Un metodo fisico per la lisi delle cellule microbiche del suolo basato su alte pressurizzazioni con CO2

La biomassa microbica del suolo (BMS), una piccola ma altamente dinamica frazione della sostanza organica del suolo, svolge un ruolo chiave nell’ecosistema suolo e pertanto le sue dimensioni e la sua attività sono determinanti per la fertilità e la qualità del suolo[1]. Perciò, lo studio e la caratterizzazione della MBS sono essenziali per la valutazione della qualità del suolo. I due metodi ancora ampiamente utilizzati per la determinazione della MBS sono la fumigazione-incubazione (FI) e la fumigazione-estrazione (FE)[2][3]. Entrambi i metodi si basano sulla fumigazione dei campioni di suolo con cloroformio (CHCl3) per lisare le cellule microbiche e determinarne il materiale citoplasmatico. L'uso del CHCl3, tuttavia, solleva diverse criticità, prima fra tutte la sua tossicità per l'uomo e per l'ambiente[4] e inoltre, diversi autori hanno dimostrato che il CHCl3 non è completamente efficiente nel lisare le cellule microbiche[5][6][7]. Pertanto, lo scopo di questo studio è stato quello di sviluppare un nuovo approccio per lisare le cellule microbiche, che possa essere più affidabile e sicuro per l'ambiente rispetto alla fumigazione. Il metodo proposto, chiamato CO2HP (CO2 - High Pressure), si basa su un'elevata pressurizzazione del suolo con CO2, seguita da una rapida depressurizzazione. Per mettere a punto il metodo CO2HP, sono state testate diverse combinazioni di pressione e durata di pressurizzazione e, per valutare la capacità del metodo CO2HP di lisare le cellule microbiche del suolo, è stato effettuato un confronto con i metodi classici FI e FE. I risultati dimostrano che il nuovo metodo CO2HP è più efficiente del CHCl3 nel lisare le cellule microbiche del suolo. La combinazione più efficiente è risultata essere 600 psi per la pressurizzazione della CO2 e 32 ore di durata.Soil microbial biomass (SMB), a small but highly dynamic pool of living organic matter, plays a key role in nutrient cycling, and therefore its size and activity are crucial determinants of soil fertility and quality[1]. For these reasons, the study and characterization of soil microbial biomass (SMB) are essential for soil quality assessment. The two methods still widely used for SMB determination are chloroform incubation (FI) and chloroform extraction (FE)[2][3]. Both methods rely on the ability of chloroform (CHCl3) to lyse soil microbial cells so as to determine the cytoplasmic material. The use of CHCl3, however, raises several critical issues, chief among them that it is toxic to humans and the environment[4]. In addition, several authors have shown that CHCl3 is not completely efficient in lysing microbial cells[5][6][7]. Therefore, the aim of this study was to develop a new approach, possibly more reliable and environmentally safe than the CHCl3-based method, for lysing soil microbial cells. The proposed method is based on high pressurization of the soil with CO2, through the use of a steel reactor, followed by rapid depressurization through gas release. Hereafter, we will call this approach CO2HP (CO2-High Pressure). To set up CO2HP method, different combinations of pressure and duration of pressurization were tested, and to evaluate the ability of the CO2HP method to lyse soil microbial cells, a comparison was made with the classical FI and FE methods. The results indicate that the new CO2HP method is more efficient than CHCl3 in lysing soil microbial cells. The most efficient combination was found to be 600 psi for CO2 pressurization and 32 hours duration

