Biotransformation of the LDPEoxo films in the VMS and HMS
The initial low hydrophobicity (day 0) of the LDPEoxo films in both microcosm systems was due to the treatment with O2 plasma , which incorporates oxygen, polar and hydrophilic functional groups on the LDPEoxo, which favoured the material surface transformation [12, 18, 49]. However, after plasma treatment, the sheets can regain hydrophobicity after 10 days of exposure to O2 plasma . In this work, the LDPEoxo sheets post-plasma treatment and after 135 days with P. ostreatus in the microcosms did not return to their initial SCA value. Due to a synergy between photodegradation and biodegradation generated by the fungus enzymatic activity. In the VMS, the contact between the microorganism and the plastic sheets was limited, compared to HMS. In HMS, the mixture homogeneity and the contact surface increased as a consequence of fungus-free radial growth that extends to the width. P. ostreatus colonised the material, forming a network of hyphae that prevented the material from regaining its initial hydrophobicity. The carbonyl and hydroxyl groups generated on the surface of the material after exposure to O2 plasma changed the surface charge of the polymer, favouring the interaction between the cationic functional groups of the fungal wall and the anions on the LDPEoxo surface. Therefore, facilitating the adsorption and subsequent adhesion of P. ostreatus to the LDPEoxo sheets , also increasing the roughness. Additionally, after adhesion, fungus secretes oligomers and polysaccharides that covalently bind to the material surface, which is relevant since these substances function as carriers of the enzymes produced by P. ostreatus to the LDPEoxo. Furthermore, the enzymatic attack of polyphenol oxidase (C.E. 126.96.36.199) and peroxidase (C.E. 188.8.131.52) generates free radicals on the surface of LDPEoxo, which are vulnerable to oxidative attack [12, 13, 25]. The enzymatic attack and subsequent oxidative attack, break the bonds of the LDPEoxo, causing the material to maintain the acquired hydrophilicity after the plasma discharge. Since the biodegradation of the material responds to oxidative reactions, there must be a wide contact surface between the available oxygen and the fungus. Oxygen is involved in the breathing process by P. ostreatus and the oxidation of substrates, as this process is a reaction coupled with the reduction of O2 . In HMS, the contact surface between oxygen, the microorganism and the substrates were greater; for this reason, the SCA value was lower and maintained for 107 days, compared to the SCA results of VMS at day 135, the SCA in VMS was lower. These results show that both microcosm systems had a synergy between plasma and fungal, thus indicating that the system geometry influences the trend and standard deviation of the response variables associated with the plastic.
Plasma treatment is particularly effective on LDPEoxo sheets, as the pro-oxidant additives favour the formation of pores in the plastic structure; facilitating the penetration of the plasma through the structure; generating hydrophilicity on the material surface , and increasing its roughness, as observed in the AFM images (Fig. 3).
Evaluation of transformation systems in microcosms
Biotransformation of solid organic and inorganic materials (with different complexity degrees and chemical structure) is a strategy for LCB transformation or LCB and plastics co-transformation and also for the production of edible ligninolytic fungi. A strategy that is often carried out by SSF. SSF can be an open, closed, static or rotary manner [14, 21, 26, 31, 51]. In this work, a modified SSF was used to improve the process conditions, favour the fungus metabolism, and increase biotransformation and growth efficiency [12, 20, 52]. In terms of process improvement and control of microcosm systems, it is possible to modify the way LCB is mixed (napkins, brewery by-products, LDPE plastic, etc.) and distributed with other materials within the closed system. To treat the LCB can horizontally be arranged, to perform a thin layer with greater surface area, to accelerates the colonization and growth of the fungi, simulating the Dutch production system for edible fungi or Tray (Tray bioreactor) type reactors [27, 35]. On the other hand, the LCB also can be vertically arranged within a closed system or in plastic bags to form a cylinder of greater height and variable diameter, to simulates the French production system or the packaged bed reactor for SSF [22, 27, 34].
