1. Introduction
Although the application of phosphorus (P) fertilisers is accompanied by a number of negative environmental effects [
1], modern agricultural production cannot do without them, and it is hard to imagine that this dependence will change if we take into account the biological importance of phosphorus [
2] and the need to meet the food needs of the fast-growing world human population [
3]. Mogollón et al. [
4] forecast that the global P input by fertilisers in croplands will increase from 14.5 million tonnes per year in 2005 to 22–27 million tonnes per year in 2050, and 4–12 million tonnes per year would be needed in 2050 for global intensively managed grasslands to maintain fertility.
The issues of P scarcity, its squandering and environmental pollution with P compounds call for a sustainable, circular economy of this element, which is based on the prudent use of mined resources, limited P accumulation in agricultural soils and enhanced P use efficiency and recycling [
5].
There have been attempts to replace non-renewable phosphate rocks with P-rich secondary raw materials [
6]. The attention of scientists and technologists has been focused on sewage sludge ash (SSA). The P content in dry matter of SSA ranges from less than 10% to less than 20% [
7], which is comparable to the content of this element in commercial phosphate rock (10.9–16.13% P) as reported by the International Fertiliser Development Centre [
8]. According to recent estimates, the annual global production of SSA is about 1.7 million tonnes and is expected to increase in the future [
9]. As the treatment of wastewater and management of process by-products are now another major global issue [
10], the use of SSA as a fertiliser may also alleviate this dilemma. Some EU countries have already introduced or intend to introduce a mandatory legal obligation to recover P from sewage sludge and slaughterhouse waste [
11].
The direct application of SSA into the soil would be the simplest and cheapest recycling method, but the raw material may contain significant amounts of toxic elements [
12]. European Directive 87/278/CEE establishes limit values for the concentration and annual load for specific elements, which are often exceeded in SSA. Moreover, some countries have even stricter limits which hinder the reuse of SSA without pre-treatment [
12]. It should also be noted that P contained in SSA is not plant-available due to its strong bonds in ash minerals [
13].
In numerous scientific centres, research on the use of SSA as a raw material for fertiliser production has been carried out [
7,
14,
15,
16,
17]. Many products were tested for their agronomic utility, but primarily plant availability and crop-enhancing efficiency were evaluated, mainly in pot experiments [
18]. Although the results appear promising [
14,
17,
19], some weak points of SSA-based fertilisers, e.g., low solubility of P compounds, were also reported [
16,
18].
The inclusion of phosphorus-solubilising microbes (PSM) into SSA-based fertilisers is an innovative approach, initiated to activate P from raw material [
20].
Bacillus megaterium has been found to be one of the most effective PSM [
21]. Generally, this strain is a component of soil edaphon but it is characterised by high ecological plasticity and has been found in different environments [
22]. The biology of
B. megaterium is quite well known. P solubilisation by these bacteria is performed thanks to the production of weak organic acids [
23]. Through solubilisation and other biological mechanisms, PSM can also work as a plant growth-promoting microorganism (PGPM) [
24]. PSM, including
B. megaterium, were already used to increase the efficiency of P fertilisers from primary raw materials [
25] and they had been applied independently to mobilise soil P reserves [
26]. It could be expected that PSM introduced into soil along with fertiliser would also be involved in soil P solubilisation, which would allow to reduce the fertiliser dose. It is worth recalling that in some industrialised countries, soil P reserves (resulting from intensive P fertilisation in the 1970s and 1980s) may be substantial [
27]. One requirement for their use is to transform them into bioavailable forms [
4]. However, since the use of PSM and PGPM in agriculture as environmental-friendly tools to increase crop yield is becoming increasingly common, the issue of ‘non-target’ effects of these organisms on native soil microbial communities is gaining prominence. To date, this matter has been rarely studied [
28].
Whether recycling fertilisers, including the PSM-activated ones, will be able to replace or supplement traditional fertilisers will be determined not only by their effectiveness in providing satisfactory yields in terms of quantity and quality but also by the lack of significant changes in the soil environment following their use. With regard to the latter, there is little research, especially under field conditions.
