Composition of Organic Fertilizers Containing Microorganisms and Their Effect on Soil Microbiological Activity and Plant Growth

Abstract

The conversion of livestock manure and peat into value-added fertilizers provides an environmentally sustainable approach to nutrient recycling and waste management. In this study, organic fertilizers were formulated from poultry, pig, and cattle manure mixed with peat and wood ash, with or without inoculation of the phosphate-solubilizing bacterium Priestia megaterium. Their efficiency was evaluated through plant growth and soil microbiological experiments involving conifer seedlings, herbaceous crops, and ornamental plants. Germination and growth trials with Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) revealed clear species-specific responses: spruce seedlings performed best in substrates containing poultry or cattle manure, while pine showed enhanced growth with pig manure combined with bacterial inoculant. In pansies (Viola × wittrockiana), growth responses varied by cultivar; cattle manure enriched with bacteria increased leaf projection area, whereas poultry manure markedly suppressed growth. For cucumbers, basil, barley, radish, and garden beans, yields were lower than with mineral fertilizers, yet bacterial inoculation significantly influenced soil microbial activity by modifying respiration rates and hydrolytic enzyme intensity in plant- and manure-specific ways. The results demonstrate that microbial supplementation can alter soil biological processes and nutrient turnover, though its effects on plant productivity remain inconsistent. Further research is required to assess long-term performance under field conditions, as practical application will depend on achieving stable and reproducible results.

1. Introduction

Global efforts to address pressing environmental challenges have increasingly emphasized sustainable approaches in agriculture. Within the European Union, the Common Agricultural Policy (CAP) Strategic Plans approved by the European Commission include nutrient management measures such as precision agriculture, restrictions on mineral fertilizer use, promotion of organic fertilizers (e.g., manure, slurry, compost), and the implementation of fertilization plans to improve efficiency (Approved 28 CAP Strategic Plans, 2023–2027).

A recent meta-analysis of 537 experiments across grasslands and croplands worldwide highlighted organic fertilization as a nature-based solution that enhances forage production and soil carbon storage without reducing plant diversity [1]. Manure use also reduces livestock waste accumulation and returns organic matter to soil [2]. However, risks must be considered. Fecal contamination of soil and water poses a major global health concern, with E. coli and enterococci serving as key indicators [3]. Manure application can also disseminate antibiotic-resistant bacteria and resistance genes in agroecosystems [4]. Composting is therefore essential, as storage and treatment reduce typical gut-associated microorganisms (e.g., Enterobacteriaceae, anaerobes, and pathogens) while increasing environmental taxa such as Firmicutes and Actinomycetota [5].

Peat, a slowly renewable organogenic material formed under anaerobic conditions in wetlands, plays a central role in horticulture. Composed primarily of lignocellulosic residues from sphagnum mosses, sedges, grasses, and woody plants, peat persists due to low oxygen availability, low temperatures, and the presence of phenolic compounds that inhibit decomposition. Its unique physical and chemical properties—including high water retention, aeration, low bulk density, absence of pathogens and weed seeds, high cation exchange capacity, and controllable pH—make peat one of the most widely used soil improvers and growing substrates worldwide [6,7]. Peat substrates are often enriched with fertilizers to further enhance plant growth [8].

The increased use of wood biomass for energy production has led to greater availability of wood ash, a byproduct rich in essential minerals. Wood ash contributes to improved nutrient availability and soil pH stabilization, thereby supporting plant growth [9]. Except for nitrogen, which is primarily lost in flue gases, ash retains significant nutrient concentrations, averaging 20% phosphorus, 55% potassium, and more than 95% calcium compared with complex mineral fertilizers [10].

Microorganisms also play a vital role in sustainable fertilization strategies. Biofertilizers—classified as nitrogen, phosphorus, or potassium fixers, as well as plant growth-promoting rhizobacteria—enhance both yield and long-term soil fertility [11]. Among these, Priestia megaterium (formerly Bacillus megaterium) is a widely studied Gram-positive, spore-forming bacterium commonly isolated from soil and plant environments. It grows optimally at ~30 °C and is valued for its phosphate-solubilizing capacity, rhizosphere colonization, and multifaceted plant growth-promoting functions [12,13]. These functions include (i) acting as a biofertilizer through phosphate solubilization, (ii) producing phytohormones such as indole-3-acetic acid and other growth regulators, and (iii) functioning as a biocontrol agent via antifungal activity [14].

The aim of the present study was to develop fertilizing formulations combining cattle, pig, or poultry manure with peat and wood ash, with selected variants supplemented with Priestia megaterium. These formulations were evaluated for their effects on soil microbiological activity and the growth of various cultivated herbaceous and woody (coniferous) plants.

2. Materials and Methods

2.1. Fertilizer Preparation

Raw materials included peat, livestock manure (cattle, pig, poultry), and wood ash. A moderately decomposed transitional woody–herbaceous peat (H4–H6 on the Von Post scale) was used. Chemical analyses were conducted on each raw material prior to formulation. Fertilizer mixtures were prepared in nitrogen-balanced proportions (

Table 1. Composition of fertilizer formulations expressed as percentage of wet mass. Mixtures were prepared in nitrogen-balanced proportions.

Fertilizer	Manure	Peat	Ash
Peat + poultry manure + ash	16	76	8
Peat + pig manure + ash	20	72	8
Peat + cattle manure + ash	37	56	7

). After mixing, the formulations were composted for four months, after which Priestia megaterium bacteria were added. The finished fertilizers were then stored under controlled conditions: winter temperatures of +2 to +6 °C in closed storage and summer temperatures of +10 to +18 °C in shaded shelters. Table 1 presents the composition of fertilizer formulations on a fresh-mass basis.

P. megaterium MSCL 895 (strain obtained from the Microbial Strain Collection of Latvia) was cultured in yeast extract–peptone broth (10 g/L peptone, 8 g/L yeast extract, 10 mg/L MnSO₄·H₂O) under aerobic conditions at 22 ± 1 °C for 5 days, with shaking at 150 rpm. The five-day incubation period was selected to ensure that cultures reached a stable late-exponential phase characterized by maximal sporulation and metabolite formation, consistent with protocols for biofertilizer production [15]. This extended growth period was necessary to obtain a physiologically mature inoculum suitable for integration into organic substrate formulations. The strain used in this study originated from previous in-house screening of rhizosphere isolates, where it demonstrated strong phosphate solubilization capacity and stable growth in organic substrate environments. One litre of bacterial suspension was added to 100 kg of fertilizer to reach a concentration of 10⁷ colony-forming units (cfu)/g.

To illustrate the overall experimental design and the relationships among the results, Figure 1 presents a schematic workflow summarizing the fertilizer preparation steps, plant systems tested, and the main measured parameters.

