Valorization of Spent Co ﬀ ee Grounds, Biochar and other residues to Produce Lightweight Clay Ceramic Aggregates Suitable for Nursery Grapevine Production

: The valorization of agro-industrial by-products is one of the key strategies to improve agricultural sustainability. In the present study, spent co ﬀ ee grounds and biochar were used as pore forming agents in the realization of lightweight clay ceramic aggregates that were used as sustainable fertilizers, in addition to tailored glass fertilizer containing phosphorous (P) and potassium (K) and nitrogen (N) synthetic fertilizer, for nursery grapevine production. The obtained fertilizers were assessed in a pot experiment for the fertilization of bare-rooted vines. Unfertilized (T0) and fertilized plants (T1, using NPK-containing commercial fertilizer) were used as controls. Plants fertilized by spent co ﬀ ee grounds and spent co ﬀ ee grounds + biochar-containing lightweight aggregates and added with 30 wt% of the above-mentioned glass and N fertilizers (T2 and T3, respectively) recorded higher values of plant height, shoot diameter, leaf and node numbers. Moreover, T2 treatment induced the highest chlorophyll content, shoot and root dry weights. The present study shows that lightweight clay ceramic aggregates containing spent co ﬀ ee grounds and glass and N fertilizers can be used for nursery grapevine production, in turn improving the agricultural sustainability.


Introduction
The increasing request of grapevine (Vitis vinifera L.) planting materials, due to the expansion of global viticulture and vineyard renewal, is encouraging research studies to increase sustainability of this crop already starting from the nursery phase [1].
Nowadays, different grapevine planting materials are available in commerce. For the planting of a new vineyard, the 1-year-old dormant bare-rooted vines are the most adopted material [2]. It is produced by bench grafting at the end of winter-early spring the dormant one-bud cuttings of the scion onto hardwood cuttings of the rootstock collected during plant dormancy phase, subsequently growing the grafted vines in a greenhouse to achieve the callusing and thereafter transferring them in the open field for the growth and development of roots and shoot. Finally, after excavation and roots and shoot pruning, bare-rooted plants are commercialized.
to degradation and mineralization. A further advantage of the storage of biochar in the ground is the reduction of CO 2 emissions in the atmosphere [29]. Indeed, the carbon sink process occurring in the soil rescues carbon dioxide from the cycle of carbon. In the last 15 years the use of biochar as a soil or substrate improver has been studied, while its application in building materials is starting to gain more attention recently. The use of biochar contributes to reducing the raw material consumption and the energy saving of the LAW firing process due to the residual carbon content present [30]. Therefore, the aim of the present study was the assessment of spent coffee grounds and biochar in the production of innovative LWAs in combination with a tailored glass-fertilizer based on cattle bone flour ash, K 2 CO 3 and packaging glassy sand and their use in the sustainable production of potted grapevine planting materials.

Lightweight Aggregates Production and Characterization
Lightweight aggregates were obtained by powder sintering of a local (Modena, Italy) ferruginous red clay (85 wt%) with the addition of spent coffee grounds (SCGs) or spent coffee grounds (Modena, Italy) + biochar (B) (15 wt%) (Emilia Romagna Region, Italy) as poring agents to reduce both density and sintering temperature. Biochar was derived from gasification of woody biomass from river maintenance. The raw materials were dried in an oven at 105 • C for 24 h and subjected to a grinding and sieving process in order to reach a particle size <100 µm for clay and <250 µm for B and SCGs. A 30 wt% of a powdered glass (100 µm) containing the nutrients phosphorus and potassium, previously developed by the authors [21], based on cattle-bone flour ash (CBA), packaging glass cullet and K 2 CO 3 and obtained by melting the mixture at 1400 • C in an electric furnace and quenching in water, was added to the clayey body in order to confer fertilizing effect to the materials. Spherical shape samples of 1.5-2.0 g of weight and a medium diameter of 1-1.5 cm were hand-pelletized by the addition of water to obtain the adequate plasticity required for shaping. Samples were dried at 105 • C for 24 h to reduce their moisture content and then subjected to a firing process in a laboratory electrical kiln (Lenton mod. AWF13/12, Hope Valley, United Kingdom) that caused sintering of the grains and changes in their density and porosity. In order to imitate the thermal shock produced in the rotary furnace, the samples were introduced and kept in the furnace at 1000 • C for 1 h and finally allowed to cool through natural convection. The samples were codified as reported: red clay (C) + pore forming agent [spent coffee ground (SCG) or biochar (B) followed by a number indicating the percentage introduced 15% + fertilizer glass (FG) followed by a number indicating the percentage of fertilizer glass added 30%].