A high CO2 pressure-based method for soil microbial cells disruption

Soil microbial biomass (SMB), a small but highly dynamic living organic matter pool, plays a pivotal role in nutrient cycling and thus its size and activity are major determinants for soil fertility and quality. Indeed, SMB performs more than 90% of the key functions in the degradation and recirculation of organic matter and nutrients[1]. Moreover, due to its nature, SMB quickly responds to biotic and abiotic factors, thus becoming a sensitive indicator of most disturbances and changes occurring in the soil ecosystem[2]. For all these reasons, the study and characterization of soil microbial biomass (SMB) is essential for the assessment of soil quality. The two methods still widely used for determining microbial biomass are the chloroform-fumigation incubation (FI) and chloroform-fumigation extraction (FE) methods[3][4]. Both methods are based on the ability of chloroform (CHCl3) in lysing soil microbial cells, so that nutrients held by them can be then determined by different techniques. The use of CHCl3, however, rises several critical issues. Not to be forgotten, it is an alkyl halide toxic to humans and the environment[5]. Furthermore, CHCl3 has been shown not to be very efficient in lysing the microbial cells. Badalucco et al. [6][7] demonstrated that amounts of phenol-reactive C and anthrone-reactive C represented higher proportions of total extractable C after CHCl3 fumigation than before, thus arguing that some non-biomass sugars were solubilized during the 24 h fumigation. Later, Badalucco et al.[8], showed that the efficiency of CHCl3 in lysing microbial cells appeared to be inversely related to the stability of soil aggregates, due to its lower diffusion in more clayey soils. Also, Toyota et al.[9] by using a sandy loam soil showed that approximately 10% of bacterial colony forming units survived a 5-day CHCl3 fumigation. This percentage could have been much higher when fumigating a clayey soils. Finally, Alessi et al.[10] demonstrated that significant concentrations of CHCl3 were adsorbed, and thus retained, by the clay fraction of soils. Therefore, the major problem in the indirect assessment of the microbial biomass inhabiting soil still persists and consists in that there is not yet a highly efficient, and possibly environmentally safe, disruption technique of microbial cells for subsequent extraction and quantification of intracellular matter. Thus, the purpose of this study was to set up a new method, possibly more reliable and environmentally safe compared to the CHCl3 based one, for lysing soil microbial cells. The method proposed here is based on a remarkable pressurization of the soil with gaseous CO2, through the use of a steel reactor, followed by rapid depressurization via the gas release. Hereafter, we call this approach CO2HP (CO2 – High Pressure). With increasing pressurization, it is expected that the microbial cells are gradually penetrated and filled with the gas. After being saturated by the gas, the applied pressure is suddenly released so that the absorbed gas will rapidly expand inside the cells; therefore, the cells are mechanically ruptured like a popped balloon. This technique, which was first reported by Fraser in 1951[11], has been implemented by several workers, applying it on pure microbial cultures as a novel sterilization/inactivation method for heat-sensitive materials like foods[12][13] but never in soil. One agricultural and one forest soil were used to set up the CO2HP method. The ability of the new method in lysing the soil microbial cells was assessed by determining, in soils pressurized or fumigated by chloroform, either the CO2-C released during 10-d incubation at controlled conditions or the KCl extractable C. In order to evaluate the most efficient pressure and time of pressurization, soils were pressurized with CO2 at pressures ranging from 400 to 800 psi and from 2 to 32 hours as duration. Results indicate that the new CO2HP method is more efficient than CHCl3 in lysing soil microbial cells. The most efficient combination was found to be 600 psi for CO2 pressurization and at 32 hours as duration. Bibliography [1] Singh, J. S., & Gupta, V. K. (2018). Soil microbial biomass: a key soil driver in management of ecosystem functioning. Science of the Total Environment, 634, 497-500. [2] Laudicina, V. A., Dennis, P. G., Palazzolo, E., & Badalucco, L. (2012). Key biochemical attributes to assess soil ecosystem sustainability. Environmental protection strategies for sustainable development, 193-227. Springer, Dordrecht. [3] Jenkinson, D. S., Powlson, D. S., 1976. The effects of biocidal treatments on metabolism in soil, a method for measuring soil biomass. Soil Biology and Biochemistry, 8, 209- 2013 [4] Vance, E. D., Brookes, P. C., Jenkinson, D. S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry, 19, 703-707 [5] Lionte, C., 2010. Lethal complications after poisoning with chloroform—case report and literature review. Human & Experimental Toxicology, 29(7), 615-622. [6] Badalucco, L., Nannipieri, P., Grego, S., Ciardi, C., 1990. Microbial biomass and anthrone-reactive carbon in soils with different organic matter contents. Soil Biology and Biochemistry 22, 899-904 [7] Badalucco, L., Gelsomino, A., Dell'Orco, S., Grego, S., Nannipieri, P., 1992. Biochemical characterization of soil organic compounds extracted by 0.5 M K2SO4 before and after chloroform fumigation. Soil Biology and Biochemistry 24, 569-578. [8] Badalucco, L., De Cesare, F., Grego, S., Landi, L., Nannipieri, P., 1997. Do physical properties of soil affect chloroform efficiency in lysing microbial biomass? Soil Biology and Biochemistry, 29 (7), 1135–1142 [9] Toyota, K., Ritz, K., Young, I.M., 1996. Survival of bacterial and fungal populations following chloroform-fumigation: effects of soil matric potential and bulk density. Soil Biology and Biochemistry 28, 1545-1547. [10] Alessi, D.S., Walsh, D.M., Fein, J.B., 2011. Uncertainties in determining microbial biomass C using the chloroform fumigation–extraction method. Chemical Geology, 280 (1-2), 58-64 [11] Fraser, D., 1951. Bursting bacteria by release of gas pressure. Nature, 167 (4236), 33-34. [12] Nakamura, K., Enomoto, A., Fukushima, H., Nagai, K., Hakoda, M., 1994. Disruption of Microbial Cells by the Flash Discharge of High-pressure Carbon Dioxide. Bioscience, Biotechnology, and Biochemistry, 58 (7), 1297–1301 [13] Garcia-Gonzalez, L., Geeraerd, A.H., Spilimbergo, S., Elst, K., Van Ginneken, L., Debevere, J., Van Impe, J.F., Devlieghere, F., 2007. High pressure carbon dioxide inactivation of microorganisms in foods: The past, the present and the future. International Journal of Food Microbiology, 117, 1-2

