Microwave pyrolyzed sewage sludge: influence on soil microbiology, nutrient status, and plant biomass

Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 https://doi.org/10.1186/s40538-022-00354-8 RESEARCH Microwave pyrolyzed sewage sludge: influence onsoil microbiology, nutrient status, andplant biomass Kamila Lonova1, Jiri Holatko2,3, Tereza Hammerschmiedt2, Ludmila Mravcova4, Jiri Kucerik4, Adnan Mustafa2,4,5*, Antonin Kintl2,6, Muhammad Naveed7, Jakub Racek8, Marie Grulichova1, Marta Miklasova4 and Martin Brtnicky2,4* Abstract Background: Sewage sludge (SS) has been considered a potent source of soil nutrients. However, its direct application to agricultural soils have been discouraged owing to its toxic nature. Therefore, conversion and modification of SS to decrease its toxicity has resulted in advanced methods. Co-pyrolysis of SS with other amendments is an ideal treatment resulting in an environmentally safe and nutrient rich final products with additional properties to sequester carbon. In the present study, a novel biochar was produced through the microwave pyrolysis of SS mixed with zeolite and sawdust. The pyrolysis product was thus characterized for elemental composition, polycyclic aromatic hydrocarbons, via Fourier Transform Infrared Spectroscopy (FTIR), and for its effects on soil microbial characteristics, soil health and plant biomass after soil application. Results: Results revealed that, the SS modification resulted in stable product with higher nutrients which further depend on the type and ratio of feedstock used. Its application to soil significantly improved soil chemical and microbiological properties and altered lettuce biomass. Conclusions: We concluded that sawdust feedstock promoted nutrient availability in the resulting biochar and induced higher activity of nutrient mineralizing enzymes, whereas zeolite slowed down the release of nutrients from soil and putatively immobilized enzymes. This joint effect of sewage sludge biochar, sawdust and zeolite benefited the plant acquisition of nutrients in comparison with the microbial nutrient uptake. We thus conclude that microwave pyrolyzed SS could be used as a soil enhancer. Keywords: Sustainable agriculture, Soil quality, Bio stimulants, Nutrient cycling, Pyrolysis © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access *Correspondence: [email protected]; [email protected] 2 Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Faculty of AgriSciences, Mendel University in Brno, 613 00 Brno, Czech Republic Full list of author information is available at the end of the article Page 2 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 Introduction Currently, municipal waste management has emerged as a serious societal issue. In this regard, a challenging issue is the increase in the volume of sewage sludge waste (SS), the treatment of which needs energy-intensive and costly processes and safe disposal methods [1]. The Commission by the member states of the Europe reported that more than 10 million tons of dry biosolids including sewage sludge (SS) are being generated in EU annually [2]. This amount is the result of the implementation of European Union Directive, the Urban Wastewater Treatment 91/271/EEC and introduction of advanced technologies in development of wastewater treatment plants (WWTP). In European Union (EU), nearly, 37% of the annually produced dry biosolids (~ 3.6 million tons) have been recycled in agricultural activities [2] directly through conventional methods used for sewage sludge (SS) disposal or incinerated, compost etc. Currently, recycling to land (directly or via composted SS) has been considered among the most beneficial and economical way for municipal SS management [3]. Nevertheless, despite of many positive impacts of recycling, there are many risks of SS application including the introduction of emerging contaminants (mainly high concentration of heavy metals, organic toxic compounds, pathogens and microplastics) in soil which, may contaminate the food chain or harm the environment by surface runoff into receiving waters [4]. Therefore, there is an increasing interest of developing alternative methods of SS treatment, in particular thermal processing methods such as monoincineration, coincineration, gasification, hydrothermal carbonation, and pyrolysis have gained momentum in the current era [5– 7]. Pyrolysis in this sense is a process of thermal anoxic conversion of organic materials producing gas, oil and solid pyrolyzed residue. This technology of SS processing can be highly advantageous, since it reduces up to 50% of the waste volume [8] and stabilizes the organic matter (OM) in SS. In addition, the liquid and gaseous products can be used as a fuel, whereas the carbon-rich, solid by-product (char) have various agricultural