The biodegradation of total petroleum hydrocarbon (TPH) in soil is very challenging due to the complex recalcitrant nature of hydrocarbon, hydrophobicity, indigenous microbial adaptation and competition, and harsh environmental conditions. This work further confirmed that limited natural attenuation of petroleum hydrocarbons (TPHs) (15% removal) necessitates efficient bioremediation strategies. Hence, a scaling-up experiment for testing and optimizing the use of biopiles for bioremediation of TPH polluted soils was conducted with three 500-kg pilots of polluted soil, and respective treatments were implemented: including control soil (CT), bioaugmentation and vermicompost treatment (BAVC), and a combined application of BAVC along with bioelectrochemical snorkels (BESBAVC), all maintained at 40% field capacity. This study identified that at pilot scale level, a successful application of BAVC treatment can achieve 90.3% TPH removal after 90 days. BAVC\uffe2\uff80\uff99s effectiveness stemmed from synergistic mechanisms. Introduced microbial consortia were capable of TPH degradation, while vermicompost provided essential nutrients, enhanced aeration, and, potentially, acted as a biosorbent. Hence, it can be concluded that the combined application of BAVC significantly enhances TPH removal compared to natural attenuation. While the combined application of a bioelectrochemical snorkel (BES) with BAVC also showed a significant TPH removal, it did not differ statistically from the individual application of BAVC, under applied conditions. Further research is needed to optimize BES integration with BAVC for broader applicability. This study demonstrates BAVC as a scalable and mechanistically sound approach for TPH bioremediation in soil.15N-labeled N solution (Nsol) and a 33P-labeled P solution (Psol) were used to fertilize the soil and create the different treatments. A commercial vermicompost of bovine manure produced in Northwestern Italy was used in this study (Fig. S1). The commercial vermicompost was air-dried and milled to <2 mm. The vermicompost was characterized using the official methods of the Regione-Piemonte (1998). The residual humidity content of the dry vermicompost was 432 g kg-1, the pH in a water suspension (1:10) was 9.9, the Corg value in dry matter was 198 g kg-1 DM , the total P was 9 g kg-1 DM , and the total N was 14.8 g kg-1 DM. Ammonium sulfate ((NH4)2SO4) and potassium phosphate (KH2PO4) were used to prepare separate aqueous solution of 80.3 \u00b5g N ml-1 and 28.5 \u00b5g P ml-1, respectively. The Nsol was prepared by dissolving 9.57 mg of (NH4)2SO4 and 9.53 mg of 10 atom% 15N((NH4)2SO4 into 50 ml of Milli-Q water, resulting in a N solution with 5.5 atom% 15N abundance. On the same day of sowing, the Psol was prepared by dissolving 625 mg of KH2PO4 into 50 ml of Milli-Q water, and labeled by adding carrier-free 33P orthophosphate (Hartmann Analytics) solution to reach a specific activity of 10.7 kBq mg-1 P. Although creating a granular or pelletized OMF would have been ideal for testing potential physical interactions between vermicompost and the mineral fertilizers, this effect was not addressed in this research because of the difficulties in producing and OMF labelled with a radioisotope P tracer. Therefore, the vermicompost and the fertilizer solutions were used to mimicking an OMF granule by mixing them together in the soil. Treatments included two mixtures of vermicompost with mineral fertilizers at a ratio between Corg \u2013 N \u2013 P205 ratio of 7.5 \u2013 20 \u2013 10 (OMF7.5C) and 15 \u2013 20 \u2013 10 (OMF15C). Controls included unfertilized soil (N0P0), soil fertilized with only mineral N (MFN), only mineral P (MFP), mineral N and P (MFNP), and vermicompost at the same rates as OMF7.5C (OF7.5C) and OMF15C (OF15C). With the Pmin fertilization (Fig. S2), soils from the pot experiment received an activity of 314 Bq g-1 soil. The soil for the experiment was collected from the experimental station of Tetto Frati of the University of Turin, in NW Italy (44\u00b0 53\u2032 N, 7\u00b0 41\u2032 E; elevation 245 m). Soil was collected from the first 0.2 m of the top layer of a plot managed with maize monoculture, regularly plowed and fertilized as the typical agronomic management of the area. The soil was sieved to 5 mm and air-dried for approximately four months prior to the start of the experiment. The soil chemical characteristics measured before the beginning of the experiment indicated a low content in both plant-available N and P. Before starting the pot experiment, the bulk soil was fertilized with nutrient solutions adding 300 mg K, 60 mg Ca, 50 mg Mg, 1 mg Zn, 0.1 mg Mo, 1 mg Fe, 1 mg B, 2 mg Mn, 2 mg Cu and 0.1 mg Co per kg-1 soil to avoid any possible