Effect of Keratin Hydrolysates Obtained from Feather Decomposition by Trichophyton ajelloi on Plant Germination, Growth and Biological Activity of Selected Arable Soils under Model Conditions

Abstract

The use of fertilizers based on organic waste as a result of microbial biodegradation and biotransformation is becoming increasingly common practice in plant cultivation. This is to limit the chemicals used in agriculture and thus protect the environment and consumer health. The aim of this study was to evaluate a hitherto unexplored effect of hydrolysates obtained after biodegradation of chicken feather waste by a soil strain of the keratinolytic fungus Trichophyton ajelloi on germination, early growth, and development of plants, in particular with high sulfur requirements, and to verify, in model conditions, their impact on soil biological activity and the total pool of soil DNA. Undiluted and diluted keratin hydrolysate generally stimulated seed germination as well as root and shoot growth of the Lepidium sativum L. (garden cress) and Brassica napus L. var. napus (oilseed rape) on sandy soil (Cambisol I), loamy soil (Cambisol II), and Chernozem. In the model experiment, in the variants with and without oilseed rape on sandy soil and Chernozem, the introduction of keratin hydrolysate generally increased the total abundance of microorganisms. In sandy soil, feather hydrolysate mostly increased respiratory activity, dehydrogenase activity, and alkaline phosphatase activity by an average of approx. 38% and the total DNA pool by 15% and 23% in the variant with and without plants. The activity of protease and acid phosphatase increased by an average of 4% and 6% only in the variant without oilseed rape. Respiratory and enzymatic activity in Chernozem, in the variants with and without oilseed rape, tended to show a downward trend, despite periodically recorded increases. The total DNA pool has increased by 8% in the oilseed rape variant. Oilseed rape biomass was almost two-fold higher after adding feather hydrolysate to both soils, and its yield was higher by 98% in Chernozem than in sandy soil. The results have demonstrated that keratin hydrolysate can be used as a biofertilizer.

1. Introduction

Unfavorable climate change has been forcing environmental protection measures for years, including limiting the use of chemical fertilizers and plant protection products. Preparation on the basis of organic waste subjected to microbiological biodegradation and biotransformation are an alternative, environmentally friendly and non-threatening form of fertilizers for consumers. Wastes with high potential for reuse include, among others, lignocellulosic waste due to their high organic C content and humus-forming potential, as well as keratin waste with high nitrogen and organic sulfur content [1]. As shown by Brandelli et al. [2], these wastes, especially feather waste, after appropriate processing, become valuable products applied in various industries. Możejko and Bohacz [3] have reported that processed keratin waste is used as a bioproduct with high fertilizer and feed potential, and keratinolytic enzymes of microbial origin find applications in medicine, pharmaceuticals, industry, and bioenergetics. Studies by Bohacz [4], Bohacz and Korniłłowicz–Kowalska [5], Bohacz et al. [6,7], and Możejko and Bohacz [3] have shown that soil fungi of the genera Chrysosporium, Arthroderma, Trichophyton, and the species Aphanoascus keratinophilus, isolated from composts, release soluble proteins, peptides, and amino acids, as well as mineral forms of sulfur and nitrogen during the biodegradation of waste native keratin, which is important from the fertilization perspective. Fertilizers and microbial-based fertilizers produced through microbiological processing are increasingly used in agriculture. For example, composted lignocellulosic waste provides compost with a high fertilizing value which is safe in sanitary and phytosanitary terms [8]. This waste is used as an ecofriendly biosorbent for the application of heavy metal ion sorption and processed in energy recovery and ethanol production processes [9].

Despite the progressive development of waste management, including waste of animal origin, methods of applying and testing the effectiveness of fertilizers produced by microbiological biodegradation of chicken feather waste remain underdeveloped in practice. Korniłłowicz-Kowalska and Bohacz [1] and Brandelli et al. [2] reported that hydrolysates obtained by enzymatic microbial biodegradation of feather keratin could be useful in agriculture as nitrogen fertilizers or soil modifications. Such fertilizers produced on the basis of microorganisms and/or their metabolites, containing plant extracts and humic substances, are described by many authors as biopreparations, biostimulants, or microbiological vaccines [10]; other authors [11] define them as biofertilizers.

As de Silva [12] has pointed out, biopreparations in the form of protein hydrolysates for agriculture use exert a beneficial effect on soil fertility and act as plant biostimulants. The study of Santi et al. [13] showed that protein hydrolysates stimulated the growth of maize more effectively than mineral nitrogen fertilization. Other authors [14] reported that the treatment of wheat seeds with 5.0% keratin hydrolysate obtained chemically and physically from sheep’s wool increased wheat growth by an average of 15%. Keratin hydrolysates, apart from being introduced into the soil, can also be used for foliar fertilization because they stimulate plant growth and development, as written by Gaidau et al. [15]. These authors have emphasized that a special role in this process is played by the low-molecular-weight protein fractions of the hydrolysate, as well as high free amino acid content. In addition, the action of protein hydrolysates is associated with the supply of nutrients readily available to microorganisms, intensification of their circulation in the soil, and thus stimulation of its biological activity [13,16]. Enriching the soil with mineral and organic compounds affects the abundance and diversity of microorganisms inhabiting this environment [17,18], as well as their biochemical activity, including enzymatic and respiratory processes [19,20]. Therefore, this practice can exert both stimulating and limiting effects on biological activity [21].

According to Możejko and Bohacz [3], keratin hydrolysate obtained under optimized Trichophyton ajelloi culture conditions could be considered a mineral–organic biofertilizer because it had a high content of soluble proteins, peptides, and amino acids, as well as ammonium and sulfate ions. As a result, it could be considered as a potential mineral-organic fertilizer, especially in the cultivation of plants with high sulfur requirements.

Global annual production of oil plants is estimated at 550 million Mg [22]. Oilseed rape plays a significant role in this type of production, as it has the largest cultivated acreage in the world second to soybean [23]. Oilseed rape is a plant with high sensitivity to soil sulfur deficiency, which has a direct impact on yields [24]. Groth et al. [22] reported that representatives of the families Brassicaceae and Liliaceae have the highest requirements for this element. Sulfur plays an important role in plant yielding because its deficiency reduces nitrogen uptake by the plant, i.e., the most important macronutrient that determines and promotes plant growth [25]. Sulfur is also important in the plant metabolic processes, as it is part of proteins and enzymes that play a key role in the synthesis of compounds important for plants, including Brassicaceae, such as proteins, lignins, or fatty acids; moreover, in the metabolism of nitrogen compounds and sugars, it affects the amount and quality of fat in oilseed rape, as well as the taste and aroma of some plants [22,26]. In addition, the particularly high demand of Brassicaceae members in relation to sulfur is due to the presence of sulfur compounds in the biomass of these plants, i.e., glutathione (GSH), sulfolipids, glucosinolates (GSL), phytoalexins, and alliins [26]. Adequate supply for oilseed rape and other sulfur-sensitive crops with this element should be the basis of the fertilization plan, as its availability determines the yield and resistance of plants to biotic and abiotic stresses [23,26,27,28].

Keratin hydrolysate obtained by microbiological biodegradation, due to the high content of soluble proteins, peptides, and mineral forms of sulfur and nitrogen, is ideally suited for use as a biofertilizer in plants’ cultivation, especially those with high sulfur demand. There are little data in the literature on the use of keratin hydrolysates, especially of fungal origin, for soil and plant fertilization. Therefore, the aim of the present work, consisting in model experiments, was to evaluate the effect of not previously studied hydrolysates obtained after chicken feather waste biodegradation by the soil strain of the keratinolytic fungus Trichophyton ajelloi on germination, early growth, and development of plants, particularly those with high sulfur demand (Brassica napus L. var. napus), and to study their phytotoxicity and influence on soil biological activity based on microbiological, biochemical, and enzymatic indices.

2. Materials and Methods

2.1. Fungus Strain

A strain of Trichophyton ajelloi (Vanbreus.) was used to obtain feather hydrolysates. The strain, designated as No. XII, was selected from among three fungal strains of this species, isolated by the keratin bait method (chicken feathers) from brown soil with particle size distribution of light loamy sand, classified according to World Reference Base Classification [29] as Cambisol [30]. The strain was selected for its ability to biodegrade chicken feathers after 21 days of static culture of the three tested strains in a liquid medium containing 1.0% feather waste as the only source of C, N, S, and energy [3]. The maximum amounts of ammonium ions and sulfate ions produced as products of feather biodegradation, important from the fertilization point of view, were considered as a selection criterion.

2.2. Production and Chemical Composition of Feather Hydrolysate

In order to obtain keratin hydrolysate, liquid cultures of the test strain No. XII Trichophyton ajelloi were established on a medium with the composition provided in the work of Możejko and Bohacz [3], with an initial pH of 4.5 and the addition of 1.0% feather waste as the only source of carbon, nitrogen, sulfur, and energy. The cultures were carried out at 28 °C under static conditions. The hydrolysate obtained after 21 days of the fungus culture was separated from feather residues and mycelium by filtering using sterile, hydrophilic PVDF syringe filters (pore diameter: 0.22 µm, filter diameter: 33 mm). The resulting fertilizer biopreparation contained 0.03% N, 443 mg kg⁻¹ K, 677.99 µg ml⁻¹ NH₄⁺, and 0.57 mg ml⁻¹ SO₄²⁻.

Detailed characteristics of the keratinolytic potential of Trichophyton ajelloi strain No. XII used in this study and the two other Trichophyton ajelloi strains designated as No. III and No. XIV, as well as the hydrolysates obtained on their basis, were described in the previous article of Możejko and Bohacz [3].

2.3. Determination of Keratin Hydrolysate Phytotoxicity

2.3.1. Soils Used for Phytotoxicity Tests of Feather Hydrolysates

Phytotoxicity of keratin hydrolysates was determined on three soil samples. Soil I and II were classified as Cambisols with particle size distribution of light loamy sand and sandy loam, respectively. These soils in the study of Bohacz et al. [30] were conventionally defined as sandy (Cambisol I) and loamy soil (Cambisol II), respectively. Soil III was classified as Chernozem with the texture of silt loam. Soil samples were collected from the depth of 0–20 cm of the arable layer of agriculture fields of individual farms located in the Lublin Province (southeastern Poland). Cambisol I was characterized by the following chemical parameters: 0.59% organic C, 0.059% N, 102.0 mg kg⁻¹ P, and 80.0 mg kg⁻¹ K; Cambisol II contained 1.02% organic C, 0.107% N, 67.0 mg kg⁻¹ P, and 140.0 mg kg⁻¹ K, and Chernozem contained 3.16% organic C, 0.301% N, 212.0 mg kg⁻¹ P, and 472.0 mg kg⁻¹ K [30]. The exact location of soils and soil sampling method are described in the study of Bohacz et al. [30].