and technological applications [6]. In particular, it is called biochar when applied to soil [9], while when applied technologically, it is called charcoal or coke [10]. Using biochar as the soil amendment or fertilizer is the subject of many recent studies. It is known that biochar can modulate the plant uptake of different nutrients or potentially toxic chemical substances and elements, such as heavy metals. Reduction of heavy metals concentrations and their leachability from fishpond sediment enriched with biochar was established by Mehmood etal. [11]. In the same study, it was observed that biochar addition could also increase the concentration of plants available macronutrients, such as phosphorous, nitrogen or potassium. This beneficial effect of biochar could lead to higher yield of plant biomass production as well as the Graphical Abstract Page 3 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 potential use of contaminated soil for the crop production due to immobilization of toxic elements, such as the cadmium [12]. The efficiency of this effect is dependent on the properties of biochar which are derived from the source of biochar, biochar particle surface, pH and the way of biochar production. Conditions of pyrolysis, such as temperature, residence time, heating rate, feedstock particle size determine both the physical and chemical parameters of resulting biochar [13–15]. The properties of feedstock influence the trace element content (contaminants or aromatic substances) and also its potential to be used in agriculture [16–18]. Depending on conditions of pyrolysis and type of SS [15], biochars obtained from SS pyrolysis are highly macroporous, with the small volumes of the mesoand micro-pores [19] among others, e.g., wood-derived biochar. However, one should bear in mind that the pyrolysis conditions regulate the availability and toxicity of emerging contaminants [20, 21]. For instance, it has been reported that, pyrolysis at lower temperature of 300°C resulted in the significant reduction in DTPA (diethylenetriaminepentaacetic acid)-extractable metals in the SS-derived biochar [14]. Another study showed that SS conversion to biochar significantly reduces the content of PAHs (polyaromatic hydrocarbons) and their toxicity [22]; however, pyrolysis increased trace metals content in resulting product due to a decrease in resulting mass (Pb, Cd, Zn, Cu, Ni and Cr) [20, 23]. Other studies also reported reduced concentration of volatile organic compounds at higher pyrolysis temperatures (up to 600°C). In addition, increased temperature led to increased content of stable aromatic carbon, ash, some macro- (Ca, Mg, P, and K) and micronutrients (Cu and Zn) and increased alkali reaction [20]. Nevertheless, a suitable temperature of the SS pyrolysis can be used to transform bioavailable heavy metals into less soluble forms [21, 23]. Addition of zeolite (organo-mineral sorbent) to sewage sludge feedstock before pyrolysis represents efficient approach for improving the quality of SS biochar. Similar enrichment of (composted) SS with zeolite brought the higher water-soluble and total macro-nutrient content, as well as lower phytotoxicity of the obtained blended organo-mineral matter [24]. Co-pyrolysis of pre-biochar feedstock and bentonite or kaolin increased chemical and thermal stability, recalcitrancy, aromatic structures in biochar [25, 26], other copyrolyzed organo-mineral clays and biochar improved sorption ability of products: the produced biochars efficiently bound ciprofloxacin [27], removed Cr(VI) from aqueous solution [28], mitigated greenhouse gas emissions (GHG) by sorption of CH4 and N2O emitted from soil [29]. Another approach to decrease the toxicity of SS-contaminating heavy metals is co-pyrolysis of SS with other biomass [30–35]. Co-pyrolysis of SS with wooden (bamboo sawdust, willow sawdust) or other lignocellulotic (rice straw) organic materials reduced the mobility and bioavailability of heavy metals in the final biochars [31, 33, 34]. However, concurrent effect of the addition of co-pyrolyzed biomass reduced yield, thermal stability, surface area, and pore volume of biochars, although the contents of organic matter and carbon in biochars significantly increased [31, 33]. Jin etal. showed the increased number of P–H (phosphorus– hydrogen) bonds (in phosphine) under co-pyrolyzed biochars as revealed by Fourier-Transform Infrared Spectroscopy (FTIR) analysis [33]. The feedstock consisting of SS and pinewood sawdust (1:1 w/w) [30] or bagasse [35] reduced vaporization of gaseous carbonaceous products (aromatic compounds, ketones, CO2), while volatilization of nitrogen-containing products (i.e., NH3) and sulphur compounds was minimized [35]. Currently, the interest in producing SS biochar has gained momentum. The research