A research consortium from Poland has collaborated on P fertiliser production technology using cheap renewable raw materials and PSM [
20]. One proposal is a suspension preparation from SSA with the addition of
B. megaterium bacteria (SSAB). Its performance was tested in field trials in comparison to traditional P-fertilisers. The results were compared to full chemical plant protection (+PP) and no plant protection (−PP). It was shown elsewhere [
29] that SSAB was not inferior to the commercial fertilisers in terms of its effect on wheat yield volumes, the uptake of P by wheat and the sanitary condition of wheat fields, especially when they were grown-protected from weeds, pathogens and pests.
This paper focuses on the assessment of SSAB effect on selected soil environment properties with a test plant, i.e., soil moisture and temperature, soil pH, content of toxic elements (As, Cd, Cr, Ni and Pb) in the soil, abundance of heterotrophic bacteria and fungi and occurrence of earthworms (
Lumbricidae). The research was based on the following assumptions: (i) soil moisture, temperature and pH, as well as the abundance and availability of P, have a direct bearing on the level of microbial activity in the soil [
30] and on the activity of
B. megaterium introduced with SSAB, (ii) the form of P fertiliser added to the soil can affect soil acidity, principally through the release or gain of H
+ ions by the phosphate molecule depending on soil pH [
31] and because, under P stress conditions, the plants can change the pH of the substrate through the release of organic acids which dissolve the poorly soluble phosphates [
32], (iii) the potential presence of heavy metals and other toxic elements in SSAB may increase their accumulation in soil [
33], (iv) the introduction of
B. megaterium, as an ingredient of SSAB, to the soil environment could modify soil biology due to an increase in the strain population size followed by the reorganisation of the microbial community structure [
34] and the modification of the chemical parameters of the soil environment (acid production) [
30], (v) the intensity of microbiological processes [
35] and the possible stimulation of crop growth resulting from the application of SSAB could indirectly lead to changes in soil moisture and temperature, (vi) changes in habitat parameters could affect the abundance of earthworms which are soil health bioindicators [
36].
It was hypothesised that SSAB would not deteriorate the properties of the soil environment, i.e., that its impact would be similar or more favourable than that of traditional P-fertilisers and that plant protection would not modify SSAB effect on the analysed parameters.
2. Materials and Methods
2.1. Experimental Design and Agronomic Management
A field experiment was conducted in 2015 in Bałcyny (Poland, 53.60°N, 19.85°E). The test plant was a spring cultivar of common wheat (
Triticum aestivum ssp.
vulgare Mac Key, cv. Monsun). In the experiment, SSAB was confronted with two commercial fertilisers: superphosphate (SP) and phosphorite (PR). Ten variants of P fertilisation were compared (
Table 1).
SP (Gdańsk Phosphate Fertilisers Plant ‘Fosfory’, Gdańsk, Poland) was purchased on the market. PR (bought at the Luvena, Luboń, Poland) was supplied by the Institute of New Chemical Synthesis in Puławy. SSAB was manufactured at the Institute of New Chemical Syntheses in Puławy (Poland), according to a concept developed at the Wrocław University of Science and Technology (Poland). SSA for the SSAB production was obtained from the Łyna Municipal Wastewater Treatment Plant in Olsztyn (Poland). The
B. megaterium strain was obtained from the Polish Collection of Microorganisms at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław (Poland). The SSAB production process consisted of the following steps: comminution of the raw material, preparation of the culture medium for
B. megaterium (mixing particular components, high-temperature sterilising, cooling down the solution to 35 °C), inoculation of the solution with
B. megaterium, solubilisation for 7–10 days (incubation at 35 °C with mixing), stabilisation of the suspension with bentonite. The content of the culture medium for
B. megaterium and the details of the SSAB production procedure were described by Rolewicz et al. [
37].
Moreover, in the experiment, two variants of plant protection were adopted: no protection (−PP) and full protection (+PP).
