Investigating the Effect of Anaerobic Digestion Residue on Basil Growth, Secondary Metabolite Synthesis, and Growing Substrate Properties

Abstract

To assess digestate’s efficacy as a fertilizer for basil development, a two-year pot experiment was established, comprising four fertilization treatments: namely, mineral fertilizer (F), digestate (D), combined mineral fertilizer and digestate (1:1, FD), and unfertilized control (C). Key metrics assessed included plant height, chlorophyll concentration index (CCI), total biomass (TB), leaf production (LP), essential oil yield, and composition. Post-harvest analysis evaluated nutrient and heavy metal content and pathogen contamination in the growing substrate and leaves. FD treatment produced the highest TB (68.2 g plant⁻¹) and LP (52.7 g plant⁻¹). Digestate application substantially enhanced substrate nutrient availability, increasing extractable phosphorus by 68.5%, potassium by 134.4%, and organic matter by 54.7%. The essential oil yield was significantly higher in the control plants. whereas different fertilization regimes altered secondary metabolite synthesis. Specifically, fertilization with digestate favored sesquiterpenes synthesis, inorganic fertilization enhanced methyleugenol and β-farnesene synthesis, and the control showed higher limonene, eugenol, and linalool. Heavy metal accumulation in the growing substrate was negligible, remaining well within regulatory limits. Salmonella spp., were not detected. Pathogen concentration in the growing substrate was low, while Enterococcus faecalis levels were marginally below EU safety limits (100 cfu g⁻¹) on the leaves. Continuous monitoring of soil chemical properties and plant products after digestate application is essential to ensure soil health and food safety.

1. Introduction

Agricultural sustainability in the Mediterranean region is facing significant issues due to soil degradation and desertification, attributed to climate change and land mismanagement [1]. Simultaneously, a considerable reduction in precipitation and an increase in salinization are expected to occur, undermining soil fertility in the Mediterranean region [1,2]. To address these challenges, digestate, the by-product of anaerobic digestion (AD), has emerged as a promising organic fertilizer that can restore and enhance soil quality.

Digestate differs from other organic amendments, such as compost or green manure, in several key respects. Specifically, unlike compost, which undergoes aerobic decomposition over extended periods, or green manure, which involves the decomposition of freshly incorporated plant matter in soil, digestate is produced through anaerobic digestion [3,4,5]. Anaerobic digestion aims to produce renewable energy in the form of biogas, while generating a nutrient-rich liquid residue known as digestate [3]. While the primary aim of most organic amendments is to increase soil organic matter content and, to a lesser extent, enrich the soil with nutrients, digestate contains nutrients in mineral forms that are readily available for plant uptake [3,4,5]. Consequently, it can be utilized effectively as a standalone fertilizer [3].

Fertilization with digestate has the potential to increase soil fertility by stimulating the propagation of beneficial microorganisms, promoting microbial community diversification, and enhancing carbon sequestration [6]. Moreover, digestate application improves soil physical structure by increasing the number and mean weight diameter (MWD) of 5–10 mm and 1–5 mm water-stable aggregates, while reducing the smaller ones (<1 mm). These improvements in soil aggregation have been observed to persist for up to four years post-application, demonstrating digestate’s potential as a soil enhancer [7].

Furthermore, long-term digestate application significantly enhances soil health and mechanical properties. Holatko et al. (2023) [8] reported that digestate application increased soil sulfur content, enhanced the activity of key enzymes involved in nutrient cycling (including arylsulfatase, N-acetyl-β-D-glucosaminidase, and phosphatase), and improved nitrogen sequestration over extended periods. Didelot et al. (2023) [9] demonstrated that continuous digestate application resulted in significantly higher dissolved organic carbon (DOC) fluxes than mineral fertilizers or raw pig manure, highlighting the superior soil-enhancing capacity of digestate. Additionally, digestate application positively influenced nutrient retention and recycling, making it a more efficient fertilizer option for diverse cropping systems [9].

However, digestate application raises potential safety concerns. Specifically, digestate produced from animal manure can contain pathogens, such as Salmonella spp., E. coli, and Enterococcus faecalis, which are sometimes able to withstand the digestion process [10,11]. Carraturo et al. (2022) [12] reported that thermophilic digestion sanitizes digestate more effectively than mesophilic digestion, due to the elevated temperatures present during the thermophilic AD process. This indicates that digestates, and particularly those from mesophilic conditions, require monitoring to ensure sufficient elimination of pathogenic microbes.

Heavy metal contamination presents another concern, as animal feed can introduce elevated concentrations of metals such as Cd, Cr, Ni, Pb, Zn, and Cu [13]. This risk is further exacerbated by digestate’s relatively low nutrient content, necessitating higher application volumes to achieve nutrient levels comparable to mineral fertilizers [14]. Consequently, repeated or excessive digestate application may lead to heavy metal accumulation in the soil [11]. The introduction of excessive pollutant loads through low-quality waste applications can adversely affect soil fertility, threaten groundwater quality, and contaminate the food chain [15,16].

Consequently, evaluating digestate’s performance on diverse crops is essential to assessing both the agronomic benefits and the safety challenges. Digestate has been successfully employed as fertilizer for various medicinal and aromatic plants (MAPs), including rosemary, peppermint, lemongrass, and basil, with positive effects on both biomass production and the synthesis of secondary metabolites [17,18,19]. In particular, Rowe et al. (2023) [18] reported that the fertilization of lemongrass with digestate increased the overall biomass production eightfold, compared to the unfertilized control. Ronga et al. (2018) [17] found that the combined application of solid digestate as a growing medium and liquid digestate as the nutrient solution increased shoot dry weight, total dry weight, and the relative amount of sesquiterpenes in basil and peppermint plants grown hydroponically. Moreover, digestate application led to significant increases in the number of leaves, total number of branches, plant dry weight, and essential oil content in rose-scented geranium plants (Pelargonium graveolens L’Hér.) [19].

Basil (Ocimum basilicum L.) is one of the most economically important MAPs, widely used in Mediterranean cuisine and across the globe [20]. Basil is consumed fresh or dried as a culinary herb, while its essential oil is used in the food, pharmaceutical, and flavoring industries [20]. Essential oil from basil exhibits exceptional medicinal properties, including antiviral, antibacterial, antioxidant, antifungal, and anticancer activities [21].

The primary constituents of basil essential oil are linalool, eucalyptol, estragole, and eugenol, alongside numerous other compounds present in varying concentrations [21]. Understanding how fertilization influences the synthesis of these bioactive compounds is critical for maximizing both the medicinal properties and the commercial value of the oil. The study of Ndah et al. (2022) [22] demonstrated that nutrient availability can significantly influence terpene synthesis by either increasing the overall metabolic capacity or the number of functional glandular trichomes in subarctic shrubs. Panuccio et al. (2019) [23] reported enhanced synthesis of compounds with anti-inflammatory and anti-carcinogenic properties only in digestate-amended cucumber plants. Consequently, the complex nutrient matrix of digestate could potentially enhance the chemical and medicinal profile of basil essential oil.

