A circular economy process has been developed to convert polyurethane waste into biofertilizing microorganisms through a sequential chemical/biological process. The chemical phase involves the complete depolymerization of polyurethane using ozone attack, generating an aqueous extract (OLE) composed of small, bioavailable molecules such as polyols, isocyanate derivatives, and carboxylic acids. The biological phase utilizes OLE for the generation of biomass with biofertilizing functional activity through Rhodococcus pyridinivorans fermentation. The metabolic-proteomic expression during the biodegradation of OLE involves the synthesis of numerous enzymes such as cutinases, hydrolases, proteases, esterases and oxidoreductases, which participate in the degradation of chemical compounds like benzene derivatives, phenols, or plastic polymers. OLE has been converted into microorganisms with biofertilizing properties, including nitrogen fixation, phytohormone production and siderophores. This process contributes to sustainability by diverting polyurethane waste from landfills, reducing the environmental impact of chemical fertilizers and promoting a more sustainable agricultural system.

We have studied the combined effect of temperature and moisture on a soil contaminated with the oxyfluorfen herbicide at a dose of 4 l ha-1 in terms of the biochemical activity (dehydrogenase activity and ergosterol content) of said soil, as well as on the concentration of said herbicide over a 90-day period in a laboratory setting. Two degrees of moisture were used: (1) irrigated soils maintained at 60% of their water retention capacity and (2) non-irrigated soils (non-watered soils, without irrigation). These treatments were subjected to three temperatures (4 degrees C, 25 degrees C, and 40 degrees C, respectively). In all cases, the oxyfluorfen caused a toxic effect on both the dehydrogenase activity and ergosterol content of the soil. However, this toxic effect was greater in non-watered soils, possibly due to the combined effect of the herbicide toxicity and the soil drought conditions. With regard to temperature, the results indicate that dehydrogenase activity and ergosterol content in soil were more negatively affected when samples were incubated at higher temperatures (40 degrees C). Statistical analysis suggests that temperature may have a greater specific weight or influence than soil moisture, since a variation in temperature causes a variation in soil moisture.

Currently, the use of biostimulants is considered an environmentally friendly alternative to synthetic fertilizers. In this work, we investigated the use of a biostimulant obtained from winemaking sludge by enzymatic hydrolysis processes in peat enrichment. The aim was to observe how this biostimulant affects the growth and development of green pepper (Capsicum annuum L. cv. Italian) seedlings. The biostimulant was applied in three doses (1, 2, and 3 g/L) for a total of four applications. Seedlings were harvested at a height of 8-16 cm and with 5-7 leaves. Plant height, weight of aboveground and belowground parts, macro- and micronutrient contents, photosynthetic pigments in leaves, and enzymes related to plant stress were determined. The results obtained indicated that plant height, weight of the aboveground and belowground parts, and macro- and micronutrient content in the aboveground and root parts were higher in plants treated with the highest dose of biostimulant. Compared with the control treatment (without amendments), the contents of chlorophyll a, chlorophyll b, and total carotenoids increased by 57%, 51.9%, and 53.1%, respectively, in plants treated with the highest dose of biostimulant. These results suggest that peat enrichment with this biostimulant could be useful for improving bell pepper quality.

A novel, non-toxic plant biostimulant has been developed through the depolymerization of polyurethane (PU) foams via an aqueous ozonolysis process. This method yields a water-based extract rich in small, soluble molecules, referred to as oxidized liquid extract (OLE), which acts as a growth-promoting substrate for pepper plants (Capsicum annuum). This approach not only enables the conversion of PU waste into plant biomass but also represents an innovative model for circular economy implementation. When applied at low concentrations (diluted 1:5 and 1:10), OLE elicits a hormetic response, enhancing photosynthetic performance, protein content, and photosynthetic pigment levels. Additionally, it stimulates the accumulation of industrially relevant secondary metabolites such as terpenoids and capsaicinoids an effect likely linked to the extract's high nitrate content. Although short-chain polyols were detected in the fruits of plants treated with the 1:5 dilution, these compounds are biodegradable and widely used in pharmaceutical and cosmetic applications. Moreover, OLE induces favorable shifts in rhizospheric microbial communities, promoting the enrichment of biofertilizing bacterial genera capable of transforming extract derived compounds and enhancing the adsorption of biomolecules secreted by plant roots. This sequential chemical and biological transformation facilitates the complete valorization of a highly recalcitrant polymeric waste, resulting in a bioactive extract that supports plant growth and development. Taken together, this strategy offers a promising and sustainable solution that integrates waste management with agricultural productivity, aligning with the core principles of the circular bioeconomy.