2.2. Chemical Analyses

All analyses were carried out at the Latvian State Forest Research Institute “Silava” laboratory. pH was determined in H₂O solution. Concentrated HNO₃-extractable elements (K, Ca, Mg, Zn, Fe, Mn, P, Al, Na, and S) were determined after microwave digestion (CEM MARS 6 iWave, Matthews, USA) using an inductively coupled plasma optical emission spectrometer (ICP-OES; Thermo Fisher Scientific iCAP 7200 Duo, Waltham, MA, USA) in accordance with ISO 11885:2009 [16]. Total C and N were determined by dry combustion (Elementar El Cube, Langenselbold, Germany) according to ISO 10694:2006 [17] and ISO 13878:1998 [18] respectively.

2.3. Culture-Dependent Analyses of Microorganisms

Ten grams of each raw material and fertilizer sample were suspended in 90 mL sterile water, shaken at 150 rpm for 30 min at 20 ± 1 °C, and serially diluted. Dilutions were plated on selective media: Endo agar (Millipore, Darmstadt, Germany) for E. coli, Aesculin bile azide agar for enterococci, R2A agar (Millipore, Darmstadt, Germany) for total bacterial counts, and Malt extract agar (Millipore, Darmstadt, Germany) for fungi. Colony counts were expressed as cfu per gram of wet sample. Isolates were purified by streaking and identification of bacteria was performed by morphological features and with biochemical/physiological kits (BBL^® Crystal^® Enteric/Nonfermenter ID kit and Gram-Positive ID kit; Becton & Dickinson, Sparks, NV, USA). Fungal genera were identified by macroscopic and microscopic appearance [19]. Analyses were conducted immediately after mixing and after two years of storage. Identification was based solely on morphological and biochemical characteristics; no molecular (16S rRNA or ITS) sequencing was conducted. Consequently, species-level attribution cannot be confirmed. Therefore, taxonomic resolution was limited, and the results should not be interpreted as a comprehensive representation of community composition. Future work will include molecular verification (e.g., P. megaterium–specific qPCR or 16S rRNA gene sequencing) to confirm inoculant survival and its potential re-isolation from mature fertilizer mixtures.

2.4. Plant Experiments

2.4.1. Conifers—Norway Spruce and Scots Pine

The effectiveness of organic fertilizers was assessed through two experimental approaches: (i) cultivation of seedlings in L-120/40 containers produced by AKB Industrial (Grobina, Latvia) and (ii) raising containerized plants in large rectangular 60 L vegetation vessels, simulating growth conditions in fertilizer-enriched peat substrates comparable to the plough method for bare-root seedlings with enhanced root systems. A fixed mixing ratio of peat to fertilizer (1:1, w/w) was employed. To ensure homogeneous mixing, the substrates were prepared using an electric construction-type concrete mixer. The same concrete mixer was used for all organic substrates. After mixing of each variant, the mixer was cleaned. To minimize cross-transfer of microorganisms between formulations, substrate batches were prepared in a fixed sequence from least to most microbiologically complex mixtures (peat and peat + ash controls first, followed by pig manure, cattle manure, and poultry manure formulations). After each batch, the mixer drum and paddles were rinsed thoroughly with pressurized tap water, brushed to remove adhering material, and wiped with 70% ethanol. Residual wash solution was discarded, and the mixer was allowed to drain and air-dry before the next mix. No intermediate buffer batches were used. These steps were implemented to reduce the risk of carry-over of viable microorganisms between fertilizer variants.

The mixtures were subsequently distributed into seedling growing containers (L-120/40) with side ventilation gaps. Care was taken not to overpack in order to minimize compaction during watering. Tree seeds were sown at a depth of 0.5–1.0 cm. Immediately after sowing, containers were gently watered to avoid seed displacement, and moisture was maintained consistently throughout the cultivation period to prevent desiccation. For each substrate treatment, two replicate containers were established per seed origin. Each treatment consisted of two replicate containers per seed origin, and each tray contained 40 seeds (5 × 8 cells; 120 mL per cell). Thus, a total of 80 seeds per treatment per year were tested for germination. Germination was recorded one month after sowing, and a second assessment of germination and survival was conducted at the end of the vegetation period.

Norway spruce and Scots pine seedlings were raised for an additional season in large vegetation vessels (60 L volume). Each vessel was filled with a mixture of 7 kg peat and 7 kg fertilizer, homogenized using the concrete mixer. The mixtures were placed into plastic construction-type containers, perforated along the bottom perimeter to allow drainage of excess water from irrigation or precipitation. During filling, the substrate was moistened to ensure adequate water content. Each treatment included 10 seedlings planted in three vessels in two rows of five (30 seedlings in total). These numbers represent biological replicates for statistical analysis. The planted vessels were placed in an outdoor experimental plot under natural conditions (Figure 2).

During the experiments, temperature and relative humidity were continuously monitored. On average, greenhouse temperatures were 2–3 °C higher than in the outdoor plot. Humidity differences between the controlled greenhouse environment and field conditions were more pronounced, with greater variability outdoors. While temperature and relative humidity were continuously recorded, light intensity and photoperiod were not measured. Data on air temperature and number of sunny hours during vegetation seasons of 2023 and 2024 from the nearest meteorological stations are available (Supplementary Materials Table S1). Given the observed between-year differences in germination and growth, these unmonitored parameters could have contributed to variability in seedling emergence and development. In future work, logging of photosynthetic photon flux density and photoperiod by season and site should be included to enable more robust interpretation of interannual variability.

At the end of both growing seasons, survival and height increments of Norway spruce and Scots pine seedlings, including both containerized plants and those raised in vegetation vessels, were assessed across treatments. Tree height was measured to the nearest 0.05 cm, while survival rates are expressed as percentages of the initially planted seedlings.

2.4.2. Cucumbers, Basil, Barley, Leaf Radish and Garden Beans

To assess the effects of different fertilizer types and microbial inoculation on the growth of cucumbers (Cucumis sativus cv. ‘Berlioz’ F1), basil (Ocimum basilicum cv. ‘Tuscany’), barley (Hordeum vulgare cv. ‘Katnis’), and leaf radish (Raphanus sativus var. sativus cv. ‘Sango’), experiments were conducted using vegetation pots of varying sizes according to plant requirements. Basil plants were transplanted once, from 1 L to 2 L pots. Cucumber plants underwent three transplantations, progressing from 1 L to 3 L, and finally to 10 L pots. Each pot contained a single cucumber or basil plant. Barley and leaf radish were sown in 5 L pots, with five plants per pot. Following the harvest of leaf radish, garden beans (Phaseolus vulgaris cv. ‘Sundance’) were sown in the same pots without additional fertilization, allowing assessment of residual fertilizer effects.