The present study aimed to investigate the effect of digestate on key agronomic characteristics and the growing substrate of basil. Notably, it is the first study to investigate the effect of digestate on the essential oil composition of basil plants cultivated in a solid growing substrate.

2. Materials and Methods

2.1. Plant Material and Experimental Design

The experiments were conducted at the Institute of Plant Breeding and Genetic Resources (IPGRB), in Thessaloniki, Greece (Latitude 40°36′58.75″ N, Longitude 22°49′51.16″ E). The experiments were conducted in a net-house with a transparent polycarbonate roofing (10 mm thickness, 2.10 m × 6.00 m). The roofing was placed to ensure protection from rainfall and controlled irrigation conditions. Notably, plants were grown under natural light throughout the experimental process.

Seeds of a large-leaved sweet basil (Ocimum basilicum L.) germplasm (preserved in the IPGRB collection) were sown in 96-cell plastic trays containing peat substrate in mid-March 2023 and 2024. Approximately 30 days after sowing (DAS), seedlings were individually transplanted into 2.5 L polyethylene pots at the four-leaf stage.

The growing substrate comprised soil and perlite at a 2:1 ratio (v:v). Soil was collected from the top 0–30 cm layer of a field near the IPGRB campus, used for MAP cultivation. After collection, the soil was sieved with a 2 mm mesh to remove rocks, large aggregates, and other extraneous materials. The soil was classified as sandy loam (SL) with a pH of 8.5, low in heavy metals, and free of pathogens (

Table 1. Physicochemical properties and heavy metal and pathogen contents of the soil and digestate used in the current experiment, pre-cultivation.

Physicochemical Properties	Units	Soil	Digestate
pH	-	8.5	7.8
Electric Conductivity (EC)	mS cm−1	0.24	2.42
Organic Matter (O.M.)	%	1.81	1.08
Soil Texture	-	Sandy Loam (SL)	-
Clay	%	14.1	-
Silt	%	30.8	-
Sand	%	55.1	-
Dry Matter (TS)	%	-	1.81
Volatile Solids (VS)	%	-	1.08
Total Nitrogen	%	-	0.2
Phosphorus (P)	mg kg−1	11.87 E	310 T
Nitrate Nitrogen (N-NO3)	mg kg−1	25.77 E	<500
Calcium (Ca)	mg kg−1	3610 E	710 T
Magnesium (Mg)	mg kg−1	290 E	260 T
Potassium (K)	mg kg−1	320 E	1430 T
Zinc (Zn)	mg kg−1	2.70 E	18 T
Iron (Fe)	mg kg−1	5.09 E	73.8 T
Manganese (Mn)	mg kg−1	3.53 E	11.9 T
Boron (B)	mg kg−1	0.19 E	2.31 T
Sodium (Na)	mg kg−1	113 E	560 T
Heavy Metals
Cadmium (Cd)	mg kg−1	0.18 T	0.007 T
Chromium (Cr)	mg kg−1	75.5 T	0.35 T
Mercury (Hg)	mg kg−1	0.05 T	0.02 T
Copper (Cu)	mg kg−1	23.7 T	0.40 T
Lead (Pb)	mg kg−1	14.0 T	0.09 T
Arsenic (As)	mg kg−1	7.90 T	0.05 T
Pathogens
Salmonella spp.	cfu g−1	N.D.	N.D.
Enterococcus faecalis	cfu g−1	N.D.	1.8 × 102
Escherichia coli (E. coli)	cfu g−1	N.D.	45 est.

^T: Total, ^E: Extractable, cfu: colony forming units, est.: estimated.

).

To assess the efficacy of digestate as an alternative organic fertilizer for developing basil, a randomized complete block design (RCBD) experiment was conducted with 3 replicates, comprising 6 plants per replication and 4 fertilization regimes, for a total of 72 plants. The fertilization treatments applied were: (1) digestate (D); (2) combined digestate with inorganic fertilizer (FD), where plants received 50% of their nitrogen requirements from digestate and 50% from inorganic fertilizer; (3) liquid fertilizer (F); and (4) unfertilized control (C).

The digestate used in the current study was obtained from an anaerobic digestion batch experiment performed in the Soil and Water Resources Institute (SWRI) in Thessaloniki, Greece [22]. The experiment entailed the use of rice straw and swine slurry at a percentage ratio of 30%:70% (on a volatile solids basis), as feedstock for an anaerobic digester operating under mesophilic conditions (37 °C). This feedstock ratio (30% rice straw: 70% swine slurry, on a volatile solids basis) was selected based on our previous study [24]. According to our results, co-digestion of pretreated rice straw and swine slurry in a 30%:70% ratio exhibited the highest methane production, with the least amount of rice straw. While the 50%:50% ratio achieved maximum methane production, our techno-economic analysis revealed that the 30%:70% ratio (exhibiting the second-highest methane production) would be more practical for commercial biogas plants due to seasonal availability and high costs associated with rice straw collection, transportation, and pretreatment [24]. The digestate used in this study, therefore, represents the most realistic output from a commercial-scale biogas power plant. The physicochemical characteristics of the digestate, as well as its heavy metal and pathogen content, are presented in Table 1. Prior to fertilization, the digestate’s pH was adjusted to neutral (7), using 1 M HCl. This adjustment was performed to avoid excessive substrate pH, which could result from combining alkaline digestate with alkaline soil components, in order to achieve optimal nutrient availability and minimize the risk of ammonia volatilization and pH stress.

The inorganic fertilizer used in the current experiment (7-3-6 NPK) is commercially available and designed for green leafy plants (Compo, Münster, Germany). It contains 7% total Nitrogen, comprising 2.6% N-NO₃, 1.5% N-NH₄, and 2.9% N-NH₂. Moreover, it includes 3% P₂O₅, 6% K₂O, and other micronutrients.

During experimentation, all plants were irrigated twice per week using tap water and maintained at 70% Water Holding Capacity (WHC), estimated gravimetrically. All fertilized plants received equal amounts of nitrogen (2.5 g plant⁻¹), although the source of nitrogen differed among treatments. Fertilizers were applied with each irrigation event (twice weekly) in equal portions throughout the growing period, beginning one week after transplanting. Nitrogen fertilization was calculated as 250 kg N ha⁻¹ [23], with a plant density of 10 plants per square meter.

Although most studies recommend lower nitrogen rates for basil (150–200 kg N ha⁻¹), we selected the highest of the recommended nitrogen fertilization (250 kg N ha⁻¹) based on the study by Bufalo et al. (2015) [25], who investigated organic versus conventional fertilization in basil under greenhouse conditions. This higher rate was deliberately chosen to accentuate both the agronomic benefits and potential risks (heavy metal accumulation, pathogen contamination, and salt stress) associated with digestate application, making them more pronounced and easier to detect.

