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

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. & 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. & Lo Meo, P., 2021. Agronomy, 11(4), 615. [3] Hardie, M., Clothier, B., Bound, S., Oliver, G., & 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., & Kumar, S., 2020. Sorption of volatile organic compounds on non-activated biochar. Bioresource Technology, 297, 122469

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

Polysaccharide-based biodegradable films for agricultural mulching

In the last 20 years, the global population has blowout growth from 6.0 billion to 7.2 billion and will reach over 8.0 billion around 2046 [1]. Consequently, food shortage has drawn attention, and the demand for agricultural products has increased annually. To meet this need, the excessive and prolonged use of mulching films based on low-density polyethene resulted in significant environmental pollution events, leading to serious side effects on human health [2]. Due to the thickness of the plastic film and the difficulty of recovery, some mulch films were discarded in agricultural soils intentionally or unintentionally. Mulch film residue is a direct source of farmland meso- and microplastics (MMPs), which constitute a global environmental issue, as they accumulate even in the food chain [3]. MMPs' further degradation into nanoscale particles can endanger human health [4]. To provide agricultural sustainability, there is a great interest in developing biodegradable bio-based polymeric films for agriculture mulching, which can be tilled directly into the soil after use. Based on the above issues, this study aims at (i) the preparation and characterisation of biodegradable bio-based composite films and (2) their enrichment with plant nutrients, which could be efficiently released into the water to sustain their application as mulch films on the soil. Sodium carboxymethyl cellulose (CMC), chitosan (CS) and sodium alginate (SA) were combined in the presence of glycerol as a plasticiser to produce composite films by solvent casting. Composition (i.e., concentrations and mass ratios between the precursors) and cross-linking agent (CaCl2) effects on films' properties were evaluated. In the first stage, we investigated the structure of the formed films through Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy, the thermal and mechanical properties by thermogravimetric analysis (TGA) and dynamic mechanical analysis (DMA), and some water-interaction properties (degree of swelling and solubility in water). This approach allowed identifying the best quality films, which were enriched with NH4H2PO4, as N and P are generally the most deficient nutrients in the soil. Moreover, the release kinetics in the water of this salt was studied. The latter aspect is of great importance as the release of N and P helps to improve the nutrient supply to the soil, thus reducing the use of synthetic fertilisers. [1] B. Chieng et al. J. Appl. Polym. Sci. 130 (2013) 4576-4580 [2] H. M. S . Akhtar, et al. Int. J. Biol. Macromol. 118 (2018), 469-477 [3] M.C. Rillig, M. C. Environ. Sci. Technol. 46 (2012), 6453-6454 [4] I. Ali et al. J. Clean. Prod. 313 (2021) 12786

Towards Sustainable Agriculture: Preparation and Characterization of Biodegradable Composite Films for Agricultural Mulching

Over the past 20 years, the world's population has grown exponentially1. Consequently, the demand for agricultural products has increased annually. To meet this need, the prolonged use of mulching films based on low-density polyethene resulted in significant environmental pollution events, leading to serious side effects on human health2. Based on this, there is a great interest in developing biodegradable polymeric films that can be tilled directly into the soil after use thus improving their environmental sustainability. Here, we present the preparation and characterization of biodegradable sodium carboxymethyl cellulose, chitosan, and sodium alginate-based composite films in the presence of glycerol as a plasticizer and calcium chloride as cross-linker and their enrichment with the NH4H2PO4 salt, as N and P are generally the most deficient nutrients in the soil. The effects of the composition and the cross-linking agent on some water interaction properties and the thermal and mechanical properties were evaluated. To rationalise the macroscopic behaviour of the films, infrared spectroscopy, and X-ray diffractometry were applied to gain information on the interactions and structural changes induced by the salt and the cross-linker. This approach allowed for the identification of the best quality films for which the release kinetics of NH4+ and PO43- ions as a function of film thickness were studied. The latter aspect is of great importance as the release of N and P helps to improve the nutrient supply to the soil, reducing the use of synthetic fertilisers. Bibliografia 1 Chieng, B. et al.; J. Appl. Polym. Sci., 2013, 130, 4576-4580 2 Akhtar, H.M.S. et al.; Int. J. Biol. Macromol., 2018, 118, 469-47

Il biochar può influenzare negativamente la capacità del cloroformio di lisare le cellule microbiche del suolo?

