Discussion

The presented results provide a comprehensive overview of the influence of fungal colonisation on the tensile behaviour of additively manufactured PLA/wood material combinations. Based on these findings, the mechanical research question defined in Section 1 can now be addressed.

Influence of fungal colonisation on mechanical response (RQ1)

Across all investigated material combinations, the fungal-colonised specimens exhibited statistically significantly lower mean E and mean UTS values than the corresponding untreated specimens. The reduction in mean Young’s modulus ranged from approximately 7.1 % to 10.5 %, while the reduction in mean UTS ranged from approximately 2.2 % to 6.8 %. Although the magnitude of the reductions varied between material combinations, the overall trend was consistent across all investigated PLA/wood material combinations.

The statistical evaluation presented in Fig. 18 supports these observations. For Young’s modulus, highly significant differences (p<0.0001) were identified between untreated and fungal-colonised specimens for all investigated material combinations. Similarly, statistically significant reductions in mean UTS were observed for all material combinations, confirming that the measured differences are unlikely to result from random experimental variation alone.

The stress–strain curves further demonstrated that fungal-colonised specimens generally exhibited lower initial slopes and lower maximum stress values compared with their untreated counterparts. Since mean Young’s modulus was determined from the initial linear region of the stress–strain curves, the observed reductions indicate a decrease in tensile stiffness in the fungal-colonised specimen groups. Likewise, the reduction in mean UTS demonstrates that fungal colonisation also affected the maximum load-bearing capacity of the investigated material combinations.

A pronounced stress peak followed by a distinct post-peak softening region was particularly observed for the untreated and fungal-colonised 40 wt.% wood-particle material combinations (PLA_W40 and PLA_W40_WM). In both cases, the stress increased rapidly to a maximum value before decreasing towards a more stable plateau region at higher strain levels. Since no in-situ damage monitoring was performed during tensile testing, the mechanisms responsible for this behaviour cannot be identified directly. However, the pronounced peak indicates that local damage initiation and load redistribution processes may occur shortly after the maximum stress level is reached. Additional investigations combining mechanical testing with advanced microstructural characterisation would be required to establish the origin of this characteristic stress–strain response.

Microscopic observations confirmed successful surface colonisation of all investigated material combinations by Fomes fomentarius. Hyphal structures were observed on specimen surfaces and within surface-adjacent regions after incubation. However, the present study does not allow direct conclusions regarding the underlying mechanisms responsible for the observed reductions in mechanical performance. While fungal growth was clearly visible on the specimen surfaces, additional investigations using techniques such as scanning electron microscopy (SEM), micro-computed tomography (_μ_CT), or interfacial characterisation would be required to establish direct relationships between fungal colonisation and changes in the internal composite structure.

An additional limitation of the present study concerns the potential influence of the fungal colonisation protocol itself on the mechanical response of the investigated PLA/wood material combinations. Although successful fungal growth was confirmed by microscopy, fungal hyphae were primarily observed on the specimen surfaces and within surface-adjacent regions. Furthermore, no sham-treated control specimens were included to isolate the individual effects of incubation, moisture exposure, drying, and specimen handling. Following fungal colonisation, all specimens were oven-dried at 55 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ$$\end{document}C and subsequently vacuum sealed prior to mechanical testing. Nevertheless, the moisture content of the specimens was not quantitatively determined before or after colonisation, and the relative humidity during the incubation period was not monitored. Consequently, the observed reductions in mean Young’s modulus (E) and mean ultimate tensile strength (UTS) should be interpreted as the mechanical response of specimens subjected to the fungal colonisation protocol rather than being attributed exclusively to fungal activity. Future investigations should therefore include sham-treated controls together with quantitative moisture measurements to distinguish moisture-related effects from the direct influence of fungal colonisation.

An additional limitation of the present study concerns the potential influence of moisture introduced during the fungal colonisation process. PLA and lignocellulosic fillers are known to exhibit hygroscopic behaviour, and moisture uptake may affect the mechanical response of PLA/wood composites. Following fungal colonisation, all specimens were oven-dried at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$55^\circ$$\end{document}C to terminate fungal activity and subsequently vacuum sealed prior to mechanical testing. However, the moisture content of the specimens was not quantitatively determined before or after colonisation, and the relative humidity during the incubation period was not monitored. Consequently, the observed reductions in mean Young’s modulus (E) and mean ultimate tensile strength (UTS) cannot be attributed exclusively to fungal colonisation, as moisture-related effects may also have contributed to the measured mechanical response. Future investigations should therefore include quantitative moisture measurements and moisture-controlled reference specimens to distinguish between moisture-related and biological effects.

This interpretation is consistent with our previous work14, in which specimens subjected to a fungal cultivation protocol likewise exhibited measurable changes in mechanical behaviour. As discussed in that study, environmental conditions associated with the cultivation process, including incubation and moisture exposure, may also contribute to changes in mechanical properties. Related studies on mycelium-based material combinations have similarly demonstrated that fungal cultivation can alter the structural characteristics and mechanical response of bio-based material combinations depending on the fungal species, substrate composition, and cultivation conditions43. Although direct quantitative comparisons between studies remain challenging due to differences in material combinations, specimen geometries, incubation conditions, and testing methodologies, the available literature collectively indicates that fungal cultivation protocols can influence the performance of engineered composite materials.