At 60 DAS, plant inflorescences were removed to promote the formation of lateral branches [26]. Inflorescences were manually removed as soon as they appeared to promote vegetative growth and maximize leaf production. This standard cultural practice prevents the plant from entering reproductive development, thereby increasing leaf biomass and essential oil yields [27].

The plants were harvested at 90 DAS by cutting the stems at the growing substrate’s surface. The leaves were removed and stored at −20 °C for subsequent analyses of essential oil yield, essential oil composition, and concentration of pathogens. Additionally, at the end of experimentation, the growing substrates from all of the pots and from each treatment were combined and homogenized. Three samples (1 kg each) per replicate were collected and stored at −20 °C for nutrient, heavy metal, and contaminant analysis. The experimentation was conducted over two consecutive growing seasons (2023, 2024). Each year, the experiment was reproduced with fresh growing substrate, new seedlings, and identical fertilization regimes. This design allowed us to assess the repeatability of digestate effects under similar environmental conditions rather than examining residual or after-effects from the previous year’s application. Data from both years were pooled for combined analysis after confirming that year effects were not statistically significant.

2.2. Environmental Conditions

Temperature and relative humidity were continuously monitored using a HOBO Micro Station (Onset Computer Corporation, Bourne, MA, USA). The climatic conditions for 2023 and 2024 are presented in Figure 1.

2.3. Growth and Physiological Parameters

2.3.1. Growth Parameters

Prior to harvest, plant height was recorded weekly, at 39, 49, 55, 62, 69, and 76 DAS, by measuring the stems from the ground level up to the tip of the plants. At harvest (90 DAS), total fresh aboveground biomass (TB) and leaf production (LP) were determined gravimetrically (g plant⁻¹).

2.3.2. Physiological Parameters

Chlorophyll content (CCI) was measured on the two youngest fully developed leaves of all plants using a portable chlorophyll content meter (CCM-200, Opti-Sciences, Tyngsboro, MA, USA) at 39, 49, 55, 62, 69, and 76 DAS.

2.4. Analytical Methods

2.4.1. Analytical Methods for Physicochemical and Microbiological Characteristics

The determination of volatile solids (VS) and total solids (TS) was performed according to standard methods [28]. To determine the heavy metal concentration, the samples were decomposed in acid at a high digestion vessel pressure with the aid of a Milestone Ethos Up microwave oven (Milestone Srl, Sorisole, Italy), and the resulting solution was analyzed using an Agilent 7850 ICP-MS (Agilent Technologies, Santa Clara, CA, USA) equipped with the ORS4 collision cell. Quality control measures included blanks to monitor contamination, duplicate sample analyses (10% of samples) to assess precision (RSD < 5%), and certified reference materials to verify accuracy (recovery rates 95–105% for all elements) [28]. Electrical conductivity was evaluated on approximately 10 g of a sample, which was placed into a 250 mL plastic container with 50 mL of deionized water. The sample was stirred for 1 h, followed by filtration. In the filtrate, the electrical conductivity was measured at 25 °C. Total nitrogen (organic and ammoniacal) was calculated according to the standard Kjeldahl method. Nitric nitrogen (N-NO₃) was determined separately by spectrophotometry (HACH DR 3900, Hach-Lange GmbH, Düsseldorf, Germany). Total calcium carbonate was determined by gravimetric analysis. The method was based on the reaction of HCl with calcium carbonate and the gravimetric loss of CO₂ from the sample [29].

At the end of experimentation (90 DAS), the growing substrates from all pots within each treatment were combined and homogenized. Three replicate samples (1 kg each) were collected per treatment and stored at −20 °C until pathogen analysis. For pathogen detection, samples were thawed, and appropriate dilutions were prepared in sterile saline solution before plating or PCR analysis. Quality control measures included negative controls (sterile saline solution) to confirm the absence of contamination and positive controls (reference bacterial strains) to validate detection methods across all assays. The detection of Salmonella was conducted according to Midorikawa et al. (2014) [30]. The number of enterococci was determined by plating known amounts of the sample on the surface of a selective medium (Enterococcus agar) prepared according to the method described by Slanetz and Bartley (1957) [31]. After 48 h of incubation at 44 °C, colonies exhibiting typical morphology were enumerated. The method of detection and enumeration of Escherichia coli in the digested material was based on the ISO 16193:2013 standard [32].

2.4.2. GC-MS/FID Analysis

Leaf samples from the aerial parts were collected from all the plants per treatment and combined to constitute three bulk samples (200–300 g each). A Clevenger-type apparatus was used to determine the essential oil (EO) content (mL 100 g⁻¹ on a fresh weight basis). The fresh leaves (100 g of pooled sample per treatment) of basil were subjected to hydrodistillation for 3 h, with a distillation rate of 3–3.5 mL min⁻¹. The EO was collected in 4 mL glass vials, dried over anhydrous sodium sulfate, and stored at 4–6 °C until further analysis. The fresh biomass was distilled in triplicate, and the % yield was expressed as the mean of three independent replicates.

Essential oil samples were diluted in pentane before they were injected for analysis. A Shimadzu 17 A Ver. 3 gas chromatograph interfaced with a QP5050A mass spectrometer and holding a capillary Agilent HP-5MS 30 m, 0.25 mm, 0.25 µm column, and supported by the GC/MS Solution ver. 1.21 software was employed for the analysis of the EO. The conditions of analysis followed were previously described by Sarrou et al. (2023) [33]. The abundance of each compound was calculated as the percentage of the total chromatographic area. The identification of the compounds was performed through the comparison of (a) their retention indices (RI) with those of n-alkanes (C7–C22) [34], (b) with the corresponding literature data, and (c) by matching their spectra with those of the MS libraries (NIST 98, Willey). For EO constituents’ quantification, GC/FID analysis was carried out on a Shimadzu Nexis GC-2030 series with flame ionization detector (FID) and a Shimadzu AOC-20i auto injector using a Crossbond MEGA5MS column (30 m × 0.25 mm, film thickness 0.25 µm) coated with 95% methyl polysiloxane. The oven temperature was as follows: 55 °C (hold time 1 min), 55–110 °C (rate 1.5 °C min⁻¹), 110–150 °C (3 °C min⁻¹), 150–220 °C (8 °C min⁻¹), and 220 °C for 10 min. The injector temperature was set at 260 °C and the detector temperature at 280 °C. Injection volume was 1 μL, Helium (He) was used as the carrier gas (1 mL min⁻¹), and the split ratio was 1:30.