The use of biostimulants in the bioremediation of polluted soils in order to eliminate/reduce the toxic effects of pesticides on the soil is currently a very common environmental practice. In this study, we studied the bioremediation effect of three biostimulants obtained from sewage sludge by way of an enzymatic hydrolysis and fermentation process in a diuron-contaminated soil for 55 days under laboratory conditions. During this period of time, the enzymatic activities, bacterial community and the evolution of diuron in the soil were analyzed. Compared with the unpolluted soil, the application of diuron decreased the dehydrogenase, β-glucosidase and phosphatase activities by 60%, 40.7%, and 60.6%, respectively. The Gram-positive bacterial population was decreased by 48.5%, while the Gram-negative population was decreased by 57.7% and the fungal population was decreased by 54.3%. The application of the three biostimulants to the soil decreased the diuron concentration. However, this decrease was higher when the biostimulant obtained by enzymatic hydrolysis was applied. This may be due to the fact that this biostimulant contains a higher quality of low molecular weight proteins than the other two biostimulants obtained by fermentation processes.

Polyurethane (PU) is a widely used polymer with a highly complex recycling process due to its chemical structure. Eliminating polyurethane is limited to incineration or accumulation in landfills. Biodegradation by enzymes and microorganisms has been studied for decades as an effective method of biological decomposition. In this study, Tenebrio molitor larvae (T. molitor) were fed polyurethane foam. They degraded the polymer by 35% in 17 days, resulting in a 14% weight loss in the mealworms. Changes in the T. molitor gut bacterial community and diversity were observed, which may be due to the colonization of the species associated with PU degradation. The physical and structural biodegradation of the PU, as achieved by T. molitor, was observed and compared to the characteristics of the original PU (PU-virgin) using Fourier Transform InfraRed spectroscopy (FTIR), Thermal Gravimetric Analysis (TGA), and Scanning Electron Microphotography (SEM).

The soil moisture content can vary the behavior of biochemical activity and its incidence on herbicides. The objective of this manuscript was to assess, under controlled laboratory conditions, whether a prolonged 75-day drought can affect the behavior of 4-chloro-2-methylphenoxyacetic acid (MCPA) herbicide and biochemical properties in three agricultural soils (Typic Xerofluvent, SA, Typic Haploxeralf, SB, and Vertic Chromoxert, SC). During the 75 experimental days, two irrigation levels were maintained: (1) watered soils during this experimental period, and (2) non-watered soils, where no water was supplied during the experimental period. The evolution of the herbicide and the biochemical properties were different depending on the soil moisture status. In the SA, the biochemical properties decreased until day 35 after initiating the experiment, whereas in the SB and SC, the biochemical parameters decreased until days 25 and 45, respectively. The application of herbicide to the non-watered soil increased the inhibition of biochemical properties. In non-watered SA, MCPA degradation occurred at day 45 after initiating the experiment, whereas in SB and SC, MCPA degradation occurred at days 35 and 60 after starting the incubation process, respectively. These results suggest that the soil persistence of MCPA under drought conditions increases, and consequently increases soil contamination.

Currently, biostimulants obtained from protein hydrolysates are considered essential agronomic tools to promote sustainable agriculture without the use of chemical fertilizers. This study aimed to investigate the effectiveness of a biostimulant obtained from slaughterhouse sludge via enzymatic hydrolysis processes on green pepper crops in a greenhouse. The biostimulant was administered through both root and foliar applications at two different doses (0.7 and 1.4 g L−1), with a total of four applications made over the 140-day experimental period. Throughout the crop growth period, various parameters were assessed, including plant height, the number of flowers and fruits, macro- and micronutrient content, and photosynthetic pigments in the leaves. Additionally, the nutritional content and vitamin C levels in the harvested fruits were determined. The results obtained indicated higher values of these parameters in the pepper plants when the biostimulant was applied at a higher dose and through root application. These higher values are likely a consequence of the increased plant absorption of the low-molecular-weight amino acids and nutrients derived from the biostimulant.

