The Effects of Spent Mushroom Substrate on the Yield and Nutritional Value of Festulolium braunii (K. Richt.) A. Camus

Abstract

The aim of the experiment was to assess the effects of mineral and mushroom substrate nitrogen on the yield and selected parameters of Festulolium braunii. To this end, a two-year field experiment was conducted between 2017 and 2018. Spent mushroom substrate was applied at three levels (10, 15 and 20 Mg·ha⁻¹) and mineral nitrogen at four (30, 68, 105 and 180 kg·ha⁻¹). Mineral phosphorus and potassium were used at one level only, with 60 and 150 kg·ha⁻¹, respectively. The research also included the effects of the interaction between organic and mineral fertilizers and the determination of an optimal amount of nitrogen resulting in good-quality forage and a high yield. The plant used in the experiment was Festulolium braunii, an intergeneric hybrid of the Sulino variety. Treatment combinations significantly affected its yield, the content of total and digestible protein, as well as the energy values. For most forage parameters, the beneficial effects of mushroom substrate nitrogen and mineral nitrogen interaction were demonstrated, compared to the effects of mineral nitrogen applied on its own. The largest significant increase in the amount of biomass, was by 16% in comparison to plants treated with mineral fertilizers only (N₄PK); a significant increase in energy level (by 17%), was recorded for grass treated with 15 Mg·ha⁻¹ of mushroom substrate together with 68 kg·ha⁻¹ of mineral nitrogen (SMS₂ + N₂PK). The highest dose of nitrogen contained in mushroom substrate applied together with the smallest amount of mineral nitrogen (SMS₃ + N₁PK) resulted in the highest increase in total and digestible protein concentrations in the grass, 18% and 21% more, respectively, than in plants treated with mineral fertilizers only. The interaction of mineral and mushroom substrate nitrogen did not significantly affect the digestibility of the forage and its concentration of net energy of lactation (NEL).

1. Introduction

The increasing demand for good-quality feedstuffs requires an accurate determination of their nutritional value to properly balance feed rations. Apart from the content of organic components, such as total and digestible protein and crude fiber, the basic measure of the nutritional value of forage is the values of its net energy of lactation (NEL), digestibility and energy efficiency. The nutritional value of grass depends to a large extent on its species, variety and the phenological stage at which it is harvested.

An intergeneric hybrid of meadow fescue (Festuca pratensis) and Italian ryegrass (Lolium multiflorum), Festulolium braunii is a valuable forage grass of significant importance [1]. Vog [2] confirms that it can produce good quality forage. It is grown both on its own and in mixtures [3,4,5]. Resistant to droughts, it has high yield potential and competitive ability, with good growth and development in subsequent years of use [6]. Ghesquičre et al. [7] and Baert et al. [8] argue that Festulolium combines high yields and good digestibility, two important factors in forage production.

The type and dose of fertilizer treatment affect forage quantity and quality. Nitrogen supply is of particular importance, it determines the yield and contributes to an increase in total and digestible protein content. The production of mushrooms in Poland currently amount to approx. 335 thousand tons per year. To produce 1 kg of mushrooms, about 5 kg of compost is needed, which means over 1.6 million tons of spent mushroom substrate to be developed each year [9]. Spent mushroom substrate is a good fertilizer material and a particularly valuable source of soil organic matter. Its nutrients are gradually released into the soil solution as a result of mineralization and are available to plants in various forms [10]. According to Kalembasa and Wiśniewska [11] and Wiśniewska-Kadżajan [12,13], mushroom substrate can be applied to arable, fruit and vegetable crops, but is also applied to lawns and used to maintain urban green areas.

The aim of the studies was to determine the effects of mineral and mushroom substrate nitrogen on the yield and selected parameters of Festulolium braunii nutritional value. Before the research, the following hypotheses were adopted: (1) the interaction of mineral and spent mushroom substrate nitrogen will have a better effect on the yield and nutritional value of Festulolium braunii than mineral nitrogen used at its full dose; (2) the interaction of mushroom substrate nitrogen at the highest level with the lowest amount of mineral nitrogen will have a better effect on Festulolium yield and the nutritional value than nitrogen contained in other doses of substrate.

The results were to show to what extent spent mushroom substrate could replace or reduce the level of mineral fertilizers without a negative impact on the Festulolium braunii yield and its nutritional value.

2. Materials and Methods

2.1. Experimental Conditions

The two-year experiment was conducted in field conditions between 2017 and 2018. The grass was sown in the autumn of 2016, but the research data were collected in 2017 and 2018. The experiment was established on the experimental plot of the University of Natural Sciences and Humanities in Siedlce, (52°10′ N, 22°17′ E), Poland, in a completely randomized design and with plots of 6 m² and three replications. It was founded on soil with a granulometric composition of loamy sand, belonging to the order of anthropogenic soils, culture-earth type and hortisole subtype [14]. The chemical characteristics of the soil were as follows: organic carbon concentration of 14.5 g·kg⁻¹DM, total nitrogen concentration of 1.4 g·kg⁻¹DM, the C:N ratio of 10.6:1 and pH of 6.7. The concentration of absorbable forms of phosphorus (170.0 mg·kg⁻¹DM) and magnesium (84.0 mg·kg⁻¹DM) was high, but potassium (114.0 mg·kg⁻¹DM) moderate.