Specific tests of release were performed according to both Italian and European regulations [31] to evaluate the release capacity of P and K nutrients as well as other elements that could be harmful for the environment. Distilled water and citric acid solution 2 vol.% were used as reaction media; the values were monitored in a period from 7 to 90 days to verify controlled release. The aggregates were tested in whole size, to simulate the conditions occurring in the soil. The liquid solutions derived were analyzed by inductively coupled plasma mass spectrometry (ICP-MS Agilent 7500a, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, USA).

Nursery Greenhouse Experiments
The potting experiment was performed in a nursery greenhouse, located at Reggio Emilia (Italy) under controlled temperature ranging from 19 to 25 • C (day/night) and relative humidity varying from 60% to 70%. Plants were grown under long-day conditions (15 h light, 9 h dark; light intensity 280 µmol m −2 s −1 ).
The scion/rootstock combination was 'Lambrusco Salamino' cultivar grafted onto 'Kober 5BB' as rootstock. Bare-rooted vines were manually transplanted (one plant per pot) in plastic pots of 4.5 L capacity and filled with commercial neutralized peat (Fondolinfa Universale, Linfa Spa, RE, Italy), containing 70% organic matter, 35% organic carbon, 0.6% nitrogen (N) and having pH 6.0. Pots were arranged in a completely randomized design with five replicates and manually irrigated every two days in order to ensure the water holding capacity of the substrate. Pests were controlled according to the integrated production rules of the Emilia-Romagna Region (Italy). Plants were pruned according to one-stem training system.
Fertilization was performed only at planting, administering, in total, 6 g of N, 1.4 g of P 2 O 5 and 1. In order to supply the expected nitrogen rate (6 g per pot) ammonium nitrite and urea (2.9 and 3.1 g of N, respectively) were applied in combination with LWAs.
Growing media (GM) were weekly monitored for the different parameters: GM water content, temperature and electrical conductivity (using a sensor Teros 11/12, Meter Group, Washington, USA). At day 314 of the year (DOY) the following traits were measured: leaf chlorophyll (Chl) and flavonoid (Flav) contents, nitrogen balance index (NBI) as ratio between Chl and Flav [30], leaf anthocyanin content (Anth), basal shoot diameter, shoot height, number of leaves and nodes. Chl, Flav, NBI, and Anth were measured on the youngest fully expanded leaf by Dualex 4 Scientific (Dx4, FORCE-A, Orsay, France), an optical leaf-clip meter for non-destructive assessment of the physiological status of the plants [33]. Leaf temperature was recorded on the fully expanded leaf using an infra-red thermometer (Everest Instruments, Chino Hills, CA, USA). Moreover, parameters characterizing plant growth were recorded: shoot fresh weight of the scion (SFW), above-ground rootstock fresh weight (RSFW), root fresh weight (RTFW), total fresh weight (TFW), shoot dry weight (SDW), above ground rootstock dry weight (RSDW), root dry weight (RTDW), total dry weight (TDW). Using these parameters, the following indexes were calculated: fraction of total dry weight allocated to shoot (FTS) ((SDW/TDW)* 100), fraction of total dry weight allocated to above-ground rootstock (FTRS) ((RSDW/TDW)* 100), and fraction of total dry weight allocated to root (FTR) ((RTDW/TDW)* 100).
In order to compare N use across the treatments, the nitrogen efficiency index was calculated from the measured plant total dry matter (g pot −1 ) and the amount of N applied (g pot −1 ) and was expressed as g dry biomass g −1 N [34].

Data Analysis
All recorded data were analyzed using GenStat 17th software (VSN International, Hemel Hempstead, UK) for analysis of variance (ANOVA). Significant means were separated by Duncan's test at p ≤ 0.05.