Abstract There is currently a great deal of information available about the toxic effects of oxyfluorfen on soil biochemical activity and microbial biodiversity. However, there is no information about how this herbicide affects plant growth promoting bacteria (PGPB) or their properties, such as biofilm formation, nitrogen fixation, siderophore and auxin production, and phosphate solubilisation. As such in this study an agricultural soil was polluted with the oxyfluorfen herbicide every 30 days at a dose of 4 L ha−1 for a total period of 90 days. During this experimental period, the dehydrogenase activity was determined, a count and isolation of cultivable bacteria was performed, and the PGPB and their properties were characterized. The results indicated that oxyfluorfen inhibits the dehydrogenase activity, with this inhibition increasing with herbicide concentration in the soil (69.9% compared to non-polluted soil). It also causes changes in the population diversity of cultivable bacteria in soils. Regard to the evolution of isolated PGPB, it was found that oxyfluorfen induces the growth of nitrogen-fixing, biofilm-forming, and siderophore-producing bacteria, while negatively affecting the growth of auxin-producing and phosphate-solubilising bacteria. These results suggest that oxyfluorfen modulates the properties of PGPB in a concentration-dependent manner.

Currently, the use of biostimulants is widespread in sustainable agriculture because they represent an alternative to chemical fertilizers. In this manuscript, we investigate the response of a greenhouse tomato crop grown in pots to a biostimulant obtained from slaughterhouse sludge applied continuously to the substrate under which the tomato crop was grown or applied directly via the foliar route. Two doses of biostimulant (0.7 and 1.4 g L−1), applied four times throughout the crop growth period (120 days), were used. During this period, the height of the plants, number of flowers and number of fruits obtained were measured. After 90 days of the growth period, the nutrient contents in leaves as well as the chlorophyll a and b contents were analyzed. For fruits, nutrient and lycopene contents were determined, and the equatorial diameter and average fresh and dry weight were measured. The results indicate a higher content of nutrients and chlorophyll a and b in the leaves of plants treated with the highest dose of biostimulant and applied continuously to the substrate. This higher content of photosynthesis pigments in the plant is possibly responsible for a greater absorption of N by the plant and, consequently, for better growth.

We report a study of the bioremediation of two biostimulants obtained from sewage sludge by fermentation processes in a oxyfluorfen-polluted soil over a 72-day period under laboratory conditions. The effects thereof on enzymatic activities, bacterial community and the evolution of oxyfluorfen in soil are determined. At the end of the experiment, and compared with the non-polluted soil, dehydrogenase, urease, β-glucosidase, and phosphatase activities in the oxyfluorfen-polluted soil decreased by 58.3%, 30.4%, 44.7%, and 48%. The application of oxyfluorfen decreased the relative abundance of the Firmicutes (24.5%) and Acidobacteria (8.3%) phyla, and increased the relative abundance of the Gaiellales order (Actinobacteria phylum). The application of both biostimulants to the soil increased the relative abundance of the Bacteroidetes, Chloroflexi and Proteobacteria phyla and decreased the relative abundance of the Acidobacteria and Actinobacteria phyla. The relative abundance of the Chloroflexi, Firmicutes and Proteobacteria phyla increased when applying the experimental biostimulants in the polluted soil. Application of both biostimulants decreased soil oxyflurofen concentration, suggesting that the use of both biostimulants with higher amounts of low molecular weight proteins and peptides could be very useful on the remediation of oxyfluorfen-polluted soils.

Abstract The objective of this study was to investigate the effect of foliar fertilization of two biostimulants (BS1 and BS2) obtained from sewage sludge by fermentative processes using Bacillus licheniformis during four consecutive seasons in an olive tree crop applied at a dose of 6 L ha-1 divided into three applications (before flowering, beginning of flowering and fruit set). BS2 had a higher number of low molecular weight peptides the BS1. The contents of macro and micronutrients in leaves, photosynthetic pigments and olive yield were higher in plants fertilized with BS2 than BS1. With respect to the control treatment and for the 2017 and 2019 seasons, the olive yield increased by 20–22% in plants fertilized with BS2 and by 28–29% for the plants foliar fertilized with the BS1. For the 2018 and 2020 seasons, the olive yield increased by 35–36% in the plants fertilized with the BS1 and by 42–43% for the plants fertilized with the BS2. These results suggest that the foliar use of these biostimulants, that contain a higher number of low molecular weight peptides, could be of great interest to farmers as regards improving olive crop yield.