complementary nutrient deficiency. After fertilization, the soil was humidified to 45 % of its water holding capacity (corresponding to 109 g per kg of dry soil) and pre-incubated during 10 days at 22 \u00b0C to boost soil microbial activity. After pre-incubation, the pots were filled with the equivalent of 1 kg of air-dried soil and fertilized according to treatments. For the fertilization, two holes of 2 cm of depth and 0.5 cm of diameter were made in each pot, and on day 0, each of them was fertilized. Immediately after fertilization, 0.75 g seeds of Italian ryegrass (Lolium multiflorum var. Gemini) were distributed uniformly over the soil and then covered with 100 g of pure sand. The pots were kept in a greenhouse at 24 and 20 \u00b0C, with 12 hours light, and 65% air humidity. Soils were irrigated daily based on weight loss. To satisfy the crop requirements, irrigation was increased to keep 60 % of field capacity during the first 2 weeks, and then up to 70 % of field capacity until the final harvest. The first harvest was made 4 weeks (Fig. S3) after sowing and a second harvest was made after 4 further weeks. The harvest consisted in cutting the whole biomass at approximately 1 cm above the soil surface. Each treatment had 4 replicates. Pots were completely randomized three times per week. Incubation Experiment Setup An incubation experiment was performed to assess the influence of the vermicompost on the nutrient availability and flow from the mineral fertilizers in the soil. Soil fertilizers used were the same as in the pot experiment, but no plants were sown. The treatments for the incubation were MFNP, OMF7.5C and OMF15C. The incubation set-up and soil sampling was adapted from Sica et al. (2023), and consisted in using plastic cylinders of 18 mm of height and 60 mm of diameter. Each experimental unit had two cylinders placed one above the another and was filled with 148.6 g of soil in total. The two cylinders were separated by a nylon net with 45 \u00b5m mesh size that allowed soil solution flow. The top cylinder was fertilized replicating vermicompost, Nsol, and Psol quantities and procedures as for one hole of the pot experiment. On the day of the Pmin fertilization, the Psol had a specific activity of 3.5 kBq mg-1 P.\u00a0 With the Pmin fertilization, soils from the incubation experiment received an activity of 313.5 Bq g-1 soil.\u00a0 The soil in cylinders was humidified to 70 % of field capacity. Experimental units were placed in a box covered with a plastic sheet that did not allow vapor and light flows and kept at the same temperature conditions as the pot experiment for 10 days. Each treatment had 6 experimental units and they were completely randomized. After the incubation, the soil from the top cylinder (topsoil) was collected entirely, while from the bottom cylinder additional soil was collected from the mesh to 6 mm depth (bottom soil). Soil from two randomly chosen experimental units was mixed to reach a higher amount of sample to be analyzed, thus leaving a total of 3 replicates per treatment. Measurements on Plants In the pot experiment, at each harvest, Italian ryegrass shoot biomass was cut and dried at 40 \u00b0C for 72 hours, and then weighted to calculate dry matter yield. Afterwards, all shoot biomass was milled in a rotational miller and stored until analysis. A chemical element analyzer (Vario Pyro cube, Elementar, Germany), coupled to a mass spectrometer (IsoPrime100 IRMS, Isoprime, United Kingdom) was used to analyze total C, total N and 15N/14N from shoot biomass. For determination of P concentrations in shoot tissues, 0.25 g of milled ryegrass shoot biomass were ashed at 450 \u00b0C during 100 min. Subsequently, ashes were dissolved in 3 ml of 15.6 M nitric acid and then the volume was brought up to 25 ml with Milli-Q water. Total P concentration in the extracts was analyzed by colorimetry with malachite green (Ohno & Zibilske, 1991). The 33P radioactivity in biomass was determined using a liquid scintillation counter (TRI CARB 2500 TR, Packard) by mixing 2 ml of extract or solution with 5 ml of a scintillation liquid (Ultima Gold AB, Packard). Values were corrected for quenching and for radioactive decay back to the day of pot fertilization. Measurements on Soil Soil samples of the incubation experiment were dried at 40\u00b0C for 3 days and then ball-milled and stored until analysis. Soil samples were analyzed for concentration of total N and 15N/14N ratio with the same method and instruments as for plant samples. The 15N enrichment of total soil N was then related to the 15N enrichment of the fertilizer and decreasing 15N enrichment of soil N interpreted as less fertilizer N having moved in the respective soil zone/layer (Frick et al., 2022). For determining