Effect of P3HB on soil microbial activity
The microbial behavior after addition of P3HB was similar as after PHBV addition  with specifics caused by dilution of soil or SOM by increasing sand content. The effect of P3HB on 16S rDNA and 18S rDNA was ambiguous, as P3HB addition reduced, increased, or did not affect bacterial and fungal content (Fig. 1A, B). The results suggest that the mechanism of their interaction and resulting effect may be related to changing physical and chemical properties of soil (see further discussion). PHB-degrading microorganisms (Fig. 1C), on the other hand, were predominantly positively affected by P3HB addition to soil. This increase is probably directly related to the supply of the preferred energy source in the form of a biodegradable polymer [5, 39]. Due to the suppression of the influence of P3HB in the sandy (100%) substrate due to the assumed negligible content of SOM and microbiota (Fig. 1), we do not discuss this variant in this chapter.
The individual enzymatic activities shed light on the processes occurring in soil after addition of P3HB. Ure is an important extracellular enzyme that hydrolyzes urea and regulates the early nitrification process in soil and is closely related to the SOM content . Therefore, it is related to the N availability. Ure is the only enzyme that showed disproportional decrease with increasing sand content (Fig. 2) as in soils without P3HB. This indicates that the P3HB increases activity and abundance of nitrifying microbes which cause enhanced Ure activity independently of SOM content. Thus, P3HB can replace (temporarily) the SOM in sandy soil.
The P3HB is composed of C, O and H, therefore the immobilization of essential nutrients (mainly N and P) is necessary for microbial degradation [15, 41]. This comes from the notion that an optimal C:N ratio in soil is around 25.0 ; above this value N is immobilized and below this value is N mineralized . In this work, P3HB application increased soil C:N ratio. During biodegradation, this increase caused N shortage around PHB particles, and the microbes immobilize N from their environment . This generally results in a decrease in plant-available N in soil. If there is no N to immobilize, microbial growth is slowed down. By addition of P3HB is the C:N disturbed, while the disruption seems to be the most pronounced in samples with low content of SOM. Noteworthy, the imbalance in C:N ratio influences the soil organisms in a different way; fungi have wider C:N ratios in their tissues than bacteria and archaea and therefore, they can grow more efficiently on low N substrates and will thus mineralize N more readily. This is also the case of PHB, which is biodegraded preferably by fungal communities at the phylum level dominated by Ascomycota . The product of urea hydrolysis is CO2 and NH4+; the latter is either used by plants or microbes, which can eventually convert it into NO3- via nitrification processes  However, nitrification rates are typically low as the nitrifiers are relatively poor competitors for NH4+ in the soil solution and occurs when the NH4+ supply exceeds plant and other heterotrophs demand . As the plant growth was largely suppressed under P3HB addition (Fig. 4), it can be anticipated that majority of NH4+ was used for growth of soil microbes, especially of PHB-degrading microorganisms (Fig. 1C). This enhanced consumption of ammonium nitrogen putatively more decreased the content of plant available, inorganic nitrogen, similarly as referred to polylactic acid (PLA) contaminated soil during vegetative state of bean . This hypothesis is supported by the enhanced Ure activity (Fig. 2D) and high correlation of Ure activity with the total degradation level indicated by DHA and PHB-degrading microorganisms (Additional file 1: Fig. S1). In addition, Zhou et al.  reported an increased activity of Acidobacteria and Verrucomicrobia phyla in soils degrading PHBV. In fact, neither Acidobacteriota  nor Verrucomicrobia  are involved in soil N-cycle processes such as nitrification, denitrification, or nitrogen fixation. Thus, it can be concluded that N (or its vast majority) is used for growing of microorganisms’ population.
This finding confirms the earlier assumption of Hoshino et al.  who explained better correlation of the biodegradable polymers degradation with the total N content than with the total C content by the necessity of soil N for the degradation by microorganisms as it is absent in bioplastics.