2.2. The Fertilizer and the Plant

The organic material used in the experiment was spent mushroom substrate (SMS), from a horticultural company producing white mushrooms (Agaricus bisporus) in the Mazowieckie Voivodeship, central and eastern Poland. The starting materials for the production of the mushroom substrate used in the experi-ment were cereal straw, chicken manure, low peat and gypsum. The content of dry matter was 30%, organic carbon 355.0 g·kg⁻¹, with 25.1 g·kg⁻¹ of nitrogen, 9.5 g·kg⁻¹ of phosphorus, and 11.6 g·kg⁻¹ of potassium. The pH value of mushroom substrate was 6.8. It was used once, as pre sowing application, in the autumn of 2016. Mushroom substrate was used at three levels: SMS₁-10 Mg·ha⁻¹; SMS₂-15 Mg·ha⁻¹; SMS₃-20 Mg·ha⁻¹, in accordance with the guidelines of the Nitrates Directive specifying the maximum amounts of nitrogen that can be introduced into the soil in an organic form. The doses of the substrate introduced the following amounts of nitrogen into the soil.: SMS₁-10 Mg·ha⁻¹ with 75 kgN·ha⁻¹); SMS₂-15 Mg·ha⁻¹ with 112 kgN·ha⁻¹; SMS₃-20 Mg·ha⁻¹ with 150 kgN·ha⁻¹. In each year of research complementary mineral nitrogen was applied to plots with mushroom substrate at four levels (N₁-30; N₂-68; N₃-105; N₄-180 kgN·ha⁻¹). Phosphorus and potassium were applied at one level: P-60 kg∙ha⁻¹ and K-150 kg∙ha⁻¹. Thus, the treatment combinations were as follows: control (with no treatment); N₄PK; SMS₁ + N₃PK; SMS₂ + N₂PK; SMS₃ + N₁PK.

The proportions of mineral and mushroom substrate nitrogen supplied to the treated plots were different, but the total amount was the same. Annual doses of mineral nitrogen (in the form of NH₄NO₃) and potassium (K₂SO₄) were divided into three equal parts. The first was applied in the spring before the start of the growing period, the next ones before the second and third grass growth cycles. Phosphorus fertilizer (in the form of Ca(H₂PO₄)₂ was applied once in the spring.

The plant used in the experiment was Festulolium braunii, an intergeneric hybrid, sown in the autumn of 2016 in accordance with the seeding standard. During the two years of full use, three harvests of the grass species were collected during each growing period to determine its yield and assess forage parameters. Immediately after the harvest, fresh matter from each plot was weighed and 0.5 kg was sampled and dried naturally in a room with ventilation. When dry matter content was determined, the plants were shredded and the homogeneous sample was crushed.

2.3. Chemical Analysis, Calculation and Statistical Processing

In the samples prepared this way, the content of total protein and crude fiber was determined by near-infrared reflection spectroscopy (NIRS), using the NIRFlex N-500 autoanalyser of the Swiss company Büchi with INGOT ready-made calibrations for dry feed. The method is described in detail in Polish Standard PN-EN ISO 12099:2010 and in the literature [15,16]. To assess selected parameters of the energy value, the DGL system, one of the most adapted to Polish conditions and containing the rules of estimating the nutritional value and feed ration, was used [17].

The content values of digestible protein, net energy of lactation (NEL), dry matter digestibility and energy yield were calculated according to formulas used in the literature:

Digestible protein after Pawlak [18] and Ostrowski [19]:

DP = −29.78 + 9.56 TP

where:

DP—digestible protein (g·kg⁻¹DM),

TP—total protein (% DM);

Net energy of lactation (NEL) after Ostrowski [20]:

NEL = 6.998 − 0.061·CF + 0.014·TP

where:

NEL—net energy of lactation (MJ·kg⁻¹DM),

CF—crude fiber content (% DM),

TP—total protein (% DM);

Dry matter digestibility after Pawlak [18]:

DMD = 103 − 1.2 CF

where:

DMD—dry matter digestibility (%),

CF—crude fiber content (%);

Forage energy yield after Domański [21]:

PE = P·100·(0.968 − 0.0063·CF + 0.033·TP)

where:

PE—forage energy yield (JP/ha),

P—dry matter yield (10 tons/ha),

CF—crude fiber content (% DM),

TP—total protein content (% DM).

The following research variables were considered in the experiment: (A)—treatments (five levels), (B)—growing seasons (two levels) and (C)—grass growth cycles (three levels). One factor analysis of variance for replicates in the experiment did not show any significant differences between them.

