Utilisation of waste wool from mountain sheep as fertiliser in winter wheat cultivation

ABSTRACT Coarse wool obtained from mountain sheep has low economic value and is treated as a troublesome by-product of sheep farming. To find ways to utilize it, waste separated from the better-quality wool during sheep shearing was used as a fertilizer in winter wheat cultivation. During the preliminary tests, it was stated that unwashed wool does not contain excessive amounts of heavy metals and environmentally unfriendly contaminations and can be safely used as a fertilizer. Then, the raw wool was cut into shorter segments and mixed with the soil, and its influence on winter wheat growth during two seasons was examined. Simultaneously, the progress of wool biodegradation and the nitrogen content in the soil were analyzed. It was stated that, during vegetation, nitrogen compounds are slowly released into the ground, and the content of nitrogen in the soil is strictly correlated with the progress of wool biodegradation. Released nitrogen positively impacts wheat growth in various stages, which is manifested by higher tillering degree, more intense leaf color, higher stems and finally, greater yield. Wool added into the soil reveals its positive influence on wheat development up to two harvests. Mountain sheep wool can be successfully used as a nitrogen-rich, organic fertilizer in organic farming. This enables utilization of coarse wool, which is not suitable for textile processing, according to the zero-waste strategy.


Introduction
Sheep wool is one of the natural fibers that are well known to mankind since ancient times.For centuries, it has been used to produce carpets, blankets, apparel and interior textiles.For a long time, wool was the most valuable sheep product that generated the highest income for the farmers.In the last two decades, wool prices decreased considerably and production of wool has become less profitable.In Poland and some other European countries, the cost of sheep shearing has outweighed the price of wool.In this situation, large volumes of this material became underrated, underused and often treated as a troublesome by-product of sheep husbandry.The lack of interest from the industry or craft sector applied especially to coarse wool obtained from local sheep breeds (Haugrønning et al. 2022).Nowadays, this wool is often stored unscoured on farms, discarded into the woods or deposed in local, not always legal, landfills.
In addition to the above-mentioned, use of wool in agriculture and horticulture as mulch, soil amendment or fertilizer (Petek and Marinšek Logar 2021;Sharma, Sahoo, and Chand 2019) has been gaining much interest.
As a mulch, it is spread on the soil surface around trees, shrubs, or plants.During plant growth, it works as thermal insulation, limiting the daily and seasonal temperature fluctuations.Additionally, it reduces evaporation of water from the soil surface, retains moisture and suppresses the growth of weeds.Compared to other organic materials, wool ensures better insulation and moisture maintenance.Moreover, wool is more resistant to biodegradation than other mulch organic materials and can be used longer, even for two vegetation seasons (Bijalwan and Singh 2020;Jungić, Turk, and Benko 2020).
Mixed with soil, wool improves its physical properties, retains water and serves as a source of nutrients necessary for plant growth.It notably influences the soil bulk density, increases its porosity and stability.Due to its chemical structure and the presence of numerous and available hydrophilic groups, wool attracts water molecules easily.In the soil, it effectively absorbs and retains moisture, facilitating water conservation (Kadam et al. 2014).Additionally, wool improves the infiltration capacity and hydraulic conductivity of the soil, contributes to a deeper drainage and reduces the risk of soil erosion (Abdallah et al. 2019;Mubarak et al. 2009;Zoccola et al. 2015).
As a protein fiber, wool is composed of carbon (ca.50%), nitrogen (15%-16%) and sulfur (3%-4%) -elements that play an essential role in plant nutrition.It also contains several trace elements (iron, cobalt, copper, manganese, molybdenum and zinc) which are equally important for plant growth (Healy, Bate, and Ludwig 1964).In a humid soil environment, wool keratin decomposes into shorter water-soluble peptides and into particular amino acids in the final stage (Broda et al. 2016;Korniłłowicz-Kowalska and Bohacz 2011).As a result, nitrogen-rich compounds are gradually released into the soil.Then, the compounds undergo ammonification and nitrification whereby organic nitrogen is gradually transformed into ammonium and nitrate forms.These forms are available to plants and serve as nutrients promoting their intense growth (Bernhard 2010;Broda and Gawlowski 2018).
In contrast to conventional mineral fertilizers, nitrogen-rich compounds are delivered to plants systematically and slowly.Consequently, nutrients are supplied over a long time during the whole vegetation season, even for the following two to five harvests.The gradual release of the nitrogen compounds is also favorable to the environment and protects water resources from contamination due to excessive nitrogen content.
Studies on the fertilizing effect of wool were carried out both for field (Abdallah et al. 2019) and greenhouse crops (Górecki and Górecki 2010;Zheljazkov et al. 2009).The investigations were performed mainly for the waste wool generated in various stages of wool processing and recycled fibers obtained from shredding post-consumer waste.The literature presents studies into the application of unprocessed and unwashed wool obtained by sheep shearing (Zheljazkov 2005), wool recycled from shredded carpets (McNeil, Sunderland, and Zaitseva 2007), waste derived from scouring and carbonization (Abdallah et al. 2019) and wool sheared from animals intended for slaughter (Mubarak et al. 2009).
Investigations presented herein focus on the coarse wool obtained from sheep raised in Polish mountains.The waste which remained after separating better quality material to be used in a smallscale technological line to spin rug yarns was applied as a fertilizer (Kobiela-Mendrek et al. 2022).For safety reasons, the concentration of heavy metals in the greasy wool was assessed during the preliminary tests.Then, the wool collected right after sheep shearing was cut into short segments and mixed with the soil in a field.The fertilizing efficiency of the wool and its impact on winter wheat growth during two vegetation seasons were examined.Simultaneously, the progress of wool destruction caused by biodegradation in actual conditions in the soil was monitored and the changes in nitrogen content in the soil were analyzed.