Zeolites for the nutrient recovery from wastewater

To meet the growing food demand of the world population, excessive use of chemical fertilizers is occurring to improve soil fertility and crop production. The excessive use of chemical fertilizers is not economically and environmentally sustainable. Indeed, from one hand, due to the increasing demand of fertilizers is rising their costs whereas, on the other hand, the accumulation of fertilizers in wastewaters is altering the homeostasis of the ecosystems thus causing serious damages to human health [1,2]. The recovery of nutrients, such as nitrogen (N) and phosphorus (P), from wastewaters is a good option to counteract both economic and environmental issues raised by the excessive use of fertilizers [3]. Adsorption is among the most widely used methods for nutrient recovery from wastewaters due to its efficiency and simplicity. The choice of appropriate adsorbent materials is a key issue for ensuring high performance and low costs of the process [4]. Over the years, several materials have been studied to absorb nutrients from wastewaters. Zeolites, both natural and modified, have attracted great attention due to their relevant specific capacity, selectivity, safety, and stability [5]. However, considering that in municipal effluents the inorganic P exists as the anionic forms of dihydrogen or monohydrogen phosphates (H2PO4 − and HPO42−, respectively) and N in both cationic (ammonium, NH4+) and anionic (nitrate, NO3−) form [6], natural zeolites can be only used for the direct recovery of NH4+

Soil bioindicators and weed emergence as affected by essential oils extracted from leaves of three different Eucalyptus species