Il biochar è un materiale carbonioso derivante dalla decomposizione termochimica della biomassa in condizioni di limitazione dell'ossigeno[1]. Negli ultimi decenni, il biochar è stato ampiamente applicato come ammendante grazie ai suoi effetti benefici sulla fertilità del suolo, sulla produttività delle colture e sulla biomassa microbica. Grazie alla sua struttura porosa e all'elevata superficie specifica, il biochar è in grado di adsorbire composti organici volatili, tra cui il cloroformio (CHCl3)[2]. Pertanto, tale proprietà del biochar potrebbe interferire con i due metodi più utilizzati per la determinazione della biomassa microbica, basati sulla capacità del CHCl3 di lisare le cellule microbiche[3][4]. Inoltre, nel corso degli anni, sono state sollevate diverse criticità legate all'uso di CHCl3 a causa della sua tossicità[5] per l'uomo e l'ambiente e della sua scarsa efficienza nel lisare le cellule microbiche del suolo[6][7][8]. Lo scopo di questo lavoro è stato quello di valutare la capacità del CHCl3 di lisare le cellule microbiche in terreni ammendati con biochar. A tal fine, il C e l’N della biomassa microbica (BMC e BMN) determinata con i metodi di fumigazione-incubazione e fumigazione-estrazione è stata confrontata con la BMC e la BMN valutate in suoli trattati con alte pressurizzazioni di CO2 (CO2HP). Quest'ultimo è un nuovo approccio fisico basato sulla lisi delle cellule microbiche del suolo mediante un'elevata pressurizzazione con CO2, al posto della fumigazione con CHCl3. I risultati hanno confermato l'ipotesi che il biochar interferisca con i metodi per la determinazione della biomassa microbica del suolo basati sulla fumigazione. Inoltre, l'approccio CO2HP è più efficace per la lisi delle cellule microbiche nei suoli modificati con biochar.Biochar is a carbonaceous material deriving from the thermochemical decomposition of biomass under oxygen-limiting conditions[1]. During the last decades, biochar has been extensively applied to soil as amendment due to its beneficial effects on fertility, crop productivity and microbial biomass. Due to its porous structure and high specific surface area, biochar is able to adsorb volatile organic compounds, including chloroform (CHCl3)[2]. Thus, we hypothesized that such a biochar property might interfere with the two most widely used methods for the determination of microbial biomass based on the ability of CHCl3 in lysing microbial cells[3][4]. Moreover, over the years, several critical issues related to the use of CHCl3 have been raised due to its toxicity[5] to humans and environment, and scarce efficiency in lysing soil microbial cells[6][7][8]. The aim of this work was to evaluate the ability of CHCl3 to lyse microbial cells in soils amended with biochar. To this aim, microbial biomass C and N (MBC and MBN) determined by the fumigation-incubation and fumigation-extraction methods were compared to MBC and MBN assessed in high CO2-pressurized soils (CO2HP). The latter is a new physical approach based on the lysis of soil microbial cells by high pressurization of soil with CO2, instead by CHCl3 fumigation. Results confirmed the hypothesis that biochar interfere with the fumigation-based methods for soil microbial biomass determination. Moreover, CO2HP approach is more effective for lysis of microbial cells in soils amended with biochar

Water reuse of treated domestic wastewater in agriculture: Effects on tomato plants, soil nutrient availability and microbial community structure

