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Article

Yield, Nutritional Quality, and Microbial Safety of Microgreens Grown in Insect Frass and Vermicompost-Based Growing Substrates

by
Giuseppe Di Cuia
1,
Massimiliano D’Imperio
1,*,
Federico Baruzzi
1,
Alessia Marzulli
1,
Francesco Gai
2 and
Angelo Parente
1
1
Institute of Sciences of Food Production (ISPA), National Council of Research (CNR), Via G. Amendola, 122/O, 70125 Bari, Italy
2
Institute of Sciences of Food Production (ISPA), National Council of Research (CNR), Largo Braccini, 2, 10095 Grugliasco, Italy
*
Author to whom correspondence should be addressed.
Agronomy 2026, 16(2), 158; https://doi.org/10.3390/agronomy16020158
Submission received: 26 November 2025 / Revised: 22 December 2025 / Accepted: 5 January 2026 / Published: 8 January 2026
(This article belongs to the Section Horticultural and Floricultural Crops)
Versions Notes

Abstract

Microgreens have gained increasing popularity due to their cooking versatility, ease of cultivation, and high nutritional value. The use of alternative organic substrates, such as vermicompost and insect frass, offers a promising alternative to peat. This study has evaluated the integration of Tenebrio molitor and Hermetia illucens frass, along with vermicompost, in a microgreen production, while assaying several concentrations (25%, 50%, 75%, and 100%) as replacements by weight. After a preliminary assay aimed at determining the optimal frass and vermicompost levels, we assessed the agronomic, nutritional, and microbiological performances of microgreens. The preliminary results revealed phytotoxic effects of T. molitor frass, while the addition of H. illucens frass or vermicompost did not significantly impact microgreen production. In the second experiment, the interaction between plant species and substrate composition significantly influenced the leaf area, plant height, and mineral content. Partial replacement of peat with H. illucens frass or vermicompost enhanced leaf area and plant height, with a notable increase in iron content in the mizuna microgreens grown with H. illucens frass, compared to the control with peat. Additionally, microbiological safety was ensured, and a complete absence of Salmonella spp. and E. coli was observed in the plants, in accordance with European food safety regulations.

1. Introduction

Despite the increasing pressure from European regulations aimed at reducing its extraction, peat continues to be the most widely used growing medium for soilless culture in both professional and amateur sectors [1]. Peat is a non-renewable resource whose extraction adversely affects natural habitats. Rockwool, which is produced industrially through the melting and extrusion of basalt at temperatures that exceed 1500 °C, involves significant energy consumption [2]. Both substrates present environmental challenges related to their extraction, which incurs high economic costs and has a substantial environmental impact. Indeed, the excessive extraction and commercialization of peat has been leading to its gradual depletion and rising prices; consequently, alternative substrates are gaining popularity as more sustainable and cost-effective alternatives [3].
In recent years, microgreens have emerged as an interesting new category of soilless vegetable products, and they are valued for their culinary versatility, easy cultivation, and high nutritional content. These small, nutrient-rich plants, which are abundant in vitamins and minerals, are typically grown in controlled environments, such as greenhouses, tunnels, or growth chambers, which allow a notably short growth cycle of 7 to 21 days from seed germination. This characteristic thus makes microgreens an ideal model for assessing the effects of various substrates on the nutritional quality of a product [4,5].
The increasing popularity of microgreens is further driven by the ability to tailor their nutritional profile to meet consumer needs and by the option of marketing them along with the growing substrate, thereby ensuring enhanced freshness for consumers who can harvest the microgreens at the moment of consumption.
Insect frass and vermicompost seem to be promising candidates as alternative peat substrates. According to European Union (EU) Regulation No. 2021/1925, which implements EU Regulation No. 142/2011 [6], insect frass, that is, a mixture of insect excrement, a feeding substrate, insect body parts, and dead eggs, is considered an organic fertilizer that has the potential to enhance plant growth, soil fertility, and the soil structure [7]. The application of insect frass in agriculture could help support a circular economy and sustainable agricultural practices [8]. However, frass requires a thermal treatment at 70 °C for one hour to prevent the transmission of plant and human pathogens [9,10,11]. Despite its promising potential, the use of insect frass in the cultivation of microgreens has raised some concerns about its microbiological composition. Recent studies have demonstrated that the microbial composition of black soldier fly (Hermetia illucens) larva frass can be influenced by the diet of the larvae and the adopted processing methods [12,13,14]. Specifically, high levels of lactic acid bacteria, Enterobacteriaceae, and aerobic endospores have been found in black soldier fly frass. High viable counts of coagulase-positive staphylococci have also been detected, although Salmonella has not been isolated (limit of detection 2.0 log CFU/g). A thermal treatment at 70 °C for 60 min has been found to effectively reduce Enterobacteriaceae to below 10 CFU/g, although in these experiments, the aerobic endospore and Clostridium perfringens counts were only slightly reduced [11].
Vermicompost is a product that is derived from a mesophilic, bio-oxidative process, and it can be obtained from different types of organic matter, such as animal manure [15], paper waste [16], agro-industrial waste [17], or municipal solid waste [18]. The result of the joint action of earthworms and microbes is a dark-colored, peat-like organic fertilizer with a high nutritional content and water-holding capacity, which is widely used as part of organic substrates for plant growth [19,20]. The application of vermicompost in agriculture has proved to be promising in promoting plant growth, as well as in mitigating the response of plants to abiotic and biotic stress [21,22].
Based on these premises, the present study is aimed at investigating the possibility of reducing the concentration of nutrient solutions prepared with mineral fertilizers and of replacing peat with insect excrement from Tenebrio molitor L., H. illucens L., or with vermicompost in the production of microgreens. The study has in particular investigated the effects of different proportions of frass and vermicompost added to the substrate on microgreens production, on the tissue mineral content, and on the potential microbial safety. Two experimental trials were conducted to evaluate the effects of different growing medium compositions on the performance of mizuna (first trial) and mizuna and rapini (second trial) (Brassica rapa L.) microgreen cultures.