For the statistical processing of the results, an analysis of variance for three-factor experiments was used [22].

y_ijlp = m + a_i + g_j + e^/1/_ij + b_l + ab_il + e^/2/_ijl + c_p + ac_ip + bc_lp + abc_ilp + e^/3/_ijlp

where:

y_ijlp—the value of the variable for the i-th level of factor A and p-th level of factor C for the j-th replicate,

m—the mean of research,

a_i, b_l, c_p—the effects of factors,

g_j—the effect of the j-th replicate,

ab_il, ac_ip, bc_lp—the effects of the interaction of two factors,

abc_ilp—the effect of the interaction of three factors,

e^/1/_ij, e^/2/_ijl, e^/3/_ijlp—the effect of random factor,

i = 1,2,…,a  a—the number of levels of factor A,

j = 1,2,…,n  n—the number of replicates,

l = 1,2,…,b  b—the number of levels of factor B,

p= 1,2,…,c  c—the number of levels of factor C.

The significance of differences between means was estimated with Tukey’s test at the significance level α ≤ 0.05. Statistica 6.0 was used for the calculations [23].

2.4. Weather Conditions

Information about weather conditions (

Table 1. Average monthly air temperature (°C), monthly precipitation (mm) and monthly Sielianinov’s coefficient in growing periods.

Year	Month
	IV	V	VI	VII	VIII	IX	X	Means
	Temperature (°C)
2016	8.2	12.3	16.5	18.7	21.0	14.5	6.5	14.0
2017	6.9	13.9	17.8	16.9	18.4	13.9	9.0	13.8
2018	13.1	17.0	18.3	20.4	20.6	15.9	9.5	16.4
	Means
	9.4	14.4	17.5	18.7	20.0	14.8	8.3	14.7
	Multiannual means
	8.5	14.0	17.4	19.8	18.9	13.2	7.9	14.2
	Precipitation (mm)
2016	30.0	100.2	43.3	62.6	11.9	77.1	39.0	52.0
2017	59.6	49.5	57.9	23.6	54.7	80.1	53.0	54.1
2018	34.5	27.3	31.5	22.4	24.5	27.4	23.3	27.3
	Means
	41.4	59.0	44.2	36.2	30.4	61.5	38.4	44.5
	Multiannual means
	33.0	52.0	52.0	65.0	56.0	48.0	28.0	47.7
	Sielianinov’s coefficient (K)
2016	1.22 (md) a	2.63 (sw)	0.87 (d)	1.08 (md)	0.18 (ed)	1.46 (o)	1.94 (mw)	-
2017	2.88 (sw)	1.15 (md)	1.08 (md)	0.45 (sd)	0.96 (d)	1.92 (mw)	1.90 (mw)	-
2018	2.63 (sw)	0.16(ed)	1.72 (mw)	1.10 (md)	1.19 (md)	1.72 (mw)	2.43 (w)	-

^a Values are ratd as: K ≤ 0.4 extremely dry (ed), 0.4 < K ≤ 0.7 severely dry (sd), 0.7 < K ≤ 1.0 dry (d), 1.0 < K ≤ 1.3 moderately dry (md), 1.3 < K ≤ 1.6 optimal (o), 1.6 < K ≤ 2.0 moderately wet (mw), 2.0 < K ≤ 2.5 wet (w), 2.5 < K ≤ 3.0 severely wet (sw), K > 3.0 extremely wet (ew).

) was obtained from the Hydrological and Meteorological Station in Siedlce. To assess the effect of the temporal variability of those conditions on plant growth and development, Sielianinov’s hydrothermal coefficient [24] was calculated. Values of Sielianinov’s hydrothermal coefficient are based on the total monthly rainfall (P) and the monthly sum of daily mean air temperatures (Σt), using the formula: K = P/0.1Σt [25].

3. Results

3.1. Yield

The yield of Festulolium braunii (

Table 2. The effect of fertilizer treatment, growing period and growth cycle on Festulolium braunii yield (Mg·ha⁻¹DM).

Growing Period	Growth Cycle	Treatment					Means
		*O	N4PK	SMS1 + N3PK	SMS2 + N2PK	SMS3 + N1PK
2017	I	3.20	4.00	4.20	4.70	4.20	4.06
	II	3.15	4.20	3.90	4.60	4.50	4.07
	III	2.90	3.80	3.40	4.45	4.10	3.73
	Sum
		9.25	12.0	11.50	13.75	12.8	11.86 B ***
2018	I	2.80	3.80	4.50	5.00	5.20	4.26
	II	3.30	4.10	4.80	4.70	4.50	4.28
	III	3.10	3.50	4.20	4.50	4.30	3.92
	Sum
		9.20	11.40	13.50	14.20	14.00	12.46 A
	Means across growing periods
		9.22 e **	11.70 d	12.50 c	13.97 a	13.40 b	12.16
	Means across growth cycles
	I	3.00	3.90	4.35	4.85	4.70	4.16 A
	II	3.22	4.15	4.35	4.65	4.50	4.17 A
	III	3.00	3.65	3.80	4.47	4.20	3.82 B