2.5. Statistical Analysis

ANOVA was conducted for data analysis using MSTAT-C ver. 1.41 (Michigan State University, East Lansing, MI, USA). Experimentation time (runs) and the interaction between the time by experimental treatment (T) were not significant; hence, data from the two runs were pooled and subjected to combined over-year ANOVA analysis (Supplementary Tables S1–S6). Fisher’s least significant difference (LSD) was used to determine significant differences among the examined treatments, at p ≤ 0.05 (n = 6).

3. Results and Discussion

3.1. Morpho-Physiological Parameters

During the first week of measurements (39 DAS), all treatments exhibited similar heights (12.5–12.8 cm). In the second week, plants receiving inorganic fertilization, either exclusively or partially (F and FD), were significantly taller than both the D-treated and control plants. However, from the third week onwards, all plants receiving fertilization did not show significant differences among them, while the control consistently presented the shortest plants (Figure 2).

Our results concur with those of other researchers who have reported that digestate supports plant growth as effectively as conventional inorganic fertilizers. Specifically, Ronga et al. (2018) [17] reported that using liquid digestate as a nutrient solution and solid digestate as a growing medium in hydroponic basil cultivation significantly increased plant height compared to mineral fertilization. Similarly, Asp et al. (2022) [35] reported that 50% substitution of growing substrate with digestate in basil cultivation performed analogously well to conventional fertilized treatments, consistent with our findings.

At 39 DAS, F-treated plants exhibited the highest CCI values (18), compared to all other treatments (13–14). At 49 and 55 DAS, F-amended plants continued to present the highest CCI values (25), while the FD plants ranked second. D-treated plants exhibited intermediate values, and control plants demonstrated the lowest CCI values (13). From 62 DAS onwards, the F and FD plants did not show significant differences, D-treated plants followed, and control plants consistently presented the lowest CCI values (Figure 3).

The variation in CCI values according to treatment reflects the differences in nutrient availability depending on the fertilization regime. Specifically, the inorganic fertilizers provide readily available nitrogen that plants utilize for rapid chlorophyll production [36], while organic fertilizers release nitrogen slowly as they rely on microbial decomposition [37]. Digestate has readily available nitrogen primarily in ammonium form, but basil plants absorb nitrogen mainly in nitrate form [38]. The procedure of nitrification is a two-step process carried out by specialized soil microorganisms and is therefore a time-consuming process [39], resulting in delayed nitrogen availability to basil plants compared to inorganic mineral fertilization. Hence, at the beginning of our experiment, plants receiving solely inorganic fertilizer exhibited higher CCI values, while plants treated exclusively with digestate exhibited lower CCI values.

In contrast, basil plants receiving the inorganic–digestate mixture (FD) performed considerably better than D-treated plants and, from 62 DAS onwards, exhibited values comparable to the F-treated plants. Therefore, inorganic and organic fertilizers appeared to act complementarily. The readily available nitrogen from the inorganic fertilizers facilitated immediate chlorophyll synthesis, while digestate provided a slower but continuous stream of nitrogen.

Similar results were reported by Li et al. (2023) [40], who studied the effect of an inorganic fertilizer, digestate, and their combination on tomato plants. According to their results, digestate addition, whether partial or exclusive, led to significant increases in the leaf chlorophyll content of the tomato plants.

TB production showed significant differences among the four fertilization regimes (Figure 4). Specifically, the FD-treated plants produced the highest TB, equal to 68.2 g plant⁻¹, while the F-treated plants followed. D-treated basil plants exhibited lower biomass production (59.3 g plant⁻¹), but significantly higher than the control. Unfertilized plants (C) presented the lowest biomass production (40.1 g plant⁻¹), approximately 41.2% less than the FD-treated plants and 32.4% less than the D-treated plants (Figure 4).

The FD-amended plants presented the highest total biomass compared to all other treatments, likely owing to the complementary effects of inorganic fertilization and digestate. Analogous results were reported by Adekiya et al. (2024) [41], who observed enhanced maize production through the application of mineral urea combined with digestate. Other researchers have noted that the substitution of mineral nitrogen fertilizers with digestate can increase fruit yield and plant growth considerably. In particular, Li et al. (2023) [40] demonstrated that inorganic fertilization combined with digestate markedly increased tomato yield, compared to mineral fertilization. Jamison et al. (2021) [42] reported enhanced plant growth in kai choy plants with 50% substitution of mineral fertilizers with digestate. Brychkova et al. (2024) [43] found that partial or complete inorganic fertilizer substitution with digestate increased grassland quality and yield by 1.5 to 2 fold.

Plants amended solely with digestate exhibited significantly lower TB than F and FD plants, albeit performed considerably better (approximately 48%) than the control. These observations are in accordance with Ronga et al. (2018) [17], who reported that digestate can serve as an effective alternative fertilizer for basil cultivation, especially in organic cultivation.

Hence, the markedly higher production of digestate-amended plants compared to the control highlights digestate’s strong potential as a nutrient source for organic cultivation systems.

Digestate is an untapped source of revenue for biogas plants [24], as its use as an alternative organic fertilizer would benefit the environment and enhance the biogas power plants’ profitability. According to Jurgutis et al. (2021) [44], the average daily value of digestate produced from a 1 MW power plant can reach up to EUR 1518, providing a considerable source of income for biogas-producing units and a valuable organic fertilizer for agricultural production. However, making digestate a marketable product necessitates a techno-economic analysis to fully estimate digestate’s potential as an alternative fertilizer for basil cultivation.

Nevertheless, there are certain constraints linked to its usage, such as low concentration of nutrients, elevated water content, considerable transportation and storage costs, and regulatory limitations [45]. Moreover, increases in pH, salinity, and electric conductivity (EC) [46], as well as the risk of pollution from heavy metals and pathogens [14,47], pose a considerable concern.

Concerning the effect of the four fertilization regimes on the LP, our analysis showed that FD-treated plants exhibited significantly higher leaf production compared to all other treatments (Figure 5). Specifically, FD-treated plants produced 52.7 g plant⁻¹, while F-treated plants followed. In contrast, plants receiving solely digestate exhibited significantly lower LP, and unfertilized plants presented the lowest LP, equal to 28.9 g plant⁻¹ (45% less than FD) (Figure 5).

The superior leaf production in FD-treated plants aligns with findings from other researchers who studied different basil species using combined mineral-organic fertilization strategies. Kalita et al. (2018) [48] observed enhanced leaf yield with a combination of vermicompost and mineral fertilization (NPK) compared to the use of solely vermicompost in O. gratissimum. Likewise, Chandel et al. (2024) [49] reported significantly higher leaf numbers and leaf area in O. gratissimum under combined inorganic–organic N fertilization. These results support our findings and highlight the synergistic effect of organic and inorganic fertilizers.