The experiments were carried out in soil with a pH of 7.5 and the following mineral element composition (mg/L): N 35, P 458, K 250, Ca 18,600, Mg 3100, S 60, Fe 1950, Mn 170, Zn 9.5, Cu 5.5, Mo 0.05, B 0.7. Three types of organic fertilizers were tested, cattle manure, poultry manure, and pig manure, each combined with peat and wood ash. Fertilization rates were adjusted to ensure a uniform nitrogen input of 0.064 g/L (11.5 g of manure compost per 1 L), although N:P:K ratios varied by treatment. A mineral fertilizer served as the control. The same dosage was applied at each transplantation event. Inoculation with P. megaterium was included in selected treatments, while others were left uninoculated to serve as controls. Each experimental treatment was replicated six times.

2.4.3. Garden Pansies

Garden pansy (Viola × wittrockiana) cultivars ‘Mega Star Yellow with Blotch’ and ‘Carneval Special Beacon Rose’ were cultivated. Plants were grown in the commercial substrate “Urban mix” (Nordspring Ltd., Valmiera, Latvia), with 0.5 L of substrate allocated per pot. The fertilizer dosage was 0.25 kg/L of substrate for all treatments, except for pig manure ± bacteria variants, where 0.3 kg/L was applied. Each treatment consisted of 15 plants. During the experiment, one supplementary fertilization was performed using a water-soluble fertilizer (NPK 7-15-27). A solution was prepared by dissolving 50 g of fertilizer in 10 L of water, and a total of 20 L of solution (equivalent to 100 g fertilizer) was applied across the entire experiment. Plants were watered at the time of potting and subsequently 1–2 times per week as needed to maintain adequate substrate moisture.

Pansy growth was assessed by measuring the leaf projection area. Leaf projection area (A) of each plant was determined by measuring two canopy diameters (D₁ and D₂) intersecting at right angles. The projected area was calculated using the equation: A = $\mathsf{\pi }\times {\mathrm{D}}_1\times {\mathrm{D}}_2/4$

2.4.4. Assessment of Soil Microbiological Activity

Substrates, i.e., the medium that supports plant roots, were collected from each vegetation pot in which leaf radish, barley, cucumber, basil, and garden beans were cultivated. Initial measurements included soil dry matter (DM) content, determined after drying samples at 105 °C, and pH. Soil microbiological activity was evaluated using two indicators: respiration rate and fluorescein diacetate (FDA) hydrolysis intensity.

Basal respiration rate was determined by placing 25 g of fresh substrate in sealed jars, each containing a small beaker with 5 mL of 0.1 M KOH solution to capture evolved CO₂. The jars were incubated at 30 °C in the dark for 24 h. Following incubation, the KOH solution was removed and titrated with 0.1 M HCl to quantify the amount of CO₂ evolved during microbial respiration. Results are expressed as mg CO₂ per 100 g soil per hour, according to ISO 14240-1:1997 [20].

To determine the activity of hydrolytic enzymes in soil, the method of evaluating the intensity of hydrolysis of FDA was used [21]. 2.5 g of soil was placed in a test tube with 10 mL buffer solution and 0.2 mL of 0.1% FDA solution. Samples were incubated at 30 °C in the dark for 1 h. After incubation, absorbance was measured spectrophotometrically at 465 nm, and the concentration of fluorescein produced was calculated and expressed as μg fluorescein per g dry soil per hour, following standard microbiological methods for assessing soil quality.

2.5. Statistical Analysis

Means ± standard deviations (SD) were calculated. Significance was tested by two-way ANOVA (p < 0.05). Pearson correlations were calculated between respiration rate and FDA activity.

3. Results

3.1. Chemical Composition of Fertilizers

Peat was acidic (pH 3.6), whereas ash was strongly alkaline (solution pH 12.8). After mixing all raw materials (peat, ash, and manure), the resulting formulations exhibited pH values of 8.4–8.9. Following one week of composting, the pH decreased slightly to 8.3–8.4. The chemical composition of the prepared fertilizers is summarized in

Table A1. Chemical composition of fertilizers, g/kg dry mass.

Fertilizer, Manure	C	N	K	Ca	Mg	Zn	Fe	Mn	P	Al	Na	S	B	pHH2O
Peat	522.7	11.1	0.226	3.324	0.891	0.012	0.641	0.021	0.182	0.594	0.093	1.140	0.001	4.05
Pig	333.0	10.8	9.909	58.962	8.196	0.455	4.403	0.817	5.004	4.773	1.085	8.523	0.050	7.53
Pig + bacteria	298.6	11.2	12.352	55.653	8.832	0.354	3.127	0.695	3.348	3.178	1.113	7.630	0.047	7.91
Cattle	295.8	11.3	12.136	47.774	8.030	0.305	2.446	0.643	3.037	2.673	1.073	6.838	0.044	7.80
Cattle + bacteria	321.1	11.1	8.913	52.212	8.109	0.363	3.315	0.688	4.086	3.468	1.012	7.209	0.044	7.77
Poultry	312.0	14.1	12.122	54.027	7.538	0.343	4.053	0.618	3.466	4.773	0.976	8.460	0.046	6.52
Poultry + bacteria	297.6	13.4	11.361	51.522	7.916	0.337	3.252	0.617	3.798	3.504	0.945	6.696	0.042	6.60

.

3.2. Culture-Dependent Microbiological Profiling of Animal Manure (Culturable Fraction Only)

The four types of manure examined—young chicken, laying hen, pig, and cattle—contained markedly different bacterial populations. Total plate counts ranged from 6.8 × 10⁶ cfu/g in pig manure to 2.8 × 10⁹ cfu/g in young chicken manure (

Table 2. Microbiological composition of fresh manure, expressed as cfu/g. Measurement error did not exceed ±0.5 log₁₀ cfu/g. Values reflect culture-dependent counts on selective media; taxonomic identities of isolates were assigned by biochemical kits (BBL Crystal) and morphology; no 16S/ITS sequencing was undertaken.

Manure	E. coli	Entero-Cocci	Total Plate Count	Fungi	Predominant Phyla (Identified Species)
Young chicken	1.0 × 109	3.8 × 108	2.8 × 109	<100	Actinomycetota (Corynebacterium spp., Micrococcus luteus)
Laying hen	2.2 × 106	1.4 × 108	3.7 × 108	<100	Actinomycetota (Kytococcus sedentarius, Rothia kristinae), Pseudomonadota (Chryseomonas luteola)
Pig	<100	1.9 × 105	6.8 × 106	<100	Pseudomonadota (Chromobacterium violaceum, Vibrio sp.)
Cattle	2.9 × 105	2.9 × 106	4.0 × 108	<100	Actinomycetota (Streptomyces spp.), Pseudomonadota (Chromobacterium violaceum, Enterobacter cloacae), Bacteroidota (Chryseobacterium indologenes)

). Cattle manure was distinguished by its high abundance of Actinobacteria (1.3 × 10⁵ cfu/g). Enterococci counts varied from 1.9 × 10⁵ cfu/g in pig manure to 3.8 × 10⁸ cfu/g in young chicken manure, whereas Escherichia coli ranged from <100 cfu/g in pig manure to 1.0 × 10⁹ cfu/g in young chicken manure. No fungi were detected in any of the manure samples (<100 cfu/g). The results in this section refer to cfu/g recovered on specific media and to taxa obtained from cultured isolates; they do not include unculturable populations or sequencing-based community profiles, and the listed ‘predominant phyla/species’ reflect representative isolates recovered rather than whole-community abundances.