*O—control (with no treatment); N₄PK—mineral fertilizers (180, 60, 150 kg·N·ha⁻¹; SMS₁ + N₃PK—spent mushroom substrate (10 Mg·ha⁻¹) + mineral fertilizers (105, 60, 150 kg·N·ha⁻¹); SMS₂ + N₂PK—spent mushroom substrate (15 Mg·ha⁻¹) + mineral fertilizers (68, 60, 150 kg·N·ha⁻¹); SMS₃ + N₁PK—spent mushroom substrate (20 Mg·ha⁻¹) + mineral fertilizers (30, 60, 150 kg·N·ha⁻¹); ** means marked with the same small letters in columns (a, b, c, d, e) do not differ significantly at p ≤ 0.05 for treatments; *** means marked with the same capital letters in rows (A, B) do not differ significantly at p ≤ 0.05 for years and harvests.

) significantly varied depending on the treatment, growing period and harvest. The highest dry matter yield was collected from the plot treated with the medium amount of mushroom substrate (SMS₂-15 Mg·ha⁻¹) supplemented with mineral nitrogen at the medium dose (SMS₂ + N₂PK). The lowest yield was on the control plot.

The yields from plots treated with both mushroom substrate and mineral fertilizers were compared to those from the plot with mineral fertilizers only. A 6% increase was recorded on the SMS₁ + N₃PK plot, 16.0% on the SMS₂ + N₂PK plot and 13% on the one with SMS₃ + N₁PK. Across growing periods, 5.0% more biomass was recorded in the second year than in the first. The second yield (of the second growth cycle) was 18.0% greater than the third.

3.2. Total and Digestible Protein

Forms and doses of nitrogen significantly affected Festulolium braunii total protein content (

Table 3. The effect of fertilizer treatment, growing period and growth cycle on Festulolium braunii total protein content (g·kg⁻¹DM).

Growing Period	Growth Cycle	Treatment					Means
		*O	N4PK	SMS1 + N3PK	SMS2 + N2PK	SMS3 + N1PK
2017	I	127.3	135.0	127.8	145.4	169.8	141.1
	II	134.9	137.9	137.4	153.7	165.9	145.9
	III	137.0	136.3	167.6	163.2	151.9	151.2
	Means
		133.1 d	136.4 cd	144.3 bc	154.1 ab	162.5 a	146.1
2018	I	126.2	138.5	125.8	135.9	181.6	141.6
	II	129.7	140.7	130.4	152.0	165.1	143.6
	III	134.6	138.9	153.8	149.9	159.6	147.4
	Means
		130.2 c **	139.4 bc	136.7 bc	145.9 b	168.8 a	144.2
	Means across growing periods
		131.6 c	137.9 bc	140.5 bc	150.0 ab	165.7 a	145.1
	Means across growth cycles
	I	126.8 c	136.8 bc	126.8 c	140.7 b	175.7 a	141.3
	II	132.3 c	139.3 c	133.9 c	152.9 b	165.5 a	144.8
	III	135.8 b	137.6 b	160.7 a	156.6 a	155.8 a	149.3

*O—control (with no treatment); N₄PK—mineral fertilizers (180, 60, 150 kg·N·ha⁻¹; SMS₁ + N₃PK—spent mushroom substrate (10 Mg·ha⁻¹) + mineral fertilizers (105, 60, 150 kg·N·ha⁻¹); SMS₂ + N₂PK—spent mushroom substrate (15 Mg·ha⁻¹) + mineral fertilizers (68, 60, 150 kg·N·ha⁻¹); SMS₃ + N₁PK—spent mushroom substrate (20 Mg·ha⁻¹) + mineral fertilizers (30, 60, 150 kg·N·ha⁻¹); ** means marked with the same small letters in columns (a, b, c, d) do not differ significantly at p ≤ 0.05 for treatments, treatments × years, treatments × harvests.

). The greatest amount (average across years and harvests) was recorded on the plots with the highest (20 Mg·ha⁻¹) dose of mushroom substrate supplemented with mineral phosphorus, potassium and the lowest dose of mineral nitrogen (SMS₃ + N₁PK). The smallest amount of protein was in control plants. As the dose of spent mushroom substrate increased, total protein content also increased. On the SMS₁ + N₃PK plot, it was 2% higher than the effect of N₄PK mineral fertilizers applied on their own, 8% higher on the SMS₂ + N₂PK plot and 17% higher on the one with SMS₃ + N₁PK. Statistical analysis also showed the interaction of treatments with years of research and of treatments with growth cycles.

The differences in total protein content (average across treatments and growth cycles) were insignificant. Similarly, when considering harvests (average across years of research and treatments), the differences in Festulolium total protein content were small (5% more in the third harvest than in the first).

Treatment combinations significantly affected digestible protein content (

Table 4. The effect of fertilizer treatment, growing period and growth cycle on Festulolium braunii digestible protein content (g·kg⁻¹DM).