2. Materials and Methods

2.1. Vermicompost and Frass Production

Peat (Pe, Brill type 3 special) was purchased by Agrochimica (Bolzano, Italy).
Vermicompost (VC, Agrilombricò, Fattoria Gallo Rosso, Basilicata, Italy) was produced using Eisenia fetida and E. andrei earthworms fed on cattle manure in windrows for 18 months. Once the earthworms had been removed, the vermicompost (VC) was air-dried for three months.
Insect frass (IF) was collected from Hermetia illucens L. (Hi) and Tenebrio molitor L. (Tm) larvae, which had been fed wheat and barley bran for 20 days in two separate production batches. The IF was air-dried for three days. Both the IF and VC were sifted to obtain particles ranging from 10 mm to 10 μm in size and were then stored at room temperature. Electrical conductivity (EC) and pH of the peat (Pe), IF and VC were analyzed on a water-soluble extract (1:10 w/v). Briefly, 10 g of sample was added to 90 mL of distilled water, shaken for 30 min at 200 rpm and, after 1 h, the suspension was filtered through Whatman no. 40 paper filters to obtain the water extract.
The Pe, VC, and IF samples were kept in a forced-air oven at 105 °C until they reached a constant weight for the determination of the dry matter (DM). The ion (Al, K, Ca, Fe, Mg, Na, B, Zn) content was determined as reported below (Section 2.5 “Analysis of the Inorganic Elements”).

2.2. Plant Materials and Experimental Conditions

Trials were conducted in a walk-in growth chamber, with internal dimensions of 260 × 284 × 253 cm (length, width, and height, respectively) located at the Institute of Sciences of Food Production (ISPA-CNR of Bari).
The seeds were uniformly distributed over the substrate surface at a density of five seeds per cm2. During the first three days, the lights were kept off to allow germination. On day 4, the seedlings were exposed to artificial lighting conditions, that is, 14 h of light and 10 h of darkness. The lighting period included 1 h of dawn, with a photosynthetic photon flux density (PPFD) of 125 µmol/s/m2, followed by 12 h of full light at a PPFD of 250 µmol/s/m2, with an RBG ratio of 36:37:36%, and 1 h of dusk with a PPFD of 125 µmol/s/m2. The Daily Light Integral (DLI) was 11.70 mol. The temperature was maintained at 20 °C during the day and at 16 °C during the night, at a constant relative humidity of 65%.
Two experiments were performed. In the first experiment, mizuna (Brassica rapa L.) microgreens were grown in plastic trays (84 cm2, 70 × 120 × 45 mm) filled with ten different mixtures (percentages expressed as w/w ratio on a dry weight basis): (i) T1—100% peat (Control; Pe); (ii) T2—75% (Pe) 25% (Hi); (iii) T3—50% (Pe) 50% (Hi); (iv) T4—25% (Pe) 75% (Hi); (v) T5—75% (Pe) 25% (VC); (vi) T6—50% (Pe) 50% (VC); (vii) T7—25% (Pe) 75% (VC); (viii) T8—75% (Pe) 25% (Tm); (ix) T9—50% (Pe) 50% (Tm); (x) T10—25% (Pe) 75% (Tm). A second experiment was conducted, on the basis of the results of the first trial, in which only the best performing growing media (T1, T3, and T6) were employed for mizuna and rapini (Brassica rapa L.). Thirty and eighteen experimental units were used in the first and second trials, respectively, in which a randomized block experimental design was adopted, with three replicates, and each experimental unit consisted of six growing trays.
The control trays were irrigated with a half-strength Hoagland nutrient solution (112 mg/L N, 117.5 mg/L K, 80 mg/L Ca, 31 mg/L P, 16 mg/L S, 12 mg/L Mg, 0.135 mg/L B, 0.56 mg/L Fe, 0.055 mg/L Mn, 0.0655 mg/L Zn, 0.016 mg/L Cu, and 0.025 mg/L Mo). The half-strength Hoagland nutrient solution (NS) was replaced with distilled water for the growing media containing IF or VC.

