Use of Spent Mushroom Substrates in Radish (Raphanus ssp.) Microgreens Cultivation

Abstract

This study evaluated the effects of incorporating spent mushroom substrates (SMS) derived from Agaricus bisporus, Pleurotus ostreatus, and Lentinula edodes into peat-based growing media on the morphological traits, photosynthetic parameters, and mineral composition of radish and black radish microgreens. Six substrate mixtures were tested, with 2.5–30% SMS and two composting durations (97 and 153 days). The results showed that a low proportion of A. bisporus SMS (2.5–5%) significantly enhanced biomass production, plant length, and leaf area, particularly in radish. In contrast, higher proportions (20–30%) of P. ostreatus and L. edodes SMS, especially when short-time composted, inhibited plant growth and photosynthetic performance (Fv/Fm, PIabs), likely due to phytotoxic compounds, high salt content, or nutrient imbalances. Mineral analysis revealed substantial increases in K, Fe, and Zn accumulation in microgreens grown on selected SMS media, particularly Agaricus 5% and Lentinula 30, while also highlighting the risk of excessive Na or heavy metal content in some treatments. Differences between the species were observed: black radish produced higher dry mass and accumulated more minerals, suggesting greater adaptability to suboptimal substrates. These findings support the potential use of well-composted SMS as a sustainable growing media component for microgreens, provided proper substrate selection, composting, and dosage control are applied.

1. Introduction

With the increasing demand for sustainable agricultural technologies and the efficient use of agricultural by-products, the management of spent mushroom substrate (SMS or Spent Mushroom Compost SMC) is attracting growing attention. The application of SMS in vegetable and microgreen production supports circular economy principles by recycling agricultural and food waste, thereby reducing the environmental burden associated with waste disposal and peat extraction [1,2].

For many decades, peat has been an essential component of horticultural substrates, valued for its favorable physical and chemical properties, including its high water-holding capacity, suitable structure, and low levels of chemical impurities [3,4]. Thanks to these characteristics, it is widely used in the greenhouse cultivation of vegetables and ornamental plants, as well as in seedling production. However, peat resources are limited, and extracting them is associated with the degradation of valuable peatland ecosystems and the emission of significant amounts of carbon dioxide into the atmosphere [5]. Consequently, there is increasing attention being given to the search for alternative, renewable raw materials that could partially or completely replace peat in horticultural substrates. One potential solution is the use of spent mushroom substrates, or SMS, which are available in abundance in Poland and could form part of a circular economy strategy [6,7].

SMS is generated during the cultivation of edible mushrooms, such as the white button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). The composition of SMS varies depending on the raw materials used to prepare the primary substrate and the mushroom species cultivated. It typically contains residual wheat straw, horse or chicken manure, peat, calcium, and enrichment additives [8]. SMS also contains valuable organic matter, macro- and micronutrients, and beneficial microflora. Compared to peat, SMC had a higher pH value, higher salt content, lower concentrations of macro- and micronutrients, and lower water-holding capacity, but much higher air capacity potential. As a component of growing media, SMS has been successfully used in the cultivation of vegetables such as tomato, cucumber, eggplant, and pepper [1]. Adding mushroom substrate (SMS) to peat substrates at a volume percentage of 25–50% improved the emergence and growth of lettuce (Lactuca sativa L.) and cucumber (Cucumis sativus L.) without reducing their yield [6]. Ahlawat et al. [9] described using SMS to cultivate tomatoes (Solanum lycopersicum L.) and peppers (Capsicum annuum L.) in greenhouses. Adding 20–40% SMS to the soil improved its structure and increased the availability of nitrogen and potassium, resulting in a 10–15% increase in yield. Uzun [10] investigated the application of SMS to carrot (Daucus carota L.) and radish (Raphanus sativus L.) crops, finding that yields were comparable to or higher than control variants with peat alone when SMS made up 30% of the soil mass. In contrast, Günes et al. [11] reported improvements in soil organic matter content and water-holding capacity following the application of SMS in onion (Allium cepa L.) cultivation, as well as an increase in plant vitamin C content.

Its physical properties—such as improved soil structure, increased water retention, and enhanced aeration—make it an attractive material for horticultural applications [12]. Studies have shown that mixing SMS with peat or coconut fiber (up to 75%) improves soil physicochemical properties, increases NPK content, and stimulates plant growth [2]. However, the use of fresh SMS as a growth medium for salt-sensitive plants is not advised due to a high salt concentration and high salinity [13]. Excessive application of SMS can result in substrate salinization and phytotoxicity; therefore, moderate dosages and prior composting are recommended [9].

Although SMS presents a promising alternative to peat, its high salt content can adversely affect seed germination and crop yield in some species [6]. However, blending SMS or other organic waste composts with conventional substrates (e.g., peat or coconut coir) can maintain or even enhance vegetable growth and nutritional quality [6]. Research by Poudel et al. [14] suggests that properly balanced substrates (SMS + peat/coconut fiber) can improve the yield and nutritional value of pea and radish microgreens, while reducing reliance on non-renewable peat.

Microgreens are young seedlings of vegetables and herbs, typically harvested 7–21 days after germination, when they have developed cotyledons and their first true leaves [15,16]. Owing to their short cultivation cycle, high concentration of bioactive compounds, and attractive sensory properties, microgreens are gaining popularity as functional foods [17].

Species of Raphanus are among the most commonly cultivated microgreens, valued for their rapid growth, high yield, and intense flavor. They are often grown under controlled conditions using artificial light and various substrates such as coconut fiber, peat, cellulose mats, or hydroponic systems [16,18]. In addition to environmental factors such as light and temperature, the optimization of mineral fertilization and substrate composition can further enhance the yield and nutritional quality of microgreens [19].

The nutritional value and health-promoting properties of radish microgreens are characterized by high concentrations of vitamins (C, E), minerals (nitrogen, potassium, iron), protein, and various bioactive compounds, including phenols, flavonoids, anthocyanins, carotenoids, and chlorophyll [18,20,21]. Their nutritional value often surpasses that of mature plants, making them an attractive component of functional diets [22].

Despite growing interest in the circular economy and sustainable agriculture, the potential of spent mushroom substrate (SMS) as a growing medium for vegetables, particularly short-cycle crops such as radish microgreens, remains largely unexplored. While existing studies have largely focused on the disposal, composting, or application of SMS as a bulk soil amendment, there has been limited emphasis on its direct use in soilless or partially soilless systems. In Poland, where the mushroom production sector is significant and large quantities of SMS are generated annually, there is a lack of experimental evidence assessing optimal SMS ratios, nutrient interactions, and limitations such as salinity or heavy metal content. This knowledge gap hinders the development of practical recommendations for integrating SMS into sustainable microgreen production systems. The present study provides new insights into the direct application of spent mushroom substrates (SMS) in microgreen radish cultivation, offering a practical solution for managing this abundant by-product of the mushroom industry. The objective of this study was to evaluate the suitability of substrates (SMS) derived from the cultivation of three edible mushroom species for the growth of Raphanus spp. microgreens.

2. Materials and Methods

2.1. Experimental Site, Plant Species, and Growing Substrates

The experiment was conducted under controlled environmental conditions in the growth chamber of the Faculty of Agronomy, Horticulture, and Biotechnology (52_240200 0 N and 16_510350 0 E), Poznan University of Life Sciences, Poland.

Seven growth media were tested for the production of radish (Raphanus sativus var. sativus L.) and black radish (Raphanus sativus var niger L.). The experiment used six substrate variants with specific percentages of used substrate after mushroom cultivation and a peat substrate as a control substrate (C). The substrate was made from ground high peat with the addition of neutralizing minerals and PG Mix N:P₂O₅:K₂O fertilizer in a ratio of 14:16:18 with a pH of 5.5 to 6.5 (in H₂O).