is focused on the wide range of SS biochar applications. One of these approaches is the use as a soil amendment. Thus, biochar produced from SS blended with other type of biomass may be improved in its nutrient content and binding properties. This biochar used as soil amendments, could have the beneficial effect to reduce leaching of soil nutrients, to enhance the fertilizing properties, and nutrient retention capacity in degraded soils [36–40]. Based on many of the studies, it seems that using of SS biochar in agriculture as the soil amendment or fertilizer is the potentially beneficial strategy. The review by Xiao et al. (2022) [41] mentioned positive effects of SS biochar: decrease of desorption capacity in soil for PAHs, increase of the soil N retention, immobilization of heavy metals—Cu [42], Pb and Cd [43, 44] etc. Taking this background into account, this work aimed to use the sawdust-blended SS to produce biochar with decreased availability of toxic contaminants and to utilize the carbon-enriched final biochar for improvement of soil chemical and biological properties, plant nutrition and crop growth. It was intended to compare the effect of co-pyrolyzed SS and zeolite on the resulting biochar properties. Recently, negative or “no effect” of SS biochar on soil was also reported, e.g., no effect on available nutrient concentration [45] or phytotoxic impact of volatile organic compounds in SS (unless they were removed by short washing or weathering) [46]. Hence, the need for further research on biochar SS as a potential soil activator and evaluation of its benefits might be highly desireable. Therefore, the specific objectives of this work were to (i) assess the efficacy of value addition of zeolite and sawdust in co-pyrolyzed SS in terms of increased soil Page 4 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 nutrient contents and reduced PAHs and (ii) evaluate the effects of soil applied co-pyrolyzed SS on microbial soil health indicators and plant biomass. To achieve the objectives, following hypotheses were tested. Materials andmethods Biochar preparation andcharacterization Treatments of biochar were prepared by microwave pyrolysis from SS mixed with zeolite or/and sawdust, as shown in Table1. Biochar was applied to soil in 3 doses corresponding to the indicated weight percentages of biochar in soil: 2.5%, 5% and 7.5% (25, 50, 75 t⋅ha−1). The sludge was obtained from the municipal WWTP. Sewage water andsludge characterization andprocessing The WWTP has a capacity of around 530,000 population equivalents. WW is predominantly municipal WW originating from households. Only 12–15% are industrial influents, but generally this WW mostly has the character of typical municipal WW. Despite the relatively low industrial WW ratio, SS tends to contain relatively high concentrations of heavy metals. The anaerobically digested SS was dried using a contact blade paddle dryer at temperature below 100°C. Tested raw dried samples of SS (Fig.1) had dry solids around 91% and output fraction from dryer was a powder-like material with particle fraction 1–8mm. Random tests revealed that the hygroscopic water content was below 2.0%. Pelletized feedstock The mixtures of dried raw SS with very fine sawdust (from softwood) and zeolite were pelletized by industrial pelletizing press (Fig.2). In this work, a synthetic zeolite (Purmol 13) was chosen—zeolite-type ZSM-5 with admixtures of other zeolites (faujasite, wassalite) with a fineness of about < 100µm. This synthetic zeolite has demonstrated the efficiency of the process of microwave depolymerization of lignocellulosic biomass [47]. For these experiments, the pelletization process used an extrusion die having diameter of 6.4mm. The temperature during pelletization was measured on the metal matrix of the pelletizer. The pyrolyzed feedstock were approx. 6.4mm diameter pellets of mixed SS with additives made by pelletizing press (Fig.3). Table 1 Treatments amended with biochar prepared from sewage sludge, sawdust, and zeolite Treatment Composition of biomass for pyrolysis Abbrev. 2.5 wt% biochar (sewage sludge + zeolite) 95 wt% SS + 5 wt% zeolite 2.5% BC (Z) 5 wt% biochar (sewage sludge + zeolite) 95 wt% SS + 5 wt% zeolite 5% BC (Z) 7.5 wt% biochar (sewage sludge + zeolite) 95 wt% SS + 5 wt% zeolite 7.5% BC (Z) 2.5 wt% biochar (sewage sludge + sawdust) 75 wt% SS + 25 wt% sawdust 2.5% BC (SD) 5 wt% biochar (sewage sludge + sawdust) 75 wt% SS + 25 wt% sawdust 5% BC (SD) 7.5 wt% biochar (sewage sludge + sawdust) 75 wt% SS + 25 wt% sawdust 7.5% BC (SD) 2.5 wt% biochar (sewage sludge + sawdust + zeolite) 75 wt% SS + 20 wt% sawdust + 5 wt% zeolite 2.5% BC (SD + Z) 5 wt% biochar (sewage sludge + sawdust + zeolite) 75 wt% SS + 20 wt% sawdust + 5 wt% zeolite 5% BC (SD + Z) 7.5 wt% biochar (sewage sludge + sawdust + zeolite) 75 wt% SS + 20 wt% sawdust + 5 wt% zeolite 7.5% BC (SD + Z) Fig. 1 Raw dried sewage sludge Fig. 2 Pelletized raw material Page 5 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 Microwave pyrolysis unit Experiments were performed by slow microwave pyrolysis unit which works at low pressure 800hPa. Microwave was generated by magnetron with 3.0kW input power, regulated output power, and with 2.45GHz. This unit works discontinuously, and the maximum capacity is approx. 