2.3. Yield and Analysis of the Growing Plants

Microgreens were collected at the first true leaf stage, 18 days after sowing, in both trials. The yield [expressed as kg of fresh weight (FW) m−2], leaf area and average height of the plants in nine trays were evaluated. Fresh leaves (FW) were maintained in a forced draft oven at 105 °C until constant weight was reached for the measurement of dry matter (DM) content. The analysis of the growth (epicotyl length and leaf area) was conducted using image analysis software (ImageJ, Fiji version 1.53e). The epicotyl length and leaf area were measured from photographs taken at harvest time. Images were captured using a smartphone camera (64 megapixels) positioned 50 cm above the ground. Nine plants from each experimental unit and species were sampled from each growing medium for the epicotyl length measurements. The leaf area was measured non-destructively by photographing 3 trays placed on a black surface alongside a ruler. Each generated image was analyzed using ImageJ [23].

2.4. Determination of Escherichia coli and Salmonella spp.

Microgreen samples were collected for microbiological determinations using sterilized scissors, thereby avoiding contamination with the substrates as much as possible. About 10 g of vegetable edible parts was collected from 9 trays per treatment to obtain a representative sample of about 100–110 g.
The determination of Salmonella spp. was conducted in conformity with UNI EN ISO 6579-1, whereby three 12.5 g portions were sub-sampled from the microgreen bulk that had been suspended in 112.5 g of Buffered peptone water (BPW, Biolife, Monza, Italy). After incubation (24 h at 37 °C under static conditions), 1 mL of the enriched microbial population was incubated for a further 24 h at 37 °C in 9 mL of selective Rappaport Vassiliadis Broth (RVS). The presumptive Salmonella spp. were enumerated on selective Xylose Lysine Desoxycholate Agar (XLD agar; Sigma Aldrich, Milano Italy) after an additional 24 h at 37 °C.
The detection of E. coli was conducted according to UNI ISO 16649-2, that is, 2 g of microgreen bulk was homogenized using a Stomacher (Stomacher basic, Generon S.p.A., San Prospero (MO)—Italy) in 18 mL of a sterile saline solution for 4 min. β-glucuronidase-positive strains, grown as blue colonies on Tryptone Bile X-Gluc (TBX) agar plates, were enumerated as presumptive E. coli after 24 h incubation at 37 °C.

2.5. Analysis of the Inorganic Elements

The Cl, NO3, and SO4 ions were determined by means of the ion exchange chromatography technique (IC-Dionex DX120, Dionex Corporation, Sunnyvale, CA, USA) with the conductivity detector performed as reported in [24]. Briefly, DW samples were extracted, with an Na2CO3 (3.5 mM) and NaHCO3 (1 mM) solution, for 30 min at room temperature in an orbital shaker (50 rpm). The extracts were then diluted and filtered using 0.45 µm (regenerated cellulose, RC), followed by Dionex OnGuard IIP (Thermo Scientific, Milano, Italy) filters, to remove any organic compounds from the sample matrices. The obtained solutions were analyzed by IC with a conductivity detector, using an IonPac AG14 precolumn and an IonPac AS14 separation column (Thermo Scientific) at 35 °C, with a 1 mL/min flow (3.5 mM Na2CO3 and NaHCO3 1 mM) and 50 mA current.
Microgreen DW samples were digested in a closed-vessel microwave digestion system (MARS 6, CEM Corporation, Matthews, NC, USA), with 10 mL of HNO3 (Pure grade, Carlo Erba), for the determination of Al, K, Ca, Fe, Mg, Na, B, Ba, Cd, Co, Cr, Cu, Li, Mn, Mo, Ni, Pb, and Zn. The digestion procedure was carried out in two steps: the first 15 min to reach 200 °C and then 10 min maintained at 200 °C (power set at 900–1050 W; 800 psi). Each solution was diluted to a volume with ultrapure H2O (Milli-Q Millipore 18 M Ω/cm) and filtered using a 0.45 μm filter. Samples were analyzed by means of Inductively Coupled Plasma—Optical Emission Spectrometry (ICP-OES; 5100 VDV, Agilent Technologies, Santa Clara, CA, USA) to measure Ca, K, Mg and Na in radial mode and Al, Fe, B, Ba, Cd, Co, Cr, Cu, Li, Mn, Mo, Ni, Pb, Zn in axial mode [25].