The other combinations were supplemented with substrate from the cultivation of the dicotyledonous mushroom (Agaricus bisporus) in two quantities of 2.5% (A-2.5) and 5% (A-5) of the total substrate volume and from the cultivation of the oyster mushroom (Pleurotus ostreatus) and the edible sclerophyllaceae (Lentinula edodes) in quantities of 20% (P-20, L-20) and 30% (P-30, L-30) of the total substrate volume. The substrate used for Agaricus cultivation was created by mixing wheat straw (1000 kg), poultry manure (750 kg), gypsum (80 kg), and water (3000 kg). Shiitake mushrooms were grown in a mixture of beech and oak sawdust at a ratio of 1:1 (v/v) with the addition of bran (10%) and flour (10%), while the substrate for oyster mushroom cultivation consisted mainly of wheat straw and additionally wheat bran (20%). The total duration of mushroom cultivation was approximately 11 weeks for A. bisporus, 12 weeks for L. edodes, and 10 weeks for P. ostreatus. Fresh SMS after commercial production was used to prepare substrates. The substrates were shredded, mixed, moistened with water, and composted for 97 days for cycle one (I) and 153 days for cycle two (II) before sowing the plants. During the composting process, the substrates were covered with a thick foil, and the process itself took place under natural conditions, in a covered area sheltered from sunlight and rain.

The substrate after commercial mushroom production, without heat treatment/sterilization after cultivation, was used in the study. The nutrient content of all substrates used in the study is presented in

Table 1. Macro- and micronutrient concentrations, pH, and EC of different substrates used in the study.

Nutrients (mg L−1)	Substrate
	C	A-2.5	A-5	P-20	P-30	L-20	L-30
N-NH4	21	śl	11	7	śl	4	33
N-NO3	252	245	242	172	4	śl	151
P	87	83	97	98	71	76	168
K	239	286	343	968	1537	202	272
Ca	14.57	13.99	14.73	13.76	12.49	12.84	13.36
Mg	166	156	158	146	139	142	201
S-SO4	22.1	25.3	43.1	23.6	20.6	12.3	16.8
Fe	27.4	21.7	24.1	18.1	14.1	17.4	23.4
Zn	1.4	1.6	2.1	2	2.4	1.9	3.6
Mn	4.9	4.3	4.6	2.5	10.9	8.4	18.7
Cu	0.4	0.3	0.4	0.4	0.3	0.4	0.4
Cl	20	25	27	57	35	13	17
Na	50	37	45	45	52	34	52
pH (H2O)	5.83	6.01	6.01	6.24	6.81	6.45	6.04
EC (mS cm−1)	1.223	1.362	1.565	1.494	1.322	1.449	1.094

.

Substrate samples were collected for analysis of pH, EC, and macro- and micronutrients just before microgreen cultivation. The substrate samples (500 mL) were collected in a plastic zip-lock bag during substrate preparation.

The samples were analyzed using the universal method, as described by Czerwińska-Kaiser et al. [23]. The extraction of macronutrients (N-NH₄, N-NO₃, P, K, Ca, Mg, S-SO₄), Cl, and Na was carried out in 0.03 M CH₃COOH, at a proportion of 1:10 (20 cm³ of sample to 200 cm³ of extraction solution). After 30 min of extraction, the following determinations were made: N-NH₄ and N-NO₃ by micro distillation according to Bremer’s method as modified by Starck [24]; P by colorimetric method with ammonium vanadomolybdate; K, Ca, and Na by photometry; Mg by atomic absorption spectrometry (AAS) using a Carl Zeiss-Jena apparatus; S-SO₄ by nephelometry with BaCl₂; and Cl by nephelometry with AgNO₃. Micronutrients (Fe, Mn, Zn, and Cu) were extracted from the samples using Lindsay’s solution containing 1 dm³ of 5 g EDTA (ethylenediaminetetraacetic acid); 9 cm³ of 25% NH₄ solution; 4 g of citric acid; and 2 g of Ca (CH₃COO)₂·2H₂O. Micronutrients were determined by ASA technics [25]. Salinity was identified conductimetrically as electrolytic soil conductivity (EC in mS·cm⁻¹), and pH was determined by potentiometry (soil: distilled water = 1:2) [26].

2.2. Plant Material, Growing Conditions, and Harvest

Radish and black radish seeds came from the ‘W. Legutko’ company, Jutrosin, Poland. There were high-quality seeds for microgreen cultivation. The plastic tray vessels (30 × 50 × 5 cm; 7.5 L) for each substrate were seeded with 3 g of seeds. After sowing, the seeds were covered with a thin layer of sand and the trays were covered with special lids to maintain high humidity during emergence. The prepared trays were placed on tables in the growth chamber. During emergence, the temperature was kept at 23 °C for the first three days after sowing and no lighting was used.

Neonica LED 240 lamps were used as the light source in the vegetation period. The photosynthetic photon flux density (PPFD) was 480 µmol m⁻² s⁻¹, and the contribution of individual colors was 383 µmol m⁻² s⁻¹ (80%) for red (R), 33 µmol m⁻² s⁻¹ (7%) for green (G), and 64 µmol m⁻² s⁻¹ (13%) for blue (B). The light period was 16 h. The air temperature in the growth chamber was maintained at 21 °C during the light period and 18 °C during the night. The relative humidity was approximately 60%.

During cultivation, the plants were watered with tap water using a watering can; the microgreens were not additionally fertilized, and no plant protection products were used. The cultivation cycle ended when the microgreens developed their first true leaves. As the plants had different growth dynamics, the plants growing in the control medium were used as a proxy. Plants were cut flush with the substrate using scissors. Each cycle of cultivation lasted 15 days, from sowing to harvesting.

On the last two days of each cultivation cycle, chlorophyll content index (CCI) was measured using an Opti-Sciences OSI CCM-200 Plus chlorophyll meter (Hudson, NH, USA); 20 measurements were taken for each combination. Fluorescence measurements were also taken. Before measurement, the microgreens were kept in the dark for at least 30 min using FP 110 leaf clips. Measurements were then taken using a FluorPen FP 110 fluorimeter (Photon Systems Instruments Ltd., Drásov, Czech Republic) (12 measurements were taken for each combination). Both sets of measurements were taken from the first true leaf. The following individual parameters related to fluorescence were analyzed: F_V/F₀ (maximum water splitting efficiency on the donor side of PSII), F_V/F_M (maximum photochemical yield of PSII), and PI_ABS (PSII function index). F_V/F₀ is a parameter that determines oxygen release efficiency, while F_V/F_M is mainly used to determine whether plants were under stress conditions. A reduced value of this parameter indicates reduced electron transport efficiency. The PI_ABS index relates to a plant’s ability to assimilate CO₂; a reduction in this index may therefore indicate reduced assimilation of this gas [27].

Biometric measurements were taken after the plants were cut. The following were measured: plant height, the fresh weight of a single plant, and the dry matter content. Measurements were taken from 20 plants in each tray (the material used for the measurements and analysis was taken from the center of the tray). Dry matter content was determined using the dry-weight method [28]. The plants were weighed after cutting, dried at 105 °C for 24 h, and then weighed again. The height was measured using a ruler, and the weight was measured to the nearest 0.01 g using an electronic laboratory scale. The plants were also scanned, and then the Skwer program was used to calculate the plant area.

2.3. Mineral Analysis of Microgreens and Substrates

Sample processing:

Microgreen and substrate samples were dried at 50 ± 3 °C for 48 h in an electric oven, then ground in a laboratory mill (PM 200, Retsch, Germany). A mass of 0.400 ± 0.001 g of dry sample was digested with concentrated nitric acid in closed Teflon containers using a microwave digestion system (Mars 5 Xpress, CEM, Matthews, NC 28104, USA). The samples were then filtered through paper filters and diluted with water to a final volume of 15.0 mL. After digestion, the samples were filtered and diluted with water to a final volume of 15.0 mL. Each sample was analyzed in triplicate. The contents of mineral elements are expressed in milligrams per kilogram of dry plant matter. An Agilent 5100 ICP-OES inductively coupled plasma optical emission spectrometer (Agilent, Santa Clara, CA 95051, USA) was used to determine the elements under standard conditions. Commercial analytical standards (ICP, Romil, Blackburn, Lancashire, UK) were used for calibration. The selected wavelengths and validation parameters are given below. The detection limits obtained follow 3-sigma criteria at a level of 0.0 mg kg^–1 dry matter (DM). The uncertainty for the complete analytical process (including sample preparation) was 20%. Traceability was checked using the following standard reference materials: CRM S-1 = loess soil; CRM NCSDC (73,349) = bush branches and leaves; CRM 2709 = soil; CRM 405 = estuarine sediments; and CRM 667 = estuarine sediments. Recovery (80–120%) was acceptable for all elements determined. Each sample was analyzed in triplicate.