Taxonomic profiling indicated that the predominant species in both young and laying hen manure belonged to Actinomycetota. In contrast, pig manure was dominated by Pseudomonadota, while cattle manure contained representatives of Actinomycetota, Pseudomonadota, and Bacteroidota.

3.3. Microbiological Composition of Fertilizer

For the preparation of poultry manure fertilizer, only laying hen manure was used rather than young chicken manure, since laying hen manure contained 2.7 log₁₀ fewer E. coli and 63% fewer enterococci compared with young chicken manure. Henceforth, references to poultry manure in fertilizer formulations refer specifically to laying hen manure.

Neither the raw materials (peat and processed manure) nor the prepared fertilizers contained detectable E. coli (<100 cfu/g) (Table 3). Peat did not contain fecal indicator organisms but was rich in bacteria (9.0 × 10⁷ cfu/g) and fungi (4.0 × 10⁴ cfu/g), with Actinobacteria, Penicillium, and Mucor spp. predominating. Ash alone was not microbiologically analyzed. Compliance with EU Regulation 2019/1009 (<10³ cfu/g for E. coli and enterococci) is summarized in Table 4 to provide a concise overview of microbiological safety across fertilizer variants.

Table 3. Microbiological composition of freshly prepared peat and peat-based fertilizers (upper row) and the same materials after two years of storage (lower row), expressed as cfu/g. Measurement error did not exceed ±0.5 log cfu/g. *—significant difference compared with freshly prepared fertilizer. Values in bold exceed the permissible limits for organic fertilizers according to [22].

Fertilizer	Year	E. coli	Enterococci	Total Plate Count	Bacillus spp.	Fungi	Microscopically Identified Microorganisms
Peat	First Second	<100 <100	<100 <100	9.0 × 107 4.8 × 106 *	9.0 × 106 <100 *	4.0 × 104 1.1 × 105	Actinobacteria, Penicillium, Mucor Actinobacteria, Penicillium, Mucor
Peat + ash	First Second	<100 <100	<100 <100	2.8 × 107 1.2 × 107	<100 <100	1.2 × 104 3.0 × 105 *	Mucor Trichoderma, Mucor
Peat + ash + bacteria	First Second	<100 <100	<100 <100	2.8 × 107 1.6 × 107	6.0 × 106 3.6 × 106	1.0 × 104 6.0 × 105 *	Bacillus, Mucor Bacillus, Actinobacteria, Mucor
Peat + poultry manure	First Second	<100 <100	6.4 × 104 7.0 × 103 *	3.6 × 108 9.8 × 107	1.1 × 108 <100 *	8.0 × 104 1.3 × 106 *	Penicillium, Mucor Penicillium, Mucor, Geomyces
Peat + poultry manure + ash	First Second	<100 <100	1.8 × 104 <100 *	2.2 × 107 5.1 × 107	2.0 × 106 <100 *	1.0 × 104 5.0 × 104	Mucor Mucor, Penicillium
Peat + poultry manure + bacteria	First Second	<100 <100	2.0 × 102 <100 *	4.8 × 108 7.0 × 106 *	1.3 × 107 7.0 × 106	1.0 × 105 1.9 × 105	Actinobacteria, Penicillium, Mucor Actinobacteria
Peat + pig manure	First Second	<100 <100	4.8 × 103 6.0 × 102	4.0 × 108 9.7 × 107	1.2 × 108 <100 *	8.0 × 104 1.7 × 105	Penicillium, Trichoderma, Mucor Actinobacteria, Penicillium
Peat + pig manure + ash	First Second	<100 <100	2.0 × 104 <100 *	4.8 × 107 2.5 × 107	2.0 × 106 <100 *	1.4 × 104 1.0 × 104	Actinobacteria, Penicillium, Mucor Bacillus, Trichoderma, Mucor
Peat + pig manure + bacteria	First Second	<100 <100	1.0 × 104 <100 *	3.2 × 107 2.0 × 106 *	1.2 × 107 3.0 × 105 *	1.6 × 105 7.0 × 105	Penicillium, Mucor Actinobacteria, Penicillium
Peat + cattle manure	First Second	<100 <100	1.0 × 103 6.0 × 102	1.3 × 108 5.5 × 107	2.4 × 107 <100 *	1.2 × 107 3.0 × 106	Penicillium, Mucor Actinobacteria, Penicillium, Geomyces
Peat + cattle manure + ash	First Second	<100 <100	1.4 × 104 <100 *	4.0 × 108 3.6 × 107	4.0 × 106 <100 *	1.2 × 104 1.6 × 104	Mucor Actinobacteria, Mucor, Trichoderma
Peat + cattle manure + bacteria	First Second	<100 <100	<100 <100	1.9 × 107 1.9 × 107	1.9 × 107 2.0 × 106	6.0 × 105 1.4 × 106	Actinobacteria, Penicillium, Trichoderma, Cladosporium, Mucor Actinobacteria, Trichoderma, Cladosporium

Table 3. Microbiological composition of freshly prepared peat and peat-based fertilizers (upper row) and the same materials after two years of storage (lower row), expressed as cfu/g. Measurement error did not exceed ±0.5 log cfu/g. *—significant difference compared with freshly prepared fertilizer. Values in bold exceed the permissible limits for organic fertilizers according to [22].