The reuse of treated wastewater (TWW) in agriculture for crop irrigation is desirable. Crop responses to irrigation with TWW depend on the characteristics of TWW and on intrinsic and extrinsic soil properties. The aim of this study was to assess the response of tomato (Solanum lycopersicum L.) cultivated in five different soils to irrigation with TWW, compared to tap water (TAP) and an inorganic NPK solution (IFW). In addition, since soil microbiota play many important roles in plant growth, a metataxonomic analysis was performed to reveal the prokaryotic community structures of TAP, TWW and IFW treated soil, respectively. A 56-days pot experiment was carried out. Plant biometric parameters, and chemical, biochemical and microbiological properties of different soils were investigated. Shoot and root dry and fresh weights, as well as plant height, were the highest in plants irrigated with IFW followed by those irrigated with TWW, and finally with TAP water. Plant biometric parameters were positively affected by soil total organic carbon (TOC) and nitrogen (TN). Electrical conductivity was increased by TWW and IFW, being such an increase proportional to clay and TOC. Soil available P was not affected by TWW, whereas mineral N increased following their application. Total microbial biomass, as well as, main microbial groups were positively affected by TOC and TN, and increased according to the following order: IFW > TWW > TAP. However, the fungi-to-bacteria ratio was lowered in soil irrigated with TWW because of its adverse effect on fungi. The germicidal effect of sodium hypochlorite on soil microorganisms was affected by soil pH. Nutrients supplied by TWW are not sufficient to meet the whole nutrients requirement of tomato, thus integration by fertilization is required. Bacteria were more stimulated than fungi by TWW, thus leading to a lower fungi-tobacteria ratio. Interestingly, IFW and TWW treatment led to an increased abundance of Proteobacteria and Acidobacteria phyla and Balneimonas, Rubrobacter, and Steroidobacter genera. This soil microbiota structure modulation paralleled a general decrement of fungi versus bacteria abundance ratio, the increment of electrical conductivity and nitrogen content of soil and an improvement of tomato growth. Finally, the potential adverse effect of TWW added with sodium chloride on soil microorganisms depends on soil pH

Soil biodegradation of nutrients enriched cellulose- and chitosan-derived mulching films for sustainable horticulture

In 2019, global plastics production reached 370 million tons, of which 58 million tons were in Europe[1]. If the plastic use in agriculture accounts for 2% of the global production[2], more than 7 million tons of plastic were used in 2019 in the agricultural sector. Mulch films represent the major source of plastic contamination in agricultural soils[3]. The agricultural surface area covered by plastic films in Europe is four times larger than that covered by greenhouses and six times that of low tunnel hoops. Over the past decades, biodegradable biopolymer mulching films (BPMFs) have been developed to reduce soil pollution by non-biodegradable plastic debris[4] and to expand the circular bioeconomy[5]. In Europe, since 1999, low density polyethylene mulches (LDPMs) have to be dismissed after their use to remove source of pollutants that can reach up to 200 kg ha-1[6] and decline soil quality, crop growth, and yield[7]. BPMFs are a sustainable alternative to conventional LDPMs. Unlike LDPMs, BPMFs, at the end of their lifetime, are tilled into soil where they are expected to be biodegraded by soil microorganisms[8]. Moreover, BPMFs show an estimated saving of about 500 kg of CO2 equivalent per hectare in comparison with LDPMs. Conversely, the impact of LDPMs in intensive horticulture could result higher than weed control by herbicides as by life cycle assessment (LCA)[9]. BPMFs can be obtained by thermo-plasticizing, solvent casting and spraying processes by using renewable and biodegradable raw materials such as starch, cellulose, chitosan, alginate, glucomannan[10] and glycerin as plasticizer[11]. Cellulose and chitosan, being the two most abundant natural biopolymers on Earth, have been proposed as the best candidates for BPMFs production. Unfortunately, the high tendency for intra- and intermolecular hydrogen bonding confers undesirable mechanical properties. The addition of plasticizer as well as fillers overcome this problem[12] modifying mechanical and functional properties of the materials. To sum up, biopolymer blending is an effective strategy to reuse cellulose and chitosan-containing by-products and develop materials with novel mechanical characteristics[13]. Moreover, the functional properties of these materials can be tuned by doping them with suitable compounds[14]. Based on what stated above, and considering that soil fertility, crop growth and yield, are generally N and P limited, the core idea of this project is the preparation of N- and P-enriched BPMFs for soil mulching, in order to slowly release soluble nutrients into soils upon their biodegradation. The latter aspect is of great importance because a proper C:N:P ratio can lead to an increase of soil-dwelling organisms thus contributing to nutrient cycling in the soil-plant system, soil C sequestration and biological fertility status[15]. Moreover, repeated additions of BPMFs over long term can increase the amount of nutrients, thus reducing the use of external inputs (e.g. synthetic fertilizers) within a circular economy perspective. The specific aim of the proposed research are: i) to set up a method for the preparation of suitable BPMFs enriched with N and P; ii) to characterize novel BPMFs and evaluate their structure, degradation kinetics, and isotopic composition iii) to assess the impact of the innovative BPMFs on soil nutrient cycling and crop growth and yield; iv) to evaluate the effect of the innovative BPMFs on soil prokaryotes and micro-arthropods communities; v) to speed-up the biodegradation of the innovative BPMFs by spraying them at the end of their lifecycle with selected microorganisms and by adding the recipient soil with earthworms; vi) to evaluate the innovative BPMFs using the LCA methodology and to investigate its role within the circular economy. Bibliography [1] Plastic Europe, 2020. Website https://www.plasticseurope.org/it/resources/publications/4312-plastics-facts-2020 accessed on 05.01.2021 [2] Vox, G., Loisi, R.V., Blanco, I., Mugnozza, G.S., & Schettini, E. (2016). Agriculture and Agricultural Science Procedia, 8, 583-591. [3] Wenqing, H., Enke, L., Qin, L., Shuang, L., Turner, N., C. & Changrong, Y. 2014. World Agriculture, 4, 3236. [4] Sanchez-Hernandez J.C., Capowiez Y. & Ro K.S., 2020. ACS Sustainable Chemistry & Engineering, 8, 4292-4316. [5] Karan, H., Funk, C., Grabert, M., Oey, M., & Hankamer, B., 2019. Trends in Plant Science, 24, 237-249. [6] Razza, F., Guerrini, S., & Impallari, F.M., 2019. Acta Horticulturae, 1252, 77-84. [7] Hou, L., Xi, J., Chen, X., Li, X., Ma, W., Lu, J., Xu J. & Lin, Y. B, 2019. Journal of Hazardous Materials, 378, 120774. [8] Kyrikou, I., & Briassoulis, D., 2007. Journal of Polymers and the Environment, 15, 125–150 [9] Tasca, A. L., Nessi, S., & Rigamonti, L., 2017. 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Volume estimation of soil stored in agricultural terrace systems : a geomorphometric approach