2.6. Statistical Analysis

The effects of the different treatments were tested using an analysis of variance (ANOVA), followed by mean separation with Fisher’s protected least-significant difference (LSD) at p = 0.05. STATISTICA 10.0 statistical software (StatSoft, Tulsa, OK, USA) was used for the analysis.

3. Results

The chemical and physical properties of the four substrates used for the composition of the growing medium mixtures are reported in Table 1.
The peat (Pe) and vermicompost (VC) showed similar pH values (near neutrality) and moderate EC, thus indicating low salinity. As far as the insect frass (IF) samples are concerned, the Hermetia illiucens (Hi) frass had a slightly acidic pH and the lowest EC, while the Tenebrio molitor (Tm) frass showed a markedly low pH and high EC, thereby indicating an elevated salinity. As far as the plant nutrient content is concerned, the Tm frass contained high levels of NH4+ and K, along with considerable quantities of Mg and Zn. Table 2 presents the yield, dry matter, leaf area, and plant height of the mizuna microgreens from the first experiment. No treatments involving Tm frass (T8, T9, and T10) are shown, as severe phytotoxicity of all the considered concentrations prevented germination, and thus no measurements were made of these parameters.
The presence of Hi frass or VC in the mixtures did not significantly affect the yield, leaf area, or the plant height, with average values of 0.93 kg m−2 FW, 346 cm2, and 4.30 cm, respectively, thus suggesting that these substrates did not impact the overall vegetative growth or productivity under the tested conditions. In contrast, the dry matter content showed highly significant differences among the treatments: specifically, the plants grown on the T4 mixture showed the highest dry matter content, while T1 exhibited the lowest value of dry matter content, which was significantly different from the others.
The results of the first experiment were considered to design the second trial, in which the use of treatments T3 and T6 (both with 50% Pe substitution) was based on two key factors: (i) substrate composition and Na management: Hi and VC have a higher sodium content than peat (Table 1). A 50% substitution allowed to obtain a substrate with an increased, yet controlled and similar, sodium level between the two mixtures (T3 and T6), reducing the potential risk of ionic imbalances compared to higher substitution rates (e.g., the 75% in treatment T4); (ii) alignment with best sustainability practices and the scientific literature: the choice to test a 50% Pe substitution aligns with the most established approaches in the literature on sustainable substrates. Partial substitutions around this percentage are widely reported as a balanced and effective strategy to significantly reduce Pe use while mitigating the risks (such as physical instability or phytotoxicity) associated with its complete replacement. Successful cultivation with various organic amendments at similar substitution levels has also been documented for microgreens [26,27,28]. Therefore, selecting T3 and T6 represented a pragmatic and well-supported step towards sustainability for the second trial. The effects of the substrate mixtures on the production parameters are shown in Table 3.
The statistical analysis revealed significant effects of both the species and substrates, as well as some significant interactions between the species and growth media. Indeed, the species had a highly significant influence on the yield, leaf area, and plant height, but not on the dry matter content, which was similar between species, and averaged around 10%. The rapini in fact exceeded the mizuna, in terms of yield (1.3-fold), leaf area (+31%), and plant height (+15%), thus highlighting its greater overall vegetative growth. Regarding the mixtures, T3 and T6 produced significantly higher yields (1.28 kg/m2 on average) than T1, which, along with T6, showed the highest value of dry matter content (10.7 g/100 g FW on average), thus suggesting a compromise between water content and yield. The interaction between species and substrate was statistically significant for the leaf area and plant height, which suggested that the effect of the substrate depended partly on the grown species. Again, the rapini from T3 and T6 exhibited a significantly larger leaf area, with an increase of 42%, compared to mizuna plants grown on T1. However, an increased plant height was only observed in the rapini grown on the first substrate. The mizuna plants cultivated on T1 and T6, as well as the rapini plants grown on T1 instead showed a significantly reduced height compared to all the other treatments.
As shown in Table 4, a significant interaction between species and treatments was observed for the mineral composition, except for Mn, whose concentration was similar among the species and treatments (6.76 mg/kg of FW on average). The calcium content was highest for the mizuna in the T1 treatment, followed by the rapini in T1. The lowest concentrations were observed in T3 for the mizuna and in T6 for the rapini. The rapini grown in the T3 and T6 treatments exhibited the highest K content, followed by the mizuna grown in the T6 and T1 treatments. In addition, the mizuna grown in T1 and the rapini grown in the T3 and T6 treatments had the highest P content, whereas the mizuna grown on Pe and NS showed the highest B value.
The Fe content was approximately one-fold higher in the mizuna grown with the T3 substrate than the mizuna grown with the other treatments, while the rapini grown with the same treatment had the lowest Fe content. After substitution with VC (T6), the greatest increases in Mg and Na were observed in the rapini, and they were followed by the T3 treatment in the mizuna. The mizuna exhibited a higher concentration of Al than the rapini, with the maximum value being observed in T3. The Zinc content was similar for the two species, with the highest value being recorded in the T6 treatment.
We assessed the safety evaluation of microgreens considering the risk of pathogens, in line with EU regulations (EC Commission Regulation No 2073/2005). Although E. coli counts of up to 100–1000 CFU/g are allowed in 2 out of 5 samples, Salmonella spp. must be absent in 5 aliquots of 25 g. During the trial, no presumptive Salmonella spp. or E. coli colonies were detected on the XLD or TBX media, respectively. A non-selective BPW enrichment step, as per ISO 6579, confirmed that E. coli was below the detection limits. Indeed, the absence of β-glucuronidase-positive colonies indicated that E. coli was not present in the 25 g of the edible samples.