Growing Period	Growth Cycle	Treatment					Means
		*O	N4PK	SMS1 + N3PK	SMS2 + N2PK	SMS3 + N1PK
2017	I	91.9	99.3	92.4	109.2	132.5	105.1
	II	99.2	102.0	101.6	117.1	128.8	109.7
	III	101.2	100.5	130.4	126.2	115.4	114.8
	Means
		97.4 c **	100.6 bc	108.1 b	117.5 a	125.6 a	109.9
2018	I	90.9	102.6	90.5	100.1	143.8	105.6
	II	94.2	104.7	94.9	115.5	128.0	107.5
	III	98.9	103.1	117.2	113.5	122.8	111.1
	Means
		94.6 c	103.4 bc	100.9 bc	109.7 b	131.6 a	108.0
	Means across growing periods
		96.0 c	102.0 bc	104.5 bc	113.6 ab	128.6 a	108.9
	Means across growth cycles
	I	91.4 c	100.9 bc	91.4 c	104.7 b	138.2 a	105.3
	II	96.7 c	103.4 c	98.2 c	116.3 b	128.4 a	108.6
	III	100.0 b	101.8 b	123.8 a	119.9 a	119.1 a	112.9

*O—control (with no treatment); N₄PK—mineral fertilizers (180, 60, 150 kg·N·ha⁻¹; SMS₁ + N₃PK—spent mushroom substrate (10 Mg·ha⁻¹) + mineral fertilizers (105, 60, 150 kg·N·ha⁻¹); SMS₂ + N₂PK—spent mushroom substrate (15 Mg·ha⁻¹) + mineral fertilizers (68, 60, 150 kg·N·ha⁻¹); SMS₃ + N₁PK—spent mushroom substrate (20 Mg·ha⁻¹) + mineral fertilizers (30, 60, 150 kg·N·ha⁻¹); ** means marked with the same small letters in columns (a, b, c) do not differ significantly at p ≤ 0.05 for treatments, treatments × years, treatments × harvests.

), which turned out to be similar to total protein content. The most digestible protein was in plants from the SMS₃ + N₁PK plot. This amount was 21% higher than in grass treated exclusively with mineral N₄PK. The amounts of digestible protein had an upward trend as the dose of mushroom substrate nitrogen increased and the dose of mineral nitrogen decreased. The data also showed the interaction of this parameter with other experimental factors.

3.3. Crude Fiber

No significant interaction between crude fiber content and experimental factors was found (

Table 5. The effect of fertilizer treatment, growing period and growth cycle on Festulolium braunii crude fiber content (g·kg⁻¹DM).

Growing Period	Growth Cycle	Treatment					Means
		*O	N4PK	SMS1 + N3PK	SMS2 + N2PK	SMS3 + N1PK
2017	I	256.0	253.9	266.9	300.7	288.8	273.3
	II	287.0	280.6	289.4	289.6	287.0	286.7
	III	315.0	276.5	279.8	289.4	301.8	292.5
	Means
		286.0	270.3	278.7	293.2	292.5	284.2
2018	I	273.0	279.8	299.8	298.1	307.5	291.6
	II	314.4	299.0	310.8	319.5	289.2	306.6
	III	309.5	312.8	279.8	314.6	306.5	304.6
	Means
		298.9	297.2	296.8	310.7	301.1	300.9
	Means across growing periods
		292.5	283.8	287.7	301.9	296.8	292.5
	Means across growth cycles
	I	264.5	266.8	283.3	299.4	298.1	282.4
	II	300.7	289.8	300.1	304.5	288.1	296.6
	III	312.2	294.6	279.8	302.0	304.1	298.6

*O—control (with no treatment); N₄PK—mineral fertilizers (180, 60, 150 kg N·ha⁻¹; SMS₁ + N₃PK—spent mushroom substrate (10 Mg·ha⁻¹) + mineral fertilizers (105, 60, 150 kg N·ha⁻¹); SMS₂ + N₂PK—spent mushroom substrate (15 Mg·ha⁻¹) + mineral fertilizers (68, 60, 150 kg N·ha⁻¹); SMS₃ + N₁PK—spent mushroom substrate (20 Mg·ha⁻¹) + mineral fertilizers (30, 60, 150 kg N·ha⁻¹).

). Its greatest amounts were recorded in the grass from the plot with 15 Mg·kg⁻¹ of mushroom substrate supplemented with mineral nitrogen in the N₂PK dose. It was 6% more than the lowest content recorded in grass treated only with mineral N₄PK. Festulolium fiber content (average across treatments and harvests) was 5% higher in the second year than in the first. Statistically insignificant differences were found across harvests. In the third harvest, a 5% higher content of crude fiber was recorded than in the first.

3.4. Digestibility of Dry Matter

The digestibility of Festulolium braunii forage (

Table 6. The effect of fertilizer treatment, growing period and growth cycle on Festulolium braunii digestibility (% DM).