2.3.2. Plants Used for Phytotoxicity Tests of Feather Hydrolysates

The effect of keratin hydrolysate obtained by fungal keratinolysis on the growth, and development of the test plants was carried out using the commercial Phytotoxkit kit (Tigret, Poland).

The phytotoxicity of feather hydrolysate was analyzed against two test plants, i.e., cress (Lepidium sativum L.), also known as garden cress, and oilseed rape (Brassica napus L. var. napus). Lepidium sativum L. was selected for the study because seeds of this plant are used in toxicity tests due to their rapid germination, growth, and high sensitivity to toxic metabolites [31]. Both plants belong to Brassicaceae, i.e., plants with high sulfur requirements [24,26]. The seeds of winter oilseed rape of the variety Hugo were derived from an individual farm in southeastern Poland, while garden cress seeds were attached to the Phytotoxkit kit as a reference material (Tigret, Poland).

2.3.3. Phytotoxicity Determination

Air-dry soil samples in the amount of 100.0 g were applied on prepared phytotoxicity test plates and moistened with undiluted and 3-times diluted hydrolysate to 70% whc. Plates with soil moistened with distilled water to the same moisture level were used as controls. The moistened soils were spread evenly over the entire surface of the test plates to obtain a soil layer of uniform thickness, and a paper filter was placed on the surface of the soaked soil (control and experimental). Subsequently 10 seeds of both tested test plants were laid out, i.e., garden cress (Lepidium sativum L.) and oilseed rape (Brassica napus L. var. napus). The plates were closed with lids, and the thus prepared phytotests were incubated for 3 days at 25 °C in the dark in type ST 350/350 incubators manufactured by Pol–Eko, Poland.

On the basis of the obtained data, seed germination capacity (G_C), root growth inhibition coefficient (I_R), and plant shoot elongation coefficient (I_S) were calculated according to Czop et al. [32], and the germination index (G_I) according to Carvalho Neves et al. [33] using the following equations:

$${\mathrm{G}}_{\mathrm{C}} (\%)=\frac{{\mathrm{N}}_{\mathrm{G}}}{{\mathrm{N}}_{\mathrm{S}}} \times 100$$

where:

N_G—number of germinated seeds; N_S—number of seeds sown

$${\mathrm{I}}_{\mathrm{R}} (\%)=\frac{{\mathrm{L}}_{\mathrm{C}}-{\mathrm{L}}_{\mathrm{S}}}{{\mathrm{L}}_{\mathrm{C}}} \times 100$$

where:

L_C—average plant root length in control (mm); L_S—average plant root length in the experimental sample (mm)

$${\mathrm{I}}_{\mathrm{S}} (\%)=\frac{{\mathrm{L}}_{\mathrm{C}}-{\mathrm{L}}_{\mathrm{S}}}{{\mathrm{L}}_{\mathrm{C}}} \times 100$$

where:

L_C—average plant shoot length in control (mm); L_S—average plant shoot length in the experimental sample (mm)

$${\mathrm{G}}_{\mathrm{I}} (\%)=\frac{{\mathrm{R}}_{\mathrm{S}}}{{\mathrm{R}}_{\mathrm{C}}} \times \frac{{\mathrm{G}}_{\mathrm{S}}}{{\mathrm{G}}_{\mathrm{C}}} \times 100$$

where:

R_S—average root length in sample (mm); R_C—average root length in control (mm); G_S—average number of germinated seeds in sample; G_C—average number of germinated seeds in control.

The G_I value exceeding 80% [34] indicates the absence of phytotoxic effect of organic fertilizers, especially composts, on plant growth and development.

The mean root to shoot length ratio was also calculated using the formula of Czop et al. [32]:

$$\frac{{\mathrm{L}}_{\mathrm{R}}}{{\mathrm{L}}_{\mathrm{S}}} ~ 1$$

where:

L_R—average root length (mm); L_S—average shoot length (mm).

This ratio is an indicator of the influence of the substrate on the disproportions in plant development. It should have a value of approx. 1, which indicates that the root and shoot grow proportionally to each other and reach equal value [32].

2.4. Determination of the Effect of Keratin Hydrolysates on Biological Activity of Soils and Plant Growth

2.4.1. Soils Used in the Study

In order to investigate the effect of keratin hydrolysates on soil biological activity and plant growth, a pot experiment was set up on two soils, i.e., sandy soil (Cambisol I) and Chernozem. Soils were selected based on differences in chemical properties and phytotoxicity results.

2.4.2. Experimental Variants, Test Plant, and Experimental Conditions

Oilseed rape Brassica napus L. var. napus was selected to study the effects of feather hydrolysates on microbiological, biochemical, and enzymatic soil parameters and plant growth.

The experimental model included four experimental variants for each of the two tested soils, in triplicate:

• Variant I (control 1): soil, water;
• Variant II (control 2): soil, water, oilseed rape;
• Variant III: soil, keratin hydrolysate;
• Variant IV: soil, keratin hydrolysate, oilseed rape.

The experiment was carried out for 60 days in a Biogenet FD147 Inox growth chamber in pots with an area of 64 cm². Each pot contained 400 g of soil on which 25 oilseed rape seeds were placed. The plants were maintained at 23.0 °C during the day and 17.5 °C at night with 85.0% air humidity. The set photoperiod at that time was 15 h light/9 h dark.

Periodically, the soils in each of the variants were moistened to 60% of the total water capacity with a water hydrolysate solution or only with water (controls).

The total amount of ammonium N and sulfate S introduced into the studied soils was calculated based on the concentration of ammonium and sulfate ions in the resulting hydrolysate. A total of 1095.2 × 10⁻⁷ kg of N—ammonium and 395.2 × 10⁻⁷ kg S—sulfate was introduced per pot of sandy soil during the experiment, which was equivalent to 171 kg ha⁻¹ N and 62 kg ha⁻¹ S. To Chernozem, a total of 1158.4 × 10⁻⁷ kg of N—ammonium and 418 ×10⁻⁷ kg of S—sulfate was introduced, and it corresponded to 183 kg ha⁻¹ N and 65 kg ha⁻¹ S.

Periodically, i.e., after 3, 30 and 60 days, the biological activity of the soil in the experimental combinations was determined based on the microbiological, enzymatic, and biochemical indicators, as well as the content of the total dsDNA pool in soil samples. The biomass of the tested plant was also determined.

2.4.3. Abundance of Soil Microorganisms

Determination of the number of microorganisms was carried out by plating decimal dilutions of the tested soil samples using standard methods commonly applied in soil microbiology. The total number of bacteria was determined on a soil extract medium (30.0% of the final medium volume) with the following composition [g dm⁻³]: yeast extract (1.0), K₂HPO₄ (1.3), KH₂PO₄ (1.0), KNO₃ (0.5), agar (20.0), and pH = 7.0. The total abundance of soil fungi was determined on Martin medium [35] containing [g dm⁻³]: glucose (10.0), peptone (5.0), KH₂PO₄ (1.0), MgSO₄ 7H₂O (0.5), and agar (20.0); [mg dm⁻³]: rose bengal (33.3), streptomycin (30.0), and chlortetracycline (2.0). The abundance of proteolytic microorganisms was determined on Frazier’s medium with gelatin as the main N source with the following composition [g dm⁻³]: NaCl (3.0), K₂HPO₄ (1.5), KH₂PO₄ (0.5), gelatin (6.0), glucose (0.05), peptone (0.1), agar (15.0), broth (1 cm³), and pH = 7.0. The number of cellulolytic bacteria and fungi was determined using cellulose filter paper (Whatman 1) as the only source of C and energy. The mineral medium for cellulolytic bacteria contained [g dm⁻³]: KNO₃ (1.0), K₂HPO₄ (0.5), MgSO₄ 7H₂O (0.2), NaCl (0.2), CaCO₃ (5.0), agar (15.0), trace amount of MnSO₄, FeSO₄ 7H₂O, and (NH₄)₆Mo₇O₂₄, and pH = 7.0. Cellulolytic fungi were cultured on a medium with the following salt composition [g dm⁻³]: NH₄NO₃ (2.0), KNO₃ (1.0), KH₂PO₄ (1.0), KCl (0.5), MgSO₄ (0.5), FeSO₄ (0.01), agar (15.0), trace amount of CuSO₄ and NaCl, and pH = 5.5.

Microbial cultures were conducted in Petri dishes in type ST 350/350 incubators (Pol–Eko, Poland). Cultures for bacteria were carried out at 28 °C for 3 days, and for fungi at 26 °C for 5 days. After this time, the grown colonies were counted using a colony counter (LKB 2002, Pol–Eko, Poland). Proteolytic microorganisms were counted after pouring Frazier’s reagent (HgCl₂ −15 g, HCl_con. −20 cm³, H₂O_dest. −100 cm³) over the medium with grown colonies. Only those colonies that were surrounded by a clear zone of degraded protein were counted.

In all cases, microbial count determinations were performed in triplicate. The results are given in cfu kg⁻¹ d. m. of soil.

2.4.4. Respiratory and Soil Enzyme Activity

Respiratory activity was determined according to the methodology of Rühling and Tyler [36] The obtained results were converted into mg CO₂ kg⁻¹ d.m. soil 24 h⁻¹. Dehydrogenase activity was determined using the method of Thalmann [37] with 1.0% 2,3,5 triphenyltetrazolium chloride (TTC) as a substrate dissolved in Tris–HCl buffer at pH 7.4. TPF (triphenylformazan) was measured spectrophotometrically (RayLeigh UV–VIS–1800 spectrophotometer, China) at 485 nm. The results are given in mg TPF kg⁻¹ d.m. of soil 24 h⁻¹. Acid and alkaline phosphatase activity was measured against a 0.1% solution of PNPNa (4–nitrophenyl phosphate disodium salt hexahydrate) in a modified universal buffer at pH 6.5 and 11.0, respectively, at 400 nm [38]. The results are given in mmol PNP kg⁻¹ d.m. of soil h⁻¹. Protease activity in soil samples was determined spectrophotometrically at 578 nm according to the method of Ladd and Butler [39] using a 1.0% solution of sodium caseinate dissolved in 0.1 M Tris–HCl buffer at pH = 8.1. The results are given in mg of tyrosine kg⁻¹ d.m. of soil h⁻¹.