3.2. Essential Oil Production and Composition

Essential oil content in basil leaves was significantly affected by the different fertilization regimes. Specifically, all the fertilized samples exhibited lower EO content than the control (Figure 6). These results align with previous studies, which state that excessive nitrogen fertilization leads to a reduction in essential oil production. Kordi et al. (2020) [50] noted that the relationship between nitrogen fertilization and EO production is inversely proportional. Alami et al. (2024) [51] reported that unfertilized plants produce more EOs to alleviate environmental and nutritional stresses. Herms et al. (1992) [52] described the mechanism behind plants’ nutrient allocation strategy, when determining whether to allocate nutrients towards vegetative growth or secondary metabolite synthesis. Under excessive nitrogen fertilization, basil plants prioritize vegetative growth and biomass accumulation, directing photosynthates toward protein synthesis and structural development rather than secondary metabolite production [53]. This resource allocation results in higher leaf mass but lower essential oil concentration per unit of biomass. Additionally, excessive nitrogen promotes rapid vegetative growth, creating a dilution effect whereby essential oil compounds synthesized by the plant are distributed across a larger biomass volume, reducing the concentration of essential oils per unit of dry matter [54]. Conversely, moderate nutrient stress can trigger enhanced secondary metabolite biosynthesis as a stress-response mechanism, resulting in higher essential oil concentrations [53]. Hence, the high fertilization used in the current experiments led to reduced EO production in the leaves of the fertilized plants.

However, the FD-treated plants had the highest LP, while the control plants had the lowest. Therefore, if we estimate the essential oil yield of each treatment per hectare, we observe that the highest production is derived from the FD plants. Specifically, the FD leaves produced 24.77 kg ha⁻¹, the F 22.10 kg ha⁻¹, the D leaves 21.74 kg ha⁻¹, and the control 21.67 kg ha⁻¹. Thus, to maximize EO production, a combined fertilization is preferable.

The dominant constituent in the EO of the basil leaves, regardless of treatment, was linalool, with concentrations ranging from 45.50 to 49.61%. The control plants demonstrated significantly higher linalool, limonene, and eugenol content, compared to all other treatments (

Table 2. Composition of the essential oil (%) in the leaves of basil plants treated with four different fertilization regimes.

Peak Number	Compound	RT	F	D	FD	C
1	Limonene	15.91	0.80 b	0.80 b	0.78 b	0.86 a
2	Eucalyptol	16.07	9.02 a	7.43 a	8.40 a	7.41 a
3	trans-β-Ocimene	17.34	2.10 b	2.77 a	2.67 a	1.69 c
4	Linalool	21.41	45.50 b	46.86 b	46.48 b	49.61 a
5	Bornanone	24.58	0.81 a	0.74 a	0.80 a	0.64 b
6	Terpinen-4-ol	27.45	3.75 a	3.89 a	3.67 a	3.33 b
7	Eugenol	42.23	6.13 b	8.17 b	7.41 b	10.11 a
8	β-Elemene	44.47	1.46 b	1.64 a	1.48 b	1.49 b
9	Methyleugenol	45.31	3.47 a	2.05 b	2.18 b	1.93 b
10	α-Bergamotene	47.05	6.29 a	5.87 a	5.56 a	4.61 a
11	α-Humulene	47.93	0.83 a	0.80 a	0.78 a	0.61 b
12	β-Farnesene	48.22	1.31 a	1.02 b	0.97 b	0.92 b
13	Germacrene D	49.40	2.31 d	3.21 a	2.59 b	2.48 c
14	τ-Cadinol	55.36	6.29 b	7.57 a	6.36 b	7.18 a
	Total		90.07	92.82	90.13	92.87

RT: Retention time, D: Digestate, F: Inorganic fertilizer, FD: Combined 50% inorganic fertilizer and 50% digestate, C: Control (no fertilization). Values are means over two years. In each line, means with different letters indicate statistically significant differences between treatments at p ≤ 0.05.

).

The concentrations of linalool, limonene (monoterpenes), and eugenol (phenylpropanoid) are increased in plants to enhance their stress tolerance. These terpenes improve salt [55], cold and drought tolerance [56], and show excellent antibacterial, antifungal, antioxidant, and antineoplastic activity [57,58,59]. Therefore, the stress induced by the lack of nutrient availability likely facilitated the increase in these stress-alleviating components in the control plants.

Digestate fertilization stimulated sesquiterpene synthesis and led to a statistically significant increase in the concentrations of two specific compounds. Specifically, D-treated plants exhibited the highest concentration in β-elemene and germacrene D (1.64 and 3.21%, respectively) (Table 2). Germacrene D is a volatile hydrocarbon and a key intermediate for the biosynthesis of several sesquiterpenes, including cadinenes [60]. Similar results were reported by Burducea et al. (2018) [61], stating that basil fertilization with biosolids led to significant increases in the concentration of β-elemene and germacrene D. Ronga et al. (2018) [17] reported enhanced sesquiterpene production in basil and peppermint plants grown hydroponically with solid digestate as the growing medium and liquid digestate as the nutrient solution.

There is no clear mechanism linking the synthesis of these sesquiterpenes with digestate fertilization. However, we hypothesize that the abundance of specific nutrients in digestate, including macronutrients such as nitrogen (NH₄⁺ and NO₃⁻), phosphorus, potassium, calcium, and magnesium, and micronutrients such as iron, zinc, manganese, and boron, coupled with organic matter, may have influenced the metabolic pathways of secondary metabolite synthesis, facilitating the preferential shift toward sesquiterpene production (β-elemene and germacrene D) observed in digestate-amended plants [22,62].

Specifically, Ndah et al. (2022) [22] demonstrated that increased nutrient availability (N, P, K) significantly enhanced sesquiterpene emissions (by 140%) in subarctic dwarf shrubs, thereby providing evidence that nutrient availability can modulate sesquiterpene biosynthesis. Similarly, Sile et al. (2022) [62] reported that nutrient availability influenced the sesquiterpene profile of Glechoma hederacea, with terpenoid composition varying in response to ecological and growing conditions. Panuccio et al. (2019) [23] found that digestate application in cucumber cultivation not only enhanced total phenol and flavonoid synthesis but also triggered the production of neohesperidine, hesperitin, naringin, and narirutin. Notably, these compounds were exclusively synthesized in response to digestate fertilization.

In contrast, plants receiving combined fertilization showed balanced results, exhibited higher content in four monoterpenes (eucalyptol, trans-β-Ocimene, bornanone, and terpinen-4-ol) and two sesquiterpenes (α-bergamotene and α-humulene). Nevertheless, in most cases, the differences with the remaining fertilization treatments were not statistically significant.