Materials
The wool was obtained from mountain sheep bred in southern Poland, in the Beskid Mountains -the westernmost part of the Carpathian range.The material used for the examinations was collected during summer shearing and comprised waste fibers separated from the better quality wool designed for yarn spinning.The waste was a mixture of fibers with different thickness of 35-65 μm, which included heavily contaminated and partially felted wool as well as short wool sheared from sheep legs, tails and heads.To avoid problems with wool-soil compounding, the unwashed wool was cut with a prototype cutting device into short, few-millimeter long segments.
The fertilizing efficiency of wool waste was examined during field tests carried out on winter wheat crops.The tests were performed in a field with arable soil of average quality with pH of 5.5, in a hilly area.The experimental site was situated in southern Poland at an altitude of 360 m above sea level, in a temperate climate zone, at the foothills of the Beskids.The field was prepared with a low tillage system and a field cultivator.Four experimental plots of 10 m 2 each were marked.One was the control plot and three were plots with different amounts of wool: 0.5%, 1%, and 2% to the weight of the 15 cm thick soil layer.The cut wool was spread out on the surface of each plot and mixed with soil using a rotary tiller.The entire field was then sown with winter wheat, the Kilimanjaro variety, in the amount of 250 kg/ha.The preparatory works and sowing were performed in November 2020, at the end of the Polish vegetation season.In spring, the land around the experimental plots was fertilized with a mineral nitrogen fertilizer (ammonium sulfate), in the amount of 120 kg/ha.
In the following year (2021), four new experimental plots were established on the other side of the field.Like in the previous year, the cut wool was spread on the soil surface and the mixing procedure was repeated.In the next stage, the whole field including both old and new plots was sown with the Sacramento variety of winter wheat.