Soil dry matter determinations were carried out at 105 °C using a balance with a built-in dryer (MAC 50/NH, RADWAG, Radom, Poland).

The soil from each experimental variant (from pot) was sieved through a sieve with a diameter of 2 mm and then weighed in the appropriate amount for each analysis in accordance with the methodology provided.

2.4.5. DNA Concentration Measurements

DNA was isolated from each tested variant from all experimental time points. Isolation of soil DNA was carried out using the commercial GeneMATRIX Series DNA kit (EURX, Gdansk, Poland) and followed the attached procedure, and dsDNA concentration was measured using a UV–VIS NanoDrop One spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).

According to the literature data [40], genetic material with the A₂₆₀/A₂₈₀ ratio between 1.80 and 2.00 was assumed as pure DNA of soil samples.

2.4.6. Oilseed Rape Biomass Determination

As one of the parameters determining the effect of soil bio-fertilization on the growth and development of oilseed rape, the yield of fresh oilseed rape biomass was periodically determined by weight (g), i.e., after 3, 14, 21, and 30 days. For this purpose, the plant biomass was separated from the soil by shaking the roots and then weighed (PS 210/C/2, RADWAG, Radom, Poland).

2.4.7. Determination of Soil Chemical Parameters

The pH of the tested soil samples was measured potentiometrically in 1 M KCl using a pH meter (pH meter CP–504, Elmetron, Zabrze, Poland) on day 3, 30, and 60 of the experiment.

Soil total organic C content (TOC) was determined on a non-dispersive NDIR infrared gas analyzer (TOC–VCSH Analyzer, Shimadzu, Kyoto, Japan). Total nitrogen (N) content was analyzed by the Kjeldahl method, after digestion at 420 °C in an automatic digester (Tecator DigestorTM Auto 2520, FOSS, Denmark), followed by distillation and titration in an analyzer (KjelROC Analyzer, OPSIS LiquidLINE, Kävlinge Municipality, Sweden). A spectrophotometric method of determining the intensity of blue color of phosphate–molybdenum (UV–1800 Spectrophotometer, Shimadzu Corporation, Kyoto, Japan) was used to determine the content of bioavailable phosphorus (P). Potassium content was measured using flame atomization atomic absorption spectrometry (FAAS) (SpektrAA 280FS with SPS–3 autosampler and SIPS diluter, Varian, Australia). Determinations of TOC, N, P, and K contents were carried out in all experimental variants at the beginning, i.e., day 3, and at the end, i.e., after 60 days of the experiment.

2.5. Statistical Analysis

The results were statistically analyzed using Statistica ver. 13.3 software (StatSoft, Kraków, Poland). A multivariate analysis of variance (ANOVA) was carried out at the α = 0.05 level of significance to determine significant differences between the microbiological, biochemical, and enzymatic parameters of soil biological activity, as well as differences in plant biomass in different experimental variants over the course of the experiment. In addition, to capture the interrelationships between the measured indicators and chemical parameters in the experimental variants, r–Pearson’s linear correlation coefficients were calculated at the significance levels of α = 0.05, 0.01, and 0.001.

Principal component analysis (PCA) was performed to demonstrate the relationship between the microbiological, biochemical, and enzymatic indicators and their changes depending on the experimental time point. The results of the analysis are presented on the score (dots) and loading (vectors) plots that should be interpreted simultaneously [41]. The loading plot shows the effect of variables, i.e., number of bacteria, fungi, proteolytic microorganisms, number of cellulolytic bacteria and fungi, respiratory activity, dehydrogenase activity, protease activity and acid and alkaline phosphatase activity on individual principal components and reveals the correlation structure against the variables. The score plot is a map of observation and presents experimental variants and analysis dates. If the observation on the score plot (individual optimized parameters and experimental variants) is in the corresponding part on the loading plot, the greater the influence of the variable on the observation.

3. Results

3.1. Phytotoxicity

Based on the seed germination capacity (G_C) of the test plants Lepidium sativum L. (garden cress) and Brassica napus L. var. napus (oilseed rape), it was found that both undiluted and diluted keratin hydrolysate stimulated seed germination (

Table 1. Phytotoxicity of feather hydrolysates in soils with different physico-chemical properties.

Soil	Plant	[%]				R:S
		GC	IR	IS	GI
	Feather hydrolysate
Cambisol I	Brassica napus L. var. napus	100.00	48.66	42.11	51.34	3.15
	Lepidium sativum L.	100.00	41.82	21.76	58.18	1.47
Chernozem	Brassica napus var. napus	100.00	17.78	21.94	82.22	2.71
	Lepidium sativum L.	100.00	−7.63	−27.81	107.63	1.71
	Feather hydrolysate dilluted 1:2
Cambisol I	Brassica napus L. var. napus	100.00	−1.63	−59.47	101.63	2.26
	Lepidium sativum L.	100.00	17.76	17.13	82.24	1.97
Cambisol II	Brassica napus L. var. napus	100.00	10.92	−2.19	89.08	2.71
	Lepidium sativum L.	100.00	−2.02	−26.25	102.02	1.38
Chernozem	Brassica napus L. var. napus	100.00	−26.55	−70.22	126.55	1.91
	Lepidium sativum L.	100.00	−13.95	−13.90	113.95	2.03

Explanations: G_C—seed germination capacity; I_R—root growth inhibition coefficient; I_S—shoot elongation inhibition coefficient; G_I—germination index; R:S—root to shoot length ratio.

). In contrast, an inhibitory effect occurred on root growth (I_R) and shoot elongation (I_S) of oilseed rape and garden cress under the influence of undiluted keratin hydrolysate introduced into sandy soil (Cambisol I). Oilseed rape turned out to be more sensitive plant, which was expressed by 16% higher inhibition of root growth and 94% higher shoot elongation inhibition of this plant compared to garden cress. In addition, the undiluted hydrolysate more strongly inhibited the shoot growth than the root of both plants tested. On the other hand, the introduction of undiluted keratin hydrolysate into Chernozem inhibited root and shoot growth of oilseed rape but not garden cress. However, compared to sandy soil, the inhibition of oilseed rape root and shoot growth on Chernozem was two times weaker (Table 1).

The triple-diluted hydrolysate introduced into all three soils: sandy soil (Cambisol I), loamy soil (Cambisol II), and Chernozem stimulated both root and shoot growth of Brassica napus L. var. napus except for root growth in loamy soil (Cambisol II) (Table 1). With regard to Lepidium sativum L., stimulation of the analyzed plant growth indices was also observed under the influence of diluted hydrolysate, except for sandy soil. The data in Table 1 show that the stimulation of oilseed rape root and shoot growth on Chernozem was 1529% and 18% higher, respectively, compared to sandy soil. In contrast, inhibition of oilseed rape root growth was recorded in loamy soil (Cambisol II).

The analyses demonstrated that the root/shoot length ratio in all experimental variants reached values above the optimum, i.e., about 1, which indicated that root growth was faster within 3 days (Table 1).

Overall, a diluted (1:2) fertilizer biopreparation had a more favorable effect on the growth and development of the tested plants. Stronger stimulation of plant growth and development after hydrolysate application was observed for rapeseed (Brassica napus L. var. napus) than for garden cress (Lepidium sativum L.) as determined by higher G_I values (Table 1).

3.2. Abundance of Soil Microorganisms

The data in Figure 1 show that the total number of bacteria in the studied soils, i.e., sandy soil (Cambisol I) and Chernozem, increased with the duration of the experiment. This effect was observed both in the control variants irrigated with distilled water and the variants with the addition of hydrolysate with and without plants. The highest significant increase (by 714%) of the abundance of this group of microorganisms in relation to the initial date was recorded after the introduction of the hydrolysate into sandy soil without oilseed rape plants (Figure 1, Table S1). The lowest, but not statistically significant (α = 0.05), increase in number of bacteria (by 27%) was recorded in sandy soil irrigated with water with the growth of the test plant (control 2). Compared to both control variants on sandy soil, the introduction of the hydrolysate stimulated bacterial growth by an average of 103% in the variant without plants and by 26% in the variant with plants (Figure 1, Table S1).

The total abundance of soil bacteria in Chernozem was significantly higher throughout the experiment and in all experimental variants compared to sandy soil. With respect to water-moistened control variants, the hydrolysate introduced into the soil increased bacterial abundance by 19% in the system with oilseed rape, while it had the opposite effect without oilseed rape plants, i.e., a decrease in the number of bacteria by 7% (Figure 1, Table S1).

In conclusion, it was found that bacterial abundance in both sandy soil and Chernozem was higher after hydrolysate application, regardless of the presence of the plant, except for Chernozem without plants (Figure 1, Table S1).

The introduction of keratin hydrolysate also had an effect on soil fungal populations in both sandy soil and Chernozem. In the first month of the experiment, there was a decrease in total fungal abundance in the control variants not fertilized with keratin hydrolysate, with the exception of the variant with plants and sandy soil (Figure 1). However, these changes were not statistically significant (α = 0.05). After this date, a further decrease in fungal abundance in the control variants without oilseed rape planting and an increase in abundance in the variants with oilseed rape in sandy soil (9%) and Chernozem (31%) were observed. The addition of the hydrolysate to sandy soil in the long term resulted in a 32% decrease in fungal abundance in the variant with plants and an 11% decrease in the variant without them (Figure 1, Table S1). In Chernozem, total fungal abundance was on average 10% lower compared to sandy soil in the control and hydrolysate-fertilized variants (Table S1). The data obtained indicated that keratin hydrolysate in both soils exerted a significantly higher and positive effect on fungal populations in the variants without plant growth. This corresponded to an increase in the abundance at an average of 22% and 31% in Cambisol I and Chernozem, respectively, (Figure 1, Table S1).

Due to the high content of organic nitrogen compounds in keratin hydrolysates, it was justified to study the dynamics of changes in the abundance of proteolytic microorganisms. The data in Figure 1 show that the number of proteolytic microorganisms in sandy soil increased in all experimental variants until day 60 of the experiment, except for the variant with the addition of the hydrolysate and plant growth. The highest significant increase (by 282%) in the number of this group of microorganisms in relation to the initial time point was recorded after the introduction of the hydrolysate into sandy soil without plants (Figure 1, Table S1). In the variant with oilseed rape plants, the abundance of proteolytic microorganisms decreased non-significantly after day 30 of the experiment (Figure 1).

