Effect of urban biowaste derived soluble substances on growth, photosynthesis and ornamental value of Euphorbia x lomi

Scientia Horticulturae 197 (2015) 90–98

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Effect of urban biowaste derived soluble substances on growth, photosynthesis and ornamental value of Euphorbia x lomi Giancarlo Fascella a,∗ , Enzo Montoneri b , Marco Ginepro c , Matteo Francavilla d a Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Unità di Ricerca per il recupero e la valorizzazione delle Specie Floricole Mediterranee. S.S. 113-Km 245.500, 90011 Bagheria (Palermo), Italy b Biowaste Processing, Via XXIV Maggio 25, 37126 Verona, Italy c Università di Torino, Dipartimento di Chimica, Via Giuria 7, 10125 Torino, Italy d STAR Research Group, Università di Foggia, Via Gramsci, 89-91, 71121 Foggia, Italy

a r t i c l e

i n f o

Article history: Received 28 June 2015 Received in revised form 22 October 2015 Accepted 25 October 2015 Keywords: Municipal biowastes recycling Leaf chlorophyll content CO2 exchange rate Plant photosynthetic activity Plant growth

a b s t r a c t Soluble bio-based substances (SBS) isolated from municipal biowastes and a commercial Leonarditebased product were applied as substrate drench or as foliar spray to grow the ornamental hybrid Euphorbia x lomi. The SBS were found more powerful than the commercial Leonardite product in enhancing plant photosynthesis, growth and aesthetic effect, improving flower quality, and optimizing water use efficiency. Enhancement factors of plant performance indicators by SBS ranged from 1.3 to 8.6 relatively to the control plants, and from 1.2 to 4.5 relatively to plants treated with the commercial Leonardite product at equal applied dose. The environmental and economic implication of these results for agriculture, the management of urban wastes, and the chemical industry are discussed. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The management of urban wastes has become a priority environmental issue, due to increasing urbanization and human consumption habits. They represent a significant cost for society. Recent work has however shown that fermented urban biowastes are a viable source of soluble bio-based substances (SBS) which may perform as efficient eco-friendly chemical auxiliaries in diversified fields; e.g., in the formulation of detergents, textile dyeing baths, flocculants, dispersants and binding agents for ceramics manufacture (Montoneri et al., 2011), emulsifiers (Vargas et al., 2014), auxiliaries for soil/water remediation (Avetta et al., 2013; Gomis et al., 2014; Montoneri et al., 2014) and enhanced oil recovery (Baxter et al., 2014), nanostructured materials for chemical (Boffa et al., 2014; Deganello et al., 2015) and biochemical catalysis (Magnacca et al., 2012), plastic materials (Franzoso et al., 2015a,b,c), soil fertilizers and plant biostimulants for horticulture (Sortino et al., 2014) and animal feed supplements (Montoneri et al., 2013; Dinuccio et al., 2013). The SBS are obtained by alkaline hydrolysis of the urban biowastes previously fermented under anaerobic and aerobic conditions. They are mixtures of molecules with molec-

∗ Corresponding author. Fax: +39 091909089. E-mail addresses: [email protected], [email protected] (G. Fascella). http://dx.doi.org/10.1016/j.scienta.2015.10.042 0304-4238/© 2015 Elsevier B.V. All rights reserved.

ular weight from 5 to several 100 kDa, comprising aliphatic and aromatic C atoms bonded to a variety of acid and basic functional groups. This is the likely reason of their multipurpose performance. The above studies prospect the substitution of synthetic chemicals by SBS for many applications, and consequently the potential reduction of the depletion of fossil sources and of the added CO2 to the environment contributed by synthetic chemicals at life end. Direct environmental implications of these substances have been described by Sortino et al. (2014) who have shown that SBS added to soil for horticulture enhance the plant photosynthetic activity, growth and productivity more than the sourcing fermented biowastes without any alkaline treatment. The same SBS are proven by Avetta et al. (2013) and Gomis et al. (2014) to enhance the photochemical degradation of organic pollutants in industrial effluents. These findings have suggested that SBS may promote either C fixation or mineralization, according to the different operational environments. In both cases it has been suggested that, by their capacity to complex Fe ions and keep them in solution at circumneutral pH, the SBS may contribute to enhance a photo-Fenton process. These findings propose SBS as a friendly interface between plant and human activities. In the present work we report the effects of three products containing organic and mineral matter on the photosynthetic activity, growth, aesthetic effect and biomass water use efficiency of ornamental hybrid Euphorbia x lomi Rauh (Euphorbia lophogona

