota and Aphelidiomycota decreased, and the unclassified fungi increased. These last can be assumed that the thermophilic phyla selected by the plastic and temperature are responsible for the shift in fungal profiles at 45°C. Thermophilic fungi are characterized by their ability to thrive at temperatures ranging from 20 to 62°C [54]; however, there are an estimated 3 million thermophilic fungal species on Earth, of which only about 100’000 have been identified, so the increase in unclassified ASVs at 45°C may be due to the presence of species yet to be classified [54].
Analyses of alpha diversity across the entire data set confirmed that the mulch plastic type was the most discriminating factor shaping bacterial communities, followed by temperature that impact also fungal communities. It was interesting to note that the bacterial community responded differentially to each source of plastics tilled in the soil. The bacterial Shannon diversity index was higher in soils treated with TMB and MB mulch residues and decreased in LDPE-amended soils as well as in controls. The introduction of available carbon sources for bacteria through biodegradable mulch residues affected the Shannon index, confirming the response of soil microbes to these inputs. [19]. According to previous studies [41], the effect of the different temperatures on the microbial community was particularly evident at the end of the experiment, e.g. by reducing the activity of mesophilic bacteria or selecting thermophilic fungi (45°C).
In addition, the beta-diversity analysis of the microbiota associated with mulch residues, investigated with PERMANOVA, showed the significant influence of sampling times that reflect the temporal dynamics of plastic degradation process. The distinct sampling stages could help to determine the interaction between microorganisms and plastic tilled in the soil. Over time, enzymes break down the plastic and microbes use the resulting fragments for their nutrition by a process involving the turnover of different populations with distinct enzyme kits [25]. Moreover, using biodegradable mulch can benefit the soil's biogeochemical cycles by promoting a dynamic microbial community, due to increased carbon intake.
Predicted enzymatic activity
In this study, particular focus is placed on genes encoding hydrolases, lipases, cutinase, and cellobiosidase, due to their significant role in natural or synthetic polymer biodegradation [55, 56].
The clustering of predicted functions demonstrated that potential gene abundances were strongly affected by sampling time since two major clusters were observed: I) samples collected after three months of incubation at 30°C and RT tilled with biodegradable mulch residues (MB and TMB), comprising three exceptions: t3-TMB_T 45°C, t6-C_T30°C and t3-LDPE_T 30°C; and II) all remaining samples. The increase in several encoding genes in cluster I, like cutinase (K08095) and esterase/lipase (K01066) suggests a potential active response to the presence of plastic residues [55, 57]. These enzymes are known to cleave the ester bonds and degrade polyurethane substrate, and therefore can act also on non-biodegradable plastics [56]. Cutinase can hydrolyze a broad variety of synthetic polymer esters, both soluble and insoluble, that are structurally related to cutin [57]. The increase of cellulose 1,4-beta-cellobiosidase (K01225, cluster I) that hydrolyze cellulose releasing cellobiose, was probably induced by the presence of biodegradable mulch residues in soil [58]. Cluster II comprises all samples at the latter sampling time, controls, and samples incubated at 45°C. In particular, the presence of MB and TMB from later sampling times where plastic degradation occurred, suggests that there may be significant differences in some metabolic activities compared to the same samples after three months of incubation. This confirms that changes in the activities of bacterial populations over time reflect the dynamics of plastic degradation in soil. Over time, polymers degrade enzymatically, and microorganisms assimilate and utilize the degradation products, potentially causing shifts in the predicted coding genes [25].
Core microbiota associated with mulch plastic residues
The microbial core investigation aimed to provide hints of potential microorganisms involved in plastic breakdown. The analysis was performed on all plastic-type soil systems at temperatures where breakdown occurred (RT and 30°C). There is still a lack of literature, on information on microbial species known for their ability to degrade plastics. Thus, core members found in this work could be evaluated based on their enzymatic kit to assess their potential action on natural and non-natural polymers. Among the identified bacterial species, Hydrogenispora ethanolica LX-B isolated from the mesophilic (35°C) anaerobic fermentation process of sludge, can ferment substrates with different carbon sources, including starch, glucose, maltose, and fructose [59], suggesting its potential involvement in the degradation of starch-based plastics. The species Thermoflavimicrobium daqui FBKL4.01, a thermophilic bacterium isolated from the Daqu used to produce Chinese liquor Moutai, can ferments pure wheat [60], confirming its involvement in the degradation of starch and cellulose. The analysis of fungal core revealed 12 different species. Among them, Solicoccozyma aeria (Cryptococcus aerius) is known for its ability to produce amylases at 30°C, to digest raw starch [61]. These three microbial species were found in soils amended with MB and TMB and could therefore be of interest for the biodegradation of starch-based plastics.