In Chernozem, the abundance of proteolytic microorganisms increased until day 30 of the experiment. After this time, it decreased in all experimental variants established on this soil. The highest decrease in the abundance of these microorganisms was recorded in the control variants without hydrolysate addition and with plants (68%) and a smaller decrease (9%) in the variant without plants (Figure 1, Table S1). Overall, keratin hydrolysate introduced into Chernozem reduced the growth of proteolytic microorganisms in both unfertilized and oilseed rape-planted variants (by 6% and 38%, respectively) relative to the unfertilized variants (Figure 1, Table S1).

On the other hand, the duration of the experiment had a positive effect on the abundance of cellulolytic bacteria colonizing the tested soils (Figure 1). A statistically significant (α = 0.05) increase in the abundance of this group of microorganisms over time was recorded for all experimental variants tested. The only exception was Cambisol I enriched with the hydrolysate, in which, after 30 days of the experiment, a significant decrease (62%) in the number of cellulolytic bacteria was recorded in the variant without oilseed rape plants. A non-significant decrease (32%) in the number of these bacteria was recorded in the variant with oilseed rape (Figure 1, Table S1). The abundance of cellulolytic bacteria generally reached higher levels in the variants where plants were included. The highest statistically significant increase of 803% in the abundance of this group of bacteria during the course of the experiment was recorded in Chernozem with oilseed rape plants and hydrolysate addition (Figure 1, Table S1). It was also found that the introduction of keratin hydrolysate had a more beneficial effect on cellulolytic bacteria inhabiting Chernozem than Cambisol I. In this soil, the number of these bacteria was significantly higher (by 171%) in the variant without oilseed rape, as well as with these plants (150%) (Figure 1, Table S1).

The abundance of cellulolytic fungi increased in all experimental variants of both tested soils (Figure 1). However, a statistically significant (α = 0.05) increase in the abundance of this group of fungi at the final stage of the experiment (182%; α = 0.05) was found only in sandy soil with oilseed rape and the absence of fertilization with hydrolysate (Table S1). After adding the hydrolysate to sandy soil, the abundance of this group of microorganisms decreased slightly in the variant with oilseed rape and increased slightly in the variant without these plants (Figure 1). With respect to Chernozem with hydrolysate treatment, the abundance of cellulolytic fungi increased compared to control samples 1 and 2 by 12% in the variant without plants and by 55% in the variant with plants (Figure 1, Table S1).

Comparing the effect of keratin hydrolysate on the two soils, it was found that it was more effective in stimulating the abundance of cellulolytic fungi in sandy soil than in Chernozem (Figure 1, Table S1).

3.3. Respiratory and Enzymatic Activity

The data in Figure 2 indicate that the amount of CO₂ released from both tested soils after 60 days of the experiment was higher compared to the initial date. Statistically significant (α = 0.05) increases in respiratory activity in sandy soil (Cambisol I), relative to the initial date of analysis, were recorded in the variants with oilseed rape without keratin hydrolysate and with the hydrolysate but without plants (433% and 75%, respectively) (Figure 2, Table S2).

The amount of CO₂ released in this soil on day 60 of the experiment was significantly the highest in the variant with the hydrolysate without plants. In Chernozem, significantly higher CO₂ release, compared to the initial time point, was recorded in the variant without rapeseed planting, irrigated with distilled water, and with keratin hydrolysate (by 108% and 204%, respectively) (Figure 2, Table S2). A slight decrease (21%) in carbon dioxide release between the first and last date of analysis was found only in the case of water-irrigated Chernozem with the test plant growth (Figure 2, Table S2). Overall, the enrichment of sandy soil with microbiologically processed feathers resulted in a visible increase in respiratory activity in the variants without oilseed rape (averaging 97% and 37%, respectively) (Table S2). On the other hand, a decrease in carbon dioxide emission was observed in Chernozem after enrichment with this keratin biofertilizer. It amounted to an average of 34% in the soil without oilseed rape and 11% in the soil with oilseed rape growth (Figure 2, Table S2). In addition, based on the average values for the entire study period, it was noted that respiratory activity, regardless of the experimental variant, was higher in Chernozem than in sandy soil. The exception was the variant with the addition of the hydrolysate without Brassica napus L. var. napus (Figure 2, Table S2).

The activity of dehydrogenases up to day 60 of the experiment gradually and statistically significantly (α = 0.05) decreased in all variants. The only but insignificant increase in the activity of this enzyme was recorded between days 30 and 60 of analyses in sandy soil with the hydrolysate and plants, as well as Chernozem moistened with water and planted (Figure 2). The decrease in the activity of these enzymes, observed in the course of the study, was stronger in Chernozem than in sandy soil. In sandy soil, the highest (96%) decrease in dehydrogenase activity was recorded in the control not fertilized with the hydrolysate and planted with oilseed rape (control 2) (Figure 2, Table S2). On the other hand, the lowest decrease in dehydrogenase activity in this soil was observed in the control moistened with water (control 1). The introduction of keratin hydrolysate to the discussed soil generally stimulated the activity of dehydrogenases by 2% in the variant without plants and by 27% with plant population (Table S2). In Chernozem, the highest decrease in dehydrogenase activity within 60 days of the experiment, amounting to 89%, was observed in the experimental variant enriched with feather hydrolysate and with plant population (Figure 2, Table S2). The lowest decrease in dehydrogenase activity, i.e., 54%, was found in Chernozem not fertilized with the hydrolysate, with oilseed rape plants (Table S2). The introduction of the hydrolysate into this soil resulted in a 3% decrease in dehydrogenase activity in the variant without oilseed rape and an 11% decrease with this plant (Table S2). Overall, it was found that dehydrogenase activity was significantly higher in Chernozem than in Cambisol I. The experiment further showed that keratin hydrolysate affected dehydrogenase activity similarly to respiratory activity; i.e., it had a stimulating effect in sandy soil, while it reduced the activity of this enzyme in Chernozem, as determined based on the mean values of this enzyme activity (Table S2, Figure 2).

During the experiment, protease activity under the influence of hydrolysate in both sandy soil and Chernozem tended to decrease (Figure 2). This was indicated by the lower values of this parameter after 60 days of the experiment in relation to the initial date. The exception was the variant with Chernozem with oilseed rape plants, in which protease activity did not significantly increase. A similar stimulating effect occurred in the control variant (control 1) without the addition of the hydrolysate and without plants (Figure 2). In sandy soil with oilseed rape plants, fertilization with the hydrolysate after 60 days resulted in a significant decrease (43%) in protease activity (Table S2). The activity of this enzyme in Chernozem in both variants fertilized with the hydrolysate was also significantly lower compared to the control variants, despite a transient increase in the activity. This effect became apparent 30 days after the introduction of keratin hydrolysate into the soil with plant growth and amounted to 49% compared to the baseline analysis (Figure 2, Table S2). Overall, the introduction of the hydrolysate into Chernozem without and with rapeseed planting resulted in a significant 21% and 19% reduction in protease activity, respectively, compared to control (Figure 2, Table S2). In sandy soil with oilseed rape, the hydrolysate effect was similar and resulted in a 21% reduction in protease activity compared to control. A beneficial effect of the hydrolysate on protease activity was noted only in the variant without plants, and it amounted to a 4% increase in their average activity (Figure 2, Table S2).

Acid phosphatase activity decreased significantly (α = 0.05) during the first 30 days of the experiment in all experimental variants in both soils tested. The exception was a sandy soil not enriched with the hydrolysate with oilseed rape planting (Figure 2). The highest (38%) significant decrease in acid phosphatase activity after 30 days was recorded in hydrolysate-enriched sandy soil with the test plants (Table S2). Over a longer period of time, i.e., after 60 days, a significant increase in the activity of this enzyme was recorded in all experimental variants on this soil (Cambisol I). Overall, the introduction of keratin hydrolysate into sandy soil resulted in a non-significant increase in acid phosphatase activity relative to the control variant (control 1). A decrease in the activity of these enzymes was recorded in the variant with plants compared to control (control 2) (Figure 2). With respect to Chernozem, the decrease in acid phosphatase activity in all variants between days 3 and 30 of the experiment was higher than in sandy soil. The lowest decrease of only 12% in the activity of the tested enzyme was determined in unfertilized soil without plants (Figure 2, Table S2). On the other hand, the highest significant decrease (i.e., 19%) in the analyzed index at that time point was found for Chernozem with plants in the absence and after the application of hydrolysate fertilization (Table S2). Later, the activity of acid phosphatase increased significantly (α = 0.05) in all tested experimental variants, except for sandy soil moistened with distilled water with plants (control 2) (Figure 2). Based on the obtained data, it was found that the introduction of keratin hydrolysate into the soil had a different effect on the activity of acid phosphatase in sandy and Chernozem (Figure 2). The use of the hydrolysate in sandy soil without oilseed rape resulted in a 6% stimulation of acid phosphatase activity in relation to the non-fertilized variant and a decrease in the activity of this enzyme by 3% in the variant with plant growth (Table S2). In contrast, an inverse relationship was noted for Chernozem, as the introduction of this biofertilizer into the soil without plants resulted in a reduction in the activity of the tested enzyme by 7%, while a 7% increase was measured in the soil with oilseed rape (Figure 2, Table S2).

It was found that the activity of alkaline phosphatase in sandy soil generally increased with the duration of the experiment (Figure 2). The exception was the control variant with plants (control 2) for which a 43% decrease in the activity of this enzyme was recorded (Table S2). However, these changes were not statistically significant. The highest increase in alkaline phosphatase activity over time, amounting to 217%, was recorded after introducing keratin hydrolysate into sandy soil without oilseed rape (Figure 2, Table S2). For Chernozem, a significant (α = 0.05) increase in the activity of the enzyme was recorded on day 60 of the experiment in the unfertilized and unplanted variant (control 1). On the other hand, a statistically insignificant increase in alkaline phosphatase activity was observed after the addition of the hydrolysate into Chernozem sown with oilseed rape (Figure 2). For both of the aforementioned variants on Chernozem, there was a 28% and 4% increase, respectively, in the activity of this enzyme over time, relative to the baseline (Table S2). However, a decrease in the activity was observed in the unfertilized variant with oilseed rape and in the variant without this plant but with the addition of keratin hydrolysate (5% and 51%, respectively) (Table S2). The experiment showed that while keratin hydrolysate stimulated alkaline phosphatase activity in sandy soil in the variant without and with oilseed rape relative to unfertilized soil variants (by 12% and 52%, respectively), it reduced the activity of this enzyme in Chernozem in analogous combinations (by 26% and 9%, respectively) (Figure 2, Table S2).