Methods
In order to measure heavy metal concentration, 0.25 g of the wool sample was weighed out, placed in mineralization vessels, flooded with 8 mL of concentrated nitric acid and 2 mL of perhydrol, covered with lids and left overnight under a fume cupboard.Then, the vessels were tightly screwed together and subjected to microwave mineralization in the Anton Paar Multiwave 3000 solv mineralizer.After mineralization, the samples were quantitatively transferred through hard filters to 50 mL volumetric flasks and made up to the mark with deionized water.The samples were then analyzed by means of Inductively Coupled Plasma Mass Spectrometry (ICP-MS), using the Elan 6100 DRC-e ICP-MS spectrometer (Perkin Elmer, Waltham, MA, USA).
Changes in the fiber morphology, resulting from cutting and biodegradation of wool in the soil, were studied with Scanning Electron Microscopy (SEM).The studies were carried out for raw cut fibers and fibers taken from sites distributed randomly in the experimental plots after harvest, at the end of the first and second vegetation season.The fibers taken from the soil were first dried and mechanically cleaned from soil particles.For the investigations, the electron microscope JEOL JSM 5500 LV operated in backscattered electron mode was applied.The microscopic observations were carried out for fibers sputtered with gold in the JEOL JFC 1200 ionic sputter.
During wheat growth, soil samples were periodically taken and the content of two nitrogen forms (NH 4 + and NO 3 − ) in the soil was determined.The measurements were carried out according to the standard research procedure of the Chemical and Agricultural Station.
Throughout the vegetation period, the wheat growing in the field was systematically monitored, while the tillering degree, height of stems and color of leaves were assessed.The measurements were carried out for 20 stems taken from points distributed randomly in the plots.The mean values of tillering degree and stem height with a 95% confidence interval were calculated for each plot.During the harvest, the yield of wheat was determined for each plot with different wool content, the control plot and other parts of the field fertilized with mineral fertilizers.

Heavy metal concentration
Concentration of six heavy metals in unwashed wool was measured (Table 1).For all metals, concentration values vary considerably between samples taken from particular animals.The differences observed are typical for unwashed wool due to its accidental contamination with exogenous contaminants, such as soil, feces and plant residues.
Concentration of all heavy metals measured in the investigated samples is relatively low.For comparison, concentration of heavy metals, determined by other authors, in wool taken from sheep raised in different countries is presented in Table 1.The trace amounts of all metals occurring in the wool from Polish mountains are significantly smaller than the values measured for wool from Greece and Syria, and even many times lower compared to the wool of sheep grazed in Morocco and Turkey in contaminated areas, i.e., near a mining area in the west of Marrakech (Morocco) and an industrial area in Van province (Turkey).The low lead content, which is even about 50 times lower than the lead concentration in wool from Turkey and Morocco, is particularly noteworthy.b Barkouch (Barkouch et al., 2008).c Tuncer (Tuncer 2019).

Fiber morphology
Cutting the wool into shorter segments made it easier to mix it with soil using the agriculture machine and obtain a uniform distribution of fibers in the soil.Simultaneously, cutting caused mechanical damage to the wool.As a result, numerous short segments of fibers with irregular cut ends were formed (Figure 1a).The scales at the fiber surface in the cutting zone were deformed and partly destroyed, while fibrils in the deeper-layered cortical cells were interrupted and partially pulled out.Additionally, cutting damaged the compact structure of the scales on the fiber surface.The scales were cracked, partially scuffed or even destroyed in many places.In certain sections, the scale fragments were bent or torn off and attached accidentally to the surface of the fibers.The detached fragments of the scales revealed the fibrillar structure of cortical cells (Figure 1b).Both, numerous cut fiber ends and the mechanical damage of the outer cuticle layer allowed enzymes to infiltrate the inner part of the fibers, which in turn facilitated the initiation of keratin decomposition.
After the harvest, the fibers pulled out from the soil were in various stages of biological destruction.The least damaged fibers contained slightly harmed scales and their pattern on the surface was visible (Figure 2a).The scales on more degraded fibers were destroyed, and the scale pattern was not observable.On some of these fibers, there were extensive and deep cavities digested by microorganisms (Figure 2b).In the medullated fibers, the cavities reached down to the inner medulla (Figure 2c).In many places, numerous isolated microorganisms or bigger microorganism colonies were attached to the damaged fibers.Biodegradation of some fibers was much more advanced and caused almost complete disintegration of the fiber structure.In this case, individual fibrils separated through defibrillation of keratin in the cortical cells were noticeable (Figure 2d).
At the end of the second vegetation season, the wool was barely visible in the soil.The remnants of fibers pulled out from the plots with the highest amount of wool showed many visible signs of destruction.The scales were no longer visible on any of these fibers.The outer cuticle layer was removed, and the inner cortical cells were exposed (Figure 3a).
The cortical layer of most fibers was heavily damaged and partially digested by microorganisms.Large cavities in the material of certain fibers were observed (Figure 3b).