2.4. Data Analyses

The study was conducted as a two-factor experiment. The first factor was the substrates (7 variants), and the second was the time of composting (2 levels). The research was performed in three replicates for each combination (a single tray constituted one repetition). The investigations were conducted in two independent cycles. The results were the means for each cycle separately. The data were analyzed using ANOVA. Differences between the means were estimated using the Newman–Keuls test at a significance level of α = 0.05. Statistical analysis was performed using the Statistica program (StatSoft, Kraków, Poland).

3. Results

3.1. Morphological Parameters

The highest fresh weight was obtained using substrate A-2.5, which contained 2.5% spent mushroom substrate (SMS) from Agaricus bisporus (A-2.5), for both radish and black radish in cycle I. This suggests that this substrate has a beneficial effect on biomass growth (see

Table 2. Morphological parameters of microgreens cultivated in different substrates.

Substrates	Fresh Weight (g)						Dry Weight (g)
	Radish			Black Radish			Radish			Black Radish
	Cycle		Mean	Cycle		Mean	Cycle		Mean	Cycle		Mean
	I	II		I	II		I	II		I	II
Control	1.09 bc	1.03 bc	1.06 c	2.43 b	2.24 b	2.34 a	0.10 c	0.13 bc	0.12 bc	0.23 ab	0.20 bc	0.21 a
A-2.5	1.82 a	1.19 b	1.51 a	2.76 a	1.74 d	2.25 a	0.20 a	0.12 bc	0.16 a	0.27 a	0.15 c	0.21 a
A-5	1.20 b	1.27 b	1.23 b	2.17 c	1.56 e	1.87 b	0.14 b	0.13 bc	0.13 ab	0.27 a	0.12 cd	0.19 ab
P-20	0.74 d	0.93 c	0.84 d	1.38 ef	1.46 e	1.42 c	0.08 d	0.10 c	0.09 c	0.16 c	0.13 cd	0.14 b
P-30	0.18 e	0.30 e	0.24 f	0.29 j	0.49 i	0.39 e	0.02 f	0.03 f	0.02 d	0.05 e	0.05 e	0.05 d
L-20	0.15 e	0.15 e	0.15 f	0.23 j	0.22 j	0.23 f	0.02 f	0.01 f	0.02 d	0.03 e	0.03 e	0.03 d
L-30	0.39 e	0.57 d	0.48 e	0.71 h	0.91 g	0.81 d	0.04 ef	0.06 de	0.05 d	0.10 d	0.08 d	0.09 c
	Plant Length (cm)						Plant Area (cm2)
Control	9.16 b	7.72 c	8.44 c	14.11 a	8.56 d	11.34 b	7.48 c	6.48 c	6.98 b	18.9 ab	17.5 b	18.2 a
A-2.5	11.68 a	9.22 b	10.45 a	14.84 a	11.56 c	13.20 a	10.2 a	9.33 b	9.76 a	18.2 ab	11.5 c	14.8 b
A-5	9.09 b	8.91 b	9.00 b	12.64 b	12.69 b	12.67 a	8.62 bc	8.51 bc	8.56 ab	20.5 a	14.7 bc	17.6 a
P-20	6.03 d	7.37 c	6.7 d	10.80 c	11.07 c	10.94 b	3.65 d	5.44 cd	4.54 c	8.59 d	9.09 d	8.84 c
P-30	3.35 g	4.56 e	3.95 f	4.71 g	6.63 ef	5.67 d	1.07 g	2.07 ef	1.57 d	2.63 f	4.44 ef	3.53 d
L-20	2.86 g	4.01 f	3.43 g	4.30 g	6.09 f	5.20 d	0.95 g	1.72 ef	1.33 d	2.10 f	2.01 f	2.05 d
L-30	4.57 e	7.39 c	5.98 e	7.03 e	7.20 e	7.12 c	3.50 e	6.88 c	5.19 c	6.64 e	8.51 d	7.57 c

Means for each species and means followed by the same letters are not significantly different at p < 0.05 A-2.5—A-SMS:Peat (2.5:97.5%), A-5.0—A-SMS:Peat (5:95%), P-20—P-SMS:Peat (20:80%), P-30—P-SMS:Peat (30:70%), L-20—L-SMS:Peat (20:80%), and L-30—L-SMS:Peat (30:70%).

). Cultivation on substrate A-5 also resulted in high fresh weight values, outperforming the control. In contrast, the lowest fresh matter values were recorded in cycle I in substrates containing shiitake substrate (L-20 and L-30) and in cycle II in the L-20 combination. The oyster substrate at a higher concentration (P-30) also showed a strong reduction in plant growth, indicating a possible phytotoxic effect or the substrates having unfavorable physicochemical properties at such a high proportion (see Figure 1).

Dry matter content generally correlated with fresh weight: the highest values were obtained in substrate A-2.5 (0.20 g for radish in cycle I), and the lowest in L-20 and P-30. A relatively high dry matter content was obtained in P-5 (black radish, cycle I: 0.27 g), which may indicate a more favorable water balance and higher tissue density.

The highest plants were recorded in A-2.5 (radish, cycle I), which were 72% and 76% taller than the shortest plants in P-30 and L-20, respectively. Notably, black radish consistently grew taller than radish across all treatments, possibly due to species-specific differences in growth rate and hypocotyl elongation.

The largest plant area of microgreens was observed in the control and A-2.5 (radish, cycle I), as well as in A-5 (black radish), confirming the beneficial effect of Agaricus SMS on shoot development. Plants grown in P-30, L-20, and L-30 showed the smallest values—often below 2 cm². Interestingly, leaf area in L-30 was much greater than in L-20, suggesting that a moderate proportion of Lentinula SMS (after composting) may partially improve plant development.

A longer composting period (cycle II) improved some growth traits in substrates of lower quality (e.g., plant weight and height in L-30), though it did not fully compensate for the negative effects of excessive SMS rates in P-30 and L-20.

3.2. Mineral Content

3.2.1. Growing Media

The growing media used for microgreen cultivation differed significantly in their mineral composition (

Table 3. Mineral content [mg kg⁻¹] in the growing substrate medium.

Element	Substrate
	C	A-2.5	A-5	P-20	P-30	L-20	L-30
Ca	25,248	25,924	26,418	23,451	21,788	23,153	24,877
K	1693	1414	1629	4692	6250	1285	1548
Mg	824.0	816.4	808.8	844.3	800.5	847.2	1093
Na	120.8	117.1	138.3	172.3	183.4	103.6	143.3
Al	414.0	490.6	445.2	383.6	378.7	345.9	426.7
B	15.15	16.11	15.06	15.00	16.40	13.71	17.53
Cd	0.167	0.150	0.167	0.174	0.162	0.134	0.154
Cr	4.179	5.649	5.216	4.346	6.051	6.139	8.072
Cu	13.10	12.86	12.17	11.76	10.75	11.07	13.74
Fe	3168	4171	3499	3179	2745	2791	3082
Mn	91.95	108.9	117.1	157.0	197.9	176.3	247.5
Mo	10.316	9.874	9.808	8.641	8.250	9.522	9.900
Pb	2.840	3.838	4.107	3.776	3.609	4.201	5.318
Pr	1.659	1.916	1.823	1.557	1.554	1.558	1.612
Rb	0.422	0.646	0.676	1.351	1.912	0.491	0.877
Si	582.2	736.6	663.6	588.5	637.5	493.4	660.6
Sr	32.57	33.93	33.74	35.29	34.36	30.87	33.81
Ti	12.41	13.15	12.64	10.88	10.91	9.81	13.01
Zn	16.46	22.54	29.50	32.73	36.82	27.07	44.25
Zr	0.247	0.262	0.239	0.219	0.235	0.198	0.258

). The highest calcium (Ca) content was found in substrates with Agaricus SMS (A-5 and A-2.5), reflecting their raw material origin—composted straw and animal manure enriched with gypsum, which are naturally calcium-rich. Elevated Ca levels were also recorded in the control peat substrate, traditionally used in horticulture.