3kg∙batch−1 of feedstock. The glass condenser attached to the pyrolyzer was used for the separation of gaseous products and the oil. For incoming and reflected waves a tuner was installed. The infrared (IR) thermometer was introduced into the center of the input feedstock. The input weight of feedstock samples was 1.0kg·batch−1. During the experiments, the output regulated power of magnetron was 1.2kW, residence time was 60min, and the temperature during the tests did not exceed 250°C. Fourier transform infrared spectroscopy (FTIR) analysis The Fourier transform infrared spectroscopy (FTIR) spectra of the obtained biochar samples was recorded on a Bruker diffused reflectance infrared Fourier transform (DRIFT) spectrometer. The spectra were collected at transmission mode between 4000 and 400 cm−1 with resolution of 8 cm−1 and 128 scans using OPUS computerbased software. Prior analysis the samples were prepared by mixing with KBr to form a homogenous mixture. Polyaromatic hydrocarbons (PAH) determination inresulting biochar The extraction of homogenized samples (1g of grounded BC sample, Retsch MM 200) was carried out by pressurized solvent extraction (one PSE, Applied Separations). Toluene was used as a solvent, the extraction was carried out at 130°C, 120bar and 3 cycles. Before extraction, internal standard (100 /10 ul, 5 deuterated PAH) was added to samples. Toluene was evaporated to approximately 1 mL of final volume. Gas chromatography with mass spectrometry (Bruker EVOQ GC-TQ) was used for the analysis of 16 EPA PAHs in the extracts. 16 EPA PAHs were separated in column DB-EUPAH (20m × 0.180mm; 0.14 um), the temperature program was 80°C for 1min, then an increase to 320°C (5min) with heating rate 15°C/min, spitless injection at 270°C, EI 70eV, SIM mode. Quantification was carried out by internal standard calibration. PO4 determination For the analysis of PO4–P in water leachate (5g of BC and 50ml of MilliQ water, filtration after 24h) was used the spectrophotometric method according ČSN EN ISO 6878 (MQuantTM Phosphate Test, Merck) [48]. Determination ofleachable heavy metals Heavy metals such as mercury (Hg), cupper (Cu), chromium (Cr), zinc (Zn), led (Pb), arsenic (As), nickel (Ni) and cadmium (Cd) were determined in the water extract using atomic absorption spectrometer with electrothermal atomization ZEEnit 60 from Analytik Jena (Germany) with Zeeman background correction and selected hollow cathode lamp by Photron (Australia) according to the method described in the work of Racek etal. (2019) [49]. Pot experiments andsampling The pot experiment with lettuce (Lactuca sativa L. var. Brilant; SEMO a.s, Czech Republic) was performed in 1-L capacity pots. The pots were filled up with 300g of commercial garden substrate TS 3 medium basic 425 standard (Klasmann–Deilmann GmbH, Germany), thoroughly mixed with a dose of biochar according to treatments reported in Table1. The substrate was a mixture of light peat (0–25mm) with wetting agent, dry matter 45%, pH 6.0, salts 1g L−1, nutrient content (according to manufacturer): N 140mg⋅L−1, P 44mg⋅L−1, K 150mg⋅L−1, Mg 100mg⋅L−1. Altogether, 10 treatments were tested (substrate amended with 9 types/doses of biochar, and unamended substrate = negative control), each treatment was prepared in four replicates. Three seeds were sown in each pot, then watered with 200mL of deionized water. After a week of germination, only one plant per pot was established. The watering was regularly carried out during cultivation to maintain the same soil moisture and plants did not wilt. The experiment was carried out for 8weeks in greenhouse under controlled conditions (day/night): temperature 22/18°C, relative air humidity 50/50%, photoperiod 14/10h. At the end of the cultivation, the chlorophyll a fluorescence in dark adapted leaves was measured and the above ground plant biomass was harvested for the analyses of pigments, Fig. 3 Biochar Page 6 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 and for plant fresh and dry biomass estimation. In addition, mixed soil sample was taken from each pot for determination of basic physical, chemical and biological soil quality indicator. Plant biomass quantification andquality parameters determination Lettuce fresh aboveground biomass (AGB) was determined gravimetrically by