The widespread use of synthetic herbicides has resulted in herbicide-resistant weeds, altered ecological balance and negative effects on human health. To overcome these problems, efforts are being made to reduce the reliance on synthetic herbicides and shift to natural products. Essential oils (EOs) extracted from plants have been demonstrated to have potential herbicide activity. EOs, composed by volatile organic compounds and characterized by a strong odor, are used in the cosmetic, pharmaceutical and food industries as they are thought to be safe compounds for humans, animals, and the environment. EOs extracted from Eucalyptus leaves have antimicrobial, antiviral, fungicidal, insecticidal, anti-inflammatory, anti-nociceptive and anti-oxidant effects. Moreover, in vitro studies have demonstrated that they have inhibitory effects on germination of seeds of many crops and weeds. The aim of this work was to evaluate the in vivo effects of EOs extracted from Eucalyptus leaves on both weed emergence and biochemical soil properties. Furthermore, since the diverse species of Eucalyptus have shown to have different biological activities, EOs were extracted from three Eucalyptus species (E. camaldulensis, E. globulus, E. occidentalis). Fresh leaves were collected from an afforested area near Piazza Armerina (province of Enna, Italy) and their EOs extracted by hydrodistillation. Soil samples were collected from the topsoil (<5 cm) of an Inceptisol within the experimental farm of the University of Palermo, air-dried and sieved at 1 cm. Five hundred grams of this soil were filled in each of 20 aluminum pots (10×20 cm). The soil samples were brought up to 100% of the water holding capacity (WHC) by adding 150 mL of tap water, followed by 70 mL of tap water containing 8 mL L-1 of one of the three extracted EOs. This experimental test was repeated for remaining two EOs. Fitoil was used as emulsifier at a concentration of 0.1% (v/v). The control consisted of the soil treated as the EO treatment but with Fitoil only. The soils were incubated in greenhouse conditions. After 2 days, the 100% WHC halved and then it was kept to this level (50% WHC) by watering soil daily. The experiment was carried out in quadruplicate. After one month the soil were brought up to 100% of WHC, plant biomass and height of germinated weeds and soil biochemical properties were evaluated. This work reports the results and discuss them

Nutrient recovery from treated wastewaters by biochar and zeolite: implications for soil fertility

The increasing demand for food due to the mounting world population, coupled with the exhaustion of phosphorus (P) mines and the rising use of more and more expensive nitrogen (N) fertilizers, impose to find alternative sources of such nutrients. The recovery of N and P from treated urban wastewaters through the use of adsorbent materials and their reuse as enriched nutrient amendments to improve soil fertility may be a valid alternative. Zeolites and biochar are suitable materials for the adsorption of nutrients from treated urban wastewaters. Zeolites are crystalline microporous tectosilicates with a negatively charged structure, due to Al replacing Si, compensated by weakly bonded exchangeable cations. They are commonly used for ammonium recovery, showing an absorption capacity higher than 93% within the first 10 minutes of contact with the liquid phase. Once the zeolites are exhausted, they can be either regenerated by washing with NaCl, allowing also the recovery of ammonium, or directly applied to soil as slow-release fertilizers. Several studies have analysed the absorption capacity of zeolites at laboratory and pilot scale, but only few at plant full-scale. On the other hand, biochar is obtained by pyrolysis of plant biomass at 300-800°C and in the absence of oxygen. Studies carried out to investigate the potential of biochar to act as absorbent for the removal of P from aqueous solutions are few and, often, contrasting each other. This is probably due to the performance and properties of biochar that are highly influenced by many factors such as temperature, heating rate and residence time during pyrolysis, the feedstock used as raw material and its particle size. For this reason, the research on the adsorption and desorption properties of biochar is in its early stages, being several questions still unanswered. Even few are studies about the role of P-enriched biochar to improve the availability of P for plants in agricultural soils. Based on the above considerations, the objectives of the PhD project, carried out within the Wider Uptake project (Horizon2020 EU project), are: (i) to identify the most suitable zeolite and biochar for the recovery of inorganic N and P from treated urban wastewaters at plant full-scale, (ii) to study the mechanisms of P absorption on biochar, iii) to evaluate the impact of P-enriched biochar and of ammonium recovered from zeolite on soil chemical and biochemical properties