Growing Period	Growth Cycle	Treatment					Means
		*O	N4PK	SMS1 + N3PK	SMS2 + N2PK	SMS3 + N1PK
2017	I	72.28	72.53	70.97	66.92	68.34	70.21
	II	68.56	69.33	68.27	68.25	68.56	68.59
	III	65.20	69.82	69.42	68.27	66.78	67.90
	Means
		68.68	70.56	69.55	67.81	67.89	68.90
2018	I	70.24	69.42	67.02	67.23	66.10	68.00
	II	65.27	67.12	65.70	64.66	68.30	66.21
	III	65.86	65.46	69.42	65.25	66.22	66.44
	Means
		67.12	67.33	67.38	65.71	66.87	66.88
	Means across growing periods
		67.90	68.95	68.47	66.76	67.38	67.90
	Means across growth cycles
	I	71.26	70.97	68.99	67.07	67.22	69.10
	II	66.91	68.22	66.98	66.45	68.43	67.40
	III	65.53	67.64	69.42	66.76	66.50	67.17

*O—control (with no treatment); N₄PK—mineral fertilizers (180, 60, 150 kg N·ha⁻¹; SMS₁ + N₃PK—spent mushroom substrate (10 Mg·ha⁻¹) + mineral fertilizers (105, 60, 150 kg N·ha⁻¹); SMS₂ + N₂PK—spent mushroom substrate (15 Mg·ha⁻¹) + mineral fertilizers (68, 60, 150 kg N·ha⁻¹); SMS₃ + N₁PK—spent mushroom substrate (20 Mg·ha⁻¹) + mineral fertilizers (30, 60, 150 kg N·ha⁻¹).

) was not significantly affected by the research factors. The greatest but statistically insignificant digestibility increase was recorded in plants treated with mineral fertilizers only. This was only 3% higher than the lowest value recorded on the plot where 15 Mg·ha⁻¹ of mushroom substrate together with the N₂PK mineral fertilizer dose was applied (SMS₂ + N₂PK). The digestibility of the grass (average from treatments and harvests) was 3% higher in the first year of research than in the second. Forage from the first harvest was the most digestible.

3.5. Net Energy of Lactation (NEL)

The net energy of lactation (NEL) of Festulolium braunii (

Table 7. The effect of fertilizer treatment, growing period and growth cycle on Festulolium braunii net energy of lactation (MJ·kg⁻¹DM).

Growing Period	Growth Cycle	Treatment					Means
		*O	N4PK	SMS1 + N3PK	SMS2 + N2PK	SMS3 + N1PK
2017	I	5.61	5.64	5.55	5.37	5.47	5.528
	II	5.44	5.48	5.43	5.45	5.48	5.456
	III	5.27	5.50	5.53	5.46	5.36	5.424
	Means
		5.44	5.54	5.50	5.43	5.44	5.47 A ***
2018	I	5.51	5.48	5.34	5.37	5.38	5.42
	II	5.26	5.37	5.28	5.26	5.46	5.33
	III	5.30	5.28	5.51	5.29	5.35	5.35
	Means
		5.36	5.38	5.38	5.31	5.39	5.36 B
	Means across growing periods
		5.40	5.46	5.44	5.37	5.42	5.42
	Means across growth cycles
	I	5.56 a **	5.56 a	5.44 a	5.37 a	5.42 a	5.47 A
	II	5.35 a	5.42 a	5.35 a	5.35 a	5.47 a	5.39 B
	III	5.28 b	5.39 ab	5.52 a	5.37 ab	5.35 ab	5.38 B

*O—control (with no treatment); N₄PK—mineral fertilizers (180, 60, 150 kg N·ha⁻¹; SMS₁ + N₃PK—spent mushroom substrate (10 Mg·ha⁻¹) + mineral fertilizers (105, 60, 150 kg N·ha⁻¹); SMS₂ + N₂PK—spent mushroom substrate (15 Mg·ha⁻¹) + mineral fertilizers (68, 60, 150 kg N·ha⁻¹); SMS₃ + N₁PK—spent mushroom substrate (20 Mg·ha⁻¹) + mineral fertilizers (30, 60, 150 kg N·ha⁻¹); ** means marked with the same small letters in columns (a, b) do not differ significantly at p ≤ 0.05 for treatments × harvests; *** means marked with the same capital letters in rows (A, B) do not differ significantly at p ≤ 0.05 for years and harvests).

) was significantly dependent on the growing period and harvest. The form and dose of nitrogen did not significantly affect it, with a slight increase of 2% as a result of N₄PK mineral treatment compared to the effect of the mushroom substrate at a dose of 15 Mg·ha⁻¹ supplemented with N₂PK. A 2% higher value of this parameter was recorded in the first year than in the second. Additionally, across treatments and years of research, a 2% higher concentration of NEL energy was recorded in the forage of the first harvest than in the third. The data also showed a significant interaction between treatments and harvests.

3.6. Energy Yield

A significant impact of all factors on the energy yield was observed (

Table 8. The effect of fertilizer treatment, growing period and growth cycle on Festulolium braunii energy yield (JP·ha⁻¹).