3.4. Total DNA Pool

The data in Figure 3 show that throughout the study period, in both sandy soil and Chernozem, the quantity of dsDNA increased significantly (α = 0.05) in all experimental variants. The exception was the control sandy soil not sown with oilseed rape (control 1), where a non-significant decrease in DNA concentration was recorded (Figure 3). In this variant, the amount of dsDNA increased up to day 30 of the experiment by 21%, after which it decreased on day 60 also by 21% relative to the first analysis time point (Table S3). Overall, i.e., within 60 days of the experiment, dsDNA levels significantly increased (by 137%) in sandy soil enriched with the hydrolysate, especially in the variant with oilseed rape (Figure 3, Table S3). For Chernozem, a significant increase in the total DNA pool during 60 days of the experiment was recorded in all experimental variants. Significantly the highest increase in dsDNA concentration, as in sandy soil, was recorded in the variant with the hydrolysate and plants.

In general, it was observed that keratin hydrolysate was more effective in increasing the concentration of dsDNA in sandy soil than in Chernozem, causing its average, significant increase by 23% and 15% in the variant without and with oilseed rape, respectively, (Table S3). In Chernozem, the addition of the hydrolysate resulted only in an 8% increase in the total dsDNA pool in the variant without the plant and a 9% decrease in the variant with the plant compared to the control variants (Figure 3, Table S3).

3.5. Plant Biomass

The data in

Table 2. Dynamic of changes in Brassica napus L. var. napus biomass.

Soil	Experimental Variants	Days of Measurement
		3 A	14 B	21 C	30 D
Cambisol A	Water A	0.88 ± 0.03 a	2.30 ± 0.02 c	4.81 ± 0.15 e	6.61 ± 0.17 b
	Hydrolysate B	1.13 ± 0.02 a	4.12 ± 0.16 d	7.00 ± 0.18 b	10.00 ± 0.32 f
Chernozem B	Water A	1.42 ± 0.07 a	4.35 ± 0.18 c	8.14 ± 0.22 e	12.65 ± 0.42 b
	Hydrolysate B	2.12 ± 0.03 a	6.59 ± 0.27 d	12.94 ± 0.20 b	19.87 ± 0.80 f

Explanations: Letters, i.e., lowercase (a, b, c, d) or capital (A, B, C, D) denote the means that form homogenous groups (HSD–Tukey post hoc test for multivariate ANOVA). When two compared means were assigned the same letter (e.g., a or A), that means did not differ significantly (at α = 0.05); if means are marked with different letters (e.g., a and b, or A and B), they differ significantly from each other (at α = 0.05).

show that at almost all measurement time points, keratin hydrolysate introduced into sandy soil (Cambisol I) and Chernozem caused a significant (α = 0.05) increase in the biomass of Brassica napus L. var. napus compared to the biomass of this plant in variants without hydrolysate fertilization. The only exception for both soils was the biomass of oilseed rape sprouts obtained 3 days after the application of the biopreparation. In comparison to the control variants, the biomass of oilseed rape was on average significantly higher by 52% in sandy soil and by 56% in Chernozem (Table 2). It was also found that in both tested soils, enriched and those untreated with the hydrolysate, the increment of oilseed rape biomass significantly (α = 0.05) increased over time.

3.6. Chemical Parameters

3.6.1. pH

Introduction of keratin hydrolysate into sandy soil (Cambisol I) within 3 days of setting up the experiment resulted in an increase in pH relative to control samples (control 1 and 2) (

Table 3. Dynamics of changes of soil pH.

Soil	Days	3	30	60	Mean
pH [−log10[H+]]
Cambisol	Water	3.71 ± 0.01	3.67 ± 0.01	3.61 ± 0.01	3.66 ± 0.01 aA
	Water + Plant	3.72 ± 0.01	3.68 ± 0.01	3.57 ± 0.01	3.66 ± 0.01 aA
	Hydrolysate	3.76 ± 0.01	3.81 ± 0.06	3.42 ± 0.01	3.67 ± 0.02 aA
	Hydrolysate + Plant	3.75 ± 0.01	3.78 ± 0.04	3.40 ± 0.01	3.64 ± 0.02 aA
	Water	6.20 ± 0.02	6.21 ± 0.08	6.17 ± 0.04	6.19 ± 0.05 bC
Chernozem	Water + Plant	6.08 ± 0.10	6.21 ± 0.12	6.06 ± 0.07	6.11 ± 0.09 bC
	Hydrolysate	6.15 ± 0.05	5.87 ± 0.07	5.16 ± 0.03	5.72 ± 0.05 aB
	Hydrolysate + Plant	6.31 ± 0.03	5.56 ± 0.26	5.11 ± 0.20	5.66 ± 0.17 aB

Explanations: Letters, i.e., lowercase (a, b) or capital (A, B, C, D) denote the means that form homogenous groups (HSD–Tukey post–hoc test for multivariate ANOVA) within each of the tested soils separately, and within both soils together, respectively. When two compared means were assigned the same letter (e.g., a or A), that means did not differ significantly (at α = 0.05); if means are marked with different letters (e.g.,: a and b, or A and B), they differ significantly from each other (at α = 0.05).

). However, the pH tended to decrease significantly (α = 0.05) over the course of the experiment in both soils tested in the variant without and with keratin hydrolysate. First, i.e., up to day 30, the pH slightly and insignificantly increased in half of the analyzed experimental variants, and then, i.e., after 60 days, it decreased again (Table 3). In sandy soil, this concerned both variants enriched with the hydrolysate, while in Chernozem both control variants. In sandy soil, the highest, i.e., 9% decrease in pH, was found during the experiment after biofertilizer introduction to the soil sown with oilseed rape (Table S4). On the other hand, in Chernozem, 3 days after the introduction of keratin hydrolysate into the soil without plants, a decrease in the reaction was recorded, while its increase was observed in the variant with plants. The most significant, 19% decrease in pH in Chernozem, analogous to sandy soil, was found in soil with oilseed rape fertilized with keratin biofertilizer (Table 3 and Table S4). The same trend was also noted in Chernozem without plant population after the introduction of keratin hydrolysate (Table 3). A significant decrease in pH after the introduction of the biofertilizer under study occurred only in Chernozem with and without plant growth. However, this relationship was not observed in sandy soil (Table 3).

3.6.2. NPK and TOC

The data in

Table 4. Content of total organic carbon (TOC) and N, P, K concentration in two types of soil in different experimental variants.

Soil	Days of Analyses	Sample	TOC (g kg−1)	N (g kg−1)	P (mg kg−1)	K (mg kg−1)
Cambisol	3	Water	5.40	0.20	32.00	348.00
		Water + Plant	6.20	0.60	31.20	485.00
		Hydrolysate	6.05	0.40	43.00	447.00
		Hydrolysate + Plant	5.95	0.40	40.60	397.00
	60	Water	6.20	0.60	29.10	483.00
		Water + Plant	5.87	0.50	44.90	352.00
		Hydrolysate	5.90	0.60	89.10	370.00
		Hydrolysate + Plant	5.73	0.30	106.80	678.00
Chernozem	3	Water	13.57	0.00	17.80	1310.00
		Water + Plant	13.75	1.50	25.40	2980.00
		Hydrolysate	13.07	1.20	33.90	2520.00
		Hydrolysate + Plant	12.97	0.40	27.30	2620.00
	60	Water	14.41	1.10	37.70	2700.00
		Water + Plant	16.41	0.40	37.60	2080.00
		Hydrolysate	14.51	0.40	99.80	2260.00
		Hydrolysate + Plant	13.49	0.50	104.60	3040.00

show differences between the studied soils in organic C (TOC), nitrogen (N), phosphorus (P), and potassium (K) content. In sandy soil (Cambisol I), organic carbon content of the experimental variants generally decreased after 60 days, except for the control variant (control 1). On the other hand, in Chernozem, where the level of this component was higher by 146%, the introduction of keratin hydrolysate tended to reduce organic carbon content in both variants, especially with plants. The highest TOC content in both soils was typically recorded in the variants with plants and without the hydrolysate (Table 4). Total N content in sandy soil, such as TOC, decreased after 60 days of the experiment, in the variants with plants. In Chernozem, this was observed in the variant only with the hydrolysate and control 2. In both soils, the highest total N content was recorded on day 3 of the experiment in the unfertilized combination with plants. On day 60, it was documented in the non-fertilized combination without plants and in sandy soil enriched with the hydrolysate without plants (Table 4). The level of available phosphorus generally increased in all soil variants 60 days after the start of the experiment. Enrichment with the hydrolysate increased the content of this nutrient in both soils compared to the non-enriched variants. On day 3 of the experiment, the highest levels of assimilable phosphorus in these soils were recorded in the variants with the hydrolysate alone and on day 60 in the variants with the hydrolysate and plants (Table 4). The introduction of the hydrolysate also altered potassium levels in the tested soils, as it increased on day 3 in the variants without plants and decreased in the variants with vegetation. After 60 days of the experiment, the opposite trend was noted in the variants with the hydrolysate and plants. Maximum potassium concentrations were recorded at the beginning of the experiment (day 3) in the variants with plants and without hydrolysate fertilization and at the end of the experiment (day 60) in the variant with the hydrolysate and plant growth (Table 4).

3.7. Statistical Analysis

3.7.1. Correlations

To better understand microbial, enzymatic, and chemical transformations and their interrelationships, the results were subjected to Pearson’s correlation analysis after the introduction of keratin hydrolysate into the studied soils differing in chemical parameters (

Table 5. The values of r–Pearson’s correlation coefficients between microbiological, biochemical, and chemical parameters.