Plants treated with mineral fertilizer exhibited significantly higher concentrations of methyleugenol and β-farnesene (3.47 and 1.31%). β-Farnesene is an important terpene with considerable and variable agricultural, pharmaceutical, cosmetic, bioenergy, and industrial applications [63]. Methyleugenol is a phenylpropanoid produced by plants as a defense mechanism for herbivores and pathogens and serves as a pollination attractant for certain insects, particularly fruit flies [64]. Hence, the terpene synthesis in F-treated plants showed that basil plants receiving inorganic fertilization focus on the synthesis of terpenes used for protection from biotic stresses and terpenes facilitating reproduction.

Notably, while F-treated plants showed the highest methyleugenol content, the control plants demonstrated the highest eugenol content. This inverse relationship suggests that, under stress conditions, plants have limited methylation capacity for eugenol and, as a result, accumulate it. Conversely, under favorable nutritional conditions, plants exhibit enhanced methylation activity, converting eugenol to methyleugenol (Table 2).

Furthermore, germacrene D content was particularly low in F-amended plants and significantly higher in D-treated plants, suggesting that mineral fertilization could have an adverse effect on germacrene D synthesis.

Therefore, different fertilization regimes can stimulate the synthesis of different secondary metabolites. Fertilization with digestate enhances sesquiterpene production and especially β-elemene and germacrene D, while inorganic fertilization facilitates the production of methyleugenol and β-farnesene. Conversely, unfertilized plants produce leaves with higher linalool, limonene, and eugenol, while FD-treated plants achieve a more balanced profile with higher EO yields.

3.3. Growing Substrate Properties

Growing substrate pH in our study was similar in the F, FD, and D-treated plants, ranging from 7.7 to 7.8, representing a substantial reduction from the initial soil pH of 8.5. Conversely, the pH reduction in the control substrate was limited and equal to 0.2 (pH = 8.3) (

Table 3. Growing substrates’ chemical properties and nutrient concentrations under different fertilization regimes, post-harvest.

Chemical and Nutrient Properties	Treatments
	Units	F	D	FD	C
pH	−	7.7 a	7.8 a	7.8 a	8.3 b
Electric Conductivity	mS cm−1	1.30 c	1.94 a	1.47 b	0.22 d
E Boron (B)	mg kg−1	0.21 c	0.25 a	0.23 b	0.17 d
E Calcium (Ca)	mg kg−1	3373 c	3868 a	3720 b	3332 c
E Phosphorus (P)	mg kg−1	13.5 c	20.0 a	17.5 b	9.5 d
E Iron (Fe)	mg kg−1	5.25 c	8.51 a	7.04 b	4.22 d
E Potassium (K)	mg kg−1	380.0 c	750.0 a	650.0 b	294.0 d
E Magnesium (Mg)	mg kg−1	310.4 c	420.6 a	380.0 b	274.3 d
E Manganese (Mn)	mg kg−1	5.20 c	6.70 a	5.83 b	3.38 d
E Sodium (Na)	mg kg−1	135.1 c	324.9 a	271.7 b	110.7 d
E Zinc (Zn)	mg kg−1	2.84 c	6.50 a	4.80 b	2.22 d
Nitrate Nitrogen (N-NO3)	mg kg−1	48.60 a	43.70 b	50.50 a	12.35 c
Organic Matter (O.M.)	%	1.86 c	2.80 a	2.53 b	1.84 c

^E: Extractable, F: Inorganic fertilizer, D: Digestate, FD: Combined 50% inorganic fertilizer and 50% digestate, C: Control (no fertilization). Values are means over two years. In each line, means with different letters indicate statistically significant differences between treatments at p ≤ 0.05.

). These results differ from those typically reported in the literature, where digestate application generally increases pH. Specifically, according to García-López et al. (2023) [46], digestate application in the cultivation of lettuce and kale resulted in considerable elevations in soil pH, significantly higher compared to the increases observed from soils fertilized with inorganic fertilizers. However, the usually high pH in digestates is beneficial to soils suffering from high acidity, because it increases their buffer capacity, protecting them from acidification, a major issue emanating from the constant addition of mineral fertilizers [65,66].

In our study, growing substrates amended with digestate showed a significant decrease in pH because the digestate’s pH was adjusted to ~7.0 prior to application. In contrast, the pH in the control exhibited a minor reduction of approximately 0.2 units, likely due to the consistent addition of irrigation water (Table 3).

Moreover, the adjustment of digestate pH from its original alkaline level (8.5) to neutral (7.0) using 1 M HCl prior to application may have influenced our results in several ways. First, adjusting the pH to neutral altered the buffering capacity and nutrient availability in the digestate. The HCl consumed the digestate’s natural pH buffers before application. Substrates amended with this pH-adjusted digestate had reduced buffering capacity and could not maintain stable pH during cultivation, becoming progressively more acidic due to root activity and nitrification.

Several macronutrients, such as phosphorus (P) and nitrogen (N), and micronutrients, such as Zinc (Zn) and manganese (Mn), present varying levels of availability depending on growing substrate pH, preferring slightly acidic substrates [67]. The use of HCl introduced chlorine ions, which may have contributed to the final electrical conductivity (EC) of the digestate. While this adjustment was performed to mitigate phenomena of alkaline toxicity and increase nutrient availability for the plants, it represents a deviation from typical digestate application practices.

EC in the growing substrate of the D-treated plants was significantly higher compared to all other treatments (1.94 mS cm⁻¹). In contrast, samples from the control plants demonstrated the lowest EC value (0.22 mS cm⁻¹) (Table 3). These results align with García-López et al. (2023) [46], who reported that increases in soil electrical conductivity were proportional to the addition of digestate. Salt content in digestate primarily derives from the substrates used for its production. The digestate used in the current study mainly comprised swine slurry, which typically has a high salt content [68]. Moreover, rice straw was treated with Na, further increasing the digestate’s sodium content.

In general, EC values exceeding 2.0 mS cm⁻¹ are considered high, requiring attention to avoid toxicity issues in salt-sensitive plants [69]. Jamison et al. (2021) [42] reported inhibitions in key growth and yield parameters in Brassica juncea plants when they used exclusively digestate from lignocellulosic biomasses for their fertilization, hypothesizing that the high EC of the media was responsible. However, proper management can alleviate any potential risks from digestate application. Ragályi et al. (2025) [69] reported that adequate irrigation post-digestate application and digestate dilution prior to implementation efficiently mitigates salt accumulation toxicity phenomena and EC increases.

In the present study, digestate-amended substrates exhibited an EC of 1.94 mS cm⁻¹, which is substantially lower than EC levels reported to cause salinity stress in basil. Morano et al. (2017) [70] demonstrated that basil grown hydroponically showed optimal growth and yield at an EC of 2.8 mS cm⁻¹, with salinity stress effects only appearing at EC values exceeding 3.1 mS cm⁻¹. Consistent with these findings, no visible salinity stress symptoms (leaf chlorosis, necrosis, or stunted growth) were observed in our digestate-treated basil plants during the cultivation period, and the EC remained well below the general threshold of 2.0 mS cm⁻¹ for salt-sensitive plants [69]. This indicates that the salinity risk from digestate application at the rates used in this study is low for basil under the experimental conditions tested. However, long-term salinity risk must be considered for repeated applications. Continuous digestate application could lead to progressive salt accumulation in the substrate, potentially exceeding safe EC levels in subsequent growing cycles.