Nitrogen content in soil
During the field tests, the control plot was not supplied with any fertilizer so the nitrogen content was kept under the measuring limit during the whole wheat vegetation period.In the plots containing wool, the contents of ammonia N-NH 4 + (Figure 4a) and nitrate N-NO 3 − (Figure 4b) nitrogen forms were significantly higher.Higher nitrogen content was observed already before the winter, shortly after the wool had been added to the soil.In December 2020, 1 month after mixing wool with the soil, the content of mineral nitrogen in the plot with the highest wool concentration was almost three times higher than in the control plot.Few months later, in late spring in May 2021, the content of both nitrogen forms in the wool-fertilized plots was significantly higher.For small wool amount, the content of both mineral nitrogen forms was two times higher, while for a higher amount of wool, the content of nitrogen was remarkably high, almost twenty-fold higher compared to the control plot.At the beginning of the second vegetation season, in  April 2022, that is 18 months after mixing wool with the soil, the level of nitrogen in the plots was still high.
It was lower compared to the first vegetation season but still much higher than in the control plot.After the second harvest in September 2022, in plots with less wool added, the content of ammonia dropped to the level recorded for the control plot.For higher wool concentration, a slightly elevated level of ammonia was maintained.At the same time, the content of nitrate was high, twice as high as in the control plot.
The impact of wool on the nitrogen content in the soil was confirmed in the new plots established in the second experimental year.Similar to the first year, addition of wool caused significant increase in nitrogen content in the soil.After the harvest in September 2022, approximately 9 months following mixing wool with soil, ammonia content was several times higher than in the control plot.The increase in nitrate content was much higher and grew significantly with increasing wool concentration (Table 2).

Growth of wheat
During field monitoring, the impact of wool on wheat development at different stages of growth was observed.In the initial stage of tiller development, the addition of wool positively influenced the tillering degree.In the experimental plots established in the first year for the Kilimanjaro variety, the greater amount of wool, the higher number of tillers, so for the highest wool content (2%) the average tillering degree was two times greater than in the control plot (Table 3).
In spring, during stem elongation, wheat growing in the soil with wool had dark green leaves and higher stems.This intense green color was visible throughout the entire growth period and was distinguishable from the pale green of the wheat growing in the control plot.The color intensity increased noticeably with the greater amount of wool.Wheat stems growing on the plots with wool were significantly higher.At the end of the head development stage, the stems grown in the plot with small amount of wool added (0.5%) were 15 cm higher than in the control plot.For higher wool concentration, the increase in stem height was greater and for the highest concentration (2%), it was as much as 20 cm (Figure 5).
In addition to positive impact on grain filling, the influence of wool on both tiller development and stem elongation resulted in increased wheat yield.The yield from plots containing wool was considerably greater.The greater amount of wool added, the greater was the yield from the experimental plots.For the highest wool content (2%), the yield level was three times higher compared to the control plot not fertilized with any fertilizers.The yield obtained from the experimental plot containing 2% of wool was also 20% bigger than the yield achieved in other parts of the field fertilized in spring with conventional mineral fertilizer.
In the second year, during the experiments performed for the Sacramento variety, similar influence of the wool on wheat development was observed.In the first year, the higher tillering, more intense green color of leaves, higher stems and greater wheat yield were detected.The impact of wool on wheat growth was well visible both in newly established plots with fresh wool added and the plots containing remnants of wool from the previous growing season.The wheat grown in both experimental plots had more intense green coloring, taller stems and was distinguishable from the wheat in the other parts of the field (Figure 6).While comparing parameters for wheat grown in plots established in the first and the second year, there was no difference in the numbers of tillers (Table 4).A large variation was found regarding the stem height.Regardless of wool concentration, the wheat was approximately 10 cm higher in plots containing fresh wool added in the second year (Figure 7).The yield harvested in the plots with fresh wool was minimally higher.