In the case of potassium (K), the highest concentrations were found in substrates with Pleurotus SMS (P-30 and P-20). In contrast, significantly lower K contents were measured in Agaricus SMS substrates, likely due to the intensive uptake of this element by fruiting bodies during commercial mushroom production. Lentinula SMS substrates (L-20 and L-30) contained the lowest potassium levels among all organic materials analyzed.

All substrates used were characterized by a relatively uniform level of magnesium (Mg) (800–850 mg·kg⁻¹), with the highest value observed in L-30, possibly due to extended use of the substrate before composting.

Sodium (Na) content varied significantly, with the lowest levels in L-20 and the highest in P-30. Excess sodium may disrupt ionic balance and hinder nutrient uptake, so its presence should be carefully monitored, especially in salt-sensitive crops.

Among the microelements, the greatest differences were observed for zinc (Zn). The highest Zn content was found in L-30 (44.25 mg·kg⁻¹), highlighting its potential for Zn enrichment. Conversely, the control substrate contained the lowest Zn level—60% less than L-30—which partly explains the lower Zn content in microgreens from the control group. High boron (B) concentrations were recorded in L-30, Agaricus substrates, and the control, ensuring adequate availability of this crucial microelement.

Aluminum (Al), though not a nutrient, serves as a key indicator of potential substrate toxicity. The highest Al content was recorded in A-2.5, while the lowest was in L-20. Elevated Al levels may limit phosphorus availability and negatively affect root growth, particularly under acidic pH conditions.

3.2.2. Radish Microgreens

The content of elements in radish microgreens showed significant differences depending on the type of substrate applied and the length of the composting period (see

Table 4. Content of selected elements [mg kg⁻¹] in radish microgreens.

Element	Control		A-2.5		A-5		P-20		P-30		L-20		L-30
Cycle	I	II	I	II	I	II	I	II	I	II	I	II	I	II
Ca	32,085 c	32,812 bc	33,348 ab	34,566 a	33,648 c	33,373 ab	16,583 f	21,617 d	10,668 g	7353 h	20,132 e	10,144 g	17,486 f	19,154 e
K	19,849 d	22,148 d	36,677 bc	49,426 a	40,758 b	45,537 a	33,122 c	32,853 c	19,822 d	19,386 d	18,649 d	11,791 e	17,240 d	33,004 c
Mg	2952 bc	3120 ab	2869 bc	3391 a	3158 ab	3234 ab	1900 ef	2588 cd	1707 f	1504 f	2206 de	1584 f	2350 d	2889 bc
Na	5302 ab	3949 c	4939 b	5647 a	3469 c	3682 c	1241 ef	2235 d	712 g	872 fg	1629 e	802 fg	1238 ef	3635 c
Al	20.1 d	22.5 c	29.5 a	27.9 b	26.0 b	16.5 ef	20.7 cd	17.8 e	15.4 fg	21.4 cd	27.5 ab	13.7 g	8.3 h	8.6 h
B	60.6 ab	57.4 bc	60.1 ab	52.3 de	62.3 a	51.3 de	48.3 ef	52.2 de	45.3 f	38.2 g	50.1 e	38.1 g	64.0 a	55.4 de
Cd	0.36 a	0.34 ab	0.28 c	0.36 a	0.29 bc	0.30 abc	0.18 ef	0.26 cd	0.17 ef	0.12 f	0.26 cd	0.18 ef	0.19 e	0.20 de
Cr	3. 4 f	3.4 f	7.3 a	3.8 ef	1.9 g	1.6 g	3.9 ef	6.7 b	3.9 f	6.6 c	7.2 a	4.1 de	1.9 g	4.5 d
Fe	262 bc	256 bc	247 bc	374 a	254 bc	233 cd	157 e	140 ef	158 e	164 e	211 d	140 ef	115 f	282 b
Mn	201 bcd	147 f	193 cd	225 ab	231 a	214 abc	72 h	43 i	186 cde	120 g	176 de	160 ef	235 a	199 abc
Mo	3.09 bc	2.67 cd	3.29 b	2.90 bc	3.86 a	2.90 bc	3.10 bc	3.72 a	3.08 bc	2.69 cd	2.34 d	1.69 e	1.88 e	1.53 e
Na	4262 b	3949 bc	4745 a	4916 a	3469 d	3682 cd	1241 gh	2235 e	712 i	872 hi	1629 fg	802 i	1238 gh	1755 f
Pb	0.34 ef	0.39 de	1.11 a	0.28 f	1.15 a	0.46 d	0.00 h	0.12 g	0.00 h	0.18 g	0.82 b	0.70 c	0.82 b	0.27 f
Pr	2.50 b	2.57 b	2.57 b	2.90 a	2.61 b	2.60 b	0.98 f	1.50 c	0.67 e	0.31 f	1.22 cd	0.44 ef	1.00 d	1.30 c
Rb	11.0 ef	12.8 de	20.8 b	18.6 c	23.0 a	22.0 ab	8.9 e	14.5 d	6.0 g	5.4 g	10.1 f	4.3 g	12.7 de	12.8 de
Si	576 a	559 ab	500 abcd	524 abcd	553 ab	486 bcd	537 abc	441 d	581 a	467 cd	464 cd	300 e	499 abcd	530 abc
Sr	53.6 b	60.2 ab	57.3 ab	62.8 a	57.3 ab	55.4 b	28.4 d	42.3 c	18.6 e	15.4 e	30.3 d	15.4 e	25.4 d	30.2 d
Ti	0.53 de	0.57 d	0.78 b	0.67 c	0.85 ab	0.43 f	0.92 a	0.14 g	0.58 d	0.80 b	0.94 a	0.46 ef	0.21 g	0.54 de
Zn	90.8 bcde	86.9 cde	88.8 bcde	100.6 abcd	100.6 abcd	102.5 abc	66.4 fg	103.2 ab	93.5 bcd	56.8 g	85.0 de	77.6 ef	116.0 a	95.9 def
Zr	0.041 a	0.038 ab	0.032 abc	0.037 ab	0.032 abc	0.024 cde	0.013 f	0.014 f	0.018 ef	0.022 de	0.027 cde	0.039 a	0.019 def	0.028 bcd

Means for each element followed by the same letters are not significantly different at p < 0.05 A-2.5—ASMS: Peat (2.5:97.5%), A-5.0—A-SMS:Peat (5:95%), P-20—P-SMS:Peat (20:80%), P-30—P-SMS:Peat (30:70%), L-20—L-SMS:Peat (20:80%), and L-30—L-SMS:Peat (30:70%).

). Particularly high values were recorded for the macroelements (Ca, K, and Mg) in the media to which substrate was added after mushroom cultivation. This is consistent with previous chemical analyses of these media.

The highest calcium content was obtained in cycle II in variants A-2.5 and A-5, as well as in the control. By contrast, very low Ca values were observed in the oyster and shiitake substrates (e.g., P-30 contained three times less Ca than the P-30 combination), reflecting the low content of this element in these substrates. The potassium (K) content of radish microgreens was exceptionally high in cycle II in variants A-2.5 and A-5. In the control combination and the shiitake and oyster mushroom substrates, the values were approximately 50% lower. The magnesium (Mg) content of the microgreens was highest in cycle II for A-2.5 and A-5. The lowest values were recorded in L-20 (cycle II) and P-30 cultivation, which confirms the lower fertilizing potential of these substrates once again. Regarding sodium (Na), variants A-2.5 and A-5 stood out, while the lowest values were found in the P-30 and L-20 cycle II (<900 mg kg⁻¹).