Growing Period	Growth Cycle	Treatment					Means
		*O	N4PK	SMS1 + N3PK	SMS2 + N2PK	SMS3 + N1PK
2017	I	3932	4808	4856	5700	5056	4833 A ***
	II	3891	5149	4708	5758	5694	4765 A
	III	3671	4734	4234	5559	5199	4893 A
	Means
		3831	4898	4599	5672	5316	4830 A
2018	I	3565	5003	5684	6681	7063	5647 A
	II	3194	3968	4646	4549	4356	4143 B
	III	2891	3268	3926	4199	4019	3663 B
	Means
		3217	4080	4752	5143	5146	4484 B
	Means across growing periods
		3524 b	4488 ab	4675 a	5407 a	5231 a **	4657
	Means across growth cycles
	I	3749	4906	5271	6191	6059	5240 A
	II	3543	4559	4677	5154	5025	4454 B
	III	3281	4001	4081	4879	4609	4278 B

*O—control (with no treatment); N₄PK—mineral fertilizers (180, 60, 150 kg N·ha⁻¹; SMS₁ + N₃PK—spent mushroom substrate (10 Mg·ha⁻¹) + mineral fertilizers (105, 60, 150 kg N·ha⁻¹); SMS₂ + N₂PK—spent mushroom substrate (15 Mg·ha⁻¹) + mineral fertilizers (68, 60, 150 kg N·ha⁻¹); SMS₃ + N₁PK—spent mushroom substrate (20 Mg·ha⁻¹) + mineral fertilizers (30, 60, 150 kg N·ha⁻¹); ** means marked with the same small letters in columns (a, b) do not differ significantly at p ≤ 0.05 for treatments; *** means marked with the same capital letters in rows (A, B) do not differ significantly at p ≤ 0.05 for years, years × harvests).

). The form and dose of nitrogen caused changes in the value of this parameter. The highest forage energy yield was on the plot where 15 Mg·ha⁻¹ of the mushroom substrate was supplemented with mineral N₂PK. It was 17% higher than for plants treated with mineral N₄PK only. The energy yield of Festulolium braunii in the first year was 13% higher than in the second. A comparison of harvest values, across treatments and growing periods, showed an 18% increase in the first harvest compared to the third.

4. Discussion

Grass-based feeding systems of ruminant animals are common in many countries with temperate climates [26]. The growth of the world’s human population entails the need to increase meat and milk production [27]. To meet this challenge, it is necessary to intensify grass-based forage production and, at the same time, reduce its possible negative impact on the environment [28]. This explains the aim of the present research and its hypotheses. Grassland forage is an important part of the diet of ruminants [29,30] because it is much cheaper than concentrates [31]. Grass-based beef and milk production systems are cost-effective if high yields and the good nutritional value of grass are efficiently used throughout the growing period [32].

As a short-lived species perfectly suited for field cultivation, Festulolium braunii can produce yields of 11.0–15.0 Mg·ha⁻¹DM [33,34,35]. According to Adamovies et al. [36], Obraztsov et al. [37] and O’Connor et al. [38], it is a species strongly responsive to nitrogen fertilizer, also in an organic form.

The impact of fertilizer treatment on grass yield is pointed out by many authors, such as Nyfeler et al. [39], Weigelt et al. [40], Vogel et al. [2] and Yin et al. [41], some of whom also argue that the frequency of harvesting is of great importance. In the present studies, an increase in the amount of nitrogen contained in the mushroom substrate (SMS₂) resulted in an increase in the yield of grass by 16% compared to the plot with mineral nitrogen at the full dose (N₄PK). According to Peyraud and Astigarrag [42], annual doses of mineral nitrogen can range from 200–250 kg·ha⁻¹ to 50–100 kg·ha⁻¹. Hennessy et al. [43] report that its high doses can increase the concentration of crude protein in the grass to an amount that may exceed animals’ dietary requirements, especially in the case of beef cattle [44].

When feeding ruminants with roughage, digestibility and total protein content are very important. Grygierzec [45] states that the minimum concentration of total protein in forage should range from 150 to 170 g·kg⁻¹DM. The protein content of grasses is a species-specific characteristic. Festulolium braunii contains about 130 g·kg⁻¹DM, which is less than in some other grasses. Kotlarz et al. [46] indicate that protein content increases with increased nitrogen doses. In turn, Adamovies and Gutmane [47] have found that an increase in a nitrogen dose contributes to an increase in protein digestibility. In the present research, Festulolium braunii responded to increased doses of nitrogen contained in the mushroom substrate with a significant increase in total protein content, in relation to the effects of mineral nitrogen in N₄PK.

Mushroom substrate nitrogen at the highest dose (SMS₃) resulted in an increase in total protein by 17% compared to the amount in the grass treated with mineral nitrogen. Similarly, nitrogen affected protein digestibility, and its largest dose in the organic form increased it by as much as 21% compared to plants treated with mineral fertilizers. The results indicated that mushroom substrate nitrogen had a more beneficial effect on this parameter than mineral nitrogen, which confirms the research hypothesis. This fact is undoubtedly of great practical and economic importance because it means that mushroom substrate application results in a favorable content of forage well-digestible protein.