It should further be noted that the investigated PLA/wood filaments originated from different commercial manufacturers and may therefore differ not only in nominal wood-particle content but also in PLA grade, additives, wood species, particle morphology, and compounding procedures. Consequently, differences observed between individual material combinations cannot be attributed exclusively to the nominal wood-particle content. Nevertheless, the influence of fungal colonisation remained consistent across all investigated material combinations.

Accordingly, the mechanical research question can be answered by concluding that post-printing fungal colonisation by Fomes fomentarius consistently reduced both mean E and mean UTS of the investigated PLA/wood material combinations. The statistically significant differences identified by the ANOVA analyses, together with the observed changes in the stress–strain response and the confirmed presence of fungal growth on the specimen surfaces, provide clear evidence that fungal colonisation influenced the tensile behaviour of the investigated materials.

Environmental implications of the investigated PLA/wood material combinations (RQ2)

The supplementary screening-level LCA provides an environmental interpretation of the investigated PLA/wood material combinations at the MEX AM printing stage. Within the defined gate-to-gate boundary, the environmental profile is mainly determined by two foreground contributions: electricity consumption during printing and material-combination-dependent material consumption. This separation between electricity- and material-related contributions is informed by the previously published XAI–LCA workflow, in which printing-stage impacts are interpreted through energy- and material-driven pathways53. Similar distinctions between process energy demand and material consumption are also discussed in LCA studies of MEX AM processes59,60.

For GWP, the most important outcome is that the total impact is dominated by the electricity-related contribution. Since all investigated specimens are produced under comparable printing conditions and show only small differences in calculated electricity consumption, the electricity term forms a nearly constant baseline across the five PLA/wood material combinations. The PLA/wood mass split changes the material-related contribution, but this effect is superimposed on the larger electricity-related baseline. Therefore, changing the wood-particle content alone has only a limited influence on total GWP within the present printing-stage boundary. This result is in line with previous MEX AM LCA studies in which process energy demand is identified as an important contributor to climate-related impacts53,60.

AP and EP provide a different environmental perspective. In contrast to GWP, these indicators are more sensitive to the material composition of the printed specimen. When the PLA fraction increases, the material-related AP and EP contributions become more pronounced. This trend reflects the different cradle-to-gate characterization factors assigned to PLA resin and wood flour in the present screening model56–58. Similar findings in LCA studies of PLA-based and wood-fibre-reinforced biocomposites show that the environmental interpretation of bio-based composites depends strongly on the selected impact category and should not be reduced to climate impact alone61. Therefore, AP and EP are retained as supplementary indicators because they reveal material-combination-dependent effects that are less visible in the GWP results.

These findings address RQ2 by showing that the environmental implications of the investigated PLA/wood material combinations are category-specific. GWP mainly reflects the electricity demand of the printing process, whereas AP and especially EP are more sensitive to the PLA/wood material split. A higher wood-particle content can reduce the PLA-related material contribution, but the environmental benefit depends on the selected midpoint indicator and on the printing-stage electricity demand. Therefore, the LCA results should be understood as an environmental profile of the investigated material combinations rather than as a single universal ranking. The relationship between these environmental findings and the mechanical performance of the printed specimens is discussed in the following section.

Relationship between mechanical performance and environmental impacts

The supplementary screening-level Life Cycle Assessment (LCA) provides additional insight into the environmental implications of the investigated PLA/wood material combinations and complements the mechanical characterisation presented in Sections 3.1 and 3.2.

The contribution analysis demonstrated that the environmental impacts of the investigated material combinations were governed by different mechanisms depending on the selected impact category. For GWP, electricity consumption during the MEX AM process represented the dominant contribution, accounting for approximately 88–92 % of the total impact. Consequently, variations in wood-particle content resulted in only relatively small differences in total GWP because all specimens were manufactured using nearly identical printing conditions and exhibited similar electricity consumption values.

In contrast, AP and EP were more strongly influenced by material composition. The contribution analysis showed that increasing wood-particle content reduced the relative amount of PLA required per specimen and therefore decreased the material-related environmental burdens. This effect was particularly pronounced for EP, where the material contribution represented approximately 95–98 % of the total impact. For AP, both electricity consumption and material composition contributed substantially to the overall results.

When the mechanical and environmental results are considered together, a trade-off becomes apparent. Fungal colonisation consistently reduced both mean E and mean UTS, as confirmed by the ANOVA results presented in Figure 18. At the same time, increasing wood-particle content generally reduced material-related environmental impacts by partially replacing PLA with a lignocellulosic filler associated with lower cradle-to-gate impacts.

However, the mechanical results indicate that increasing wood-particle content does not automatically lead to improved tensile performance. Although the investigated commercial filament combinations containing approximately 30 wt.% wood particles exhibited the highest measured values of mean E and UTS, direct comparison between all investigated materials remains limited because the filaments originated from different manufacturers and may differ in PLA grade, additives, wood species, particle morphology, and compounding procedures. Consequently, the observed trends should be interpreted as material-combination-specific behaviour rather than as evidence for an optimal wood-particle content.

Overall, within the assumptions of the present screening-level assessment, increasing wood-particle content can reduce material-related environmental burden indicators, while fungal colonisation introduces a measurable reduction in tensile performance. The results therefore highlight the importance of simultaneously considering mechanical requirements and environmental impacts when selecting PLA/wood material combinations for additively manufactured applications.