Discussion
The wool investigated was obtained from the mountain sheep raised in environmentally friendly conditions, in the site-adapted pastoral system developed over centuries in harmony with the local climate and vegetation.The system involves seasonal migration of flocks between summer highland and winter lowland pastures (Salachna 2021).According to this system, the sheep graze in the top parts of the mountains in summer.In autumn, the animals are brought down to the village and when the first frost and snow appear, they are kept in the barns.In the mountain pastures, sheep use natural vast resources of green forage and in winter, they are fed with hay without any additions of ensilage or grain food.
The mountains are far from big urban centers and motorway networks, at a safe distance from great heavy metal emitters located in Polish and Czech industrial zones.Due to such favorable location, heavy metals are absorbed from the air in minimal amounts.Concentration of heavy metals in the wool is low, it does not exceed values measured for sheep raised in different parts of the world and is much lower than the concentration of metals detected in the wool of sheep grazed in contaminated industrial and mining regions (Pereira et al. 2021;Shen, Chi, and Xiong 2019;Tuncer 2019).The wool contains low amounts of lead and cadmium, the most toxic metals that pose a serious threat to the  functioning of ecosystems and human health.The sheep are not sprayed with insecticides against lice, mites or ticks during the whole grazing season.Wool used for the tests was taken directly after shearing and was not subject to any additional chemical or environmentally unfriendly treatment.The examinations revealed that the fibers did not contain any excess of heavy metals, environmental contaminants or residues of other harmful substances.The wool fully complied with the EU Regulation No. 1069/2009 regarding health rules for animal by-products and can be safely applied as a clean soil additive and fertilizer.
The fertilizing ability of wool results mainly from the presence of nitrogen, which is a major constituent of wool keratin.Nitrogen is a vital nutrient in wheat cultivation, essential to the synthesis of proteins, nucleic acids, chlorophyll, vitamins, enzymes and several other organic compounds.Nitrogen fertilizers promote tillering, stimulate the increase in plant height and head size, and contribute to a greater wheat yield.The high demand for nitrogen begins right after sowing, in the tillering stage, and remains during the next phases of wheat growth: stem elongation and grain filling (Liu and Shi 2013;Zhang et al. 2020).
The nitrogen needed for wheat growth is acquired mainly from the soil.In the soil mixed with wool, the nitrogen content is strictly correlated with the progress of wool biodegradation.In the beginning, fragmentation of wool fibers and the uniform distribution of short wool segments in the soil coupled with mechanical fiber damage ensures easier access to proteolytic enzymes.In this way, decomposition of wool keratin starts relatively quickly and the first portion of nitrogen-rich compounds is released into the soil in a short time.The nitrogen released in the first period, right after sowing and seed germination, is consumed in the tillering stage of wheat growth.The positive influence of the fibers added is manifested by the increase in the tillering degree.In the following months, the biodegradation of the wool proceeds and the amount of nitrogen in the soil gradually increases.In spring, nitrogen transformed in the soil into mineral forms promotes quick wheat growth and intense stem elongation.As a result, wheat stems grow higher and their leaves take on an intense green color.During stem elongation and later, at the stage of head development, further wool biodegradation results in a slow release of the next portions of nitrogen.Due to it, the nitrogen present in the soil is not quickly lost through leaching, runoff and denitrification.The required amounts of nitrogen remain available for the plants and can be systematically consumed during wheat development.Once the head development is completed, especially in plots with bigger wool doses, the keratin decomposition is highly advanced and nitrogen content in the soil is still pretty high.The amount of nitrogen is sufficient to support grain filling to ensure a high wheat yield.
At the end of the first vegetation season -after the harvest, the wool biodegradation is not complete and the amount of nitrogen remaining in the soil is still relatively high.Even though the majority of fibers are heavily damaged, many of them maintain their integrity, at least partially.Damaged fibers and their remnants still contain a considerable amount of keratin, which is further decomposed into smaller nitrogen-rich organic compounds.These compounds are later transformed into mineral forms and serve as a nitrogen supply for wheat growth in the next vegetation season.In this way, wheat crops can be supplied afresh without the necessity to add extra portions of fertilizer.
In comparison to highly commercial crops, the grain yield achieved during the investigations is relatively low.The yield achieved in both vegetation seasons and for both wheat varieties is few times smaller than the yield from fertile lands.Nevertheless, taking into account the foothill area, infertile soil and unfavorable climatic conditions, the yield level is satisfactory and comparable with other wheat crops in the area.The yield achieved in the experimental wool-fertilized plots was much higher than the yield obtained in the control plot with no fertilizers.In addition, it was considerably higher compared to the rest of the field fertilized with conventional mineral fertilizers.