Zeolite – Ammonium interaction: physical-chemistry of adsorption process

Zeolites are crystalline microporous tectosilicates, either natural or synthetic. Natural zeolites were formed as a result of the interaction of volcanic rocks and volcanic ash with alkaline groundwater. Due to the formation process, there are more than 50 types of natural zeolites, the most common being clinoptilolite, which belongs to the heulandite family and has the simplified ideal formula of (Na,K,Ca)2-3Al3(Al,Si)2Si13O36·12(H2O). Geological settings and conditions during zeolite formation and geological weathering influence several parameters such as mineralogy, rock porosity/permeability and reaction rate, all of which affect their operational capabilities, hence their use in technical applications. Indeed, zeolites, due to their properties, can be used in several operations, including gas separation, adsorption, ion exchange and catalysis. In recent years, they have been playing an important role in the recovery and removal of nutrients from treated wastewater due to their ion exchange property. Their ability to adsorb cations (such as ammonium ions) comes from the substitution of Si4+ by Al3+, which increases the negative charge of the mineral lattice. The resulting negative charge is balanced by exchangeable cations such as Na+, K+ and Ca2+. The recovery of nutrients (nitrogen and phosphorus) from wastewater is necessary, as their presence in wastewater accelerates the eutrophication of receiving water bodies, creating a potentially toxic environment for fish and other aquatic life. In addition, nutrient recovery from wastewater allows solving problems i.e. the poor access to fertilizers in developing countries and the looming high cost of fertilizers; in fact, the recovered fraction of nutrients can be reused as fertilizer in agriculture promoting a circular economy approach. Furthermore, the use of natural adsorbent materials, such as zeolites, to recover nutrients from wastewater overcomes the problem associated with existing technologies. Which are often expensive and difficult to apply, limiting their use in economically poor countries due to lack of infrastructure and maintenance costs. However, the removal of ammonium by ion exchange on zeolites is influenced by the origin of the zeolites used. Previous studies on clinoptilolites with different lithological matrix have shown how the ability to adsorb NH4+ varies in clinoptilolites of different origin. For example, a Canadian clinoptilolite was capable of adsorbing about 20 mg NH4+ g-1, while a Chinese clinoptilolite did not exceed 5 mg NH4+ g-1. The original matrix may also have an influence on the treatment when natural zeolites are treated to increase the adsorption capacity. Based on the above considerations, the objectives of the PhD project, carried out within the Wider Uptake project (Horizon2020 EU project), are: i) to compare the ammonium adsorption rate on two clinoptilolites of different origin (Slovakia and Cuba), ii) to evaluate the effect that the matrix had on the treatment carried out to improve the adsorption capacit

Can the presence of Biochar negatively affect the ability of chloroform to lyse soil microbial cells?