Soil	Experimental Variant	Analyzed Parameters	TOC	N	P	K
Cambisol	Water	Soil bacteria	0.598 *	0.666 *	0.025	0.504
		Soil fungi	−0.339	–0.245	0.600 *	−0.594 *
		Proteolytic microorganisms	0.336	0.325	−0.371	0.399
		Cellulolytic bacteria	0.316	0.436	0.486	−0.034
		Cellulolytic fungi	0.047	0.213	0.742 **	−0.296
		Soil respiration	−0.145	−0.010	0.344	−0.265
		Dehydrogenase	−0.265	−0.393	−0.443	0.051
		Protease	0.122	0.306	0.728 **	−0.181
		Acid phosphatase	−0.163	−0.153	−0.426	0.190
		Alkaline phosphatase	0.487	0.426	−0.038	0.337
		pH	−0.180	−0.315	−0.620 *	0.205
		dsDNA	−0.043	0.111	0.941 ***	−0.516
	Hydrolysate	Soil bacteria	−0.159	0.826 ***	0.527	−0.337
		Soil fungi	0.184	0.227	−0.243	−0.370
		Proteolytic microorganisms	0.087	0.855 ***	0.356	−0.439
		Cellulolytic bacteria	−0.720 **	−0.434	0.856 ***	0.868 ***
		Cellulolytic fungi	−0.421	−0.063	0.807 **	0.503
		Soil respiration	−0.185	0.703 *	0.574	−0.153
		Dehydrogenase	0.547	−0.309	−0.944 ***	−0.347
		Protease	0.400	−0.436	−0.720 **	−0.187
		Acid phosphatase	0.148	0.385	–0.258	–0.501
		Alkaline phosphatase	−0.639 *	0.133	0.511	0.197
		pH	0.627 *	−0.183	−0.980 ***	−0.449
		dsDNA	−0.700 *	0.034	0.989 ***	0.585 *
Chernozem	Water	Soil bacteria	0.426	0.117	0.772 **	0.246
		Soil fungi	0.025	−0.086	−0.462	−0.143
		Proteolytic microorganisms	−0.652 *	0.745 **	−0.156	0.624 *
		Cellulolytic bacteria	0.632 *	−0.032	0.869 ***	0.134
		Cellulolytic fungi	0.141	−0.143	0.267	−0.170
		Soil respiration	−0.128	0.438	0.431	0.481
		Dehydrogenase	−0.537	0.053	−0.835 ***	−0.112
		Protease	−0.129	0.893 ***	0.153	0.884 ***
		Acid phosphatase	−0.613 *	0.316	−0.259	0.248
		Alkaline phosphatase	−0.004	0.683 *	0.625 *	0.736 **
		pH	−0.502	−0.237	−0.340	−0.371
		dsDNA	0.837 ***	0.071	0.946 ***	0.273
	Hydrolysate	Soil bacteria	0.535	−0.586 *	0.927 ***	0.281
		Soil fungi	−0.203	–0.387	0.430	0.600 *
		Proteolytic microorganisms	0.555	−0.023	0.381	−0.577 *
		Cellulolytic bacteria	0.449	−0.353	0.885 ***	0.487
		Cellulolytic fungi	0.325	−0.311	0.577 *	−0.049
		Soil respiration	0.566	−0.750 **	0.600 *	−0.316
		Dehydrogenase	−0.664 *	0.368	−0.986 ***	−0.102
		Protease	−0.171	0.263	0.121	0.519
		Acid phosphatase	−0.523	−0.020	−0.012	0.916 ***
		Alkaline phosphatase	−0.749 **	0.328	−0.441	0.695 *
		pH	−0.654 *	0.414	−0.988 ***	−0.143
		dsDNA	0.551	−0.370	0.981 ***	0.273

Explanations: r—Pearson’s correlation coefficients at three levels of significance: * (0.05); ** (0.01); *** (0.001).

). Non-fertilized and fertilized variants, with and without plants, were considered jointly. The conducted analysis generally showed that the keratin hydrolysate introduced into the soils stimulated their biological activity associated with the transformations of TOC_., N, P and K, especially in sandy soil (Cambisol I). This was expressed in an increase in the number of correlations and the significance between the number of microorganisms, enzyme activity, and chemical parameters, i.e., the content of organic C, N, P, and K.

In sandy soil not fertilized with the hydrolysate, bacteria played an important role in TOC and N transformations (Table 5). This was evidenced by a positive, significant correlation between the level of these components and the total bacterial abundance (0.598 *, and 0.666 *, respectively). However, the relationship of total fungi and cellulolytic fungi became apparent in the context of bioavailable P content. Their numbers grew with increasing concentration of this component (0.600 *, and 0.742 **, respectively). Total fungal abundance in sandy soil not enriched with the hydrolysate was also significantly but negatively correlated with potassium content (−0.594 *). Unlike fungi, total bacterial abundance was not correlated with either P or K content (Table 5). The introduction of keratin hydrolysate into sandy soil significantly increased the correlation between the abundance of total soil bacteria (0.826 ***), proteolytic bacteria (0. 855 ***), respiratory activity (0.703 *), and N content. At the same time, a negative correlation between the total DNA pool (−0.700 *), cellulolytic microbial abundance (−0.720 **), alkaline phosphatase (−0.639 *), and TOC content was recorded (Table 5). The level of the latter parameter rose with increasing pH (0.627 *). The introduction of the hydrolysate to sandy soil also increased the correlation between P content and the development of the population of cellulolytic fungi (0.807 **) and bacteria (0.856 ***). The latter relationship was not observed in the variant not enriched with the hydrolysate. An inverse significant correlation was found between the level of P and the activity of dehydrogenase (−0.944 **) and protease (−0.720 **) after sandy soil enrichment with the hydrolysate (Table 5). Moreover, the hydrolysate enhanced significantly the correlations between the content of this component, pH (−0.980 ***), and dsDNA (0.989 ***). It was found that only the number of cellulolytic bacteria (0.868 ***) and dsDNA were significantly positively correlated with potassium content after the addition of the hydrolysate (0.585 *).

In Chernozem, no significant relationships were found after the introduction of the keratin hydrolysate between TOC and the number of microorganisms (Table 5). Only a significantly negative correlation was found between the TOC level and the activity of dehydrogenases (−0.664 *), alkaline phosphatase (−0.749 **), and pH (−0.654 *). In the variant without the addition of keratin hydrolysate, the increase in N content elevated the number of proteolytic microorganisms (0.745 **) and the activity of protease (0.893 ***) and alkaline phosphatase (0.683 *). The introduction of biofertilizer to this soil in relation to N content only showed that the increase in N levels significantly reduced the number of soil bacteria (−0.586 *) and the intensity of soil respiration (−0.750 **). Considering the P content, it was found that Chernozem not enriched with keratin biopreparation was characterized by a significant positive effect on the total number of bacteria (0.772 **), cellulolytic bacteria (0.869 ***), and dsDNA content (0.946 ***). In turn, a negative relationship of this element with dehydrogenase activity was observed (−0.835 ***) (Table 5). All these relationships were strengthened after introducing the feather hydrolysate into Chernozem. In addition, the hydrolysate suppressed the significant positive correlation between P content and alkaline phosphatase activity (α = 0.05) observed in the variant without hydrolysate. However, it showed a positive relationship of this element with the number of cellulolytic fungi (0.577 *) and soil respiration (0.600 *). Potassium in Chernozem not enriched with biofertilizer showed a beneficial effect on the activity of protease (0.884 ***) and alkaline phosphatase (0.736 **). Both relationships were significantly weakened after introducing biofertilizer to the tested soil (Table 5). In the presence of the biofertilizer, a significant positive relationship between K content, the abundance of soil fungi (0.600 *), and acid phosphatase activity (0.916 ***) was observed. Fertilization of Chernozem with processed feathers also resulted in a significant reduction in the number of proteolytic microorganisms in relation to K increase (−0.577 *).

In general, the correlation analysis showed that phosphorus was the element that had the most significant influence on the biochemical and microbiological indicators in unfertilized and fertilized Cambisol I and Chernozem (Table 5). In both soils, hydrolyzed feathers strengthened the negative correlation between P availability and pH (Table 5).

3.7.2. PCA

The obtained results were subjected to principal component analysis to explain the relationships between microbial indicators, enzymatic activity, experimental variants, and the dates of the analyses, and the applied model explained 62.36% of the variability by two principal components (PC1: 38.63% and PC2: 23.73%) for Cambisol I and 61.34% of the variability by two principal components (PC1: 40.86% and PC2: 20.48%) for Chernozem. The loading plot (Figure 4) demonstrates the correlation structure of the analyzed variables, i.e., total abundance of bacteria and fungi, proteolytic microorganisms, cellulolytic bacteria and fungi, respiratory activity, dehydrogenase activity, protease, acid and alkaline phosphatase, pH, and dsDNA. Location of the dots on the score plot grouped the experimental variants, i.e., water-irrigated (W), water-irrigated and planted (WP), keratin hydrolysate-fertilized (H), and keratin hydrolysate-fertilized with oilseed rape plants (HP) after 3, 30, and 60 days of the experiment in relation to the parameters (variables) studied.

The results obtained for Cambisol I (sandy soil) demonstrated the association of protease and dehydrogenase activity with all experimental variants on day 3 of the experiment (observation–dots), as well as respiratory activity, total abundance of bacteria, proteolytic microorganisms, and cellulolytic fungi with the experimental variants on day 60 of the experiment, especially with the variant fertilized with keratin hydrolysate (H) (Figure 4A,B). In addition, this analysis indicated significantly positive correlations between these last variables.

PCA analysis demonstrated that the pH in sandy soil significantly decreased with increasing respiration activity, the total number of bacteria, proteolytic microorganisms, cellulolytic bacteria and fungi, and the total DNA pool. Factor 2 explained to a lesser extent the relationship between the variables (Figure 4A,B). The analysis showed that the activity of dehydrogenase increased significantly with acid phosphatase activity and decreased with alkaline phosphatase activity, higher number of cellulolytic bacteria, and the total pool of dsDNA, as well as with the duration of the experiment. The results showed (Figure 4A,B) that the total DNA pool was associated with the abundance of cellulolytic bacteria and fungi and the total number of bacteria and proteolytic microorganisms. A significantly positive correlation between these variables indicated a high involvement of this group of microorganisms in the transformations of C and N compounds in the soil, which results in the multiplication of the number of these microorganisms and thus an increase in DNA concentration (Figure 4A,B).

The results obtained for Chernozem demonstrated the association of high dehydrogenase activity with day 3 of the experiment in all four experimental variants (Figure 4C,D). The increased abundance of cellulolytic fungi and the total number of bacteria, in particular in variants fertilized with the hydrolysate, with and without plant growth, was significant, especially on day 60 of the experiment. PC analysis showed that the protease activity abundance of proteolytic microorganisms was significantly associated only with the control variant with plants. In addition, a strong positive correlation was demonstrated between the total abundance of bacteria, cellulolytic bacteria and fungi, and dsDNA (Figure 4C,D). However, these variables were significantly negatively correlated with soil pH and dehydrogenase activity. A significant, although statistically weaker (PC2) negative correlation between proteolytic microorganisms and acid phosphatase was also demonstrated. An insignificant, but important, relationship, in terms of the hydrolysates introduced into the soil with a high mineral N content, was shown between proteolytic microorganisms and protease activity, and a negative one with the number of fungi. This indicated the involvement of proteolytic bacteria in the transformation of mineral forms of nitrogen, correlated especially in the variants without keratin hydrolysate but with plants on day 30 and the total bacterial pool in the variant with the hydrolysate, as well as with and without plant growth on day 60 of the experiment (Figure 4C,D).