Importantly, unlike the Ure the phosphatase activity showed proportional results in terms of sand content (Fig. 2F). Phosphatase is an extracellular enzyme that mineralizes organic P into phosphate by hydrolyzing phosphoric (mono) ester bonds . Similarly to all extracellular phosphatases, enzyme expression is induced by P deficiency . Notably, Fig. 2F shows higher demand for P in soils containing P3HB and high sand content comparing to soils with lower sand content.
DHA is a basic indicator of microbial activities coupled with SOM degradation in soil . The most important function of DHA is the biological oxidation of SOM, achieved by transferring protons and electrons from organic substrates to inorganic acceptors . For this reason, DHA positively correlates with SOC and Corg as it reflects the activity of living cells and not of DHA stabilized in soil complexes . This explains its positive correlation with other microbe-related soil properties (Additional file 1: Fig. S1). In addition, significantly increased DHA values in P3HB-amended variants (Fig. 2A) assumed that PHB addition enhanced soil degradation rate due to its utilization as an energy/C source [15, 56, 57]. This assumption was confirmed especially by the prevailing increase in PHB-degrading microorganisms (Fig. 1C) and their high positive correlation with DHA (Additional file 1: Fig. S1).
ARS is involved in the S mineralization process which cleaves organosulphates  and is used as a measure of soil health and soil microbial activity . ARS activity is correlated with soil microbial biomass and the rate of S immobilization , pH and SOC . Here, similarly as phosphatase, its activity is elevated in sandy soils (> 60% sand) with P3HB (Fig. 2B), which reflects higher demand for immobilization of S in less buffered system.
NAG is an enzyme catalyzing the hydrolysis of terminal 1,4 linked N-acetyl-beta-D-glucosaminide residues in chitooligosaccharides, i.e., it is involved in degradation of chitin, the key polysaccharide of fungal cell wall . Its activity was enhanced in most P3HB-amended variants (Fig. 2C), which underlines higher demand of degrading organisms for N acquisition from available sources, but different than Ure, as suggested by their low correlation (Additional file 1: Fig. S1). Enhanced NAG activity supports the view that plants or microbes may use N-containing monomers and not only inorganic N .
GLU is an enzyme that catalyzes the hydrolysis of terminal 1,4 linked β-D-glucose residues from β-D-glucosides, including cellulose oligomers and thus it is an indicator of SOM degradation and soil C utilization . Its activity was either the same or even lower in soils with P3HB (Fig. 2E). This may be attributed to either preferable cleavage of PHB rather than cellulose or a high C:N ratio .
BR is a key indicator of aerobic catabolic activity in soil, and accessibility and degradability of organic C in SOM . The P3HB application positively influenced BR (Fig. 3A) according to the mechanism described above in the DHA-related part. Substrate-induced respiration is used to measure the activity of specific microorganisms responding to addition of substrates with specific composition. Response induced by Glc, NAG, Tre, Ala and Lys corroborated with results of BR (Fig. 3). Similar mechanisms and involvement of the same organisms is a probable reason of the grouping of all these respirations (Additional file 1: Fig. S2) and their positive significant correlation (Additional file 1: Fig. S1). On the contrary, arginine substrate showed slightly different results comparing to them.
The results of Arg.IR, an indicator of fungal respiratory activity in soil, suggest neutral-to-positive effect of P3HB as well (Fig. 3F). However, a slight deviation in the trend of values from the results of other respirations is visible (Fig. 3) and evident also from the PCA (Additional file 1: Fig. S2). The respiration decreases with increasing sand content. This indicates the relation of Arg-IR to soil texture. In sandy soils, the pores are better aerated, water content and water holding capacity are significantly reduced. These conditions probably limited the use of arginine substrate by microorganisms.
Effect of P3HB on soil fertility
The results clearly indicate an adverse effect of P3HB on both above- and below-ground biomass production of L. sativa (Fig. 4). These results are in accordance with previous works that degradation of biodegradable plastics might negatively affect plant growth [66, 67]. In general, for biodegradation of P3HB, two major explanations may be attributed to the observed effects: (i) phytotoxicity of P3HB microplastics or their degradation products and (ii) the effect on soil properties and/or inhibition of nutrients.