Among microelements, the particularly high iron (Fe) content of the microgreens grown in A-2.5 cycle II, superior to all other combinations, is noteworthy. This value may indicate the possibility of using this substrate variant in biofortification. The Fe content of the control microgreens was similar to that of A-2.5 in cycle I and A-5, with the lowest values occurring for L-20 (cycle II) and L-30 (cycle I). Low levels of iron in the herb were also found in the microgreens grown on the substrate after oyster mushroom cultivation. The manganese (Mn) content of the plants was highest for S-30, which may also be important from a nutritional point of view. High Mn content was also found in radish from the P-5 and P-2.5 (ii cycle) combination. Significantly lower values were recorded for B-20. Zinc (Zn) content was highest in L-30 (cycle I), confirming earlier observations about the potential of post-shiitake substrates to enrich plants in Zn, despite overall growth limitation. High Zn values were also found in the microgreens from A-5 and A-2.5, as well as P-20, in the second cycle.

Cadmium (Cd) concentrations were highest in microgreens from the control combination, while lower values occurred in radish from the fungal substrates, especially after oyster and shiitake. Also, lead (Pb) concentrations were lowest for microgreens from the oyster substrate and highest in A-2.5 and A-5 (more than 1.1 mg kg⁻¹—cycle I), which requires further evaluation.

Exceptionally high chromium (Cr) concentrations occurred in the plants for A-2.5 (cycle I), which may be due to the content of this element in the mushroom substrate or its interaction with other components. Low Cr values were observed in microgreens grown in pure substrate and with the addition of shiitake substrate.

Cycle II (153 days of composting) mostly favored higher elemental content (especially K, Mg, and Fe), suggesting that longer maturation of the substrate post-improves its fertilizing value and microelement bioavailability.

3.2.3. Black Radish Microgreens

The highest calcium content was found in radish microgreens grown in the control substrate in cycle I, which was statistically different from most other combinations, except for P-2.5 and P-5 in cycle I (

Table 5. Content of selected elements [mg kg⁻¹] in black radish microgreens.

Element	Control (C)		A-2.5		A-5		P-20		B 30		S 20		S 30
Cycle	I	II	I	II	I	II	I	II	I	II	I	II	I	II
Ca	42,140 a	37,511 ab	38,742 ab	36,243 b	37,513 ab	30,025 c	20,742 de	20,853 de	15,568 f	13,226 f	15,780 f	15,161 f	24,305 d	17,351 ef
K	32,247 c	35,215 c	43,837 b	49,378 ab	54,156 a	49,110 ab	42,491 b	48,719 ab	45,079 b	46,908 ab	32,529 c	32,815 c	47,757 ab	46,921 ab
Mg	2716 ab	2405 bc	2583 ab	2658 ab	2882 a	2414 bc	1884 de	2107 cd	1816 de	1674 e	1855 de	1868 de	2497 abc	2431 bc
Al	12.5 efg	7.7 h	23.5 c	17.15 d	30.9 b	10.5 gh	34.9 ab	30.7 b	34.0 ab	31.5 ab	11.2 fgh	14.3 def	15.2 de	21.6 c
B	56.5 def	48.4 fg	67.9 bc	48.8 fg	63.6 bcd	46.1 g	51.5 efg	54.9 defg	69.0 bc	59.6 cde	57.1 def	56.9 def	81.9 a	73.1 ab
Cd	0.21 a	0.22 a	0.17 c	0.21 a	0.16 b	0.14 b	0.09 cde	0.10 cd	0.11 c	0.07 e	0.15 b	0.08 de	0.14 b	0.09 de
Cr	3.8 ef	2.9 g	4.0 ef	0.59 j	2.8 g	3.2 fg	10.9 a	6.8 b	1.5 i	5.0 d	2.5 gh	4.5 de	5.9 c	1.9 hi
Cu	3.21 abc	1.94 fg	3.39 ab	2.70 ed	3.41 a	2.66 de	3.05 abcd	2.81 cd	2.31 ef	2.76 cde	1.86 fg	1.76 g	2.93 bcd	3.41 a
Fe	117 fg	125 fg	200 cd	167 de	314 b	179 d	349 a	134 fg	112 fg	226 c	104 g	131 fg	140 ef	187 d
Mn	282 a	114 f	205 c	198 cd	192 cde	170 de	70 g	50 g	183 cde	166 e	134 f	165 e	239 b	269 a
Mo	2.20 b	0.98 h	1.77 cde	1.33 fg	1.60 def	1.31 g	1.80 cd	1.68 cde	2.52 a	1.82 cd	1.91 c	1.78 cde	2.23 b	1.50 efg
Na	7715 b	6794 b	7502 b	9230 a	7772 b	7673 b	2252 ef	2676 de	1620 f	1829 ef	1502 f	1821 ef	3333 d	4376 c
Pb	0.51 c	0.47 c	0.00 d	0.00 d	0.72 a	0.51 c	0.65 ab	0.64 ab	0.66 ab	0.64 ab	0.62 b	0.00 d	0.68 ab	0.67 ab
Pr	2.41 ab	2.20 b	2.64 a	2.59 a	2.73 a	2.47 ab	1.53 c	1.44 cd	0.97 e	0.84 e	0.97 e	0.95 e	1.65 c	1.15 de
Rb	15.6 d	16.0 d	27.2 b	27.1 b	34.1 a	28.5 b	21.7 c	19.9 cd	15.7 d	15.9 d	17.9 cd	18.4 cd	36.1 a	33.7 a
Si	553 bcd	461 de	524 bcde	600 ab	586 ab	490 cde	549 bcd	582 abc	562 abc	652 a	440 e	487 cde	576 abc	554 bcd
Sr	61.0 ab	54.1 b	67.8 a	67.3 a	68.7 a	54.6 b	41.8 c	43.5 c	30.6 d	28.3 d	28.5 d	27.8 d	41.8 c	31.9 d
Ti	0.28 cd	0.09 g	0.59 a	0.42 b	0.63 b	0.17 f	0.21 ef	0.23 def	0.31 c	0.44 b	0.31 c	0.23 def	0.46 b	0.26 cde
Zn	77.7 cde	70.4 def	86.7 bc	67.8 ef	94.6 b	63.3 f	77.8 cde	68.0 ef	96.1 a	84.2 bcd	82.6 bcd	90.7 bc	117.4 a	112.9 a
Zr	0.019 cde	0.014 de	0.021 cde	0.026 bc	0.013 e	0.018 cde	0.032 b	0.061 a	0.023 bcde	0.33 b	0.022 bcde	0.020 cde	0.026 bc	0.024 bcd

Means for each element followed by the same letters are not significantly different at p < 0.05 A-2.5—ASMS: Peat (2.5:97.5%), A-5.0—A-SMS:Peat (5:95%), P-20—P-SMS:Peat (20:80%), P-30—P-SMS:Peat (30:70%), L-20—L-SMS:Peat (20:80%), and L-30—L-SMS:Peat (30:70%).

). Significantly lower Ca concentrations were observed in plants from the oyster and shiitake substrates.

Microgreens from combination A-5 in cycle I contained the most potassium, significantly more than the others. K concentrations were also high in A-2.5, A-5, and P-30 in cycle II. Plants grown on control medium and with the addition of shiitake substrates (L-20, L-30) showed lower K content. The highest magnesium levels were recorded in microgreens from A-5 in cycle I. Similar values were also obtained for P-2.5 and the control (1st cycle). In contrast, the lowest values were for plants from the oyster and shiitake substrates (except for L-30 in cycle I). Plants grown in A-5 (cycle II) had the highest sodium content. Microgreens from oyster and shiitake substrates had significantly lower sodium concentrations (below 2700).