4. Discussion

Agricultural land use and climate change are one of the main reasons for the deterioration of the natural environment [21,42]. The methods counteracting these phenomena include, among others, developing organic crops and using organic or mineral–organic fertilizers that contain microorganisms to support specific soil processes. The determinant of the dynamics of changes occurring in the soil, reflecting its current state, is the so-called biological activity of soil consisting of a number of microbiological, biochemical, and enzymatic parameters [43,44,45], as well as the yield of cultivated plants [46].

The subject of this study was to investigate the effect of feather hydrolysate with a high content of soluble proteins, peptides, and amino acids, as well as mineral forms of nitrogen and sulfur obtained during decomposition of waste keratin of chicken feathers by the Trichophyton ajelloi strain No. XII on the early growth of test plants and microbiological and enzymatic properties in two soils with different physical and chemical parameters, i.e., in the soil conventionally called sandy soil (Cambisol I) and Chernozem.

4.1. Phytotoxicity

Phytotoxicity is a negative phenomenon consisting in the harmful effect of various chemical compounds or environmental conditions on plants [47]. It can be manifested by limited seed germination, inhibited growth of plant roots and shoots, the appearance of necrosis, chlorosis, etc. [47,48]. This study found that keratin hydrolysates introduced into sandy soil and Chernozem, both undiluted and diluted (1:2), did not have a negative effect on the germination of seeds of the Brassica napus L. var. napus and Lepidium sativum L. Our observations are consistent with the results of other authors [46,49,50,51] studying the effects of native keratin hydrolysates obtained through both physicochemical and microbiological transformations. This was evidenced by the results of Nustorova et al. [49], who demonstrated the beneficial effect of the hydrolysate obtained through alkaline hydrolysis of sheep wool on the germination and growth of ryegrass, especially at its higher doses. In turn, Bhavsar et al. [50] reported that hydrothermal wool hydrolysate had no phytotoxic effect on the germination of Lepidium sativum L. seeds, as it generally oscillated around 100%, especially in the presence of diluted hydrolysates. Gousterova et al. [51] applied soil feather hydrolysate obtained using Thermoactinomycetes strains and showed stimulation of ryegrass (Lolium L.) seed germination at lower and inhibition at higher hydrolysate concentrations. Jain et al. [46] used feathers hydrolyzed by the actinomycete Streptomyces sampsonii strain GS1322 and observed stimulation of wheat seed germination (%) in the presence of all applied doses. In the present study, three-fold diluted keratin hydrolysates obtained from feather decomposition by the soil strain No. XII of the fungus Trichophyton ajelloi, contrary to undiluted hydrolysates, did not inhibit root and shoot growth of Lepidium sativum L. and Brassica napus L. var. napus in Chernozem, Lepidium sativum L. in loamy soil (Cambisol II), and Brassica napus L. var. napus in sandy soil (Cambisol I). According to Bhavsar et al. [50], undiluted hydrolysates may reduce germination due to too high osmotic salt concentration surrounding the seeds. On the other hand, the stimulating effect on plant germination observed for both diluted and undiluted hydrolysates was attributed to the presence of proteins, amino acids, and micronutrients in the hydrolysates. Keratin hydrolysates used in our study can therefore be regarded as biostimulants of root and shoot germination and growth due to their content of soluble proteins, peptides, and amino acids.

The so-called germination index (G_I) is one of important indices of the absence of phytotoxic effects of introduced organic or organic–mineral fertilizers. According to the interpretation proposed by Zucconi et al. [34] for composts, those with G_I < 80% are considered phytotoxic. Bohacz [8] showed that composts obtained on the basis of chicken feathers and lignocellulosic waste had no toxic effect on Lepidium sativum L. and even stimulated seed germination and root growth of this plant. The research conducted in this study demonstrated that only the G_I of undiluted hydrolysates introduced into sandy soil (Cambisol I) reached values below 80%. In other cases, it was higher (G_I > 80%), especially in variants with diluted hydrolysate. Moreover, the G_I after the application of diluted feather hydrolysate was generally higher for Brassica napus L. var. napus than for Lepidium sativum L.: 101.63 and 82.24 in sandy soil, and 126.55 and 113.95 in Chernozem, respectively. According to the interpretation proposed by Zucconi et al. [34], this indicated no phytotoxic effect of the diluted hydrolysate, in contrast to the undiluted preparation. The root-to-stem length ratio, whose optimal value is approx. 1.0, is also an important plant growth indicator [32], indicating the uniform growth of the root and stem and showing the substrate’s effect on developmental disproportions [32]. The current experiment showed that undiluted feather hydrolysate in sandy soil caused almost three times faster Brassica napus L. var. napus root than the shoot growth and approx. 1.5 times more intensive growth of roots than shoots of Lepidium sativum L. For Chernozem, its influence against oilseed rape was weaker, while against garden cress it was stronger. The diluted hydrolysate most strongly altered the ratio between root and shoot growth in loamy soil (Cambisol II) (R:S = 2.71), which was visible in case of oilseed rape. The R:S ratio in sandy soil and Chernozem indicated a more uniform growth of oilseed rape roots and shoots.

For the above reasons, further studies concerning the effect of feather hydrolysates on soil biological activity and plant growth focused on two soils: sandy (Cambisol I) and Chernozem, resigning from loamy soil (Cambisol II).

4.2. Abundance of Soil Microorganisms in Variants Treated and Non-Treated with Feather Hydrolysate

Microorganisms play a key role in shaping soil fertility [52]. The diversity of microorganism communities and their metabolic activity contribute not only to the good quality of the soil environment, but also allow to maintain high yielding potential [53]. The model experiment carried out in this study generally indicated the activating effect of keratin hydrolysates, formed after fungal biodegradation of chicken feathers, on soil microorganisms. This effect should be attributed to soil enrichment with readily available organic matter, nitrogen, and mineral sulfur, as noted by many authors who observed stimulation of microorganism development in soils fertilized with protein hydrolysates [16,49]. Our study also showed that the intensity of microbial growth varied depending on the soil, time of exposure, and experimental variant (with and without hydrolysate, with and without plants). It was found that populations of bacteria, fungi, proteolytic microorganisms, and cellulolytic bacteria in sandy soil developed faster in both variants with the hydrolysate than in variants not fertilized with the preparation. In Chernozem, the abundance of all studied groups of microorganisms increased, especially in the variants with plants fertilized with the hydrolysate, except for proteolytic microorganisms. Due to the significant abundance of organic carbon compounds in Chernozem, growth stimulation of microorganisms involved in the transformation of these substances, such as bacteria and fungi, including cellulolytic microorganisms, may have resulted from the supply of large amounts of readily available nitrogen with the hydrolysate. This suggestion was supported by the findings of Nustorova et al. [49], who showed that the introduction of the hydrolysate into the soil after sheep’s wool biodegradation increased the number of microorganisms, which led to an accelerated decomposition and mineralization of organic matter in the soil and thus increased the assimilability of nutrients, also for plants. The latter thesis was confirmed in this study by the decrease in organic carbon content in the tested soils after the introduction of the hydrolysate, especially in variants with plants, as well as the correlation between the total number of bacteria, cellulolytic bacteria and fungi, and proteolytic microorganisms, as well as the total DNA pool, and respiratory activity in the tested soil samples. The presence of proteolytic microorganisms in the soil reflects the intensity of mineralization processes of organic nitrogen forms, which are subsequently made available in an easily assimilable form to plants and microorganisms [54,55]. Respiratory activity is a measure of the intensity of the oxidation and mineralization processes of an organic substance. It has long been known [56] that bacteria are much more efficient than fungi in utilizing low molecular weight organic and mineral forms of nitrogen and sulfur. This suggests that in the initial period, bacterial populations were more active in the hydrolysate transformations in the studied soils. This was evidenced by the stimulation of the overall increase in the abundance of bacteria, proteolytic microorganisms, and cellulolytic bacteria during the first 30 days of the experiment, after the introduction of keratin hydrolysate into both tested soils. The subsequent decline in the abundance of the bacterial groups studied, especially in the variants with plants, may have been due to the depletion of readily available carbon and nitrogen compounds both by the microorganisms and plants. The introduced feather hydrolysate had a relatively weak effect on the populations of cellulolytic bacteria and fungi, with the exception of Chernozem with plants, where significant differences were recorded after the introduction of the hydrolysate compared to the control variant. Cellulolytic microorganisms mainly utilize cellulose as a source of C and energy, which was only available in the soil and not in the hydrolysate. The high stimulation of cellulolytic bacteria in Chernozem may have been due to favorable growth conditions for these bacteria, i.e., the presence of cellulose-rich organic matter and the optimal soil pH, as well as the availability of nitrogen, which stimulated the growth of cellulolytic microorganisms. On the other hand, the higher overall abundance and stimulation of fungal growth in sandy soil than in Chernozem, after the introduction of the hydrolysate (the first month), should be associated with the lower pH of this soil, favoring the growth of these microorganisms. The long-term decrease in fungal abundance observed in both soils, especially in sandy soil in the variant with plants, may have been due to the effect of oilseed rape root secretions with antifungal activity that inhibited fungal growth or the presence of antagonistic rhizosphere microorganisms that synthesize fungal growth inhibitors. We based this assumption on studies indicating that fungal growth was inhibited by oilseed rape root exudates [57,58], as well as reporting the stimulation of toxic nitrosamine biosynthesis in the presence of high-dose nitrogen fertilization [59].

Soil pH is considered to be an important factor in the microbiological metabolism of organic matter and its mineralization due to the formation of the products of different chemical nature. PCA analysis showed that a decrease in the pH of the tested soils in different variants of the experiment was accompanied by an increase in the number of the tested groups of microorganisms, with the exception of sandy soil in which a decrease in pH resulted in a lower abundance of fungi. However, it was not a significant relationship. According to Borymski [60], a decrease in soil pH in addition to carbonic acid secretion during root respiration, organic acid secretion by roots, and decomposition of organic matter in the soil was also caused by ammonium ion uptake and ion exchange. On this basis, it can be concluded that the introduction of feather hydrolysate with a high content of ammonium ions into the studied soils could activate the uptake of these ions by microorganisms and plants and intensify nitrification. This assumption was confirmed by the reduction of total nitrogen content in the variants with oilseed rape in both studied soils. As a result, not only would the soil become slightly acidified but also stimulate the development of many populations of soil microorganisms. On the other hand, soil acidification may also have had the effect of increased phosphorus availability. The present study found a significantly negative correlation between pH and the content of available P in all experimental variants. Phosphorus is known [61] to be necessary for the proper development of aboveground and underground plant organs; it affects the rate of plant maturation, biomass growth, fruit quantity and size, flowering intensity, and grain yield. In addition to organic compounds, this element is also found in the soil in the form of metal salts inaccessible to plants such as iron phosphate, calcium phosphate, and aluminum phosphate. Root exudates, i.e., organic acids and various compounds produced by microorganisms, among others siderophores, have the ability to complex metal ions, thereby contributing to the release of phosphorus from inaccessible associations and increasing the forms of iron available for plants [60,62,63]. In this study, the total dsDNA pool was also significantly correlated with soil phosphorus content, which was on average higher after the introduction of feather hydrolysate to the studied soils. Particularly high content of assimilable P was recorded in sandy soil and Chernozem at the end of the experiment in the variants with the hydrolysate and plants, which could indicate its release from inaccessible sources and stimulation of the general pool of soil microorganisms.