Alternative (i) was thoroughly discussed by Zhou et al.  who observed similar results as in this work by testing PHBV, which is a common derivative of PHB. The authors speculated about possible phytotoxic effect of PHB biodegradation products due to acidification of soil caused by released of 3-hydroxybutyric acid during PHBV degradation, but this speculation was in the cited work rejected. The rejection of the hypothesis is partially in line with our results showing the resulting pH above 7 in soil without P3HB (in other soils was probably increased due to sand content). Moreover, the pKa of 3-hydroxybutyric acid is 4.41  indicating that the acidification effect of this acid is very low. Influence of 3-HB effect on the phenylpropanoid pathway regulation related to reaction on abiotic stress [19, 20] was also mentioned. Liwarska-Bizukojc  used S. saccharatum, S. alba and L. sativum as phytotoxicity bioindicators of PHB and found no effect on seed germination even at concentration as high as 11.9% w/w. Nevertheless, the presence of PHB in soil caused root growth inhibition mainly on Sinapsis alba and Lepidium sativum. On the contrary, Dahal et al.  did not find any significant effect of PHBV on plant growth. Possible adverse effect of PHB due to reduced seedlings survival was indirectly suggested by .
Alternative ii) was discussed by Silveira Alves et al.  who stated that the role of PHB in plant-bacterium interactions is still poorly understood, however, their study suggested that PHB metabolism may contribute to bacterial plant growth promotion and that deletion of genes involved in the synthesis and degradation of PHB reduce the bacterial ability to enhance plant growth . Here, despite the prevailed P3HB-related promotion of microorganisms’ abundance and community structure, the suggestion of plant growth promotion must be rejected. Furthermore, due to the hydrophobic nature of PHB , a higher water repellency and increased drain-off may be expected in PHB-amended substrates. Zhou et al.  concluded that PHBV addition increased microbial activity, growth, and exoenzyme activity, changed the soil bacterial community at different taxonomical levels and increased the alpha diversity, which most likely led to the enhanced mineralization of native SOM and negatively influenced the growth of Triticum aestivum L.
Our results, i.e., serious P3HB-related growth inhibition of L. sativa (Fig. 4) and enhanced microbial activity, are supported by the results reported in . As indicated by the low negative correlations between plant biomass and BR, Lys-IR and Ure, respectively (Additional file 1: Fig. S1), growth inhibition could result from an adverse consequence of plant-microbiota interaction, such as competition for nutrients. The most likely scenario appears to be competition for N, which was probably utilized by PHB-degrading microorganisms (as discussed above). Similarly, suppressed growth of common bean shoots and roots in PLA-treated sandy soil, reported by , was likely caused by significant deficit of plant available (mainly nitrate nitrogen) and disproportion in dissolved organic carbon (DOC) and nitrogen (DON), leading to increased C:N ratio. Noteworthy, in our case, we can exclude the negative effect of drought stress caused by hydrophobicity of PHB as the design of pot experiments (i.e., regular irrigation) exclude the possibility of shortage of moisture in soils due to regular irrigation.
Effect of P3HB under changing SOM content as an implication of soil degradation
The results of multivariate analysis of variance (MANOVA) showed significant (p < 0.001) differences among experimental variants in all determined properties confirming the importance of increasing sand content in arable soils for the use of P3HB in agricultural soils. However, the consequences were variable, as they were positive, neutral, and negative.
16S rDNA and 18S rDNA (Fig. 1A, B) were very highly positively correlating (Additional file 1: Fig. S1) and followed similar pattern of overall decrease in both P3HB-amended and control substrates. Despite clear disproportionality, the decrease was probably caused by the decrease of the SOM content and number of soil microorganisms and fungi following soil dilution by sand. This also negatively affected all enzymatic activities, which showed a similar overall trend (Fig. 2). Therefore, the progressive deterioration of soil quality by the increase in the sand fraction will have a negative impact on microbial and fungal biomass and enzymatic activities, whether the soil is contaminated with P3HB or not.