Microgreens from the P-20 (cycle I) resulted in the highest Fe content. High values were also found in A-5 (cycle I). Black radish grown in the substrate and P-30 had the lowest Fe content, while microgreens from L-30 had the highest Zn content. Plants grown in the substrate and A-2.5 had significantly lower contents of this microelement. Plants from the substrate and L-30 contained the highest amount of Mn. The lowest values were found in plants from oyster mushroom substrates. Black radish microgreens from A-5 cycle I and L-30 cycle II had the highest CU content. Plants from the substrate (cycle II) and from L-30 showed the lowest content. Plants from L-30 were characterized by a high boron content. Other combinations, including A-5 cycle II, had significantly lower values.

Cadmium (Cd) was highest in black radish from the control and A-2.5, while it was lowest in plants from the oyster mushroom and shiitake substrates. The highest level of Pb resulted in radish from A-5 and the oyster substrate, while in A-2.5 and L-20, the level was minimal (0.00). Microgreens from P-20 (cycle I) were characterized by the highest Cr concentrations. The lowest values occurred in plants from A-5 and the control (cycle II)—<1.0.

3.2.4. Compare Both Species and Cycles

Analysis of the average mineral content of Raphanus ssp. microgreens (radish and black radish) showed clear differences both between species and between composting cycles of post-composting substrates (Tables S1 and S2). The averaged data for each cycle also indicated the effect of the degree of decomposition of organic material in the substrates on mineral accumulation.

Black radish had significantly higher calcium (Ca), sodium (Na), and phosphorus (P) contents than radish. The average Ca content of black radish exceeded 32,000 mg kg⁻¹, while that of radish was around 25,000 mg kg⁻¹. Similarly, higher values were recorded for sodium: 7912 mg kg⁻¹ in black radish vs. 3855 mg kg⁻¹ in radish, which may indicate the greater potential of black radish to accumulate components responsible for regulating water metabolism and cellular conductivity. Potassium (K) and magnesium (Mg), on the other hand, showed similar levels in both species, but radish had slightly higher average concentrations of these elements, especially in cycle II. Potassium averaged 41,701 mg-kg⁻¹ in radish and 39,642 mg-kg⁻¹ in black radish. This may indicate a higher efficiency of K and Mg uptake from well-decomposed organic matter by radish.

In terms of micronutrients, black radish was distinguished by a significantly higher content of iron (Fe), zinc (Zn), manganese (Mn), and boron (B) compared to radish. The average Fe content in black radish was 251 mg kg⁻¹, compared to 210 mg kg⁻¹ in radish. The differences in Mn (257 mg kg⁻¹ vs. 193 mg kg⁻¹) and Zn content (90.7 mg kg⁻¹ vs. 84.6 mg kg⁻¹) were particularly significant. There were no clear species differences for copper (Cu), with average Cu contents of 2.91 mg-kg⁻¹ in black radish and 2.89 mg-kg⁻¹ in radish.

Cadmium (Cd) and lead (Pb) values were low in both species and did not exceed acceptable safety limits. Cd content was slightly higher in radish (0.27 mg kg⁻¹ vs. 0.18 mg kg⁻¹), while Pb, conversely, reached higher levels in black radish (0.38 mg kg⁻¹ vs. 0.26 mg kg⁻¹). The data for chromium (Cr) are interesting, where radish had on average higher values than black radish (6.24 mg-kg⁻¹ vs. 5.41 mg-kg⁻¹), which may indicate selective accumulation of this element. For molybdenum (Mo), rubidium (Rb), titanium (Ti), and zirconium (Zr), black radish clearly dominated, suggesting its higher accumulation capacity for these microelements.

Analysis of the average values over the two composting cycles indicates that cycle II (153 days of composting) favored an increase in the content of many minerals, particularly K, Mg, Fe, and Zn. These differences were evident both within and between species.

3.3. Photosynthetic Parameters

Analysis of the relative chlorophyll content (CCI) and chlorophyll fluorescence parameters in microgreens of radish (Raphanus sativus var. sativus) and black radish (Raphanus sativus var. niger) showed significant differences both between substrates and between cultivation cycles (

Table 6. Selected chlorophyll fluorescence parameters of microgreens cultivated in different substrates.

Substrates	Chlorophyll Content Index (CCI) [a.u.]						FV/FO [a.u.]
	Radish			Black Radish			Radish			Black Radish
	Cycle		Mean	Cycle		Mean	Cycle		Mean	Cycle		Mean
	I	II		I	II		I	II		I	II
Control	20.6 b	18.9 c	19.8 a	9.53 e	17.3 b	13.4 cd	4.61 ab	4.45 ab	4.53 ab	4.53 a	4.19 ab	4.36 a
A-2.5	15.7 def	14.4 fgh	15.0 b	12.2 d	14.9 c	13.5 cd	4.40 ab	4.37 ab	4.38 b	4.50 a	4.50 a	4.50 a
A-5	17.4 cd	13.4 gh	15.4 b	16.5 bc	14.6 c	15.6 b	4.73 a	4.47 ab	4.60 ab	3.79 abcd	3.93 abc	3.86 b
P-20	16.9 de	15.1 efg	16.0 b	12.4 d	12.8 d	12.6 d	4.86 a	4.85 a	4.85 ab	2.76 e	4.12 ab	3.44 bc
P-30	2.09 i	12.9 h	7.48 c	10.4 de	11.9 d	11.2 e	4.06 b	3.11 c	3.59 c	3.38 bcde	4.00 abc	3.69 bc
L-20	1.93 i	15.2 efg	8.54 c	20.6 a	15.9 bc	18.3 a	3.56 c	3.43 c	3.50 c	3.29 cde	3.13 de	3.21 c
L-30	14.8 fg	25.0 a	19.9 a	11.4 de	17.5 b	14.3 c	4.51 ab	4.62 ab	4.56 ab	2.73 e	4.15 ab	3.44 b
	FV/FM [a.u.]						Piabs [a.u.]
Control	0.82 a	0.82 a	0.82 a	0.82 a	0.81 ab	0.81 a	3.92 abcd	3.10 cdef	3.51 ab	2.38 ab	2.39 ab	2.38 a
A-2.5	0.81 a	0.81 a	0.81 a	0.82 a	0.82 a	0.82 a	2.85 def	2.92 def	2.88 b	1.81 bcd	1.81 bcd	1.81 ab
A-5	0.83 a	0.82 a	0.82 a	0.79 abc	0.79 abc	0.79 b	4.14 abc	3.35 bcde	3.75 a	1.97 abc	1.85 bcd	1.91 ab
P-20	0.83 a	0.83 a	0.83 a	0.73 e	0.80 ab	0.77 bc	4.24 ab	3.63 abcde	3.94 a	1.19 cd	2.09 abc	1.64 b
P-30	0.79 b	0.76 c	0.77 b	0.77 bcd	0.80 abc	0.78 b	2.66 ef	1.25 g	1.95 c	2.12 abc	1.98 abc	2.05 ab
L-20	0.78 b	0.77 bc	0.78 b	0.76 cde	0.75 de	0.76 c	2.06 fg	1.55 g	1.8 c	3.01 a	1.23 cd	2.12 ab
L-30	0.82 a	0.82 a	0.82 a	0.73 e	0.80 d	0.77 bc	4.56 a	2.95 def	3.76 a	0.92 d	2.32 ab	1.62 b

Means for each parameter and species followed by the same letters are not significantly different at p < 0.05 A-2.5—ASMS: Peat (2.5:97.5%), A-5.0—A-SMS:Peat (5:95%), P-20—P-SMS:Peat (20:80%),P-30—P-SMS:Peat (30:70%), L-20—L-SMS:Peat (20:80%), and L-30—L-SMS:Peat (30:70%).

). The mean values of the parameters are presented separately for both species.

The highest CCI values were found in radish in L-30 medium in the second cycle, indicating a strong stimulation of chlorophyll synthesis in this organic medium. In contrast, the lowest values were recorded in L-20 and P-30 media—significantly lower than in the control, which may indicate cultivation stress or poor mineral availability under these conditions. In black radish, CCI values were generally lower and more even. However, the high CCI values in substrate L-30 and P-20 indicate that these substrates have a co-favorable effect on photosynthetic pigment synthesis, also in this species.