4. Discussion

This study has evaluated the potential of enhancing the sustainability of microgreen production by incorporating insect frass (IF) or vermicompost (VC) residues into the growing media. The choice of growing media is a crucial step that can influence the commercial and nutritional quality of microgreens, as well as the sustainability of their production [29,30]. The main objective of the first trial was to assess the feasibility of using IF or VC as novel substrates for microgreen cultivation. However, it was found that the high salt content and acidity registered for the Tenebrio molitor (Tm) frass could pose a risk of phytotoxicity, especially in sensitive plant species or when applied in large quantities Notably, Zn and B were present in moderate amounts in the Hermetia illucens (Hi) frass. Thus, in order to assess the feasibility of using these matrices as novel substrates for microgreen cultivation, we used increasing percentages (w/w) of IF and VC mixed with peat (Pe) to grow the mizuna microgreens. The results of this experiment allowed us to identify the most suitable mixtures for use in the second trial.
The phytotoxic effects observed in the plants cultivated with a high percentage of Tm IF during the first experiment might be the result of the combination of high NH4+ concentration and the elevated salinity of the substrate (EC) (Table 1). High levels of NH4+ can disrupt the cation-anion balance around seeds and can interfere with the nutrient and water transport through both the seed coat and the roots [31]. This imbalance may impair germination or result in malformed root development [32]. Excess ammonium can also compete with essential nutrients, such as K, which plays a critical role in seed germination and early growth [33]. Additionally, high NH4+ concentrations may lead to the formation of toxic compounds, such as ammonia (NH3), which damages cell membranes and organelles in seeds, thereby hindering germination. At elevated NH4+ levels, cells may struggle to regulate the ammonium influx effectively, and this can lead to cytosolic accumulation and potential cell death [34,35]. In the second experiment, the partial replacement of conventional substrates, such as Pe, enabled fresh yields to be achieved that were comparable with or even better than those reported in previous studies that had used similar substrates and the same standard nutrient solution [5].
Indeed, the T3 and T6 substrates had a positive impact on the plant growth parameters, probably due to a satisfactory supply of nutrients, especially K and Mg.
Thus, the selection of materials for a substrate could be guided by such parameters as the nutrient content, EC values, and the ammonium concentration. This would allow customized substrates to be tailored to the specific needs of plants, fully utilizing the mineral composition of the used materials. This approach could be particularly advantageous for specific species, such as endive, which can directly utilize ammonium as a nitrogen source [36].
The variations in the mineral composition of the plant tissues grown on different substrates, observed in both brassica species, support the hypothesis that the inclusion of insect frass or VC influenced the nutritional profile of the final products. Notably, the mizuna grown with Hi, and irrigated only with water, showed a 2.3-fold increase in the Fe content, compared to microgreens grown on Pe fertilized with an NS containing Fe.
Additionally, the mizuna microgreens grown on T3 showed a significant increase in the Fe content. These results align with the objectives of numerous studies aimed at improving the nutritional quality of agricultural products through the innovative management of substrates or fertilization [37]. The increase in Fe content in the mizuna and the rise in the Zn content in both species represent significant findings. These findings suggest that mizuna could have a greater capacity to mobilize and tolerate these elements under specific substrate conditions. This accumulation could result from enhanced rhizosphere acidification [38], increased root exudation of organic ligands (e.g., phenolics, citrate), or differences in the Fe transporters [39].
The partial replacement of Pe with Hi or VC yielded microgreens with a high added value. Indeed, an increase in the Zn content was observed in both species, an important result, given that this mineral is often deficient in diets.