Associated with these modifications in the present work, the effect of horizontal and vertical closed systems on the percentage of humidity in the treatment of LDPEoxo and LCB sheets at laboratory scale in microcosms was evaluated. Concerning the humidity percentage, the initial values were slightly low (68.1 ± 3.3 and 69.3 ± 1.1) %, for HMS and VMS, respectively), (Fig. 2A) if compared with the optimal values reported for a solid fermentation with ligninolytic fungi (70–80%), [26, 27]. However, this did not affect the process of colonization and growth of P. ostreatus in any of the microcosm systems; on the contrary, in the HMS, there was an increase in the percentage of humidity (71.7 ± 1.5%) at 135 days, which is related to the highest growth of P. ostreatus, since approximately 50% of the humid weight of P. ostreatus is water [14, 22]. Additionally, in HMS, the LCB formed a thinner layer than in VMS, generating more surface area, which facilitated oxygen transfer and helped dissipate other gases produced during metabolism . Maintaining humidity between 60 and 80% helps the growth of the fungus and the production of ligninolytic and hydrolytic enzymes (LiP, MnP, Lac, cellulases, hemicellulases, among others); favouring the delignification process of the LCB, under oxidative conditions [22, 26].
The conditions of initial substrate acidity (6.2 ± 0.1 and 5.9 ± 0.2) and final acidity (6.5 ± 0.2 and 6.3 ± 0.5) for HMS and VMS, respectively, were another factor that allowed the abundant development of fungal mycelium and extracellular enzyme production (Fig. 4B). The values remained similar because there was no relationship between the products associated with carbon and nitrogen metabolism. On the one hand, the enzymes endoglucanases (E.C. 184.108.40.206) and endoxylanases (EC 220.127.116.11) were able to release glucose and xylose, which once assimilated by P. ostreatus generated organic acids that slightly lowered the pH in the first days of the process. On the other hand, when the mineralization or ammoniation of the organic nitrogen source (hydrolyzed brewer’s yeast) occurred, NH4 is produced and increase the pH, without ending up in the alkalinity range (> 7.0). Yoon et al. (2014), reported that in SSF of fungi for production of cellulases must start at pH below 7.0 ± 0.2, and acidification occurs during fermentation. At the end of the process, the pH can return to neutrality and even rise to a pH of 8.0 ± 0.2 . Other authors also reported that culture of P. ostreatus in SSF and under similar conditions to those of this work, the pH should be 6.0 ± 0.2, to favour the initial production of mycelium and the later formation of the fruiting body, when the pH is between 3.5 and 5.0 ± 0.2 . The biotransformation of the LCB was demonstrated with the decrease in the percentage of TOC and increase in the production of CO2, being higher in the HMS with an initial TOC of 60.2 ± 2.1% and final of 14.2 ± 0.2%. In the VMS, the initial TOC was 60.7 ± 1.7 and the final was 20.1 ± 1.3%, (Fig. 4C and D). In both systems the mixture of residues (pine bark, paper napkins and hydrolyzed brewer’s yeast) allowed the process of colonization and growth of P. ostreatus to take place. Being a saprophytic fungus must extract nutrients from the substrate, therefore, the initial colonization and extension of its hyphae are essential for obtaining the source of carbon, nitrogen and trace elements, which is favoured when using mixtures of substrates with different degrees of complexity and not just lignin since, It represents a lower energy expenditure to hydrolyze simpler polymers to obtain carbon as an energy source, than to delignify the phenylpropane subunits strongly bound by C-C and ether bonds, to obtain aliphatic or three-carbon intermediates that can enter the Krebs cycle [14, 53]. On the other hand, one reason why the decrease in TOC was higher in HMS is that, having the LCB distributed in a thin layer P. ostreatus colonized the superficial part more quickly and later its hyphae extended towards the middle and lower part of the LCB layer, while in VMS, the LCB formed a thicker cylinder-like layer, decreasing the superficial area for colonization and despite the use of intermittent forced aeration, oxygen gradients could be formed in certain areas that could delay the growth of P. ostreatus (Fig. 4C), [22, 48].