P contained in soil, soil ashes were obtained similarly to plant biomass ashes. Soil ashes were dissolved into 50 ml of H2SO4 solution (0.5 M). Then, 5 to 10 ml of the solution was filtered with 0.2 \u03bcm syringe filters and stored at 4\u00b0C for 1 day until analysis of radioactivity. Values of 33P radioactivity in extracts were measured 32 days after fertilization following the same procedures as with biomass samples and corrected for radioactive decay by calculating back to day 0 of fertilization. The decrease of the specific activity of the soil P with distance from the fertilizer spot indicated decreasing presence of fertilizer P (as above explained for N). Statistical Analysis Both experiments had a completely randomized design. When testing for differences between treatments over the harvests, a repeated measures ANOVA was used. The incubation experiment was analyzed comparing treatments of each soil layer with a one-way ANOVA using treatment as factor. If significant differences between treatments were found a Tukey\u2019s HSD test was performed as a post hoc comparison. Some values were analyzed as the total production (sums or averages of both harvests, or both soil layers), in those cases data were analyzed by a one-way ANOVA using treatment as factor. All analyses were performed using the software R, version 4.0.5. Package multcompView was used to display post hoc results.", "keywords": ["vermicompost", "FOS: Agricultural sciences", "nutrient use efficiency", "double labeling", "organo-mineral fertilizer"], "contacts": [{"organization": "Sitzmann, Tomas Javier, Sica, Pietro, Zavattaro, Laura, Moretti, Barbara, Grignani, Carlo, Oberson, Astrid,", "roles": ["creator"]}]}, "links": [{"href": "https://doi.org/10.5061/dryad.ffbg79d23"}, {"rel": "self", "type": "application/geo+json", "title": "10.5061/dryad.ffbg79d23", "name": "item", "description": "10.5061/dryad.ffbg79d23", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main/items/10.5061/dryad.ffbg79d23"}, {"rel": "collection", "type": "application/json", "title": "Collection", "name": "collection", "description": "Collection", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main"}], "time": {"date": "2023-01-18T00:00:00Z"}}, {"id": "10.5281/zenodo.7024773", "type": "Feature", "geometry": null, "properties": {"updated": "2026-05-30T16:24:40Z", "type": "Dataset", "title": "Dataset of the paper \"Vermicomposting as a sustainable option for the management of the biomass of the invasive tree Acacia dealbata Link.\"", "description": "Data generated during an experiment of vermicomposting of Acacia dealbata fresh biomass. Four files are included: 'vermicompost_and_earthworm_data.csv' and 'readme.csv' are the raw data of different parameters measured in vermicompost samples during the vermicomposting of Acacia dealbata by the earthworm Eisenia andrei and an explanation of each parameter and the unit in which the parameter is expressed.\u00a0 'germination test.csv' and 'radicle_length.csv' are the results of an ecotoxicological test on the effect of A. dealbata biomass and vermicompost on the germination and radicle elongation in Lepidium sativum.", "keywords": ["Acacia dealbata", "ecotoxicological test", "vermicompost", "Eisenia andrei", "15. Life on land", "earthworm", "Lepidium sativum"], "contacts": [{"organization": "Quintela-Sabar\u00eds, Celestino, Mendes, Lu\u00eds A., Dom\u00ednguez, Jorge,", "roles": ["creator"]}]}, "links": [{"href": "https://doi.org/10.5281/zenodo.7024773"}, {"rel": "self", "type": "application/geo+json", "title": "10.5281/zenodo.7024773", "name": "item", "description": "10.5281/zenodo.7024773", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main/items/10.5281/zenodo.7024773"}, {"rel": "collection", "type": "application/json", "title": "Collection", "name": "collection", "description": "Collection", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main"}], "time": {"date": "2022-08-26T00:00:00Z"}}, {"id": "10.5281/zenodo.7024774", "type": "Feature", "geometry": null, "properties": {"updated": "2026-05-30T16:24:40Z", "type": "Dataset", "title": "Dataset of the paper \"Vermicomposting as a sustainable option for the management of the biomass of the invasive tree Acacia dealbata Link.\"", "description": "Data generated during an experiment of vermicomposting of Acacia dealbata fresh biomass. Four files are included: 'vermicompost_and_earthworm_data.csv' and 'readme.csv' are the raw data of different parameters measured in vermicompost samples during the vermicomposting of Acacia dealbata by the earthworm Eisenia andrei and an explanation of each parameter and the unit in which the parameter is expressed.