weighing the shoots on the laboratory scales. To determine plant dry biomass, fresh plant material was dried at 60°C to constant weight and obtained dry biomass was again estimated gravimetrically. Dry matter content in fresh plant biomass was calculated (data are not presented). Changes in the photosynthetic apparatus were evaluated using chlorophyll fluorescence parameters measured in dark adapted leaves of intact plants using the portable fluorometer FluorPen FP 100 (Photon System Instrument, Czech Republic). It was measured the fast fluorescent kinetic presented as the OJIP transient curves. Prior to drying of fresh plant biomass, 0.5 g of fresh material was taken from each plant to determine the content of leaf pigments, lyophilized and stored at −18 °C. Lyophilized samples were homogenized with 10mL acetone. Acetone extracts were analyzed with spectrophotometer (Spetronic 20 Genesys, Thermo Spectronic, USA) at wavelength 662, 645 and 470nm. The content of individual pigments was calculated according to the methodology Lichtenthaler and Buschmann (2005) [50]. Determination ofsoil quality properties The substrate from each pot was homogenized by sieving it through 2mm mesh and stored at 4°C (for determination of soil respiration), lyophilized and stored at −18°C (for determination of soil enzyme activities), and the rest were air-dried for quantification of nutrient content and pH measurement. Soil reaction, pH (CaCl2)—was determined according to ISO 10390:2005 [51], dehydrogenase (DHA) activity was measured according to Voberkova et al. [52] and expressed in µg (triphenyl formazan) TPF·g−1·h−1, other enzymatic activities—β-glucosidase (GLU), arylsulfatase (ARS), phosphatase (Phos), urease (Ure) and N-acetyl-β-D-glucosaminidase (NAG)— were measured spectrophotometrically according to ISO 20130:2018 [53] and the values expressed in µmol (p-nitrophenol) PNP·g−1·h−1 and in µmol NH3·g−1·h−1 (urease). Statistical analyses Data obtained from the determination of plant biomass, qualitative properties, and soil chemical and biological parameters were statistically analyzed using the methods of principal component analysis (PCA), one-way analysis of variance (ANOVA), Tukey HSD post-hoc test (at significance level p = 0.05), and Pearson correlation analysis via Program R, version 3.6.1 [54, 55]. For testing of the normality of distribution, it was used Kolmogorov and Smirnov test and data homoscedasticity was examined by Bartlett’s test, both at significance levels of 0.05. Besides, assumptions of all tests were also checked by different diagnostic plots. The minimal level of statistical significance for most of the used methods was 0.05. The results of Pearson’s correlation analysis were interpreted (according to the value of correlation coefficient r) as follows: 0.5 < r < 0.7 (moderate correlation) and 0.7 < r < 0.9 (high correlation) [56]. Results Biochar characteristics andresults ofFTIR analysis The concentrations of Hg, Cr, Pb, As and Cd in the water extract from biochar types were below detection limits, for Cu it was 1mg L−1, for Zn 2.2mg L−1 and for Ni 0.7mg L−1. As the concentrations of all the metals were very low and did not reach minimal inhibition/toxic levels for plants, they are not further discussed in the text. The total nitrogen (Ntot) and total hydrogen (Htot) contents were both significantly highest in the BC (SD) biochar and lowest in the BC (SD + Z) treatment (Fig.4). The mutual ratios between macroelements in the three biochar types showed significant differences too. C:N ratio was highest in the BC (SD + Z) biochar (around 12.0) and the lowest in the BC (Z) biochar (8.0). H:C ratio on the other hand was highest (> 1.0) in the BC (SD) biochar and the lowest (< 0.8) in the treatment BC (SD + Z). Similarly, O:C was highest (> 0.4) under biochar BC (Z), followed by BC (SD + Z) and BC (SD). These diverse results anticipated no significant mutual correlations between the ratio properties (Fig.5). The significantly highest content of leachable phosphate (calculated to dry biochar weight—around 600mg⋅kg−1) was detected in the water leachate of BC (SD) biochar, compared to the SD (Z) treatment (around 300mg⋅kg−1) and the lowest content was in the BC (SD + Z) biochar (< 100 mg⋅kg−1). No significant difference in the sum content of 16 EPA PAHs between all three biochar types was detected. No correlation between PAH content was observed with other biochar properties. The FTIR analysis of the three types of biochar samples revealed similarities in their composition in terms of aromatic and aliphatic moieties. Importantly, the biochar prepared from 25 wt% sawdust (Fig.5B), which showed an enhanced intensity signal at 2927 cm−1, depicting characteristic aliphatic C–H stretching as compared to other biochar types (Fig.5A, C). Moreover, unlike (25% sawdust) and (5% zeolite) derived biochar (Fig.5B, A), the (20% sawdust + 5% zeolite) derived biochar (Fig.5C) lack Page 7 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 a characteristic peak between 3900 and 3500 cm−1, representing OH-stretching of carboxyl functional groups. Furthermore, all the three types of biochar showed strong peak above 1500–400 cm−1, depicting compounds derived from polysaccharides (1057 cm−1), aromatic C–H stretches (1506 cm−1) and aliphatic amides (1557 cm−1) (Fig.5A, B, C). In addition, the C–H stretching vibrations were observed at 2970 and 2860 cm−1. This shows that the co-pyrolysis of SS biochar with different rates of sawdust and zeolite resulted in altered functional group chemistry of resultant biochar samples. Soil reaction andenzyme activities Soil reaction pH (CaCl2) value was significantly lowest in the treatments 2.5% BC (SD), 7.5% BC (SD), and 7.5% BC (SD + Z) as compared to control (Fig.6). The soil pH did not show any significant and considerable correlation with other measured parameters. The dehydrogenase activity (DHA) was increased in all treatments except for 2.5% BC (SD + Z) and 7.5% BC (SD + Z) as compared to the control (Fig.6). The highest DHA value was detected in the 2.5% BC (SD) treatments and was increased as compared to all treatments of control, with BC (Z), with BC (SD + Z), and 7.5% BC (SD) treatment. The Pearson’s correlation analysis revealed no Fig. 4 Chemical properties of biochar treatments made of sewage sludge, sawdust, and zeolite, content of A nitrogen, B oxygen, C carbon, D hydrogen, E carbon and nitrogen ratio, F hydrogen and carbon ratio, G oxygen and carbon ratio, H concentration of phosphorous in form of phosphate in water leachate of biochar (mg·l−1), I concentration of phosphate in biochar (mg·kg−1), J concentration of 16 priority PAHs according to U.S. EPA (mg·kg−1). Mean ± standard deviation (error bars) calculated from independent values (n = 4), different letters express the statistical differences at significance level p ≤ 0.05 Fig. 5 FTIR spectra of A 5% zeolite–biochar, B 25% sawdust–biochar and C 20% sawdust + 5% zeolite–biochar Page 8 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 significant and considerable correlation of DHA with any other plant or soil property. The activity of arylsulfatase (ARS) was increased in all treatments with BC (SD), under both 2.5% and 7.5% BC (Z), and 7.5% BC (SD + Z) as compared to control (Fig.7). The highest ARS value was revealed in the 7.5% BC (SD) as compared to all other treatments, while the lowest ARS was in 2.5% BC (SD + Z) and 5% BC (SD + Z) relative to all other biochar-amended treatments. The Pearson’s correlation analysis showed that ARS was Fig. 6 Soil pH and dehydrogenase activity (DHA) of substrate treatments amended with biochar made of sewage sludge, sawdust, and zeolite. Mean ± standard deviation (error bars) calculated from independent values (n = 4), different letters express the statistical differences at significance level p ≤ 0.05. Fig. 7 Soil enzyme activities—A arylsulfatase (ARS), B urease (URE), C phosphatase (PHOS), D N-acetyl-β-D-glucosaminidase (NAG), E β-glucosidase (GLU)—of substrate treatments amended with biochar made of sewage sludge, sawdust, and zeolite. Mean ± standard deviation (error bars) calculated from independent values (n = 36), different letters express the statistical differences at significance level p ≤ 0.05 Page 9 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 significantly (p ≤ 0.001) and moderately positively correlated with PHOS (r = 0.65), URE (r = 0.58), and GLU (r = 0.58) (Fig.8). The URE activity was significantly increased in treatments 2.5% and 7.5% BC (Z), 5% and 7.5% BC (SD), as well as 5% and 7.5% BC (SD + Z) as compared to the control. However, the highest urease activity was detected in the 7.5% BC (SD) treatment and the lowest was in 5% BC (Z), as compared to all other treatments. The URE showed moderate positive and significant (p ≤ 0.001) correlation to PHOS (r = 0.61) and GLU (r = 0.66), respectively (Fig.8). PHOS was significantly increased (compared to the control) in all three treatments amended with BC (SD) at 2.5%, 5%, and 7.5% BC (SD). Moreover, 5% and 7.5% BC (SD) treatments showed higher PHOS compared to any other biochar amended treatment. The highest PHOS activity was recorded in 7.5% BC (SD) and the lowest was in 2.5% BC (SD + Z) as compared to control and other treatments (Fig.6). We also observed a significant (p ≤ 0.001) correlation between PHOS and NAG (moderate positive, r = 0.51) and GLU (high positive, r = 0.79). N-acetyl-β-D-glucosaminidase (NAG) was increased in the treatments 5% BC (SD), 7.5% BC (SD), and 2.5% BC (SD + Z) in comparison with the control (Fig.7) and significantly decreased in 7.5% BC (SD + Z) in comparison with all other treatments. NAG correlated significantly (p ≤ 0.001) and moderately positively with GLU (r = 0.6). The activity of GLU was significantly increased in all 3 treatments with BC (SD) and BC (SD + Z) as well as in both 5% and 7.5% BC (Z), as compared to the control (Fig.7). The highest GLU values were observed for 5% BC (SD) and 7.5% BC (SD) as compared to controls and other treatments. Plant biomass andphotosynthetic pigments The fresh and dry aboveground (AGB) biomass showed no significant difference between all amended experimental treatments and the control as well (Fig.9). The content of chlorophyll a and b showed highly positive and significant correlation (p ≤ 0.001, r = 0.96) in our study. Their values were considerably decreased in all three treatments with BC (Z) and in 7.5% BC (SD + Z) treatment, and significantly increased only in 5% BC (SD + Z) treatment, as compared to the control (Fig.9). A significant decrease in the chlorophyll a/b ratio was detected in both 2.5% and 7.5% BC (Z) compared to the control. The total carotenoids content was decreased (as compared to the control) in all treatments amended with BC (Z) additive. The highest value was observed for 5% BC Fig. 8 Results of Pearson’s correlation analysis among soil and plant properties. Values in the cells = correlation coefficient r, calculated on the level of significance p: ≤ 0.1 (⋅), ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***) Page 16 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 the prediction that the long-term cultivation of plants could lead to observation of positive effect of the biochar addition due to the support of soil microflora and improvement of nutrient availability especially in nutrient poor soil [101]. Microwave pyrolyzed biochar—an amendment orfertilizer? Application of biochar to soil represents a strategy for sequestration of carbon, because biochar’s chemical structure is considered to be resistant to microbiological attack [102]. Simultaneously, depending on the feedstock and conditions of preparation, biochar may represent a source of macronutrients and an amendment improving water-holding capacity. The European Biochar Certificate states that, “Biochar is a charcoal-like substance that is pyrolysed from sustainable obtained biomass under controlled conditions and which is used for any purpose which does not involve its rapid mineralization to CO2” [67]. Based on this definition, Conte etal. [103] concluded that biochar may be produced only from fast growing plants, plant residues from certified forestry management, agricultural residues, and organic wastes from urban areas [103]. However, pyrolysis of sewage sludge pyrolyzed under conditions (i.e., low temperature microwave pyrolysis) used in this work appeared to produce biochar that is apparently biodegradable and intensively stimulates the activity of soil microorganisms. We attribute this observation to the interplay of two important factors: temperature of the pyrolysis and used feedstock. According to Tag etal., [104] the increasing temperature decreases both the H:C and O:C ratios in pyrolyzed biomass, such as vine pruning (VP), poultry litter (PL), orange pomace (OP) and seaweed [104]. Comparing results with those reported by Tag etal., (2016) roughly correspond to the dependence of O:C at respective pyrolysis temperature, but H:C ratio is in our case significantly lower [104]. In other words, biochar from sewage sludge is less aromatic (see Fig.5) comparing to other sources, which confirms the comparison with other authors [104–106]. In fact, feedstocks for pyrolysis are usually based on lignocellulose materials, such as wood residues, grass and others. On the contrary, sewage sludge is of microbiological origin, i.e., it contains mainly N-rich compounds from protoplasma and cell membranes, such as proteins and fatty acids. Therefore, resulting structure differs compared to lignocellulose-based biochar; the O:C is similar, but lower H:C in SS-based biochar shows that this biochar is significantly less aromatic, i.e., more aliphatic. As it is well-known that aliphatic structures are better biodegradable comparing to aromatic structures [107], biochar prepared from sewage sludge acts as more as a fertilizer instead of an amendment. This is supported by higher content of nitrogen in its structure, which seems to be also bioavailable as suggested by activity of urease. Microwave pyrolysis is fast, selective and efficient method for production of pyrolyzed materials [49, 108]. However, the conditions such a low temperature may lead to products whose properties are far from definition of biochar. As follows from the results, no every intentionally pyrolyzed organic material is suitable for carbon sequestration; nevertheless, its