Biostimulants are substances and/or microorganisms that are applied to plants or to the rhizosphere in order to enhance the natural process improving the absorption of nutrients and the quality of crops as well as the tolerance to abiotic stresses. A new biostimulant was developed from sewage sludge through its fermentation with Bacillus licheniformis as a plant growth-promoting bacteria (PGPB). The fermented product includes three classes of biostimulant components: the B. licheniformis biomass; the enzymatic secretion of said microorganism, which are mainly peptidases and amidases related to nitrogen metabolism and glucanases, related to carbohydrate metabolism; and finally, the hydrolyzed sludge organic matter, with a high content of protein hydrolysates. The biostimulant was evaluated in soil at the biochemical (enzymatic activities) and microbiological levels (metabarcoding analysis). Metabarcoding analysis revealed that the biostimulant complex, mainly the soluble fraction containing the Bacillus multienzyme complex and protein hydrolysate, induced PGPB soil bacteria, and it was detected that the inoculation in the soil of B. licheniformis remained active throughout the study. These results show the fermentation process with B. licheniformis as an interesting option for the total valorization of activated sewage sludge aimed at obtaining products of agronomic/environmental interest.

The use of organic matter is a highly accepted environmental practice among scientists for the bioremediation of polluted soils. In this manuscript we study under laboratory conditions the bioremediation capacity of a new biostimulant obtained from slaughterhouse sludge in a soil polluted by the oxyfluorfen at a rate of 4 l ha −1 (manufacturer’s rate recommended) over a 90-day period. We determined its effects on dehydrogenase, urease, β-glucosidase and phosphatase activities, the soil microbial community structure and the evolution of the herbicide in soil. Possibly due to the high content of low molecular weight proteins in the biostimulant, the enzymatic activities were stimulated mainly at the beginning of the experiment. Soil biological parameters were inhibited in oxyfluorfen-polluted soil. At the end of the experiment and compared with the control soil, dehydrogenase, urease, β-glucosidase, and phosphatase activities significantly decreased by 47.8%, 50.5%, 36.4%, and 45.5% in the oxyfluorfen-polluted soil. At 5 days into the experiment, the use of the biostimulant in oxyfluorfen-polluted soils decreased soil enzymatic activities and microbial community inhibition. At the end of the incubation period the oxyfluorfen concentration had decreased by 60% in the polluted soil and amended with biostimulants. These results suggested that the use of this biostimulant with higher amounts of low molecular weight proteins and peptides had a positive effect on the remediating oxyfluorfen-polluted soils. Therefore, this study provides the use of a new biostimulant obtained from slaughterhouse sludge by enzymatic hydrolysis processes used in the bioremediation of a soil polluted by the oxyfluorfen herbicide.

The objective of this study was to study under laboratory conditions the bioremediation effect of two biostimulants obtained from okara in a soil polluted by imazamox at a rate of 2.5 l ha-1 (manufacturer's recommended rate). These biostimulants were obtained by enzymatic hydrolysis processes using one (subtilisine) and two (subtilisine and flavourzyme) enzymes. Their effects on soil enzymatic activities, the microbial community structure and the evolution of the herbicide in soil over a 90-day period. Both biostimulants stimulated the soil biochemical activity and microbial population. However, this stimulation was greater in the soil amended with the biostimulant with a higher percentage of low-molecular-weight proteins (<300 Da). Applying the biostimulants to the polluted soil decreased the inhibition of the biological properties. This decrease was also greater when the biostimulant had a higher content of low-molecular-weight proteins content. Consequently, the biostimulant obtained by hydrolytic processes using the subtilisin and flavourzyme enzymes would be of greater interest in the bioremediation process.

Proteinaceous material constitute the main source of nitrogen in soil organic matter. Use of this nitrogen is limited by the breakdown of proteins into peptides and free amino acids. Proteases, therefore, are important in the use of protein N. The aim of this study is to assess the effect of subtilisin, which is a soil extracellular endopeptidase from Bacillus sp., both on soil microbial biostimulation and biodiversity. In addition to the protease, keratins, an insoluble-protein and low-bioavailable substrate, was added to enhance the effect of protease in soil. Protease, both on its own and in combination with keratins, has a biostimulant effect on soil microbial activity that was reflected in the protease, dehydrogenase and phosphatase activities. DNA metabarcoding analysis further revealed specific changes in soil biodiversity, mainly defined by the enrichment of plant-growth promoting microorganisms, specifically those producing hydrolase enzymes such as phosphatase and ammonifiers. Surprisingly, the addition of keratins specifically showed a rapid stimulation of soil proteolytic bacteria. Thus, the present work supports the use of subtilisin as biofertiliser and values the recycling of keratinous industrial waste in the field of biofertilisation.