Fertilizer	Year	E. coli	Enterococci	Total Plate Count	Bacillus spp.	Fungi	Microscopically Identified Microorganisms
Peat	First Second	<100 <100	<100 <100	9.0 × 107 4.8 × 106 *	9.0 × 106 <100 *	4.0 × 104 1.1 × 105	Actinobacteria, Penicillium, Mucor Actinobacteria, Penicillium, Mucor
Peat + ash	First Second	<100 <100	<100 <100	2.8 × 107 1.2 × 107	<100 <100	1.2 × 104 3.0 × 105 *	Mucor Trichoderma, Mucor
Peat + ash + bacteria	First Second	<100 <100	<100 <100	2.8 × 107 1.6 × 107	6.0 × 106 3.6 × 106	1.0 × 104 6.0 × 105 *	Bacillus, Mucor Bacillus, Actinobacteria, Mucor
Peat + poultry manure	First Second	<100 <100	6.4 × 104 7.0 × 103 *	3.6 × 108 9.8 × 107	1.1 × 108 <100 *	8.0 × 104 1.3 × 106 *	Penicillium, Mucor Penicillium, Mucor, Geomyces
Peat + poultry manure + ash	First Second	<100 <100	1.8 × 104 <100 *	2.2 × 107 5.1 × 107	2.0 × 106 <100 *	1.0 × 104 5.0 × 104	Mucor Mucor, Penicillium
Peat + poultry manure + bacteria	First Second	<100 <100	2.0 × 102 <100 *	4.8 × 108 7.0 × 106 *	1.3 × 107 7.0 × 106	1.0 × 105 1.9 × 105	Actinobacteria, Penicillium, Mucor Actinobacteria
Peat + pig manure	First Second	<100 <100	4.8 × 103 6.0 × 102	4.0 × 108 9.7 × 107	1.2 × 108 <100 *	8.0 × 104 1.7 × 105	Penicillium, Trichoderma, Mucor Actinobacteria, Penicillium
Peat + pig manure + ash	First Second	<100 <100	2.0 × 104 <100 *	4.8 × 107 2.5 × 107	2.0 × 106 <100 *	1.4 × 104 1.0 × 104	Actinobacteria, Penicillium, Mucor Bacillus, Trichoderma, Mucor
Peat + pig manure + bacteria	First Second	<100 <100	1.0 × 104 <100 *	3.2 × 107 2.0 × 106 *	1.2 × 107 3.0 × 105 *	1.6 × 105 7.0 × 105	Penicillium, Mucor Actinobacteria, Penicillium
Peat + cattle manure	First Second	<100 <100	1.0 × 103 6.0 × 102	1.3 × 108 5.5 × 107	2.4 × 107 <100 *	1.2 × 107 3.0 × 106	Penicillium, Mucor Actinobacteria, Penicillium, Geomyces
Peat + cattle manure + ash	First Second	<100 <100	1.4 × 104 <100 *	4.0 × 108 3.6 × 107	4.0 × 106 <100 *	1.2 × 104 1.6 × 104	Mucor Actinobacteria, Mucor, Trichoderma
Peat + cattle manure + bacteria	First Second	<100 <100	<100 <100	1.9 × 107 1.9 × 107	1.9 × 107 2.0 × 106	6.0 × 105 1.4 × 106	Actinobacteria, Penicillium, Trichoderma, Cladosporium, Mucor Actinobacteria, Trichoderma, Cladosporium

Table 4. Regulatory compliance summary according to EU threshold (<10³ cfu/g for E. coli and enterococci) [22]. + compliant; − non-compliant. Values in bold exceed the permissible limits for organic fertilizers.

Fertilizer	Year	E. coli Status	Enterococci Status	Enterococci Status	Overall Compliance
Peat	First Second	+ +	<100 <100	+ +	+ +
Peat + ash	First Second	+ +	<100 <100	+ +	+ +
Peat + ash + bacteria	First Second	+ +	<100 <100	+ +	+ +
Peat + poultry manure	First Second	+ +	6.4  × 104 7.0  × 103	− −	− −
Peat + poultry manure + ash	First Second	+ +	1.8 × 104 <100	− +	− +
Peat + poultry manure + bacteria	First Second	+ +	2.0 × 102 <100	+ +	+ +
Peat + pig manure	First Second	+ +	4.8 × 103 6.0 × 102	− +	− +
Peat + pig manure + ash	First Second	+ +	2.0 × 104 <100	− +	− +
Peat + pig manure + bacteria	First Second	+ +	1.0 × 104 <100	+ +	− +
Peat + cattle manure	First Second	+ +	1.0 × 103 6.0 × 102	+ +	+ +
Peat + cattle manure + ash	First Second	+ +	1.4 × 104 <100	− +	− +
Peat + cattle manure + bacteria	First Second	+ +	<100 <100	+ +	+ +

Table 4. Regulatory compliance summary according to EU threshold (<10³ cfu/g for E. coli and enterococci) [22]. + compliant; − non-compliant. Values in bold exceed the permissible limits for organic fertilizers.

Fertilizer	Year	E. coli Status	Enterococci Status	Enterococci Status	Overall Compliance
Peat	First Second	+ +	<100 <100	+ +	+ +
Peat + ash	First Second	+ +	<100 <100	+ +	+ +
Peat + ash + bacteria	First Second	+ +	<100 <100	+ +	+ +
Peat + poultry manure	First Second	+ +	6.4  × 104 7.0  × 103	− −	− −
Peat + poultry manure + ash	First Second	+ +	1.8 × 104 <100	− +	− +
Peat + poultry manure + bacteria	First Second	+ +	2.0 × 102 <100	+ +	+ +
Peat + pig manure	First Second	+ +	4.8 × 103 6.0 × 102	− +	− +
Peat + pig manure + ash	First Second	+ +	2.0 × 104 <100	− +	− +
Peat + pig manure + bacteria	First Second	+ +	1.0 × 104 <100	+ +	− +
Peat + cattle manure	First Second	+ +	1.0 × 103 6.0 × 102	+ +	+ +
Peat + cattle manure + ash	First Second	+ +	1.4 × 104 <100	− +	− +
Peat + cattle manure + bacteria	First Second	+ +	<100 <100	+ +	+ +

The addition of ash resulted in a pH increase and markedly influenced the microbial composition of the fertilizers. When added to peat, ash reduced bacterial abundance approximately threefold (from 9.0 × 10⁷ to 2.8 × 10⁷ cfu/g) and fungal abundance about threefold (from 4.0 × 10⁴ to 1.2 × 10⁴ cfu/g) (Table 3). In peat–ash–manure mixtures, ash addition reduced total plate counts 16-fold in poultry manure and 8-fold in pig manure variants, while fungal counts were reduced 8-fold and 6-fold, respectively. In cattle manure mixtures, fungal abundance declined by 1000-fold (3 log₁₀ cfu/g).

The addition of P. megaterium had little effect on bacterial total plate counts but increased fungal abundance in all three manure variants compared with ash-only treatments. In poultry and pig manure formulations, fungal abundance even exceeded levels observed without ash addition, suggesting that the bacterium counteracted or reversed the suppressive effect of ash.

After two years of storage, total bacterial counts decreased significantly (p < 0.05) in three variants: peat, peat + poultry manure + bacteria, and peat + pig manure + bacteria (Table 3). Enterococci were reduced across all variants and were no longer detectable in five variants in which they had previously been present: peat + poultry manure + ash, peat + poultry manure + bacteria, peat + pig manure + ash, peat + pig manure + bacteria, and peat + cattle manure + ash. Bacillus spp. were not detected in any fertilizers without bacterial inoculation, suggesting that survival in other variants was attributable to the introduced P. megaterium. A significant decline (p < 0.05) in Bacillus spp. counts was observed only in peat + pig manure + bacteria, from 1.2 × 10⁷ to 3.0 × 10⁵ cfu/g. Fungal abundance increased significantly (p < 0.05) in three variants (peat + ash, peat + ash + bacteria, and peat + poultry manure), while in other variants it remained largely unchanged. All peat-based fertilizers met EU microbiological safety limits (<10³ cfu/g for E. coli and enterococci) after storage. Peat, peat + ash, and bacterial inoculated variants were fully compliant throughout. Manure-containing formulations initially showed higher bacterial counts, particularly with poultry manure, but ash and Priestia megaterium addition markedly reduced contamination, restoring compliance after two years (Table 4).