D-treated growing substrates also showed significant concentrations of extractable phosphorus and boron, increasing the soil P and B content by 68.5 and 31.6%, respectively (Table 3). Conversely, samples from the control plants exhibited the lowest concentration, reducing P and B content by 20% and 10.5%, respectively. These increases in P availability with digestate application align with the research performed by Hammerschmiedt et al. (2022) [71]. The researchers reported that digestate application enhances soil P availability, particularly when different feedstocks are combined. García-López et al. (2023) [46] stated that digestate affects nutrient availability and alters the biogeochemistry of phosphorus in soil, potentially increasing both inorganic and organic P fractions.

Digestate application considerably increased Ca concentrations. Particularly, Ca levels in the growing substrates amended solely with digestate increased by 7.1%, while the FD samples presented the second-highest concentrations. In contrast, samples from F and unfertilized plants exhibited the lowest Ca levels and actually removed Ca from their substrates (approximately 7 and 8%, respectively) (Table 3). Other researchers have reported similar results. In particular, da Silva (2024) [72] noted that digestate contains many essential nutrients, including calcium, which are mineralized and readily available for plant use, potentially increasing Ca availability in the soil.

Digestate application also resulted in significantly higher concentrations of iron, magnesium, manganese, sodium, and zinc. These results are consistent with findings from other researchers, who reported that digestate is rich in minerals, and soils amended with digestate exhibit elevations in the concentration of nutrients significant for agricultural production. Moreover, according to Hammerschmiedt et al. (2022) [71], digestate enhances the micronutrient content of soil, improves plant growth, and health. Rolka et al. (2024) [73] reported that the addition of liquid digestate leads to considerable increases in K, P, Fe, Mn, Ca, Na, and total nitrogen, while the addition of solid digestate leads to major incremental changes in the concentration of available P, Mg, Mn, and exchangeable cations of Ca and Mg.

In contrast, FD and F treatments produced the highest nitrate nitrogen concentrations (96% and 88.6%, respectively) compared to the initial soil values. The increase in N-NO₃ from digestate application was significantly lower (69.6%), while the control reduced it by 52.1% (Table 3). According to García-López et al. (2023) [46], nitrogen in digestate is mostly organic, necessitating mineralization in order to become available to the plants. Hence, soils amended with inorganic fertilizers exhibited higher concentrations of N-NO₃, due to the immediate nitrogen availability from mineral fertilizers.

Substrates amended exclusively with digestate presented a significantly higher concentration of organic matter (2.80%), representing a 54.7% increase relative to the initial substrate O.M. content of 1.81% (Table 3), while FD samples followed. Conversely, samples from the F and control exhibited the lowest O.M., demonstrating limited increases equal to 2.8% and 1.7%, respectively (Table 3). The significant increases in O.M. from the use of digestate were expected, since several studies have supported this notion. In particular, da Silva (2024) [72] noted that soil amended with digestate presents considerable increases in organic matter, microbial activity, and nutrients. Villarino et al. (2025) [74] reported that constant digestate application to 14 crop fields over more than five years led to markedly higher increases in soil organic carbon (SOC), particularly in soils with low initial SOC.

Concerning the K concentration, all the fertilized growing substrates exhibited elevated K content, while the control exhibited reductions. Specifically, the highest concentration of K appeared in the substrates amended with digestate, elevating soil K content by 134.4%, significantly outperforming all other treatments. In contrast, the control samples exhibited the lowest concentration and reduced it by 8.1% (Table 3). Okoli et al. (2023) [75] reported that digestate addition to soil enhances its potassium content. Yuan et al. (2023) [76] stated that the combined application of inorganic and organic fertilizers leads to higher yields of sweet potato and enhanced K uptake, demonstrating digestates’ ability to meaningfully elevate K concentration in soil and availability to plants.

The increases in heavy metal concentrations due to digestate addition were limited (

Table 4. Growing substrates’ heavy metal concentrations under different fertilization regimes, post-harvest.

Heavy Metals	Treatments
	Units	F	D	FD	C
T Arsenic (As)	mg kg−1	8.04 a	8.18 a	8.15 a	7.85 b
T Cadmium (Cd)	mg kg−1	0.19 a	0.20 a	0.19 a	0.19 a
T Chromium (Cr)	mg kg−1	75.29 a	76.61 a	76.12 a	75.09 a
T Copper (Cu)	mg kg−1	23.74 a	24.13 a	24.06 a	23.47 a
T Lead (Pb)	mg kg−1	15.1 a	15.3 a	15.0 a	14.8 a
T Mercury (Hg)	mg kg−1	0.05 a	0.05 a	0.04 a	0.04 a

^T: Total, F: Inorganic fertilizer, D: Digestate, FD: Combined 50% inorganic fertilizer and 50% digestate, C: Control (no fertilization). Values are means over two years. In each line, means with different letters indicate statistically significant differences between treatments at p ≤ 0.05.

). These findings align with the observations of Derehajło et al. (2023) [14], who reported that digestate can contain variable trace amounts of heavy metals depending on the feedstock composition. Tang et al. (2020) [77] reported that Cd levels in soils amended with digestate remained within safe limits after five years of constant application. In the study of Baldasso et al. (2023) [78], researchers reported that digestate is safe to use for land restoration even if the concentration of heavy metals is high due to the low metal mobility in the soil profile.

Increases in Cu were limited, with the highest increases appearing in D-amended growing substrates (Table 4). These results correspond with the findings of Ferreira et al. (2023) [79], who stated that pig slurry application increases the availability of the exchangeable fraction of copper in the soil without compromising crop productivity.

Derehajło et al. (2023) [14] reported that the digestate used in their experiments exhibited a higher concentration of Hg compared to animal slurry. However, Hg content was within the limits set for organic fertilizers [80] and, therefore, safe for agricultural use. The researchers also stated that continuous monitoring of heavy metals is imperative to ensure their concentration remains within safe limits [14].

The authors of the current study concur that, even though heavy metal content in the digestate and its translocation to the growing substrate were limited, continuous monitoring is essential to ensure soil health and agricultural production free of heavy metal contamination.

According to our pathogen contamination analysis, all samples were free of Salmonella spp. (

Table 5. Growing substrates’ pathogen concentrations under different fertilization regimes, post-harvest.