\u00a0 'germination test.csv' and 'radicle_length.csv' are the results of an ecotoxicological test on the effect of A. dealbata biomass and vermicompost on the germination and radicle elongation in Lepidium sativum.", "keywords": ["Acacia dealbata", "ecotoxicological test", "vermicompost", "Eisenia andrei", "15. Life on land", "earthworm", "Lepidium sativum"], "contacts": [{"organization": "Quintela-Sabar\u00eds, Celestino, Mendes, Lu\u00eds A., Dom\u00ednguez, Jorge,", "roles": ["creator"]}]}, "links": [{"href": "https://doi.org/10.5281/zenodo.7024774"}, {"rel": "self", "type": "application/geo+json", "title": "10.5281/zenodo.7024774", "name": "item", "description": "10.5281/zenodo.7024774", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main/items/10.5281/zenodo.7024774"}, {"rel": "collection", "type": "application/json", "title": "Collection", "name": "collection", "description": "Collection", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main"}], "time": {"date": "2022-08-26T00:00:00Z"}}, {"id": "10486/717833", "type": "Feature", "geometry": null, "properties": {"updated": "2026-05-30T16:25:59Z", "type": "Journal Article", "created": "2024-03-22", "title": "Bioaugmentation and vermicompost facilitated the hydrocarbon bioremediation: scaling up from lab to field for petroleum-contaminated soils", "description": "Abstract
The biodegradation of total petroleum hydrocarbon (TPH) in soil is very challenging due to the complex recalcitrant nature of hydrocarbon, hydrophobicity, indigenous microbial adaptation and competition, and harsh environmental conditions. This work further confirmed that limited natural attenuation of petroleum hydrocarbons (TPHs) (15% removal) necessitates efficient bioremediation strategies. Hence, a scaling-up experiment for testing and optimizing the use of biopiles for bioremediation of TPH polluted soils was conducted with three 500-kg pilots of polluted soil, and respective treatments were implemented: including control soil (CT), bioaugmentation and vermicompost treatment (BAVC), and a combined application of BAVC along with bioelectrochemical snorkels (BESBAVC), all maintained at 40% field capacity. This study identified that at pilot scale level, a successful application of BAVC treatment can achieve 90.3% TPH removal after 90 days. BAVC\uffe2\uff80\uff99s effectiveness stemmed from synergistic mechanisms. Introduced microbial consortia were capable of TPH degradation, while vermicompost provided essential nutrients, enhanced aeration, and, potentially, acted as a biosorbent. Hence, it can be concluded that the combined application of BAVC significantly enhances TPH removal compared to natural attenuation. While the combined application of a bioelectrochemical snorkel (BES) with BAVC also showed a significant TPH removal, it did not differ statistically from the individual application of BAVC, under applied conditions. Further research is needed to optimize BES integration with BAVC for broader applicability. This study demonstrates BAVC as a scalable and mechanistically sound approach for TPH bioremediation in soil.The biodegradation of total petroleum hydrocarbon (TPH) in soil is very challenging due to the complex recalcitrant nature of hydrocarbon, hydrophobicity, indigenous microbial adaptation and competition, and harsh environmental conditions. This work further confirmed that limited natural attenuation of petroleum hydrocarbons (TPHs) (15% removal) necessitates efficient bioremediation strategies. Hence, a scaling-up experiment for testing and optimizing the use of biopiles for bioremediation of TPH polluted soils was conducted with three 500-kg pilots of polluted soil, and respective treatments were implemented: including control soil (CT), bioaugmentation and vermicompost treatment (BAVC), and a combined application of BAVC along with bioelectrochemical snorkels (BESBAVC), all maintained at 40% field capacity. This study identified that at pilot scale level, a successful application of BAVC treatment can achieve 90.3% TPH removal after 90 days. BAVC\uffe2\uff80\uff99s effectiveness stemmed from synergistic mechanisms. Introduced microbial consortia were capable of TPH degradation, while vermicompost provided essential nutrients, enhanced aeration, and, potentially, acted as a biosorbent. Hence, it can be concluded that the combined application of BAVC significantly enhances TPH removal compared to natural attenuation. While the combined application of a bioelectrochemical snorkel (BES) with BAVC also showed a significant TPH removal, it did not differ statistically from the individual application of BAVC, under applied conditions. Further research is needed to optimize BES integration with BAVC for broader applicability. This study demonstrates BAVC as a scalable and mechanistically sound approach for TPH bioremediation in soil.