Conclusions
Wool from mountain sheep, which is not suitable for textile processing and treated as troublesome and useless waste, was successfully applied as an organic fertilizer for winter wheat.The addition of wool into the soil is beneficial for wheat growth and productivity.Fibers cut into shorter segments can be easily mixed with the soil and provide nitrogen supply shortly after seeding.In the consecutive months, as a result of keratin decomposition, the nitrogen compounds are permanently and slowly released, providing nitrogen needed in the successive stages of wheat growth.The wool is not completely decomposed after the harvest.The remnants of fibers still contain a significant amount of nitrogen, which is further released into the soil.Released nitrogen supports re-growth of wheat and ensures high yield in the second vegetation season, with no new fertilizers added.
The application of waste wool to fertilize wheat crops is a reasonable solution to the problem of utilization of wool remnants according to a zero waste strategy.Wool used in small fields located close to sheep farms ensures better utilization of local resources.In a small-scale agriculture, the application of wool reduces the usage of mineral fertilizers and helps avoid several environmental problems connected with their production and application.Wool serves as an environmentally friendly, organic, nitrogen-rich fertilizer (nitrogen content several times higher than in most other organic fertilizers) and ensures sustainable plant cultivation.
The positive influence of wool fertilizer on wheat growth creates an opportunity to maximize valorization of coarse underutilized waste wool and its wider application in organic farming for fertilization of other agricultural and horticultural crops grown in other soils, in other climatic conditions.

Figure 1 .
Figure 1.Mechanical damage of wool fibers caused by cutting; a/cut end of fiber; b/damaged scales on fiber surface.

Figure 2 .
Figure 2. Progressive biodegradation of wool fibers at the end of first vegetation season; a/fibers with slight degree of damage; b/fibers with destroyed cuticle layer; c/fibers with cavities digested by microorganisms; d/fibers defibrillated into separate fibrils.

Figure 3 .
Figure 3. Biological damage of wool fibers at the end of second vegetation season; a/damaged fibers lacking outer cuticle layer; b/cavities in damaged fibers.

Figure 6 .
Figure 6.Field of winter wheat Sacramento variety at the stage of stem elongation with marked plots established in: 1/first year; 2/second year.

Figure 7 .
Figure 7. Stems of wheat Sacramento variety grown in plots with different wool content established in first (left) and second (right) year at the end of heading stage.

Table 1 .
Content of heavy metals in unwashed wool.

Table 3 .
Growing parameters for winter wheat crops Kilimandzaro variety in first vegetation season.
Figure 5. Stems of winter wheat Kilimandzaro variety grown in plots with different wool content at the end of heading stage.

Table 4 .
Growing parameters for winter wheat crops of Sacramento variety in second vegetation season.