Biochar is the solid product of the thermochemical decomposition of biomass at moderate temperatures (350–700 °C)[1] under oxygen-limiting conditions. It is nowadays utilized in various applications, for example, in the synthesis of new materials for environmental remediation, catalysis, animal feeds, adsorbent for odours, etc.[2]. In recent decades, interest has grown in the application of biochar as a soil amendment due to its beneficial effects on soil fertility and crop productivity. Biochar amendment is known to alter soil porosity, improve soil structure, increase soil surface area[3], cation exchange capacity, soil organic carbon content and soil microbial biomass[1]. The latter variable is one of the most widely adopted biological indicator for the evaluation of soil fertility status. In fact, the microbial component is the engine that governs energy transfers and nutrient transformations in the soil, thus playing a key role in its fertility. The most widely used methods for determining soil microbial biomass are the chloroform-incubation (FI) and chloroform-extraction (FE) methods[4][5], both relying on the ability of chloroform (CHCl3) fumigation to lyse soil microbial cells and release their contents. Over the years, several critical issues related to the use of CHCl3 have risen due to its toxicity to humans and the environment, as well as due to its not fully proved ability to lyse soil microbial cells. Toyota et al.[6] showed that approximately 10% of bacterial colony forming units in a sandy loam soil survived a 5-day CHCl3 fumigation. This percentage was much higher when fumigating a clayey soils. Alessi et al.[7] demonstrated that significant concentrations of CHCl3 were adsorbed, and thus retained by the clay fraction of soils thus negatively affecting the extractability of microbial-derived constituents. Such a controversial ability of CHCl3 to lyse microbial cells may be even more critical when applied to soils amended with biochar. Indeed, biochar, due to its porous structure and high specific surface area can adsorb several volatile organic compounds, including CHCl3[8]. Therefore, the aim of this study was to assess the ability of CHCl3 to lyse microbial cells in soils amended with two different biochars (EG) and (NB). Treatments were: soil without biochar (control), soil amended with 16 g of EG or NB biochar per kg of air dry soil (corresponding to 20 t ha-1) and soil amended with double amount of EG or NB biochar (corresponding to 40 t ha-1). The ability of the CHCl3 to lyse soil microbial cells in soils with or without biochar was assessed by quantifying either the amount of CO2-C released during incubation or the extractable C and N in fumigated soils, and comparing with the corresponding amount of C obtained from soil pressurized with CO2 (CO2HP). The latter is a new method, under evaluation, that causes lysis of soil microbial cells by high CO2 pressurization and subsequent rapid decompression. Since the CO2HP method is based on a physical approach, it should not be influenced by the presence of biochar in the soil samples being analyzed. Results showed that the amount of CO2-C emitted during the incubation of pressurized soils amended with biochar is higher than that of the same soils but fumigated, thus suggesting higher cell lysis efficiency of the CO2HP method than the CHCl3 in soil amended with biochar. Moreover, extractable C and N results suggested that the ability of CHCl3 depends on the type and concentration of biochar used. CHCl3 could be partly adsorbed and thus retained in the soil after fumigation and risks overestimating the C of the microbial biomass or does not allow for complete lysis of soil microbial cells. Bibliography [1] Brassard, P., Godbout, S., Lévesque, V., Palacios, J. H., Raghavan, V., Ahmed, A., Houge R., Jeanne T. &amp; Verma, M., 2019. Char and Carbon Materials Derived from Biomass,109-146. Elsevier. [2] Conte, P., Bertani, R., Sgarbossa, P., Bambina, P., Schmidt, H.P., Raga, R., Lo Papa, G., Chillura Martino, D.F. &amp; Lo Meo, P., 2021. Agronomy, 11(4), 615. [3] Hardie, M., Clothier, B., Bound, S., Oliver, G., &amp; Close, D., 2014. Plant and Soil, 376(1), 347-361. [4] Jenkinson, D. S., Powlson, D. S., 1976. Soil Biology and Biochemistry, 8, 209- 2013 [5] Vance, E. D., Brookes, P. C., Jenkinson, D. S., 1987. Soil Biology and Biochemistry, 19, 703-707 [6] Toyota, K., Ritz, K., Young, I.M., 1996. Soil Biology and Biochemistry 28, 1545-1547. [7] Alessi, D.S., Walsh, D.M., Fein, J.B., 2011. Chemical Geology, 280 (1-2), 58-64 [8] Kumar, A., Singh, E., Khapre, A., Bordoloi, N., &amp; Kumar, S., 2020. Sorption of volatile organic compounds on non-activated biochar. Bioresource Technology, 297, 122469