3.4. The Effect of Fertilizers on the Germination of Conifer Seeds and Seedling Production

Norway spruce germination was moderate in 2023 (≈18–72%) but markedly higher in 2024 (≈68–93%). The highest germination rates were achieved with poultry manure ± bacteria, consistently reaching ≥90% (

Table 5. Germination of Norway spruce and Scots pine from seed (%) under seven substrate amendments across two independent replicates (2023 and 2024). Values are treatment means; “±” denotes the absolute spread in percentage points within treatment. *—significant difference (p < 0.05) with the corresponding Control group.

Tree Species	Manure	1st Replicate (2023)	2nd Replicate (2024)
Norway spruce	Control	70.0 ± 12.5	67.5 ± 9.4
	Cattle	52.5 ± 7.5	75.8 ± 6.2
	Cattle + bacteria	54.4 ± 1.9	76.7 ± 12.0
	Pig	61.3 ± 10.0	89.2 ± 4.2 *
	Pig + bacteria	71.9 ± 5.6	84.2 ± 7.2 *
	Poultry	18.1 ± 5.6 *	92.5 ± 4.1 *
	Poultry + bacteria	29.4 ± 3.1 *	90.8 ± 8.2 *
Scots pine	Control	82.5 ± 7.5	26.7 ± 10.3
	Cattle	66.9 ± 8.1	58.3 ± 12.0 *
	Cattle + bacteria	57.5 ± 2.5 *	61.7 ± 18.3 *
	Pig	75.0 ± 5.0	39.2 ± 11.6
	Pig + bacteria	72.5 ± 5.0	42.5 ± 17.4
	Poultry	15.0 ± 7.5 *	35.0 ± 16.7
	Poultry + bacteria	13.1 ± 1.9 *	46.7 ± 14.3

). In contrast, Scots pine germination was generally high in 2023 (≈13–83%, with the control at 83%) but declined in 2024. The best results in 2024 were obtained with cattle manure ± bacteria (≈58–62%), whereas other treatments remained at ≤47%. The bacterial inoculant did not significantly affect germination outcomes (p > 0.05) compared with the corresponding manure treatments without inoculation.

During the first year, Scots pine and Norway spruce seedlings did not reach sizes suitable for outplanting in the forest stand; therefore, they were grown in vegetation vessels in the greenhouse for an additional season. Results at the end of both seasons exhibited similar trends (Figure 3). Across treatments, mean heights during the first year varied only slightly in both species. By the second year, however, treatment-specific differences became more pronounced. In Norway spruce, the greatest second-year mean heights occurred in cattle manure (±bacteria) and poultry manure + bacteria treatments (≈50–55 cm), whereas the control and pig manure + bacteria were lower (≈44 cm). In Scots pine, second-year heights exceeded those of spruce overall. The tallest seedlings were recorded in the control and pig manure + bacteria treatments, whereas poultry manure treatments and cattle manure + bacteria were among the lowest (Figure 3). In Norway spruce, the tallest seedlings after the second year occurred in cattle manure (with or without bacteria) and poultry + bacteria treatments, averaging ≈50–55 cm. The control and pig + bacteria variants were lower, both around 44 cm, indicating intermediate growth performance. The bacterial inoculant exerted variable effects: slightly positive in combination with cattle manure for spruce, clearly positive with pig manure for pine, and neutral to negative in other cases. These trends were reflected in overlapping error bars (p > 0.05), indicating modest and treatment-specific responses.

Seedling survival was assessed in the large-container cultivation experiment (Figure 4). Scots pine seedlings grown in containers or cassettes appeared visually more robust than Norway spruce, and in all peat–organic fertilizer mixtures seedlings remained healthy. Analysis of annual mean height increments revealed species-specific trends. Overall, there was a tendency for greater height growth in treatments where organic fertilizer was supplemented with bacterial inoculant, although these differences were not statistically significant (p > 0.05). In spruce, mean height increments were generally higher in containers where fertilizer alone was used, rather than when combined with bacteria. For pine, however, the opposite pattern was observed: across all treatments, higher increments occurred in substrates amended with fertilizer plus bacteria. For both species, height increments did not differ significantly during the first growing season, whereas in the second season, differences became more pronounced, reflecting treatment-specific growth responses.

Second-year seedling heights exceeded first-year values across all treatments and both species (Figure 3). In Norway spruce, the greatest second-year mean heights were recorded in cattle manure treatments (with and without bacteria) and in poultry manure + bacteria (≈50–55 cm), whereas pig manure + bacteria produced the lowest means (≈44–45 cm). The control treatment was intermediate (≈44 cm).

In Scots pine, absolute heights were lower than in spruce, with second-year means clustering around 29–34 cm. The tallest seedlings occurred in the control, cattle manure, and pig manure + bacteria treatments, while pig manure alone yielded the lowest values.

The bacterial inoculant showed variable effects across treatments: it was beneficial in combination with poultry manure for spruce and with pig manure for pine, but neutral to slightly negative in other cases. These patterns were reflected in overlapping error bars (p > 0.05), indicating modest treatment-specific responses.

It should be noted that cattle manure contained viable clover (Trifolium spp.) seeds, leading to spontaneous clover germination in those treatments across both container types. This represents a potential confounding factor, particularly in the vegetation vessel setups. To minimize bias from volunteer clover, emerging Trifolium seedlings were manually removed during routine maintenance of the vegetation vessels. However, complete elimination could not be guaranteed, and transient competition for light and nutrients may have affected local substrate conditions.

3.5. The Effect of Fertilizers on the Growth of Garden Pansies

The pansy cultivar ‘Yellow with Blotch’ was more responsive to organic fertilizer treatments than ‘Beacon Rose’. Relative to the control, the application of pig manure fertilizer increased the leaf projection area of ‘Yellow with Blotch’ by an average of 35%, while cattle manure fertilizer produced a 24% increase. This stronger response in ‘Yellow with Blotch’ may be attributed to its slightly later developmental pattern, allowing for more gradual nutrient uptake.

For ‘Beacon Rose’, pig manure fertilizer produced only a modest, statistically insignificant increase of 13% (p > 0.05) compared with the control. In contrast, poultry manure fertilizer reduced leaf projection area substantially in both cultivars—by 72% in ‘Beacon Rose’ and 53% in ‘Yellow with Blotch’ (Figure 5).