Results and Discussion
To produce grapevine planting materials for new vineyards or to replace diseased plants, high-standard quality attributes (such as good vigor, absence of defects, well healed graft union, in addition to the guarantees of satisfactory phytosanitary status and genetic identity of rootstock and scion), can be obtained under greenhouse conditions [35]. Nowadays the environmental sustainability of orchards or vineyards must necessarily consider the pre-planting phases, i.e., the propagation phases of the plant material (i.e., proper recycling of any plastic residues or use of biodegradable pots, low impacting irrigation, disease control and fertilization methods). For the latter agronomic practice, researchers are called to find alternative fertilizers for nursery phase ensuring good plant development and low environmental impact. Considering some previous evidences of the positive effects of spent coffee grounds and biochar on growth of horticultural and woody species [2,12,13,20,36], in the present study SCGs and B were valorized to obtain new LWAs which were for the first time tested for the fertilization of bare-rooted vines in a nursery greenhouse.

Lightweight Aggregates Characteristics
All specimens prepared using red clay together with spent coffee ground (CSCG) and mixed with biochar (CBSCG) showed good workability and a good final aggregation. Unfired samples containing biochar had a darker color but after firing it disappeared obtaining aggregates with a classic red color. The idea of mixing biochar and spent coffee ground as poring agent derives from the natural alkaline pH of the biochar (11.8) and acid pH of SCGs (5.5). Aggregates prepared with only biochar as a pore-forming agent had an alkaline pH (9.3) out of the limit. The aggregates to be used in the soil must have a pH value within the optimal range of plant comfort (6)(7)(8) and EC less than 2 mS/cm. Considering these premises, it is important to evaluate the properties of LWAs functionalized with 30 wt% of fertilizing glass (FG). Table 1 shows the results of the physical and chemical characterization. The physical characterization permitted us to classify the specimens functionalized with 30 wt% of fertilizing glass as LWAs, because their apparent density values are lower than 2000 kg m -3 (UNI-EN 13055-1:2003) and their porosity higher than 40%.
pH and electrical conductivity are the most important parameters to check for an agronomic application. As can be seen in Table 1, the CSCG15FG30 shows results in line with the soil guidelines above reported values while the pH of CBSCG15FG30 is slightly higher.
By the citric acid test, the release of the main nutrients P and K was monitored until 90 days. LWAs prepared with only SCG as a pore-forming agent showed an increase of P% release from 30 to 90% while those prepared using the mix (SCG + B) as a pore-forming agent highlighted a P% release from 30 to 82%. The K% release observed was lower than P% for the two compositions analyzed. For CSCG15FG30, the values increased from 5% (7 days) to 17% in 90 days while a higher release was observed for CBSCG15FG30 from 8% (7 days) to 24% in 90 days.
These differences are putatively due to the capacity of biochar to adsorb nutrients like P and K as already suggested by Carrey et al. in 2005 [37]. However, further studies are needed to corroborate this hypothesis. In addition, regarding the use of the proposed innovative fertilizers and their possible implication for soil-plant relationship, it is well known that nutrient availability of P and K depends on soil pH and on the interaction between soil microbiota and rhizosphere [38].