PHYTOTOXIC POTENTIAL OF EUCALYPTUS ESSENTIAL OILS FOR WEED CONTROL

The widespread use of synthetic herbicides has resulted in herbicide-resistant weeds, disturbed ecological balance and negative effects on human health. Due to this fact, it is necessary to rely on alternative weed control strategies using natural compounds released by plants, such as essential oils (EOs). EOs have a short half-life since they are biodegradable, and are safer than synthetic compounds, with little damage to the environment, without even contaminating ground water (Topal and Kocaçalıskan 2006). Essential oils from different species contain allelochemical compounds that possess significant phytotoxic activity. Azizi and Fuji (2006) demonstrated that Eucalyptus (family Myrtaceae) EOs showed strong inhibitory effects on germination of seeds of many crops and weeds. The aim of this work is to evaluate the phytotoxic potential effect of four Eucalyptus species (E. camaldulensis, E. lesouefi, E.occidentalis, E. torquata) EOs, on weed seed germination of two dicotyledons (Amaranthus retroflexus and Portulaca oleracea) and two monocotyledons (Avena fatua and Echinochloa crus-galli) which are considered among the most serious weeds for the Mediterranean crops. Fresh leaves of E. camaldulensis and E. occidentalis were collected in afforested area near Agrigento (Sicily, Italy) during November and December of 2017. The leaves of E. lesouefi and E. torquata were collected during March, April and May from Gabes, located in the South of Tunisia on 2015. The EOs were extracted from each species by steam distillation with a Clevenger apparatus according to the standard procedure described in the European Pharmacopoeia (1975), and stored at 4 °C until they were used. Weed seeds of A. retroflexus, P. oleracea, A. fatua and E. crus-galli were purchased from Herbiseed (England). To test the phytotoxicity activity of the EOs, different concentrations were prepared: 0.125; 0.25; 0.5; 1; 2; 4 µl/ml for dicotyledons and 0.5; 1; 2; 4; 8; 12 µl/ml for monocotyledons. The oils were loaded on the inner side of two layer of filter paper (73 g/m2) in Petri dishes (9 cm diameter), after sowing twenty seeds of each weed type (10 in case of monocotyledons) on the base of the Petri dishes, in two other layers of filter paper wetted with 5 ml of distilled water, in case of the dicotyledons, and 6 ml for the monocotyledons. The controls were prepared with the same quantities of distilled water. For each concentration, five replications were maintained (10 in case of monocotyledons). All the Petri dishes were kept in a growth chamber maintained alternating 30.0 +/- 0.1 °C, 16 h in light and 20.0 +/- 0.1 °C, 8 h in dark. To register germination and seedling length data, photos were taken after 3, 5, 7, 10 and 14 days, and they will be processed with Digimizer. In the poster, the results will be illustrated and discussed