The increase of CO2 production in the first 75 days of fermentation is related to the biotransformation of materials easier to degrade such as glucose, present in the solution of trace elements and part of the hydrolyzed yeast, cellulose and hemicellulose, in pine bark and paper napkins (Fig. 4D), . The increase in CO2 occurred because, after the O2 plasma, the pro-oxidant additives act by favouring the oxidation of the LDPEoxo, and low concentrations of CO2 are also released ; an amount which is in addition to those produced by the biodegradation of the LCB.
The results obtained in this work were similar to those reported by Rojas-Higuera et al. , who used a fungal consortium (Ganoderma lucidum, Pleurotus ostreatus, Trametes versicolor and Phanerochaete chrysosporium) to biotransform sawdust from Tabebuia roseae and Eucalyptus pellita at microcosm scale, observing that highest CO2 production occurred during the first 45 days; then observed a decrease . Likewise, these results are similar to those of Moreno-Bayona et al. , who used the same mixture of LCB used in the present work and observed the production of CO2 in two phases. The first phase was lower and lasted until 45 days, the second phase with higher production finished at 75 days . In this work, after day 75, a decrease in the production of CO2 was observed in both HMS and VMS because, possibly, lignin and LDPEoxo began to biodegrade since, as they are difficult to degrade compound, P. ostreatus takes longer to transform them and therefore the speed of CO2 production also decreases. In HMS, the release of CO2 behaved with some oscillations, indicating that direct and indirect humification processes were taking place, because of the metabolic activity of P. ostreatus and that the different substrates are being metabolized (Fig. 4D).
Ligninolytic enzymes increased as a function of time in both HMS and VMS. However, the trend and activities were different for peroxidases and laccase (Fig. 4E and F). In the HMS, was observed highest peroxidase and Lac activity than in the VMS. Enzyme activity attributed to two aspects, first the distribution of the LCB within the closed system. The thin layer formed by the material into the HMS allowed that fungal colonisation and vegetative growth were higher than in the VMS and promoted the increase of viable and metabolically active fungal biomass; concomitantly with high enzymes activities. In addition, the LCB, being horizontally distributed and thinner than in the VMS, allowed easier diffusion of P. ostreatus mycelium and greater access to phenolic and non-phenolic intermediates susceptible to oxidation by LiP in the absence of mediators, especially in the last two samples where the highest activity was observed .
On the other hand, polymers such as cellulose and hemicellulose (present in the LCB) were also hydrolysed easier in the HMS system. This situation favoured the formation of low molecular weight intermediates, which are susceptible to be oxidised by auxiliary enzymes involved in lignocellulose degradation. Among these enzymes are glyoxal oxidase (GLOX; EC 18.104.22.168), aryl alcohol oxidases (AAO; EC 22.214.171.124), pyranose 2-oxidase (POX; EC 126.96.36.199), cellobiose dehydrogenase (CDH; EC 188.8.131.52) and glucose oxidase (EC 184.108.40.206). These enzymes produce hydrogen peroxide, released in greater quantity during the last days of processing, allowing it to continue participating as a cofactor for LiP and maintaining the catalytic cycle of the enzyme more efficiently in HMS than in VMS .
Authors such as Bellettini et al. , reported that during the vegetative growth of P. ostreatus these are the enzymes most closely related to the initial colonisation of substrates during the first days of SSF . The second aspect is the availability of O2 within the system. Possibly in the HMS, there was a higher concentration of O2, which allowed the Lac to have a big number of final electron acceptors, to the formation of water [22, 50]. Another fact favouring Lac activity was the presence of the ABTS redox mediator, which potentiates the enzyme activity and could generate more reactive species, contributing to the oxidation of the LCB. Additionally, cellulases and hemicellulases also participated in the biotransformation process of the LCB. These enzymes release simpler monomers that can be used during primary metabolism and generate intermediates that can promote biological Fenton processes or help regenerate the H2O2 required during the peroxidase catalytic cycle .