Bare-Rooted Vines Production
The main parameters recorded for the growing media were reported in Table 2. Peat fertilized with LWA containing spent coffee grounds and biochar (T3) showed the highest values of growing media water content, temperature and electrical conductivity (+33%, +1% and +9%, on average). These three parameters are strictly related with the plant growth and its development. In fact, soil/growing media temperature and water availability play a key role in the growth and development of roots and the vegetative growth of the vineyard canopy. During the productive phase of the vineyard, to obtain high-quality grapes, an optimal soil water deficit pattern is required [36]. On the other hand, young vines in the nursery and after planting need a high water availability, allowing maximum growth to hasten the development of the vineyard canopy [39]. Furthermore, soil temperature is positively related with root growth and development. Indeed, root growth starts above a threshold of 6-7 • C and increases with temperature with an optimum occurring at around 30 • C [40]. In bare-rooted vines after planting the availability of carbohydrate reserves is presumably quite low, and roots, shoots and leaves may face competition for photoassimilates. Warmer soil increases the mobilization of root reserves and the shoot biomass and may enhance root growth through a higher rate of carbohydrate reserve catabolism and a consequent greater availability of energy and carbon, according to Clarke et al. [41]. Together with the good water availability, a warmer soil also enhances the uptake of nitrogen, phosphorus, potassium and other nutrients [42]. These considerations are consistent with the best growth observed in T3-treated vines and with higher values of root and shoot weights, plant height, number of nodes and shoot diameter of T2-treatment than the control (T0) ( Tables 3 and 4). Regarding soil electrical conductivity, by the effect of fertilization [43], Abad et al. [44] reported 0.5 dS m −1 as the threshold to achieve the highest plant growth. In our experiment a lower EC was found for T1 and T2 treatments than T0; moreover, the values detected for the overall thesis never reached the threshold indicated by Abad et al. [44].
Morpho-physiological parameters of bare-rooted vines were affected by LWA applications which, induced for the overall measured traits a better performance respect to commercial fertilizer applications (Table 3). At 314 DOY, corresponding to the "Principal growth stage 9: Senescence" of the BBCH-scale, T2-and T3-fertilized plants displayed higher values of plant height, node number and shoot diameter than T1-fertilized or T0-unfertilized ones (+9%, +21% and +22% of T2 and T3 respect to T0). Within stage 9, the degree of leaf fall ranged from 93 "beginning of leaf-fall" for the fertilized theses to 97 "end of leaf-fall" for the unfertilized one, indicating a longer growth duration for fertilized plants. Moreover, the highest value of leaf number (16 per plant) was detected in plants fertilized with LWAs containing spent coffee grounds (T2). Regarding the physiological parameters, T2-and T3-fertilized plants reported the highest values of leaf chlorophyll and flavonoid contents, NBI and leaf temperature. Moreover, for the same treatments the lowest value of leaf anthocyanins content was measured (on average, +19%, +3%, +16%, +0.4% and -24%, respectively).  Our results were in agreement with those of previous research reporting, for different species, improvement in plant development under low supplies of spent coffee ground or biochar [45][46][47][48]. The increased Chl and NBI values for T3 treatment suggested a higher rate of leaf photosynthetic activity, assimilation and use (protein synthesis) of mineral nitrogen from the substrate. These results were in agreement with those reported by Ronga et al. [2,45] and Bozzolo et al. [12]. Leaf chlorophyll, flavonoid, and anthocyanin content are suggested as indices of plant physiological status and are associated with the N uptake and leaf photosynthetic activity [33]. Furthermore, Chalker-Scott [49] suggested that high leaf Anth levels are related with an improvement of tolerance to abiotic and biotic stresses. Interestingly, T2-fertilized plants showed the highest values of Anth. T2 treatment showed lower values of Chl and higher of Flav than T0, due to higher values of all the measured morphological parameters. In fact, in the investigated treatments, the same amount of nutrients was used, hence plants with a higher development can result in a dilution of the macro-and micronutrients that are involved in the physiological activities and related parameters like Chl content [33]. On the other hand, biochar application can allow a slow and constant release of the nutrients in the soil/growing media for plant nutrition as previously reported by Zhao et al. [50] and Gwenzi et al. [51]. In addition, this slowing release can allow a higher plant physiological activity as observed in the present study in T3-fertilized plants. Hence, our results suggested that LWA based on spent coffee ground and biochar may be used to improve plant physiological performance (increasing in stress resistances), since supplying of LWA based on biochar increased leaf photosynthetic activity in grapevine nursery management.
The results regarding the main agronomic parameters were shown in Table 4. Among the investigated treatments, bare-rooted vines fertilized with T3 showed the highest values of root, shoot and total fresh weights (+15%, +24% and +11%, on average), while the highest value of RSFW (+16%, on average) was measured for T1-fertilized plants (Table 4A). A similar trend was also noticed for the biomass dry weight. Indeed, plants fertilized with LWA based on spent coffee ground and biochar (T3) showed the highest values of root, shoot and total dry weights (+23%, +19% and +10%, on average), while T1-fertilized plants showed the highest value of RSDW (+12%, on average). In any case, this value did not significantly differ from T2 treatments, as well as total dry weight of T3 and T2 treatments was statistically comparable among them (46.3 and 27.0 g plant −1 , respectively).