Wastewater treatment sludge composting

In recent years, the amount of sewage sludge generated by wastewater treatment plants (WWTPs) has increased due to worldwide population growth and to efficiency of biological treatment processes [1,2]. Sludge is an important source of secondary pollution to aquatic environments and a potential risk to human health; moreover, it represents one of the most important cost items in the functioning of water treatment plants [3–5]. About 60% of the operating costs of secondary wastewater treatment plants in Europe can be associated with the treatment and disposal of products [6]. For this reason, proper sludge management becomes increasingly important, at both national and international level, and it becomes necessary to find effective measures to limit the environmental impacts and to reuse sludge as a resource, within a circular economy vision [2,7]. Current methods of utilization of sewage sludge include agricultural application, landfilling, incineration, drying, and composting and/or vermicomposting. Composting is a widely used cost-effective and socially acceptable method for treating solid or semisolid biodegradable waste [8]. In agriculture sewage sludge is used for rehabilitation of degraded soils, reclamation, or adaptation of land to specific needs [9]. The above consideration comes from several studies showing that the application of sludges to agricultural land can improve soil fertility and, therefore, crop productivity [10–12]. This field of use is also possible due to its composition; in fact, it is rich in organic matter, nitrogen, phosphorus, calcium, magnesium, sulfur, and other microelements needed by plants and living native organisms in the soil. However, sewage sludge may contain a wide range of harmful toxic substances such as heavy metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins and dibenzo-p-furans, polychlorinated biphenyls, di(2-ethylhexyl) phthalate, polybrominated diphenyl ethers, detergent and drug residues, pharmaceutical and personal care products (PPCPs), endogenous hormones, synthetic steroids and pathogenic organisms [13,14], which can cause harm to the environment and humans. Due to the presence of those toxic elements, stabilization of sewage sludge is necessary to avoid any environmental risk [15]. Stabilization of sewage sludge is defined as “biological, chemical or thermal treatment, long-term storage or any other appropriate process aimed at reducing its fermentability and the health hazards arising from its use” [16]. This definition is found in Council Directive 86/278/EEC, which was issued to regulate the use of sludge in agriculture, the primary objective of which is the environment, in particular the soil, and the protection of human health. European Directive 86/278/EEC was implemented in Italy by Legislative Decree 99/1992 [17]. Both the European Directive and the Italian legislative decree can be considered obsolete, this is why the European Union is moving towards amending them to reflect the new needs of the sector and to keep up with technological innovations. Currently, there are several processes for sludge stabilization, including composting, which is one of the most widely used methods for stabilizing organic matter in general, reducing the number of pathogenic microorganisms and the amount of toxic elements [18]. This is possible because during the composting process the organic compounds present in the biomass to be composted are converted into chemically recalcitrant, that is, stabilized, humic substances, while pathogens are eliminated due to the heat generated during the process thermophilic phase [19,20]. During the composting of sludges, the addition of bulking agents is needed, as they ameliorate the composting performance by providing structural support that improves aeration and regulates moisture content and C/N ratio of composting mass [21,22]. Sludge composting, however, has to be focused on limiting some secondary causes of pollution related to the process itself, such as greenhouse gas (GHG) emissions and heavy metal contamination [23]. Indeed, in the last decades, the handling of sewage sludge with traditional methods has led to the release of an enormous amount of greenhouse gases. The choice of an appropriate bulking agent is, therefore, fundamental to limit the emission of climate-altering gases, and, at the same time, to increase the microbial activity thus improving the quality of the compost [24,25]. This chapter aims (1) to give an overview of the national and international legislation on sludge management and reuse, (2) to analyze the composting process and the state of the art regarding sludge composting to understand the limitations at large-scale application, and (3) to discuss the technological innovations in the field and highlight future perspectives

Dynamics of soil available carbon, nitrogen and phosphorus pools after burying innovative bio-based mulching films

The use of plastic mulching films is rapidly increasing in agriculture to enhance crop productivity and control weeds. However, their non-biodegradability and long-lasting presence in soil have raised serious concerns regarding their environmental impact. Typically composed of non-biodegradable materials like polyethylene, these films can persist in the soil for several years after use, enhancing the plastic pollution and posing challenges for sustainable agricultural practices. Consequently, there is a pressing need to explore alternative materials that are both biodegradable and environment-friendly. In this context, the PRIN mulching+ project aims to make innovative mulching films based on carboxymethyl cellulose, chitosan, and sodium alginate, enriched with N and P salts acting as slow-release fertilizers in the soil. Thus, the purpose of this study is to evaluate the effects of the degradation of these innovative films after burial in the soil on the dynamics of available N and P and on the microbial biomass C (MBC) and N (MBN)for assessing their suitability as sustainable alternatives to conventional plastic mulch films. Four types of mulch films were used in the study. They were prepared with either 1:1 or 17:3 mass ratio of chitosan to cellulose, both with and without the addition of 90% by weight of NH4H2PO4, in order to investigate the influence of material ratios and nutrient addition on the biodegradation processes and soil microbial component. The experiment involved burying 0.1% by weight of the film in pre-wetted soil, to simulate the field conditions. Soil samples were collected 30, 60, 90 and 120 days after burial to evaluate as variables MBC and MBN, available ammonium, nitrate and phosphate, but also the composition and abundance of major microbial groups in the soil. The results showed significant changes in soil parameters, with ammonium, nitrate and phosphate levels influenced by the presence of NH4H2PO4. Moreover, an increase in soil MBC and MBN over time occurred, suggesting the assimilation of film organic matter by soil microorganisms. Overall, results were promising for the use of these innovative bio-based films in agriculture. Ongoing activities include the use of 13C- and 15N-labeled films to track the fate of film-derived C and N in soil and to identify which main microbial groups are responsible for their degradation