The addition of the bacterial inoculant was most effective when combined with cattle manure. In both cultivars, bacterial supplementation with cattle manure resulted in an average 20% increase in leaf projection area (p < 0.05) (Figure 5).

3.6. The Effect of Fertilizers on Soil Microbiological Activity

Soil microbiological activity was evaluated by basal respiration rate (Figure 6) and fluorescein diacetate (FDA) hydrolysis intensity (Figure 7). The cultivated plant species exerted a strong influence on microbial activity within the substrate. In mineral fertilizer treatments, soil respiration rates ranged from 5.0 mg CO₂ per 100 g soil per 24 h in garden bean to 9.1 mg CO₂ per 100 g soil per 24 h in basil. FDA hydrolysis activity varied between 174.9 μg fluorescein/(g h) (cucumber) and 267.1 μg fluorescein/(g h) (garden bean). No statistically significant Pearson correlation was detected between respiration rate and FDA activity (p > 0.05).

The addition of bacterial inoculant to mineral fertilizer significantly reduced respiration rate (p < 0.05) in substrates where leaf radish, cucumber, and basil were cultivated, as well as in pig manure + leaf radish treatments. In contrast, bacterial addition increased respiration in pig manure treatments with cucumber, basil, or garden bean.

The inoculant also had a significant effect (p < 0.05) on FDA hydrolysis activity in all five plant substrates supplemented with pig manure. In these treatments, FDA activity was increased in leaf radish and barley, whereas it was decreased in cucumber, basil, and garden bean.

Patterns of hydrolytic enzyme activity, as measured by FDA hydrolysis, generally paralleled those observed in respiration rate but also showed treatment-specific differences (Figure 7). Compared with the control variants (K and KB), higher FDA hydrolysis intensity was observed in substrates amended with cattle manure where radish was grown, and in pig manure treatments where cucumber and basil were cultivated. The addition of bacterial inoculant increased FDA activity only in selected treatments—namely, pig manure with radish and barley, and poultry manure with radish. In contrast, bacterial addition reduced FDA hydrolysis activity in basil treatments across the control, cattle manure, and pig manure variants.

4. Discussion

4.1. Microbiological Evaluation

Moderately decomposed transitional woody–herbaceous peat forms in transitional mires and contains both herbaceous and woody plant remains. At this stage of decomposition, the peat is partly humified: individual plant fragments remain identifiable, but much of the material is amorphous and dark in colour. Such peat typically exhibits moderate porosity, improved nutrient retention compared with less decomposed peat, and suitability for agricultural use and soil improvement. Peat-based substrates and fertilizers are further characterized by high cation exchange capacity, enabling efficient storage and gradual release of nutrients [23]. In addition, peat contains minimal amounts of pathogens and weed seeds, providing a homogeneous and biologically clean substrate. These properties are particularly important in professional horticulture, where uniform and controlled plant growth is required [24].

Composting is an important process for ensuring the microbiological safety of livestock manure, as it effectively reduces the abundance of pathogens and fecal indicator bacteria, though efficiency varies with manure type and composting conditions [25]. Time and temperature are the principal factors influencing pathogen inactivation, and according to EU standards, E. coli and enterococci levels in organic fertilizers must not exceed 1000 cfu/g [22]. In our study, raw poultry and cattle manure contained high levels of both E. coli (10⁵–10⁹ cfu/g) and enterococci (10⁶–10⁸ cfu/g), whereas pig manure contained enterococci (10⁵ cfu/g) but no detectable E. coli. Total plate count represents the overall number of culturable heterotrophic bacteria grown on R2A agar and includes the number of E. coli and enterococci. In our study, the total count was always significantly higher than the number of E. coli and enterococci. Fungal colonies were absent from all manure types.

Microbial community composition varied across manure sources: poultry manures were dominated by Actinomycetota, pig manure by Pseudomonadota, and cattle manure by a mixture of Actinomycetota, Pseudomonadota, and Bacteroidota. Consistency of microbial communities across different batches of manure and peat is another important consideration. While the present study analyzed representative samples from each raw material, batch-to-batch variation is inevitable due to differences in livestock diet, storage conditions, and peat extraction sites. Therefore, the microbial composition reported here should be interpreted as indicative rather than universally representative. Future studies should assess variability across multiple batches to determine the stability and reproducibility of microbial inputs in organic fertilizer production.

It is also important to acknowledge the potential risks of manure application, as highlighted in the Introduction, where pathogens and antibiotic-resistant bacteria are noted as possible contaminants. In this study, these concerns were partly addressed through composting of raw manure prior to fertilizer formulation, which significantly reduced Escherichia coli and enterococci levels, and by monitoring microbiological composition of fertilizers after storage (Table 3 and Table 4). The data obtained show that ash and P. megaterium are two factors whose addition accelerates the reduction in enterococci in fertilizers containing manure. Nevertheless, residual microbial risks cannot be excluded, and future work should explicitly assess the fate of resistance genes and pathogenic taxa during composting and after field application. Such an approach would strengthen confidence in the safety of manure-based biofertilizers.

Similar to the effects of soil pH on microbial communities [26], pH shifts during composting or after mineral amendments influenced the microbial composition of the fertilizers. The addition of ash elevated substrate pH and reduced bacterial and fungal abundance (Table 3), while P. megaterium inoculation counteracted this effect by increasing fungal populations, though it had limited impact on bacterial total counts. During storage, both ash and bacterial inoculants contributed to reductions in enterococci.

Indigenous microorganisms present in manure are widely utilized in organic, and particularly in biodynamic, farming systems. In biodynamic preparations based on cow dung, Bacillus spp. were predominant and promoted maize growth, nutrient solubilization, and pathogen suppression [27]. Cattle manure-based fertilizer may offer added value for greening areas due to the presence of viable clover (Trifolium spp.) seeds. Spontaneously emerging clover can fix atmospheric nitrogen in the long term, thus improving soil fertility. Although initial competition with target species may occur, this is usually short-lived, and senescing clover biomass enriches the soil with nitrogen and organic matter. In future experiments, critical variants should be repeated using cattle manure confirmed to be free of viable clover seeds to rule out this confounding effect.

These findings align with literature showing that livestock manure amendments not only enhance soil fertility but also restructure microbial communities. For example, ref. [28] demonstrated that cattle manure increased nutrient uptake in oats, stimulated root growth, enriched beneficial bacterial groups (e.g., Proteobacteria, Bacteroidota, and Firmicutes), and suppressed pathogenic fungi such as Alternaria and Fusarium. Likewise, cattle manure has been shown to increase bacterial diversity and regulate community composition in tea plantations compared with mineral fertilizers [29].