Regarding the K and Mg content, which was found to be higher in the rapini microgreens in the T6 treatment, this might be attributed to the high availability of these cations in the VC. Contrary to the traditional view of K-Mg antagonism [40], recent studies emphasize a synergistic interplay between these nutrients. This synergy is particularly evident in photosynthesis and N metabolism, where Mg facilitates carbohydrate allocation and can partially compensate for K deficiencies [41]. The Na content in the brassica microgreens grown on the T3 and T6 substrates was higher than in those grown in the 100% Pe (T1), likely due to the elevated Na concentrations present in IF and VC (Table 1). Similar findings have been reported for other brassica microgreens [24] and for lettuce grown on innovative substrates derived from organic residues, such as those with high Na levels [42]. Even though the Na content was notably higher (563 mg/kg FW) in the rapini grown on T6 than the content reported to be optimal in microgreens, the daily consumption of 30 g of rapini microgreens corresponds to a modest intake of just 17 mg of sodium per day. This may represent a potentially negative indicator and could act as a deterrent for individuals who require low-sodium diets; however, the absolute Na contribution per serving remains minimal and is unlikely to pose a serious dietary concern. As far as the Al content is concerned, which was found to be below thresholds considered potentially harmful for consumers [43], the highest levels were observed in the mizuna grown on T3. In addition, the accumulation of Al in the mizuna, without any apparent toxicity, points toward an internal detoxification mechanism. Specifically, the sequestration of Al in the vacuole, via the action of ABC transporters, as identified by [44] in their study on the ALS3 gene, could explain this observed tolerance.
The higher Ca and B content found in the mizuna could be attributed to the control treatment with 100% Pe (T1), where the NS containing these elements was supplied directly to the plants. This likely enhanced the availability of Ca and B, compared to other treatments whereby these minerals were primarily available through the substrate.
The use of emerging organic alternatives to replace Pe poses several challenges related to food safety and the risk of pathogen contamination. In our study, the feeding substrates consisted of wheat and barley bran, which are safe and nutrient-rich ingredients that are used for human consumption [45]. For this reason, it is possible to hypothesize that the absence of pathogens in microgreens could be the result of the choice of a feeding substrate from the food processing industry (e.g., beer, coffee, and/or olive oil production) [46,47], which follows GHP and HACCP protocols and is therefore likely to be pathogen-free. However, the absence of pathogens in IF cannot be guaranteed when insects feed on sewage sludge, organic wastes, or poultry manure, which are well known sources of pathogens, even though larvae can suppress pathogens by producing ammonia [48,49,50].
For these reasons, the feeding substrate for insect breeding should be decided on while carefully considering the potential use of its frass. Moreover, the action of earthworms during vermicomposting intensifies competition and antagonism between vermicompost-associated bacteria and pathogens, thereby producing a fertilizer that is safe to use [51,52].

5. Conclusions

This study highlights the effectiveness of Hermetia illucens (Hi) frass and vermicompost (VC) as alternative substrates for microgreen production and suggests that they can provide a more sustainable and nutrient-rich solution for plant cultivation than Tenebrio molitor (Tm) frass, which is known to exhibit phytotoxic effects. However, Hi and VC led to a significant increase in Fe and Zn content, representing a substantial step forward in the nutritional enhancement of microgreens. Furthermore, the absence of Salmonella spp. and E. coli in the plants was in compliance with European food safety regulations. The results of this study suggest that the use of organic by-products, such as frass and VC, could support a circular economy in agriculture and reduce the environmental impact of soilless cultivation when applied on a large scale to replace peat, as demonstrated here for microgreens. However, an economic analysis is essential to assess the commercial viability of these substrates, and this should be the focus of future research.