However, the degree of sand influence is significantly affected by the presence of P3HB. Compared to control, bacterial and fungal biomass content (Fig. 1A, B) followed similar pattern of initial stagnation or decrease after P3HB addition replaced by stagnation or growth at higher (≥ 60%) sand load. In PHB-degrading microbiome, the negative effect of increasing sand content was alleviated by presence of P3HB (Fig. 1C). Moreover, the results suggest that presence of P3HB together with increasing sand content (up to 80% of sand) can even stimulate PHB-degrading microorganisms. Thus, the partial increase of 16S rDNA and 18S rDNA was probably related to this part of the microbiome. The reason may be better aeration of the substrate accompanied by the necessary presence of the P3HB energy source substituting declining SOM content, resulting in the booming of PHB-degrading microorganisms and overall shift in microbiome structure. This hypothesis is supported especially by the similar phenomenon in DHA (Fig. 2A), increasing respirations with increasing sand content (Fig. 3) and the grouping of these factors in the PCA (Additional file 1: Fig. S2).
Some other enzymes (ARS, NAG, Ure, GLU, Phos) also showed partial deviations indicating a different effect of P3HB depending on the sand content (Fig. 2). For example, at lower sand content (0–40%), the effect of P3HB on ARS (Fig. 2B) and Phos (Fig. 2F) was neutral to negative, while at higher sand content (60–100%), the P3HB effect was neutral to positive. This suggests that the expected PHB-degrading microorganisms boom related to the increased aeration had a positive effect on these enzymatic activities as well. Therefore, although P3HB acts as a potential selective microbial inhibitor in a favorable state of soil due to the dominance of different (natural) functional groups of microorganisms, in unfavorable conditions of increasing sand content, P3HB can maintain or even stimulate the activity of some enzymes associated especially with the PHB-specific microbes as an alternative energy source.
As already mentioned, predominant stimulation of BR, Glc-IR, NAG-IR, Tre-IR, Ala-IR and Lys-IR with increasing (up to 80%) sand content in P3HB-amended substrates (Fig. 3) was probably caused by better aeration coupled with P3HB utilization. This phenomenon could also be explained by the increasing rate of preferable utilization of P3HB with the increasing portion of PHB-derived C regarding the total soil organic C. This feature might be comparable to the observation of Kuzyakov and Bol , who described a metabolism switch from the hardly utilizable recalcitrant C in SOM to the easily available carbonaceous compound leading a positive priming effect. The study carried out with bean-planted PLA-contaminated sandy soil also showed increasing values of readily oxidizable carbon (POXC) with ascending content of plastics (up to 2%) . However, this can be considered as a necessary side effect rather than a cause, as it would have a similar graded positive effect on the other characteristics studied.
The increase in sand content significantly reduced the production of plant biomass in the control (Fig. 4), however, the presence of P3HB suppressed this phenomenon and limited biomass production to a minimum. Therefore, the gradual degradation of P3HB-contaminated arable soils by increasing sand content is not determining for L. sativa yields; P3HB presence limits the growth of L. sativa to the same extent as growing in pure sand. It is important to keep in mind that the possible contamination of soil by 1% of P3HB is realistic in the case of application PHB-based fertilizer coatings, delivery systems and mulching films. The SOC content in the soil was 14 g kg−1, therefore, applied dose of P3HB is very high in terms of SOM-to-P3HB ratio. As the P3HB is easily available substrate, we speculate that even significantly lower dose may have an adverse effect on plant growth. A decrease in SOM (represented here by sand dilution) can worsen this adverse effect .
In summary, the effect of changing sand content is also well reflected in the PCA (Additional file 1: Fig. S2). Sand's dominant property, high pH (Additional file 1: Fig. S3), is clearly related to the sandy variants, which form one separate group of substrates unsuitable for soil organisms and plant biomass production. The absence of P3HB and high soil content are clearly the most important factors for high L. sativa biomass production. High soil content is key for high level of bacterial and fungal biomass, as well as most enzymatic activities. The presence of P3HB and a more balanced soil:sand ratio is both crucial for high soil respiration and content of PHB-degrading microorganisms.