Priestia megaterium was added after four months of composting. However, complete data on its survival kinetics during storage are not available. Observation of Bacillus-like colonies after storage suggest that spore-forming bacteria, possibly including P. megaterium, remained viable; however, species-level confirmation was not performed, and this observation should be interpreted with caution (Table 3). This was expected given the durability of bacterial endospores [30]. No data are available on its colonization ability in the plant rhizosphere, or its potential interactions with indigenous microbial communities. These unknowns limit conclusions about the functional contribution of the inoculant.

4.2. Soil Microbiological Activity

An additional point of consideration is the conflicting responses observed in soil microbiological activity across different plant species. For example, FDA hydrolytic activity increased in cucumber treatments but decreased in basil, despite similar fertilizer amendments (Figure 7). Such opposite trends likely reflect differences in root exudate composition and associated microbial recruitment, as plant species selectively stimulate or suppress microbial groups in the rhizosphere. This aligns with the well-documented ‘soil–plant–microbe feedback’ mechanism [31]. Plant physiological traits and exudates shape microbial community structure, which in turn affects nutrient cycling and plant performance. Including these mechanisms provides a more comprehensive explanation of the observed discrepancies.

Several studies have shown that FDA hydrolysis, unlike CO₂ evolution, is unaffected by disturbances caused by soil amendments, suggesting that the two parameters reflect different aspects of soil microbial activity. The stability of organic matter plays an important role: the degree of stabilization is inversely related to microbial activity and CO₂ release [32,33,34]. Organic fertilizers—particularly pig, cattle, and poultry manure—generally enhanced FDA hydrolysis compared to mineral fertilizer alone, indicating increased microbial activity. This is consistent with previous studies showing that organic amendments stimulate microbial biomass and enzymatic functions due to their rich organic matter and nutrient content [35,36]. For example, soils amended with green manures from Lupinus spp. exhibited FDA hydrolysis rates of up to 0.82 mg fluorescein/(kg h), significantly higher than untreated soils [33].

However, the addition of bacterial inoculants produced mixed effects. In substrates cultivated with leaf radish, cucumber, and basil, inoculation reduced FDA activity when combined with mineral fertilizer, possibly due to microbial competition or shifts in community structure. In contrast, inoculants enhanced enzymatic activity in manure-amended soils with basil and garden bean, suggesting a synergistic interaction between organic matter and introduced microbial strains.

These results support findings from long-term fertilization studies, where microbial diversity and enzymatic activity were influenced by fertilizer type. Microbial composition data showed that fertilization altered soil nutrient status, which was reflected in changes in microbial communities and their enzymatic activities [37].

The absence of a uniform effect across treatments highlights the plant-specific influence on microbial activity. Plant roots strongly affected the intensity of FDA hydrolysis, largely due to differences in root exudate composition and quantity [31]. Root exudates provide substrates and signalling molecules for soil microbes, and their composition varies widely among species, selectively stimulating microbial populations with distinct enzymatic capacities [38,39]. For instance, legumes such as garden bean release nitrogen-rich compounds that promote microbial growth and enzyme production, whereas cucurbits like cucumber may produce exudates that favour less hydrolytically active communities. In this study, garden bean and basil substrates showed the highest FDA activity, suggesting that their root systems supported more active microbial populations.

These findings are consistent with broader research on compost applications. Both vermicompost and conventional compost applied at 40 Mg/ha improved microbial respiration, microbial biomass, and enzymatic activity, with no significant differences between compost types. However, a higher dose of vermicompost (80 Mg/ha) slightly reduced FDA activity, indicating that excessive organic input may not linearly enhance all microbial functions [40]. This underscores the need for balanced fertilization strategies that stimulate microbial activity while maintaining enzymatic efficiency. Overall, FDA hydrolysis proved to be a sensitive indicator of microbial activity and its responsiveness to fertilizer type and microbial inoculation. These findings highlight the importance of tailoring fertilization strategies to specific crop systems to optimize soil biological health.

Our earlier work on cucumbers and basil [2] showed that mineral fertilizers produced the highest yields. Poultry manure resulted in moderate yields, whereas cattle manure reduced cucumber yield substantially (≈42% below the mineral control). For basil, poultry manure proved most effective. In this treatment, and in all basil treatments with manure additives, hydrolytic enzyme activity in soil was lower than in control. Theoretically, FDA hydrolysis is a method for measuring total microbial activity in soils and other environmental samples [32], and the addition of organic amendments is generally expected to increase soil microbial activity [34], thereby releasing more nutrients available for plants. In our experiments, however, soil microbiological activity measurements with basil did not confirm this trend, whereas cucumber experiments did (Figure 7), even though cucumber yield was lowest with cattle and poultry manure [2].

4.3. Plant Growth

The effects of different fertilizers on plant growth varied significantly across species. Poultry manure ± bacteria improved Norway spruce germination, but reduced Scots pine germination in the first year (Table 5). Pig manure ± bacteria enhanced Norway spruce germination, with this effect becoming evident only in the second year. The addition of bacteria had no significant effect in conifer experiments overall. The large differences in germination rates between years may reflect variation in environmental conditions. In 2023, lower germination coincided with cooler spring temperatures and fluctuating humidity, whereas in 2024 more stable conditions prevailed. Although temperature and relative humidity were monitored during experiments, light intensity and photoperiod were not systematically recorded. These factors may have influenced year-to-year variation and should be considered.

Differences were also observed at the cultivar level, as demonstrated in experiments with garden pansies ‘Mega Star Yellow with Blotch’ and ‘Carneval Special Beacon Rose’. In pansy cultivation, poultry manure fertilizer reduced the leaf projection area (Figure 5), most likely due to excessive nitrogen concentrations (Table A1). A lower application rate would therefore be necessary for safe use. Among the tested variants, the only manure type where bacterial supplementation improved pansy growth was cattle manure.

Although organic fertilizers often cannot match the immediate growth effects of mineral fertilizers, they may contribute to more sustainable nutrient cycling in subsequent years [41,42]. The effects of microbial inoculants remain inconsistent: while many bacterial strains exhibit plant growth-promoting traits in vitro, their in vivo effectiveness is often limited [43]. Nevertheless, extensive evidence supports the potential of beneficial microbes to improve nutrient acquisition, stimulate root development, and produce growth-promoting compounds [44].

Taken together, the results indicate that composted manures combined with peat and ash—particularly when supplemented with carefully selected microbial inoculants—hold some promise as sustainable alternatives to conventional fertilizers. However, the variability in plant responses, along with the inconsistent agronomic benefits observed, highlights that practical application remains far from straightforward. Considerable research on plant–microbe interactions and formulation stability will still be required, and without such refinement, widespread replacement of mineral fertilizers may remain unrealistic in the near term. Future studies should include systematic recording of environmental parameters. Providing such data, alongside exact replicate numbers, will strengthen the reproducibility and reliability of the findings. Contrasting outcomes emphasize the extreme species-specificity of responses and highlight that growth suppression was frequently observed alongside isolated positive cases. Therefore, practical application requires careful evaluation of species- and cultivar-specific outcomes.