The biostimulant potential of three different organic acids (OAs) present in the rhizosphere, specifically lactic, oxalic, and citric acids, have been studied. The results showed a rapid and complete metabolism of these three acids with soil microorganisms using them as a source of carbon and energy. Biostimulation was confirmed by soil biochemical studies which showed an increase in enzymatic activities, such as dehydrogenase and phosphatase, lactic and citric acids being those that produced the greatest biostimulation. With regard to microbiota composition, amplicon sequencing of the 16S rRNA gene showed changes in the structure of soil microbial communities. Applying OAs produced a decrease in richness and diversity indices, inducing specific changes in the structure of the microbiological communities. Applying lactic acid induced rapid changes in microbiota composition at both phylum and family taxonomic levels, favoring the proliferation of microorganisms involved in its degradation and soil fertility, such as the genus Bacillus and the family Micrococcaceae. Once the lactic acid was degraded, the biodiversity tended to return to similar phyla, but specific distinctive families and genera remained, leaving a pattern of induction of taxa described as plant growth-promoting bacteria (PGPB), such as the Sinorhizobium and Lysobacter genera, and the Pseudomonaceae family. Similar behavior was found with citric acid, which favored the proliferation and dominance of microorganisms of the Clostridiaceae family, involved in its degradation, as well as microorganisms of both the Micrococcaceae and Pseudomonadaceae families which were found on day 7, leaving a similar pattern of induction as that found after the mineralization of lactic acid. On the other hand, oxalic acid induced long-lasting changes in the bacterial community composition. This was characterized by an increase in the proportion of the Burkholderiales order, which includes microorganisms involved in the degradation of this acid and microorganisms described as PGPB. This study presents evidence supporting the use of OAs as potential soil fertility inducers, due both to their effects in enhancing the dominance of taxa described as PGPB and to their stimulating soil microbial activity.

In this work okara (OK), a by-product of soy milk manufacturing, is submitted to an enzymatic hydrolysis and a fermentative process to produce different soil biostimulants (BS): EH, hydrolysate obtained by the enzymatic process; FHEB, fermentation broth with Bacillus licheniformis and the enzymes secreted during the fermentation; FHE, fermentation broth without bacteria and FH, the FHE hydrolysate in which enzymes were denatured. Enzymatic hydrolysates showed a different chemical composition compared with fermented hydrolysates and OK. It had a higher protein concentration as well as C, P and K. The proteins of OK were converted into peptides with a lower molecular weight, the fermented hydrolysates being those with the lowest molecular weight profile. The influences of hydrolysates and OK were tested in soil, finding that β-glucosidase, phosphatase and dehydrogenase activities were stimulated by every treatment. However, it was observed that EH produced a greater stimulation of dehydrogenase and phosphatase than both OK and fermented BS. The bacterial and fungal phospholipid fatty acids were also higher in soils amended with BS than those of the control and soils with OK. It has also been found that β-glucosidase, phosphatase and microbial biomass were dose-dependent in every treatment, but dehydrogenase only was dose-dependent in EH and OK treatments.

In this manuscript we studied in the laboratory the bioremediation effects of a biostimulant obtained from okara by enzymatic hydrolysis processes in a soil polluted with used motor-car oil at a rate of 1 % (w/w) over an 89-day period. The biostimulant was added to the soil 6 times during the incubation period at a rate of 2 %. Dehydrogenase activity and the evolution of polycyclic aromatic hydrocarbons (PAHs) and pseudo total heavy metals in soil were studied. The successive applications of the biostimulant to the polluted soil gradually increased PAHs degradation during the experimental period. Thus, at the end of the experiment, the application of the biostimulant decreased the concentration of naphthalene in soil by 74 %, while PAHs with 3, 4, 5 and 6 aromatic rings had been reduced by around 58 %, 44 %, 30 % and 23 %, respectively. This degradation is possibly due to the high number of low molecular weight peptides (< 300 Da) in the biostimulant which are readily available for PAHs-tolerant soil microorganisms that accelerate the degradation of the said toxins. The concentration of heavy metals in the oil used was not very high and consequently the dehydrogenase activity was not negatively affected.