Author Contributions

Conceptualization, G.D.C. and M.D.; methodology, G.D.C., M.D. and A.P.; validation: M.D. and A.P.; investigation, G.D.C., M.D., A.M. and F.B.; data curation, G.D.C., M.D. and A.P.; writing—original draft preparation, G.D.C., M.D., A.M., F.G., F.B. and A.P.; writing—review and editing, G.D.C., M.D. and A.P.; supervision, A.P.; funding acquisition, A.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the EU-PRIMA program project ADVAGROMED (ADVanced AGROecological approaches based on the integration of insect farming with local field practices in MEDiterranean countries, Prima, 2021—Section 2) and Agritech National Research Center, and it received funding from the European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR)—MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4—D.D. 1032 17/06/2022, CN00000022).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed at the corresponding author.

Acknowledgments

The authors would like to thank Ilaria Biasato and Sara Bellezza Oddon for their valuable contribution and technical support in the management of the frass.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
HiHermetia illucens L.
TmTenebrio molitor L.
IFInsect frass
PePeat
VCVermicompost

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Table 1. Chemical characterization of the materials used for the mixture preparation.
Table 1. Chemical characterization of the materials used for the mixture preparation.
ParameterUnit of MeasurePeat
Pe		Vermicompost (VC)Hermetia Frass (Hi)Tenebrio Frass (Tm)
pH 7.277.386.985.83
ECmS/cm1.261.290.295.27
DM%58.077.049.289.5
Ammonium (NH4) mg/kg DW15.46.276.36584
Nitrate (NO3)g/kg DW2.732.881.320.97
Sulfate (SO4)3.112.320.251.79
Chlorine (Cl)0.420.140.370.00
Potassium (K)2.6510.18.7724.2
Aluminum (Al)2.3214.70.590.06
Calcium (Ca)35.91045.822.35
Iron (Fe)2.2312.40.790.36
Magnesium (Mg)2.4610.71.417.75
Sodium (Na)0.351.531.840.28
Boron (B)mg/kg DW10.042.017.86.13
Cadmium (Cd)1.120.621.820.47
Copper (Cu)12.145.412.016.7
Molybdenum (Mo)2.112.240.411.39
Zinc (Zn)12523732.8136
Data are expressed as the mean values of three independent samples. DW: dry weight; pH and EC were measured in a 1:10 water extract (w/v substrate/water).
Table 2. Yield and biometric parameters of the mizuna microgreens cultivated with seven different organic substrates in the first trial.
Table 2. Yield and biometric parameters of the mizuna microgreens cultivated with seven different organic substrates in the first trial.
TreatmentsYield
(kg m−2)		Leaf area a
(cm2)		Height of Plants
(cm)		Dry Matter
(g/100 g FW)
T11.063464.018.18 c
T20.913403.7310.1 ab
T30.943524.659.44 b
T40.723304.5010.4 a
T50.933444.369.83 ab
T60.993664.399.63 b
T70.983414.479.57 b
Significancensnsns***
[T1: 100% peat (Pe); T2: 75% Pe 25% H. illucens L. frass (HI); T3: 50% Pe 50% HI; T4: 25% Pe 75% HI; T5: 75% Pe 25% vermicompost (VC); T6: 50% Pe 50% VC; T7: 25% Pe 75% VC]. a: 3 plastic trays were considered for the leaf area measurement. Significance: ns = not significant; *** p ≤ 0.001. Data are expressed as the mean values of the treatment (n  =  3). FW: fresh weight. Different letters indicate that the mean values are significantly different, according to the LSD test (α  =  0.05). The T8, T9, and T10 treatments are not shown in the table, as severe phytotoxicity of all the considered concentrations prevented germination, and thus no measurements were made of these parameters.
Table 3. Productive parameters of the mizuna and rapini microgreens cultivated with the three different organic substrates in the second trial.
Table 3. Productive parameters of the mizuna and rapini microgreens cultivated with the three different organic substrates in the second trial.