effect on soil may still be positive. In particular, it may represent a source of labile carbon that supports soil microbial processes together with macronutrients. In addition, these effects may be tuned by addition of zeolite, which either stabilizes the biochar structure or moderate release of nutrients. It remains a question, if the pyrolyzed product of sewage sludge should still be named as a biochar, as the biochar per definition, is microbiologically stable material [67]. Moreover, its application is one of the keys in the longterm strategy of increasing of soil organic carbon in soils and sequestration/storage of carbon in soil. Despite the enhanced microbial activity of soil microorganisms, the use of this particular biochar neither increased nor decreased biomass yield which can be explained as follows: i) the amount of N released from SS biochar was high enough to support the soil microorganism and no competition between soil microbiome and plant roots of Lactuca sativa occurred and ii) the length of the experiment was too short and the effect on plant could not manifest, i.e., biochar affected soil microbiological processes, but the effect on plants appears with a delay, iii) heavy metals, the higher content of which is usually a problem of the municipal SS [49], affected neither soil microbiome nor plant which confirms its fixation and immobilization in biochar structure [49]. Conclusions This study concluded that microwave pyrolysis produced biochar from sewage sludge exerted a decreased microbiological stability of carbonaceous content and was putatively less efficient in soil carbon sequestration. The produced biochar significantly affected soil chemical and microbiological properties. In particular, soil pH was significantly decreased due to application of biochar produced from sewage sludge and sawdust, whereas dehydrogenase, β-glucosidase, arylsulfatase, phosphatase, urease, N-acetyl-β-D-glucosaminidase was increased. Biochar application level was the crucial factor in governing enzyme activities. Sawdust biomass promoted nutrient availability in the resulting biochars and induced higher activity of nutrient mineralizing enzymes, whereas zeolite slowed down the release of nutrients from soil and putatively immobilized enzymes. This joint Page 17 of 20 Lonovaetal. Chem. Biol. Technol. Agric. (2022) 9:92 effect of sewage sludge biochar, sawdust and zeolite benefited the plant acquisition of nutrients in comparison with the microbial nutrient uptake. However, this effect was not accompanied with a changed lettuce biomass yield as the fresh and dry aboveground (AGB) biomass showed no significant difference between all experimental treatments. Albeit the biochar SS + SD + Z (at dose 5% of sewage sludge) determined no improvement in quantity of lettuce biomass, it showed the highest content of photosynthesis pigment (chlorophyl a, b, carotenoids) and represent an eventual approach in the production of sewage sludge-based biochar with desired traits for soil/ agricultural application. Acknowledgements Not applicable. Author contributions AM, JH and MB: conceptualization. KL, JR: methodology. TH, KL, and LM: software. MN, JK, and AK: validation. MB, MM, LM and TH: formal analysis. JR, MM and MG: resources. KL, MG, MM and AK: data curation. KL, JH: writing—original draft preparation. JK, AM, TH, MN, JR and MB: writing—review and editing. MB and JK: supervision. KL, LM and JR: project administration. KL, AK, JK and MB: funding acquisition. All authors read and approved the final manuscript. Funding The work was supported by the projects of Technology Agency of the Czech Republic TJ02000261 and TH03030319, by the Ministry of Agriculture of the Czech Republic, institutional support MZE-RO1218, MZE-RO1722 and by Ministry of Education, Youth and Sports of the Czech Republic, grant number FCH-S-22-8001. Availability of data and materials The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Department of Plant Biology, Faculty of AgriSciences, Mendel University in Brno, Brno 61300, Czech Republic. 2 Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Faculty of AgriSciences, Mendel University in Brno, 613 00 Brno, Czech Republic. 3 Agrovyzkum Rapotin, Ltd., Vyzkumniku 267, 788 13 Rapotin, Czech Republic. 4 Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic. 5 I nstitute for Environmental Studies, Faculty of Science, Charles University, Benatska 2, 12800 Prague, Czech Republic. 6 Agricultural Research, Ltd., 664 41 Troubsko, Czech Republic. 7 Institute of Soil and Environmental Science, University of Agriculture Faisalabad, Faisalabad, Pakistan. 8 AdMaS Research Centre, Faculty of Civil Engineering, Brno University of Technology, Purkynova 651/139, 61200 Brno, Czech Republic. 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