Results regarding the improved biomass production using LWA based on spent coffee ground and biochar were in accordance with those reported by Setti et al. [52], Raviv et al. [53] and Ronga et al. [2,45], who suggested that composts derived from several by-products like spent coffee grounds and biochar positively affected the growth of potted plants grown in greenhouses. Moreover, different works reported increases in grapevine root biomass under different soil amendments like biochar, compost and digestate [2,12,54,55]. The increased biomass was putatively due to the availability of some growth-promoting compounds in the LWAs both based on spent coffee grounds and biochar as suggested by different researchers who used the same fertilizers based on agro-industrial by-products [2,56,57]. Finally, vines showing high total dry weight can better overcome transplanting stresses [58] and this improved plant physiological state represents a very important goal for nursery growers. Figure 1 shows the biomass allocation to the different organs: root, above-ground part of the rootstock and shoot. T3-fertilized plants displayed the highest values of FTR (+11% and +4%, on average), while the highest values of biomass allocated to rootstock (FTRS) were observed in T1-and T2-fertilized plants (+5%, on average,). As suggested by Ericsson et al. [59], changes in the source-sink organ relationship can impact on the growth of the fruit-tree. Indeed, the same authors demonstrated that a high shoot development can negatively impact on the root growth. Anyway, this behavior was also partially highlighted in T2 treatment; instead, in our work, enhanced percentages of biomass allocation to shoot did not result in reduction of partitioning to the roots. The differences reported between our results and ones reported by Ericsson et al. [59] were probably ascribed to the use of SCGs and biochar in the present study. In fact, SCGs and biochar can stimulate the growth of the different plant organs as already reported in other studies [20,60]. This physiological behavior is much appreciated by vine-growers since they can dispose of planting material with a good development of both root and shoot ensuring early and faster plant establishment after transplanting, due to the good availability of accumulated reserves. Figure 2 reports nitrogen efficiency, representing an important parameter to assess the effect of the nitrogen fertilization on the plant growth [61,62]. T3-treated plants, as well as plant fertilized with Osmocote Dropress, showed similar nitrogen efficiency values (7.8 g of biomass produced per each g of N applied). The performance of LWAs based on spent coffee grounds was improved with addition of biochar. Our results confirmed that this kind of substrate acts synergistically on plant growth, similarly to the results of previous studies on the same fruit tree [2,12] and on tomato [20]. In fact, in the fertilization, the use of SCGs and biochar can allow a higher plant nutrient uptake as observed in the present study and in the already published study [2,12,20]. Dry matter allocation to different part of the bare-rooted vines in the compared thesis. T0 = unfertilized pot; T1 = fertilized pot with commercial NPK fertilizer (Osmocote Topdress); T2 = fertilized pot with LWA based on spent coffee ground, glass and N fertilizers; T3 = fertilized pot with LWA based on spent coffee grounds + biochar, glass and N fertilizers; FTS = fraction of total dry weight allocated to shoot; FTRS = fraction of total dry weight allocated to above-ground rootstock); FTR= fraction of total dry weight allocated to root Bars indicate standard deviations. Means followed by the same letter do not significantly differ at p ≤ 0.05. Figure 2 reports nitrogen efficiency, representing an important parameter to assess the effect of the nitrogen fertilization on the plant growth [61,62]. T3-treated plants, as well as plant fertilized with Osmocote Dropress, showed similar nitrogen efficiency values (7.8 g of biomass produced per each g of N applied). The performance of LWAs based on spent coffee grounds was improved with addition of biochar. Our results confirmed that this kind of substrate acts synergistically on plant growth, similarly to the results of previous studies on the same fruit tree [2,12] and on tomato [20]. In fact, in the fertilization, the use of SCGs and biochar can allow a higher plant nutrient uptake as observed in the present study and in the already published study [2,12,20]. Finally, the obtained results were broadly in accordance with those of several previous studies, which reported increases in plant biomass production as effect of agro-industrial by-products, such as