The experiment was set up in a parallel strip design with four replications (
Figure S1). The size of a single experimental plot was 20 m
2 (2 m × 10 m). A cereal–legume mixture was grown before cultivating spring wheat. The tillage was performed using the ploughing system. The P fertilisers were applied once, prior to wheat sowing: SP and PR were scattered by hand, while SSAB was applied by large-drop sprinkling of the soil. Along with the particular P doses, 1.0, 1.5 and 2.0 L/m
2 SSAB solutions were applied, respectively.
Nitrogen (N) and potassium (K) fertilisers were applied over the entire experiment area in the following amounts and forms: N, 110 kg/ha (ammonium sulphate, 21% N, 24% S, Grupa Azoty Puławy, Poland), K, 83 kg (potassium chloride, 60% K2O, Luvena, Luboń, Poland). The entire K dose was applied before sowing, and the N dose was divided into two parts: 60 kg was applied before sowing and 50 kg at wheat stem elongation.
Wheat was sown on April 9, at a depth of 3–4 cm, with a spacing of 15 cm. Under +PP conditions, wheat was protected: against weeds, with MCPA 300 g/L (3 L/ha, May 19, Chwastox Extra 300 SL, Ciech, Nowa Sarzyna, Poland), against fungal diseases, with azoxystrobin 250 g/L (0.6 L/ha, June 11, Amistar 250 SC, Syngenta, Warsaw, Poland) and propiconazole 250 g/L + cyproconazole 80 g/L (0.4 L/ha, June 11, Artea 330 EC, Syngenta, Warsaw, Poland), and against insects, with lambda-cyhalothrin 50 g/L (0.4 L/ha, June 10, Karate Zeon 050 CS, Syngenta, Warsaw, Poland).
Combine harvesting was performed on August 11.
2.2. Soil and Meteorological Conditions
The soil type on the experimental field was Luvisol [
38] formed from sandy loam. It contained (total contents) on average: 8.90 g/kg C, 1.35 g/kg N, 566 mg/kg P, 2.90 g/kg K, 2.01 g/kg Mg, 0.686 (max 5.795) mg/kg As, 0.292 (max 0.827) mg/kg Cd, 19.6 (max 25.0) mg/kg Cr, 8.53 (max 14.78) mg/kg Ni, and 7.99 (max 25.1) mg/kg Pb. The arable layer of soil produced an acid reaction (pH in KCl was 5.32).
The total annual precipitation was 492.3 mm, with 23.4 mm, 25.4 mm, 43.0 mm, 71.0 mm and 13.0 mm of the precipitation occurring from April to September, respectively. The mean annual temperature in the research area was 9.1 °C, with the mean monthly temperature ranging from 0.3 °C in February to 21.3 °C in September.
2.3. Sampling and Analyses
2.3.1. Soil Moisture and Temperature
The analysis was conducted three times: at (1) tillering, (2) stem elongation and (3) heading of wheat, by time-domain reflectometry (TDR) with the use of the FOM/mts meter (Field Operated Multimeter for moisture, temperature and salinity; E-Test, sole manufacturer of TDR meters and probes designed by the Institute of Agrophysics of the Polish Academy of Sciences in Lublin, Poland). The measurements were performed in layers of 0–10, 10–20 and 20–30 cm and repeated five times on each plot.
2.3.2. Soil pH
Soil samples were taken from the 0–30 cm soil layer on three dates: (1) before the start of the experiment, (2) at the wheat leaf development stage, (3) after wheat harvest. The samples were collected using a hand-held twisting probe (Egner’s soil sampler) from each plot separately at evenly distributed points. A total of about 1 kg of soil was gathered from a single plot. The collected soil material was dried at room temperature for several days, then thoroughly mixed and sieved. Afterwards, the separated portions of about 300 g each were passed to the Chemical and Agricultural Station in Olsztyn (Poland), where the pH in 1 M KCl was determined by the potentiometric method.
2.3.3. Content of Elements in Soil
Soil samples were taken twice: (1) before the start of the experiment and (2) after wheat harvest. Soil collection and preparation for elemental analysis proceeded as described in
Section 2.3.2. Samples of soil material of about 300 g were delivered to the accredited chemical laboratory (number AB 696), where the total content of C, N, P, K, Mg, As, Cd, Cr, Ni, and Pb was determined.