According to Hartmann et al. [63], ion exchange associated with the release of protons into the soil by plant roots in exchange for cations uptake, contributing to lowering soil pH, was also one of the mechanisms that helped plants take up iron, an element important, inter alia, in enzymatic processes.

The total pool of DNA isolated from the studied sandy soil and Chernozem was significantly positively correlated with all analyzed groups of microorganisms, except for the total number of fungi in sandy soil and proteolytic microorganisms in Chernozem. Wolińska et al. [64] also reported the correlation of the total DNA pool with microbial abundance. The positive correlation observed by us between DNA levels and soil respiration activity could indicate an intracellular source of microbial DNA. The process of cellular respiration, i.e., biological oxidation in microbial cells, is accompanied by decarboxylation.

4.3. Enzymatic Activity of Soils Fertilized and Not Fertilized with Feather Hydrolysate

All transformations of organic matter and biogenic elements occurring in the soil are conditioned by the activity of enzymes, which affects the availability of nutrients for plants and soil fertility [52].

Zhu et al. [65] reported that soil respiratory activity was stimulated by low nitrogen levels and inhibited by high nitrogen content. The higher content of nitrogen compounds in the studied soils caused, among others, by the introduction of feather hydrolysate, caused a periodic reduction in CO₂ release. This was particularly evident in sandy soil because it was characterized by higher N content in the initial period. In Chernozem, a decrease in respiration was recorded on day 30 of the experiment, which was also associated with high levels of total nitrogen. It was visible in all experimental variants except the control irrigated with water. In the next experimental period, a significant reduction in nitrogen content, particularly in the variants with the hydrolysate, resulted in a significant increase in CO₂ release. This effect was visible especially in Chernozem fertilized with the hydrolysate without and with plants and in sandy soil fertilized with the hydrolysate only.

Dehydrogenases are respiratory enzymes that serve as indicators of respiration intensity and carbon mineralization of organic matter in the soil environment. Their activity is determined by the availability of food substrates for microorganisms and the pH of the environment. Dehydrogenase activity also reflects the oxidative potential of the soil, as these enzymes are associated with living, metabolizing microbial cells [21]. The activity of dehydrogenases in this study, in all analyzed experimental variants, tended to gradually decrease, which could be explained by reduced soil pH. Dehydrogenases belong to enzymes showing low activity in acidic soils; while in alkaline soils, they reach high catalytic activity [66].

Keratin hydrolysate affected dehydrogenase activity similarly to respiratory activity, which was expressed as stimulation of the activity of these enzymes in sandy soil and its inhibition in Chernozem. The latter effect, due to the high activity of dehydrogenases in this soil, may have been caused by faster depletion of the readily available carbon and energy sources brought with this biofertilizer. On the other hand, with the generally low activity of dehydrogenases in sandy soil, its enrichment with the hydrolysate may have been sufficient not only to maintain, but also to stimulate the catalytic activity of these enzymes. This explanation was supported by the positive, albeit non-significant, correlation in sandy soil between dehydrogenase activity and organic C content after the introduction of feather hydrolysate and indicated that microorganisms in this soil did not utilize organic matter contained in the hydrolysate.

Just as dehydrogenases are necessary in organic C transformations in soil, proteases are key enzymes in the transformations of organic N associated with protein breakdown into simpler compounds, i.e., short-chain peptides and amino acids [67]. Nitrogen-rich feather hydrolysates, when introduced into the tested soils, tended to decrease the activity of these enzymes, with a periodic increase, especially in the variants with plants. This effect, particularly pronounced in Chernozem with high TOC content, could have been caused by catabolic repression of the synthesis of these enzymes under the influence of simple carbon bonds broken during carbon hydrolysis of organic matter [68]. As regards nitrogen, it is primarily the level of its macromolecular fraction that is important. Vranova et al. [68] reported that protease activity was dependent on both organic C and nitrogen content. For instance, Caballero et al. [69] showed that enrichment of anthropogenic soil with autoclaved poultry feathers, i.e., denatured keratin, significantly stimulated proteolytic activity of this soil. Opinions regarding mineral forms of nitrogen, i.e., N–NH₄⁺, are divided. Vranova et al. [68] reported that while some researchers believed that the addition of ammonium ion to soil stimulated proteolytic activity, others were of the opinion that it had an inhibitory effect on protease biosynthesis in soil. A similar conclusion, indicating the inhibition of soil protease activity by N–NH₄⁺, can be drawn based on the present results, after enriching the soil with a feather hydrolysate rich in ammonium ions. Additionally, PCA analysis revealed that the decrease in protease activity in the studied soils was correlated with an increase in respiratory activity and vice versa. The effect of the level of CO₂ release, as an indicator of respiratory activity, on the dynamics of protease activity was previously reported by Vranova et al. [68]. The relationship found in this study indicated that stimulation of protease activity occurred when readily available sources of organic C were exhausted as respiratory substrates (reduced respiratory activity). This “forces” the microorganisms to use the “inferior” sources of organic C, such as organic nitrogen associations (increased protease activity). In the present study, protease activity decreased, while respiratory activity increased.

This study also analyzed the activity of soil phosphatases (acid and alkaline phosphatases) due to the importance of phosphorus in maintaining the functional status of the soil and thus crop yields. These enzymes play an important role in the transformation of organic forms of phosphorus into soluble inorganic forms absorbed by plants and soil microorganisms. The activity of phosphatases depends primarily on the pH of the soil [70]. The most optimal use of phosphorus by living organisms occurs in soils with a pH in the range of 5.5–7.0 [71].

Acid phosphatase is more widespread than alkaline phosphatase in most soils, and its activity also depends on the availability of N and increases when the pool of available N rises [72]. In the present study, this could be applied to acid phosphatase, whose activity significantly increased after introducing feather hydrolysate containing soluble proteins, peptides, and amino acids into sandy soil without plants. Caballero et al. [69] showed that the introduction of denatured chicken feathers into the soil enhanced the phosphatase activity, which would confirm the beneficial effect of the available N associations on acid phosphatase activity observed in this study. The present study showed, however, that after a month of the experiment, the activity of acid phosphatase decreased in both soils, fertilized and not fertilized with feather hydrolysate, with and without oilseed rape. This effect can be explained by lower pH in the tested variants and depletion of assimilable N compounds. In this study, it was also noted that acid phosphatase activity was higher than the activity of alkaline phosphatase in all variants of the experiment. According to Saha et al. [73], the dominance of acid phosphatase activity over alkaline phosphatase activity was directly related to the reaction of the soil environment. The higher activity of acid phosphatase observed in Chernozem could be due to both the higher pH of this soil and the higher content of organic matter in this soil. Saha et al. [73] showed in their research that the highest phosphatase activity occurred in soil fertilized simultaneously and not separately with mineral and organic fertilizers (manure). Kwiatkowski et al. [74] also reported the influence of organic fertilization on the activity of acid phosphatase. The cited authors showed that organic fertilization with Humac Agro biofertilizer combined with crop rotation was more effective in stimulating the activity of acid phosphatase than conventional mineral–organic fertilization.

Alkaline phosphatase is considered to be a very good indicator of the activity and population size of soil microorganisms [52]. Its availability in soil is much lower due to its predominantly microbiological origin [75]. Acid phosphatases, which have a much broader origin, support alkaline phosphatases in making phosphorus available to plants [75]. In our study, alkaline phosphatase activity generally increased over time, especially in sandy soil (Cambisol I). The only exception was a variant of planted soil in the absence of fertilizer. Enrichment of sandy soil with waste feather hydrolysate stimulated the activity of this enzyme. In the case of Chernozem, the applied biofertilizer reduced alkaline phosphatase activity both in the variant with and without oilseed rape. Kwiatkowski et al. [74] demonstrated that conventional mineral–organic fertilization had a more negative effect on alkaline phosphatase activity in the soil compared to only organic fertilization such as the Humac Agro biopreparation. Opposite results were obtained by Chang et al. [76], who showed that this mineral fertilization reduced the activity of soil alkaline phosphatase in the soil. The data obtained in the present study have indicated that mineral–organic feather hydrolysate may have a different effect on the soil depending on its physicochemical parameters.

4.4. Oilseed Rape Biomass

This study showed a significant almost two–fold increase in oilseed rape (Brassica napus L. var. napus) biomass after the addition of feather hydrolysate to both soils. This was consistent with the results of the research by Nustorova et al. [49], who also showed an increase in fresh plant biomass (ryegrass Lolium perenne L.), especially in the variant with a higher concentration of feather hydrolysate obtained by chemical transformation. Kaur et al. [77] also demonstrated that the introduction of feathers processed by Bacillus aerius NSMk2 into the soil stimulated the increment of Vigna radiata biomass relative to the biomass estimated from the non-fertilized control.

5. Conclusions

Feathers processed by the soil strain No. XII Trichophyton ajelloi are valuable in terms of composition and mineral–organic biopreparation with a high fertilization potential. Biodegradation of chicken feather waste by Trichophyton ajelloi not only reduces their deposition in the environment and uncontrolled decomposition, but also is a safe alternative in the fertilization practice of plants with high sulfur requirements, such as Lepidium sativum L. and Brassica napus L. var. napus, due to the lack of phytotoxicity signs and the high content of sulfur products. Hydrolyzed feather keratin significantly affects both microbiological, biochemical, and enzymatic indicators of soil biological activity. The effect of the hydrolysate, however, depends on the physical and chemical properties of the soil into which it is introduced. In addition, the extracted keratin hydrolysate significantly intensifies the growth of test plants such as oilseed rape.

The tested Trichophyton ajelloi strain No. XII, due to its high ability to biodegrade keratin protein, can be used in the future to manage keratin waste other than chicken feathers and study its effect on soil biological activity and plant growth not only under model but also field conditions.