compost, digestate, and biochar adopted as fertilizers [2,12,18]. Increases in plant biomass were  Topdress); T2 = fertilized pot with LWA based on spent coffee ground, glass and N fertilizers; T3 = fertilized pot with LWA based on spent coffee grounds + biochar, glass and N fertilizers; FTS = fraction of total dry weight allocated to shoot; FTRS = fraction of total dry weight allocated to above-ground rootstock); FTR= fraction of total dry weight allocated to root Bars indicate standard deviations. Means followed by the same letter do not significantly differ at p ≤ 0.05. Figure 1. Dry matter allocation to different part of the bare-rooted vines in the compared thesis. T0 = unfertilized pot; T1 = fertilized pot with commercial NPK fertilizer (Osmocote Topdress); T2 = fertilized pot with LWA based on spent coffee ground, glass and N fertilizers; T3 = fertilized pot with LWA based on spent coffee grounds + biochar, glass and N fertilizers; FTS = fraction of total dry weight allocated to shoot; FTRS = fraction of total dry weight allocated to above-ground rootstock); FTR= fraction of total dry weight allocated to root Bars indicate standard deviations. Means followed by the same letter do not significantly differ at p ≤ 0.05. Figure 2 reports nitrogen efficiency, representing an important parameter to assess the effect of the nitrogen fertilization on the plant growth [61,62]. T3-treated plants, as well as plant fertilized with Osmocote Dropress, showed similar nitrogen efficiency values (7.8 g of biomass produced per each g of N applied). The performance of LWAs based on spent coffee grounds was improved with addition of biochar. Our results confirmed that this kind of substrate acts synergistically on plant growth, similarly to the results of previous studies on the same fruit tree [2,12] and on tomato [20]. In fact, in the fertilization, the use of SCGs and biochar can allow a higher plant nutrient uptake as observed in the present study and in the already published study [2,12,20]. Finally, the obtained results were broadly in accordance with those of several previous studies, which reported increases in plant biomass production as effect of agro-industrial by-products, such as compost, digestate, and biochar adopted as fertilizers [2,12,18]. Increases in plant biomass were Figure 2. Comparison of nitrogen efficiency in the three fertilized theses. T1 = fertilized pot with commercial NPK fertilizer (Osmocote Topdress); T2 = fertilized pot with LWA based on spent coffee ground, glass and N fertilizer; T3 = fertilized pot with LWA based on spent coffee grounds + biochar, glass and N fertilizers; NAE = nitrogen efficiency; bars indicate standard deviations. Means followed by the same letter do not significantly differ at p < 0.05. Finally, the obtained results were broadly in accordance with those of several previous studies, which reported increases in plant biomass production as effect of agro-industrial by-products, such as compost, digestate, and biochar adopted as fertilizers [2,12,18]. Increases in plant biomass were obtained by fertilizing with agro-industrial by-products and were related to the content of humic substances and some compounds able to enhance the plant growth [63]. Finally, Jindo et al. [64] reported that plant biochemical activity is improved by plant hormone-like promoters.

Conclusions
Results obtained in the present study suggested that spent coffee grounds and biochar represent useful components, due to their pore-forming role, for producing the matrix of lightweight aggregates.
To our knowledge, this was the first study investigating the suitability of spent coffee grounds, biochar and other residues in the form of glass in innovative slow-release fertilizer production. These recycled matrices fit well in with sustainable agriculture management and in circular economy contests. LWAs based on spent coffee grounds and biochar, when functionalized by the addition of 30 wt% of glass fertilizer, showed good agronomic performances comparable to those of synthetic fertilizer in the production of grapevine planting materials. LWAs here studied also proved to be able to reduce the needs to use phosphate and potassium rocks in nursery management phase. The positive effects on the physical characteristics of the substrate, especially on its water retention capacity, provided an interesting opportunity to save irrigation water. Finally, bare-rooted vines grown using LWAs resulted in better agronomic performances and in improved plant physiological status. However, further research is needed to assess the effect of LWAs in nursery greenhouse production of other fruit planting materials as well as in open field vine nursery production.