The C and N contents in soil were determined using the Vario Macro Cube Elementar (C,H,N) analyser (Elementar Analysensysteme, Langenselbold, Germany). D-phenylalanine (C = 65.44%; N = 8.48%) was used as a standard solution. The contents of other elements were determined using an inductively coupled plasma-optical emission spectrometer (ICP–OES). An appropriate mass (0.5 g) of soil samples was digested in Teflon vessels (microwave oven Milestone MLS-1200, Sorisole, Bergamo, Italy) with 10 mL of aqua regia. After mineralisation, all samples were An ICP–OES with a pneumatic nebuliser with an axial view (iCAP Duo Thermo Scientific, Waltham, MA, USA, diluted to 50 mL) was used to determine the concentration of elements in all mineralised and diluted biological samples. Determination of elemental content was carried out with all the principles of measurement traceability. Certificated reference materials were also used to check the quality and metrological traceability. The detection levels of P, K, Mg, As, Cd, Cr, Ni, and Pb in the soil material were 3.59, 2.55, 1.17, 0.5, 0.01, 0.035, 0.25, and 0.15 mg/kg, respectively.
2.3.4. Abundance of Heterotrophic Bacteria and Fungi
Soil samples were collected on two dates: at wheat tillering and at full vegetation (wheat heading), as described in
Section 2.3.2. The soil samplers were sterilised with 96% ethanol (Czempur, Piekary Śląskie, Poland) before switching to a subsequent plot. Immediately after collection, each soil sample was thoroughly mixed, while maintaining sterility. Subsequently, small portions of material from each soil sample were placed in sterile 120 mL plastic pots, which were forwarded to the microbiological laboratory.
The number of heterotrophic bacteria (colony-forming units, CFUs) was determined on tryptic soy agar (TSA, Merck KGaA, Darmstadt, Germany), containing 15.0 g/L tripticase peptone, 5.0 g/L papaic digest of soyabean meal, 5.0 g/L NaCl, and 15.0 g/L agar, and the number of fungi (CFUs) was determined on Rose-Bengal Chloramphenicol (RBC, Merck KGaA, Darmstadt, Germany) agar, containing 5.0 g/L mycological peptone, 10.0 g/L glucose, 1.0 g/L KH2PO4, 0.5 g/L MgSO4, 0.05 g/L rose bengal, 0.1 g/L chloramphenicol, and 15.5 g/L agar. TSA and RBC media were sterilised in an autoclave at 121 °C for 20 min. RBC had a pH of 7.2, and TSA had a pH of 7.3–7.5. The media were cooled to 45–50 °C, thoroughly mixed and poured in the amount of 10 mL onto Petri plates with soil solution previously deposited on them (1 mL of 10–3, 10–4 and 10–5 dilutions). Each dilution was prepared in duplicate. Heterotrophic bacteria were incubated at 30 °C for 72 h, and fungi were incubated at 28 °C for 5 days. The emergent colonies of heterotrophic bacteria and fungi were counted and expressed in terms of 1 g of soil.
2.3.5. Earthworm (Lumbricidae) Occurrence
The species composition, number and weight of earthworms (
Lumbricidae) in the 0–40 cm soil layer were determined after spring wheat harvest and expressed in terms of 1 m
2 of plot area. Earthworms were harvested mechanically: samples of the investigated soil layer were dug out, crushed and passed through a sieve, and individuals of
Lumbricidae were collected. Earthworms were anesthetised in a 30% ethanol (Czempur, Piekary Śląskie, Poland) solution and preserved in a 4% formalin (Czempur, Piekary Śląskie, Poland) and 75% ethanol solution. Their species composition was determined using an identification key to soil-dwelling oligochaetes [
39].
2.4. Statistical Analysis
The results were processed by analysis of variance (ANOVA) or the alternative Kruskal–Wallis test if the analysis of variance assumptions were not met. The normality of variable distribution was checked using the Shapiro–Wilk W-test, and the homogeneity of variance was checked using Levene’s test. The differences between objects were evaluated using Duncan’s test or a multiple comparison test. In statistical calculations, the soil element contents below the detection level (LD) were replaced by values equal to the LD. For the soil contents of toxic elements, the median and the maximum values were also determined. The calculations were performed using Statistica 12.0 software [
40]. Since the interaction between experimental factors was not significant, in the tables only the average values for the main factors are presented.