High-resolution topographic (HRT) techniques allow the mapping and characterization of geomorphological features with wide-ranging perspectives at multiple scales. We can exploit geomorphometric information in the study of the most extensive and common landforms that humans have ever produced: agricultural terraces. We can only develop an understanding of these historical landform through in-depth knowledge of their origin, evolution and current state in the landscape. These factors can ultimately assist in the future preservation of such landforms in a world increasingly affected by anthropogenic activities. From HRT surveys, it is possible to produce high-resolution Digital Terrain Models (DTMs) from which important geomorphometric parameters such as topographic curvature, to identify terrace edges can be extracted, even if abandoned or covered by uncontrolled vegetation. By using riser bases as well as terrace edges (riser tops) and through the computation of minimum curvature, it is possible to obtain environmentally useful information on these agricultural systems such as terrace soil thickness and volumes. The quantification of terrace volumes can provide new benchmarks for soil erosion models, new perspectives to stakeholders for terrace management in terms of natural hazard and offer a measure of the effect of these agricultural systems on soil organic carbon sequestration. This paper presents the realization and testing of an innovative and rapid methodological workflow to estimate the anthropogenic reworked and moved soil of terrace systems in different landscapes. We start with remote terrace mapping at large scale and then utilize more detailed HRT surveys to extract geomorphological features, from which the original theoretical slope-surface of terrace systems were derived. These last elements were compared with sub-surface information obtained from the excavations across the study sites that confirm the reliability of the methodology used. The results of this work have produced accurate DTMs of Difference (DoD) for three terrace sites in central Europe in Italy and Belgium. Differences between actual and theoretical terraces from DTM and excavation evidence have been used to estimate the soil volumes and masses used to remould slopes. The utilization of terrace and lynchet volumetric data, enriched by geomorphometric analysis through indices such as sediment conductivity provides a unique and efficient methodology for the greater understanding of these globally important landforms, in a period of increasing land pressure