TreatmentsYieldDry MatterLeaf Area aHeight of Plants
Species (S)(kg/m2) (g/100 g FW)(cm2) (cm)
Mizuna 0.73 b10.03464.55
Rapini1.65 a9.924525.25
Substrates (T)
T11.01 b10.7 a3344.21
T31.30 a9.00 b4415.73
T61.26 a10.3 a4234.77
Interaction (S × T)
Mizuna T10.6711.1314 c4.03 c
Mizuna T30.799.02369 b5.41 b
Mizuna T60.7210.0356 b4.20 c
Rapini T11.3410.3 354 b4.38 c
Rapini T31.818.97513 a6.04 a
Rapini T61.7910.5490 a5.34 b
Significance
Species (S)***ns******
Treatment (T)*********
S × Tnsns****
[T1: 100% peat (Pe); T3: 50% Pe 50% H. illucens frass L. (Hi); T6: 50% Pe 50% vermicompost (VC)]. Data are expressed as the mean of the treatment (n  =  3). a: 3 plastic trays were considered for the leaf area measurement. FW: fresh weight. Significance: ns = not significant; * p ≤ 0.05; ** p < 0.01; *** p ≤ 0.001. Different letters indicate that the mean values are significantly different, according to the LSD test (α  =  0.05).
Table 4. Mineral content of the mizuna and rapini microgreens cultivated with three different organic substrates.
Table 4. Mineral content of the mizuna and rapini microgreens cultivated with three different organic substrates.
Source of
Variation		CaKMgPBFeMnNaZnAl
Species (S)g/kg of FWmg/kg of FW
Mizuna 2.104.670.390.993.1820.06.921695.9118.2
Rapini1.805.380.421.052.238.226.592847.061.46
Substrates (T)
T12.943.720.380.983.1611.795.5725.94.573.66
T31.305.320.400.982.3618.566.982986.5617.3
T61.626.050.451.102.6111.97.733578.338.64
Interaction
(S × T)
Mizuna T13.31 a5.50 b0.35 cd1.16 a4.02 a14.0 b5.1730.5 d3.46 d6.73 c
Mizuna T31.77 c3.16 c0.47 b0.81 b2.62 bc31.8 a7.37326 b6.06 c34.0 a
Mizuna T61.22 d5.35 b0.35 cd1.00 ab2.90 b14.1 b8.22151 c8.21 ab14.0 b
Rapini T12.56 b1.93 d0.40 bc0.80 b2.29 cd9.58 bc5.9721.2 d5.68 c0.59 e
Rapini T30.82 d7.47 a0.32 d1.15 a2.09 d5.32 c6.58269 b7.05 bc0.50 e
Rapini T62.01 c6.75 a0.55 a1.20 a2.31 cd9.75 bc7.23563 a8.45 a3.28 d
Significance
Species (S)**nsns******ns********
Substrate (T)*******ns******ns***ns***
S × T****************ns*********
[T1: 100% peat (Pe); T3: 50% Pe 50% H. illucens frass (Hi); T6: 50% Pe 50% vermicompost (VC)]. Significance: ns = not significant; * p ≤ 0.05; ** p < 0.01; *** p ≤ 0.001. Data are expressed as the mean values of the treatment (n  =  3). FW: fresh weight. Different letters indicate that the mean values are significantly different, according to the LSD test (α  =  0.05). FW: fresh weight.
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Di Cuia, G.; D’Imperio, M.; Baruzzi, F.; Marzulli, A.; Gai, F.; Parente, A. Yield, Nutritional Quality, and Microbial Safety of Microgreens Grown in Insect Frass and Vermicompost-Based Growing Substrates. Agronomy 2026, 16, 158. https://doi.org/10.3390/agronomy16020158

AMA Style

Di Cuia G, D’Imperio M, Baruzzi F, Marzulli A, Gai F, Parente A. Yield, Nutritional Quality, and Microbial Safety of Microgreens Grown in Insect Frass and Vermicompost-Based Growing Substrates. Agronomy. 2026; 16(2):158. https://doi.org/10.3390/agronomy16020158

Chicago/Turabian Style

Di Cuia, Giuseppe, Massimiliano D’Imperio, Federico Baruzzi, Alessia Marzulli, Francesco Gai, and Angelo Parente. 2026. "Yield, Nutritional Quality, and Microbial Safety of Microgreens Grown in Insect Frass and Vermicompost-Based Growing Substrates" Agronomy 16, no. 2: 158. https://doi.org/10.3390/agronomy16020158

APA Style

Di Cuia, G., D’Imperio, M., Baruzzi, F., Marzulli, A., Gai, F., & Parente, A. (2026). Yield, Nutritional Quality, and Microbial Safety of Microgreens Grown in Insect Frass and Vermicompost-Based Growing Substrates. Agronomy, 16(2), 158. https://doi.org/10.3390/agronomy16020158

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