The present studies did not show a significant effect of the growing period and harvest on total and digestible protein content. Grant et al. [48] highlighted the significant impact of precipitation or droughts during the growing period on plant protein concentration. In turn, Tonn et al. [49] proved the effect of harvest time on grass total protein content.

The crude fiber content of Festulolium braunii did not significantly vary in response to experimental factors. In relation to mineral nitrogen, a slight increase was observed on plots with mushroom substrate (an increase of 6% on the SMS₂ + N₂PK plot). In the studies on Festulolium braunii conducted by Wiśniewska-Kadżajan and Stefaniak [50], there was also no significant response of fiber content to organic treatment.

Ranging from 65.0 to 72.0%, the digestibility of Festulolium braunii forage did not change significantly in response to the experimental factors. The values were comparable to forage digestibility reported by Cougnon et al. [51]. In addition, in other studies by Cougnon et al. [52] high resistance of this hybrid to droughts was recorded, which significantly determined its value.

According to Krzywiecki et al. [53], the net energy of lactation (NEL) concentration needed for the proper development of animals should be at the level of 6MJ·kg⁻¹DM. In the present experiment, Festulolium braunii turned out to be medium quality forage with a NEL ranging from 5.0 to 5.5 MJ·kg⁻¹ [54]. Nitrogen application did not significantly affect the NEL value, which was confirmed by the research of Adamovies and Gutmane [47]. However, its significant variation across harvests was observed, confirming the findings of Peratoner et al. [55]. In other reports by the same authors [56], there was no significant effect of the temperature-humidity factor (growing period) on NEL concentration, which was also found in the present research.

The energy yield of the grass was significantly dependent on all research factors. The medium dose of the mushroom substrate (SMS₂) supplemented with mineral fertilizers in the N₂PK dose contributed to the highest energy yield of Festulolium braunii although its total and digestible protein content from this plot was lower. The results also proved the importance of growing periods and their interaction with harvests in shaping forage energy values, confirmed by the research of Abas et al. [57] and Ciepiela et al. [58].

The results showed that the nitrogen contained in the mushroom substrate had a positive effect on the amount of Festuloliun braunii biomass, its total and digestible protein content as well as its energy efficiency. However, the interaction of both forms of nitrogen did not have a positive effect on crude fiber content, dry matter digestibility and net concentration of lactation energy (NEL). Replacing mushroom substrate nitrogen with mineral nitrogen would certainly improve those parameters, but it would not solve the problem of constantly increasing amounts of this waste. The possibility of using mushroom substrate to reduce the deepening deficit of soil organic matter is of great importance for the soil biophysical environment [10,11]. Partially confirming the research hypotheses, the results indicate that it is necessary to constantly search for new ways and possibilities of using nitrogen from organic waste materials for both environmental and economic reasons [12,13,59].

5. Conclusions

The treatment used in the experiment significantly affected the yield of Festulolium braunii, its content of total and digestible protein, as well as the energy yield. Increased doses of mushroom substrate nitrogen resulted in an increase in the above-mentioned parameters, which proves that not only the amount of nitrogen, but also its form is important for yield and nutritional value.

The growing period had a significant impact on the yield of Festulolium braunii, NEL concentration and energy yield. In the 2017 growing period, significantly more NEL and a higher energy yield were recorded than in 2018. In turn, in 2018, as a result of more favorable weather conditions, significantly more biomass was produced than in 2017.

The growth cycle (harvest) significantly affected the yield of the grass, NEL concentration as well as energy yield. The highest biomass yield, NEL concentration and energy yield were recorded in the first harvest, and the lowest in the third.

The results partially confirmed the research hypotheses. The interaction of nitrogen in the mushroom substrate with mineral nitrogen resulted in more favorable forage quality compared to the effects of mineral nitrogen applied with phosphorus and potassium (N₄PK). The application of nitrogen in the mushroom substrate at the highest dose (SMS₃—20 Mg·ha⁻¹) supplemented with the lowest dose of mineral nitrogen (N₁PK) resulted in the highest concentration of total protein and digestible protein. The yield of Festulolium braunii, as well as crude fiber content and energy efficiency was affected the most by the medium dose of nitrogen in the mushroom substrate (SMS₂—15 Mg·ha⁻¹) supplemented with mineral nitrogen also at the medium dose (N₂PK). The highest concentration of NEL and the highest digestibility were recorded in plants from plots treated exclusively with mineral fertilizers (N₄PK), which did not confirm the research hypothesis concerning those parameters.

The results indicate that it is possible to replace, at least partially, mineral nitrogen with nitrogen contained in the mushroom substrate. Although the studies did not demonstrate a beneficial effect of the latter on all forage parameters, further research should be conducted on the possibility of using nitrogen on grass to maintain good forage quality.