The highest Fv/Fo values were recorded for radish in substrate P-20, which may indicate the high efficiency of the PSII system. Also, the A-5 substrate and the control showed relatively high and even values, indicating good photosynthetic plant condition. The lowest values were observed in substrates L-20 and P-30, especially in the second cycle, indicating reduced efficiency of photochemical mechanisms.

Generally in black radish, the pattern was similar, although overall the values were lower than for radish. The high Fv/Fo values in substrates A-2.5 and L-30 may indicate moderate stress conditions favoring photosynthetic efficiency.

The Fv/Fm values for both species remained at levels typical of healthy plants (about 0.79–0.83), indicating that, irrespective of the substrate, the PSII system was not permanently damaged. However, the slightly reduced Fv/Fm values in the P-30 and L-20 substrates (0.76–0.78) may suggest environmental stress. In black radish, the lowest values were recorded in substrate P-20 in the second cycle (0.73), which may indicate reduced photochemical efficiency.

The highest values of PIabs for radish were obtained in substrates L-30, P-20, and A-5, indicating a high potential for energy accumulation in the electron transport chain. The lowest values (<2.0) were observed in substrate L-20, indicating that stress limited photosynthetic efficiency.

In black radish, PIabs also varied. Moderate values were recorded in the control and L-20 substrate, while the lowest PIabs occurred in the P-20 substrate in cycle I, indicating limited photosynthetic activity.

Radish generally showed higher values of CCI, Fv/Fo, and PIabs compared to black radish, especially in organic substrates such as P-20 and L-30. This indicates greater adaptability of radish to a variety of substrate conditions. Black radish showed more stable but lower fluorescence parameters, which may suggest a different adaptation strategy or lower photosynthetic efficiency under the conditions of the substrates tested.

4. Discussion

The obtained results indicate that the type of growing medium, composting duration, and plant species significantly influenced the growth and biometric parameters of microgreens of radish (Raphanus sativus var. sativus) and black radish (Raphanus sativus var. niger).

The observed differences in the morphological parameters of radish and black radish microgreens depending on the type of substrate and composting cycle were strongly associated with the chemical composition and physicochemical properties of the growing media. The highest fresh and dry biomass, as well as the most favorable morphological traits, were recorded in variants enriched with 2.5% spent mushroom substrate (SMS) from Agaricus bisporus (A-2.5), which aligns with previous findings indicating that low proportions of well-composted SMS can improve plant development by enhancing nutrient availability and the physical structure of the substrate [29,30]. The beneficial properties of the mushroom-based substrates can be attributed to moderate electrical conductivity (EC = 1.36–1.57 mS·cm⁻¹), optimal pH (6.01), and elevated levels of macroelements—particularly potassium (K), phosphorus (P), and nitrate nitrogen (NO₃⁻)—which likely supported enhanced plant development. These parameters promote vigorous plant growth and efficient nutrient uptake, as confirmed by Di Gioia et al. [30] and Fidanza et al. [29], who emphasized the favorable structure and nutrient availability of mushroom composts.

In contrast, P-30 (30% Pleurotus ostreatus SMS) and L-20 (20% Lentinula edodes SMS) substrates negatively affected growth, which was especially evident in the first composting cycle. This is likely related to high C:N ratios and low mineral nitrogen levels in these substrates (Table 1), suggesting intense microbial decomposition and nitrogen immobilization. Organic substrates rich in undecomposed lignocellulosic material can stimulate microbial activity, leading to microbial assimilation of available mineral nitrogen and limiting its availability to plants [31,32]. This interpretation is supported by low dry biomass and weak elongation of hypocotyls in microgreens cultivated in P-30 and L-20 media. Similar effects have been reported by Berić et al. [33], who observed growth inhibition of leafy vegetables grown in substrates with insufficiently composted organic amendments.

One plausible mechanism for the observed nitrogen deficiency is the intense microbial decomposition of organic matter, leading to nitrogen immobilization. In environments with abundant easily degradable carbohydrates (e.g., cellulose, lignin), microbes compete with plants for mineral nitrogen, binding it temporarily in forms unavailable to plants [34,35,36]. This phenomenon is particularly likely in substrates with a high C:N ratio and insufficient compost maturity [37]. Similar nitrogen-limiting effects on microgreen growth have been noted by Petropoulos et al. [38], who emphasized the importance of balanced nitrogen supply during early developmental stages. The lack of adequate humification and microbial competition for nitrogen may further restrict its availability to plants [39,40].

The P-30 and L-20 substrates also exhibited lower pH and higher EC, which could cause osmotic stress or phytotoxicity. Such effects have been previously described by Ashbell et al. [41] and Di Gioia and Rosskopf [42], who noted that immature or overly saline substrates may inhibit seed germination and seedling elongation. Black radish generally showed greater tolerance to suboptimal substrate conditions than radish—producing longer shoots and higher fresh biomass across all treatments—highlighting its greater adaptability to variable growth environments, relevant for selecting species for sustainable microgreen production [43,44].

Furthermore, a longer composting period (cycle II) improved morphological traits in less mature substrates. For example, in the S 30 variant, both leaf length and area increased. This supports findings by Gruda [7] and Prasad et al. [45], who emphasized the importance of compost maturity for reducing phytotoxic effects and enhancing nutrient availability, allowing for safe use in greenhouse and container cultivation.

Using organic waste-derived substrates, including spent mushroom substrate, aligns with the principles of the circular economy and sustainable agriculture [7,46]. Mushroom compost (SMC), due to its high potassium, calcium, and organic matter content, serves as a valuable soil amendment and structural enhancer [29,47].

In our research, SMC contributed to elevated K, Mg, and Fe concentrations in microgreens, consistent with findings by Di Gioia et al. [30] and Poudel et al. [14], who reported improved nutritional quality in crops grown in mature organic substrates. Although oyster and shiitake substrates had lower overall fertilizing value, they enhanced Zn and B content—microelements important for biofortification [14,48].

Prolonged composting (cycle II) significantly improved mineral availability, particularly for K, Fe, and Zn, due to enhanced humification and mineralization [41,48]. Sodium and calcium levels slightly declined, possibly due to chemical transformations and ion binding in the substrate matrix.

Both radish species demonstrated a strong capacity for mineral accumulation, with black radish showing higher concentrations of Fe, Mn, Zn, and B. These results align with observations by Xiao et al. [49], who reported species-specific variation in microelement uptake among Brassicaceae microgreens. As nutrient-dense functional foods, microgreens offer significant nutritional value in small biomass, making substrate selection critical for product quality—especially when using recycled materials [43,44,50]. High concentrations of K, Fe, and Zn in microgreens cultivated in mushroom-based substrates support Beyer’s (2011) observations that SMS retains substantial macroelement and microelement content post-harvest. SMS, when properly composted, may serve as a potent fertilizer and substrate component [47].

There was also a strong relationship between substrate mineral content and the elemental composition of microgreens. Plants grown in A-2.5 and A-5 variants showed elevated levels of calcium and magnesium, likely reflecting the high availability of these macronutrients in composted straw and gypsum-based mushroom media [45]. Conversely, microgreens cultivated in P-5 and S-30 media accumulated significantly more potassium, iron, and zinc, which corresponded to the respective substrate composition. These results suggest that SMS-based media may support targeted micronutrient enrichment (biofortification), depending on the fungal species and composting process [31].

A 153-day composting period notably increased K, Mg, Fe, and Zn levels, confirming the role of organic matter mineralization in enhancing macro- and microelement bioavailability and reducing phytotoxicity [30,41]. Studies by Abad et al. [46] show that agricultural organic waste, if properly composted, can replace conventional substrates, although shiitake and oyster substrates showed lower availability of Ca, Fe, Mn, and Mg, reflected in lower plant tissue levels.