The biotransformation of the LCB in both HMS and VMS by P. ostreatus and its enzymes were supported by the E4/E6. This ratio makes it possible to semi-quantitatively determine whether total HS formed from an initial organic matter were biotransformed by biotic and abiotic factors related to indirect humification processes . Literature reports that the E4/E6 is inversely proportional to the degree of condensation of the aromatic rings. High values in the E4/E6 suggest a low degree of condensation and the presence of a high proportion of aliphatic compounds. Therefore, by obtaining high values, it is inferred that indirect humification has started and that so far of the FS that is forming in a lower proportion may be recently polymerised SH. As the material matures, the E4/E6 stabilises and may fall below 2.0 when SH predominate .
Concerning the present work, results indicate the FS and HS in both microcosm systems after 135 days (Table 1). However, it is not adequate to believe that the LCB was transformed completely and stabilized so that the HS are predominant since the E4/E6 in the HS was not lower than 2.0 (3.07 ± 0.35 and 3.66 ± 0.92 for HMS and VMS, respectively). On the other hand, the finding of strong Ultraviolet spectra (UV) absorption could be due to compounds containing similar chromophore groups (phenolic sands, benzoic acids and anilines derived from polyenes, etc) , (Supplementary Material S2).
Biochar production and characterization
The biochar produced was classified as class III, which means that, under the experimental conditions employed, the percentage of TOC was less than 30%, the FC was 6.55 ± 0.12%, and the ash was 20.2 ± 1.9%. These results indicated that, during the thermal treatment, a high percentage of carbon and other elements were lost in the volatile fraction, decreasing the carbon fraction that should condense as part of the biochar. Additionally, the RM had other mineral-type substances concentrated in the ashes and increased the pH (6.1 ± 0.1) %. The changes produced by the thermal treatment have reported by other authors .
The biochar was the product of the co-pyrolysis of a mixture of raw materials, composed of pine bark, napkins, hydrolyzed brewer’s yeast and fungal biomass. Being a heterogeneous mixture, the biochar obtained could have different functional groups that favoured the adsorption of the MG dye at pH 4.0 and 7.0 ± 0.2. The expectation was that MG could be adsorbed by biochar in greater quantity at pH 9.0 ± 0.2 because, at this pH, the biochar surface becomes negative. However, the highest adsorption was observed at pH 4.0 and 7.0 ± 0.2 (0.321 ± 0.003, 0.311 ± 0.023 mg g− 1 for each pH), (Table 2). This highest adsorption could be related to the presence of carboxyl groups with a neutral charge and negative charge, which could come from the fungal biomass. These opposite charge groups to the MG dye could participate in the MG adsorption at acid and neutral pH [45, 46]. On the other hand, the biochar ash content could also contribute to the increase of the MG adsorption at pH 4.0 ± 0.2. When biochar containing fungal biomass in a mixture with other materials is produced, the products contain high ash concentration that favour the removal of metals and dyes in solution. Additionally, in biochar produced with pine by-products, the initial pH is close to or higher than the isoelectric point of the biochar, the hydroxyl (−OH) and carboxyl (−COOH) groups of the surface lose protons and becomes negatively charged [57, 58]. Although at the present study, the isoelectric point of the biochar was not determined, possibly the values could be slightly higher than 7.0 ± 0.2. These results, and the pseudo-second-order model, indicate that the adsorption process could occur by chemical attraction (Fig. 5C, Table 2).
Finally, another possible application of the biochar is as a substrate for the germination of seeds of ornamental plants and grass. Table 3 shows that the biochar (T4) without additives serves as a germination substrate and could replace an expensive substrate such as peat. However, the germination percentages can be increased when using PGPB and/or chemical fertilizers. The combined use and low doses of the two products in this study favour the germination process because the bacteria produce substances that promote the germination and development of seeds and seedlings. Afterwards, the germinated seeds can take the nutrients provided by the chemical fertilizer and continue their development due to the porous organic support (biochar), which helps for nutrients retaining .