Pathogens	Treatments
	Units	F	D	FD	C
	Growing substrate
Salmonella spp.	cfu g−1	N.D	N.D	N.D	N.D
Enterococcus faecalis	cfu g−1	N.D	<9.1	<9.1	N.D
Escherichia coli (E. coli)	cfu g−1	N.D	<9.1	<9.1	N.D
	Basil Leaves
Salmonella spp.	cfu g−1	N.D	N.D	N.D	N.D
Enterococcus faecalis	cfu g−1	N.D	91est.	73est.	N.D
Escherichia coli (E. coli)	cfu g−1	N.D	<9.1	<9.1	N.D

N.D: Not detected, F: Inorganic fertilizer, D: Digestate, FD: Combined 50% inorganic fertilizer and 50% digestate, C: Control (no fertilization), cfu: colony forming units, est.: estimated.

). Low levels of Enterococcus faecalis and Escherichia coli were detected in the samples of the D-treated and FD-treated plants. Nevertheless, their concentrations were very low (<9.1 cfu g⁻¹). Conversely, these indicator organisms were not detected in the samples from the inorganic fertilizer or the control.

Furthermore, Salmonella spp. also remained undetected across all leaf samples. Enterococcus faecalis was found at estimated concentrations of 91 cfu g⁻¹ and 73 cfu g⁻¹ in the leaves of basil plants amended solely or partially with digestate (D and FD) (Table 5). These results align with findings of Bonetta et al. (2014) [81], who noted that properly processed digestate typically contains undetectable levels of Salmonella spp. Muhondwa (2019) [82] reported a high survival rate of E. coli enteropathogens in mesophilic conditions, while Carraturo et al. (2022) [12] noted that thermophilic anaerobic digestion of sewage sludge leads to complete E. coli pathogen elimination. Hence, anaerobic digestion in thermophilic conditions appears to be more efficient in eliminating indicator organisms, probably owing to elevated temperatures present during the thermophilic process, while mesophilic treatment appears unable to sufficiently sanitize the digestate.

Our results indicate potential transfer of these bacteria either from the growing substrate to plant tissue or contamination during the application process. In contrast, E. coli was detected at very low levels (<9.1 cfu g⁻¹) in leaf samples from digestate-amended treatments. Several studies have reported that indicator bacteria are able to survive anaerobic digestion, especially under mesophilic conditions [47], while storage conditions also play a crucial role in mitigating pathogen survival and propagation [83]. Thus, it is safe to assume that improper digestate application and handling can contaminate plant matter and soil with pathogens, posing a risk for environmental and food safety.

The detectable concentrations of Enterococcus faecalis on basil leaves were below but close to the EU regulatory threshold for ready-to-eat precut fruit and vegetables (100 cfu g⁻¹) [84]. Specifically, leaves from D-amended plants exhibited 91 cfu g⁻¹, while FD-amended plants showed 73 cfu g⁻¹ (Table 5). Although these levels are technically compliant with food safety regulations, the proximity of these values to the threshold represents a narrow safety margin that requires attention, particularly considering that basil is typically consumed fresh without cooking.

The translocation of the pathogens Enterococcus faecalis and E. coli on the leaves of basil likely occurred through splashing during irrigation/fertilization or handling during harvest. The survival of Enterococcus faecalis in the digestate likely stemmed from the mesophilic conditions during anaerobic digestion. Several studies have noted that indicator bacteria can survive mesophilic anaerobic conditions [47], whereas thermophilic treatment (>55 °C) achieves more effective pathogen elimination [12,82]. Muhondwa (2019) [82] reported high survival rates of E. coli enteropathogens under mesophilic conditions, while Carraturo et al. (2022) [12] reported complete E. coli elimination following thermophilic anaerobic digestion of sewage sludge. Additionally, storage conditions play a crucial role in pathogen survival and propagation [83].

Therefore, implementing thermophilic anaerobic digestion (>55 °C) or post-digestion pasteurization, as mandated by European law [80], is essential to achieving significant pathogen reduction or elimination. Moreover, digestate has to be monitored for pathogen levels prior to application, especially when utilized for fresh-consumed crops.

During cultivation, the use of drip irrigation could mitigate the splash effect compared to sprinklers or other forms of irrigation that can lead to digestate and plant matter contact. According to Fabiani et al. (2025) [85], maize plants fertigated with digestate exhibited only a transient presence of indicator pathogenic bacteria, while their presence diminished over time. Therefore, adequate timing between the last fertilization event and harvest could facilitate a reduction or elimination of pathogenic microorganisms in the harvested plant matter. Moreover, using clean tools, harvesting in dry conditions, and washing tools and hands before and during harvest can mitigate pathogen proliferation [86]. Finally, implementing disinfecting protocols when using digestate to cultivate herbs and vegetables consumed fresh should be made mandatory.

4. Conclusions

This study demonstrated the efficiency of digestate as a source of nutrients for basil plant development, especially when combined with mineral fertilizers. The FD application was the optimum treatment, yielding significantly higher TB and LP, while sufficiently elevating the concentration of nutrients and organic matter (+54.7%) in the growing substrate, compared with the pre-cultivation soil analysis.

Furthermore, the use of solely digestate provided markedly higher values in all the metrics compared to the control. Hence, using digestate as fertilizer in organic basil production is worth exploring. Essential oil concentration in the control plants was significantly higher due to stress from limited nutrient availability, while control plants exhibited superior linalool, limonene (monoterpenes), and eugenol (phenylpropanoid) content. This is particularly important because linalool was the main component of the EO, and eugenol content was almost double in the control plants compared to all the other treatments. In contrast, digestate significantly increased the concentration of sesquiterpenes, suggesting that specific fertilization strategies can influence secondary metabolite synthesis.

Additionally, the application of digestate did not significantly increase heavy metal concentration in the growing substrate, suggesting that the digestate used in our study is safe to use as an organic fertilizer. Nevertheless, continuous monitoring of heavy metals in the digestate and soil is imperative to ensure soil health and food safety.

While our study monitored nutrients and heavy metals, future research should include chloride measurements, particularly when HCl is used for pH adjustment or when the feedstocks have potentially high chloride content (such as swine slurry). Moreover, it should be noted that the pH adjustment of digestate to neutral levels prior to application may have altered its natural chemical properties and buffering capacity, potentially influencing the observed effects on substrate pH and nutrient dynamics. Future studies should compare the effects of pH-adjusted versus unmodified digestate to better understand how this pretreatment influences agronomic outcomes.

Our study was performed in pots, and data retrieved from field studies are required to validate our observations. Our semi-controlled conditions may not fully represent field-scale dynamics, including drainage, nutrient leaching, and cumulative effects over multiple growing seasons. Moreover, a techno-economic analysis would further elucidate the dynamics of digestate as a replacement for mineral fertilizers and highlight whether digestate can become a competitive marketable product for basil cultivation.

Finally, the considerable level of indicator organisms (Enterococcus faecalis) on the leaves of basil plants shows that precautionary measures, such as pasteurization and disinfecting protocols, must be implemented to ensure food safety.