Species differences revealed that black radish was more effective in accumulating Ca, Fe, Zn, Mn, and Na—likely due to anatomical and physiological factors and differential transporter enzyme activity [49,51]. This underscores the importance of species selection in microelement biofortification [48,52].

The substrates used for mushroom cultivation, such as those for Agaricus bisporus, typically consist of a mixture of organic and mineral components, including cereal straw, horse or poultry manure, gypsum, peat, and mineral supplements. Some of these materials—especially manure and certain mineral additives—may naturally contain trace amounts of heavy metals originating from animal feed, water, or the soil where fodder crops were grown [53]. Since heavy metals are persistent elements, the composting process does not remove them.

In spent mushroom substrates (SMS), the concentration of heavy metals may be further increased due to their accumulation over multiple production cycles or the use of mineral supplements to enhance mushroom yields. When SMS is incorporated into the growing medium for microgreens, it can act as a source of heavy metals available for plant uptake. Radish and daikon radish microgreens have a rapid growth rate and high metabolic activity, which promotes intensive nutrient absorption, including trace elements such as heavy metals [54]. Although in the present study the levels of Cd, Pb, and Ni in the harvested microgreens were well below EU safety limits, their presence can be attributed to both the initial composition of the substrate and the plants’ natural uptake capacity from the growth medium.

In this research, the amounts of cadmium (Cd), lead (Pb), and nickel (Ni) found in radish microgreens remained below the upper limits set by Commission Regulation (EU) 2023/915 and its 2024 revision [55,56], which apply in Poland. According to the current standards for fresh vegetables, the allowable limits are 0.20 mg kg⁻¹ of fresh weight (FW) for Cd, 0.30 mg kg⁻¹ of FW for Pb, and starting January 2025, 2.0 mg kg⁻¹ of FW for Ni. The detected levels of Cd, Pb, and Ni in this investigation were well below these accepted limits, suggesting that the substrates used in this study are safe regarding heavy metal presence. Regarding copper (Cu), zinc (Zn), and manganese (Mn), even though there are no specific EU maximum limits for fresh vegetables, the levels found were within the safe consumption limits as outlined by WHO/FAO dietary guidelines [57]. These results indicate that using spent mushroom substrates under the conditions tested presents no risk of heavy metal contamination and complies with food safety regulations in Poland and the European Union.

Using spent mushroom substrates in microgreen cultivation aligns with sustainable agriculture trends and provides an eco-friendly alternative to peat-based media, whose extraction contributes to peatland degradation [40,58]. Overall, the results confirm that composted SMS can be an effective and sustainable component of microgreen cultivation substrates, provided that the type, proportion, and composting time are appropriately controlled.

The results obtained indicate that the type of substrate and its composting time (cycle I vs. cycle II) have a significant effect on parameters related to the functioning of the photosynthetic apparatus of radish and black radish microgreens. The highest values of relative chlorophyll content (CCI), as well as fluorescence parameters, were recorded in media with the addition of 30% used substrate after shiitake (L-30) and oyster mushroom (P-20) cultivation, suggesting a beneficial effect of these organic components on plant physiology.

According to the results obtained by Prasad et al. [45], the use of fungal substrates can not only improve plant growth and yield, but also affect photosynthetic parameters. In their study with strawberries, substrates containing spent fungal substrate increased chlorophyll content and PI index, which is consistent with current observations in microgreens. The authors indicate that the abundance of organic components and microelements contained in post-fungal substrates, such as nitrogen, magnesium, and iron, can stimulate photosynthetic activity by increasing chlorophyll synthesis and photosystem II efficiency.

In our study, it was noted that Fv/Fm values in most samples remained in the range of 0.78–0.83, indicating good condition of the photosynthetic apparatus, similar to literature data, where this value is considered an indicator of intact PSII [49,50,59]. Values below 0.78, such as in the P-30 substrate in radish, may indicate environmental stress, limited nitrogen availability, or disturbances in substrate water management [41,60].

It is also worth noting the PIabs parameter, which, as an integrated indicator of photosynthetic efficiency (related to the conservation of energy from photon absorption to electron acceptor reduction), showed the greatest variation. The high PIabs values in the L-30 and A-5 substrates are consistent with the results of Di Gioia et al. [30], who pointed out that the use of organic materials as substrate components can post-improve chloroplast energy management and increase plant physiological productivity. PIABS values in the range of 1.5–2.5 are considered to be characteristic of plants growing under optimal conditions [61], which coincides with the highest values obtained in this study in substrates L-30 and P-20 (radish).

In a study by Bonasia et al. [62], photosynthetic parameters of arugula grown in a soilless system with different salinity levels were compared; an increase in salinity led to lower Fv/Fm and PIabs, confirming that the high electrical conductivity (EC) of the substrate can hurt plant physiology. Similarly, the lower values of fluorescence parameters observed in radish in P-30-type substrates may be the result of salt stress or an excess of phenolic compounds in an insufficiently mature substrate.

The use of substrates containing spent fungal substrates, especially after an adequate composting period, may increase the content of readily available macro- and microelements [7,29], which support photosynthetic functions. At the same time, however, as noted in some combinations (e.g., P-30, L-20), excess salts or unfavorable physicochemical conditions can lead to a decrease in fluorescence parameters, consistent with the study of Ding et al. [63] concerning the effect of electrical conductivity (EC) of the substrate on photosynthetic efficiency. A study by Kowitcharoen et al. [64], which included microgreens of broccoli, mustard, and cabbage, found that substrate type can affect not only biometrics but also chlorophyll accumulation and photosynthetic efficiency. Microgreens grown in organic substrates such as coconut or compost showed higher chlorophyll levels than those grown in mineral substrates (e.g., rockwool), confirming current observations on the beneficial effects of substrates with the addition of spent fungal substrate.

The differences between radish and black radish in terms of CCI and PIabs may be due to species-specific conditions—radish showed significantly higher chlorophyll content and higher PSII activity. This may indicate higher physiological plasticity and better adaptation of this species to modified substrate conditions, which is also consistent with observations from previous studies on microgreens of the Brassicaceae [44,65]. The differences between radish and black radish are also confirmed by the reports of Xiao et al. [49,66], who indicated that different Brassicaceae species exhibit different abilities to accumulate chlorophyll and adapt to environmental conditions. Radish was one of the cultivars showing higher biomass values and photosynthetic pigment content compared, for example, to mustard or broccoli.

5. Conclusions

This research confirms that spent mushroom substrates (SMS), when properly selected and composted, can serve as valuable components of growing media for microgreen cultivation. Substrates containing 2.5–5% Agaricus bisporus SMS significantly enhanced plant growth and improved morphological parameters in both radish species, indicating a positive effect on biomass accumulation and canopy development. This benefit likely results from a balanced nutrient profile, favorable physical properties, and low phytotoxicity of this substrate type.

In contrast, higher inclusion rates (20–30%) of Pleurotus ostreatus and Lentinula edodes SMS, especially in short composting durations (97 days), resulted in growth inhibition and decreased photosynthetic efficiency, suggesting the presence of organic compounds or microbial activity that consumed available nitrogen or introduced stress factors. The very low nitrogen levels measured in P-30 and L-20 substrates support this hypothesis.

Black radish microgreens consistently outperformed common radish in dry mass and mineral content, particularly in less favorable substrates, suggesting better adaptation to suboptimal conditions and greater efficiency in nutrient uptake. This species-related variation should be considered when optimizing substrate formulations.

From a practical perspective, low-dose Agaricus-derived SMS (2.5–5%) and thoroughly composted S-30 represent promising alternatives to peat, promoting sustainable horticultural production while reducing peat consumption and organic waste accumulation. In contrast, the use of high-dose substrates, particularly poorly composted Lentinula and Pleurotus residues, should be limited or preceded by extended composting to avoid nutrient imbalance, phytotoxicity, or heavy metal accumulation. The presence of excessive sodium or trace heavy metals in certain SMS types highlights the need for routine chemical analysis and composting protocols to ensure safety and crop quality. These findings offer a foundation for developing standardized, sustainable substrates for controlled-environment agriculture.