G. Fascella et al. / Scientia Horticulturae 197 (2015) 90–98

Lamarck × E. milii Des Moulins). One product is a commercial formulation traded under the name of Enersoil, obtained by alkaline hydrolysis of Leonardite (Intrachem Bio Italia, 2008). The other two products are SBS isolated from the alkaline hydrolysate of two fermented biowaste materials. These are the digestate (DG) of the biogas production reactor fed with the organic humid refuse of separate source collection and the compost (CP) obtained from DG mixed with private and public gardening residues, and sewage sludge. The CP and DG were used in the previous work by Sortino et al. (2014) for tomato and pepper cultivation. The purpose of the present work was to assess whether the effects reported on horticultural species were confirmed also for ornamental plants. Consistently with their sources, the investigated products have different C, N and mineral composition, and therefore allow to evaluate the effects of the different nutrients content on the plant performance indicators. The hybrid Euphorbia x lomi Rauh plant was chosen as test plant, being perennial and so much different from many short-cycle crops as tomato and pepper. The former belongs to the Spurge family and is a succulent shrub with milky latex, long lanceolate leaves and large colored inflorescences, usually cultivated as potted flowering plant or as hedge plant for landscaping and xerogardening (Fascella and Zizzo, 2009) because of its low exigencies and tolerance to drought stress (Fascella et al., 2011). The results were expected to add further argument to the previous work on horticultural species (Sortino et al., 2014) supporting the role of SBS promoting a friendly C cycle in the ecosystems. 2. Materials and methods 2.1. SBS and Enersoil The SBS were prepared and supplied by Studio Chiono ed Associati (SCA) in Rivarolo Canavese (TO), Italy. This company obtained the SBS by hydrolysis of two fermented urban biowaste materials according to a previously reported procedure (Sortino et al., 2014; Franzoso et al., 2015a,b,c). The first material was the anaerobic digestate (DG) of the organic humid fraction of urban waste from separate source collection. The second material was the compost (CP) obtained from a mix of DG, home gardening and park trimming residues and sewage sludge, at 3.5/5.5/1 respective weight ratio, which was aged under aerobic conditions for 110 days. The two fermented urban biowaste materials were further processed by SCA as follows. The DG or CP material, separately, was hydrolyzed with KOH alkaline water at pH 13 and 60 ◦ C. The hydrolyzate was run through an ultra filtration polysulphone membrane with 5 kD cut off. The membrane retentate was dried at 60 ◦ C to yield the final SBS product as black solid in a 15–20% yield, relative to the starting material. The CP and DGSBS contained 91 and 99% dry matter, respectively. The commercial Enersoil product was supplied by Intrachem Bio in Grassobio (BG), Italy. 2.2. Greenhouse facilities and plant material The plant growth trials were conducted in 2013 in an unheated (28 ◦ C day/14 ◦ C night) double-span East–West oriented greenhouse (34 × 16 m) with steel structure and polyethylene cover (thickness 0.15 mm), located at the Research Unit for Mediterranean Flower Species near Palermo (38◦ 5 N, −13◦ 30 E, 23 m above sea level), on the North Western Sicily coastal area. Six months-old 8 cm-tall micropropagated plants of Euphorbia x lomi Rauh cv. ‘Serena’ were grown in plastic pots of 13 cm diameter (1 L capacity, 1 plant per pot) filled with a substrate of sphagnum peat (Dueemme marketing, Reggio Emilia, Italy) and perlite (Perlite Italiana, Milano, Italy) in 1:1 v/v ratio. Water, macro and micronutrients were supplied to plants through a drip fertigation system (1 dripper per

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plant, 2 L h−1 ) controlled by a computer. All plants were fed with the same nutrient solution which had the following composition (mg L−1 ): 180 total N, 50 P, 200 K, 120 Ca, 30 Mg, 1.2 Fe, 0.2 Cu, 0.2 Zn, 0.3 Mn, 0.2 B. The pH and the electrical conductivity (EC) of the nutrient solution were maintained at 5.8 and 1.8 mS cm−1 , respectively. Irrigation scheduling was performed using electronic low-tension tensiometers connected to an electronic programmer, that controlled irrigation based on substrate matric potential. The SBS and Enersoil materials were dissolved or diluted in water to yield five solutions with the following dry matter g L−1 concentrations: 45.5 CP, 31 and 15.5 DG, 18.7 and 9.4 Enersoil. An aliquot of 100 ml of each solution was applied as substrate drench or as foliar spray to each plant. Foliar sprays and drenches applications were provided to plants two times during the trial (120 days). The total applied amounts of dry matter g per plant were 4.6 for CP as substrate drench, 3.1 and 1.5 for DG as substrate drench and foliar spray, respectively, and 1.9 and 0.94 for Enersoil as substrate drench and foliar spray, respectively.

2.3. Plant growth measurements Ten plants per treatment, randomly chosen from each replicate, were harvested every 30 days and separated into stems, leaves and roots for growth measurements (plant height, number of leaves per plant, number of flowers per plant). Plant height was determined as the distance from the surface of the substrate to the top of the plant. Dry weight of the biomass was determined after 72 h in a forced-air oven (at 100 ◦ C) when harvested tissues reached a constant value. Shoot to root ratio (S/R) was calculated by dividing sum of leaf and stem dry weights by the root dry weight. Leaf area (LA) was measured using a digital area meter (WinDIAS 2; DELTA-T DEVICES Ltd., Cambridge, U.K.). Relative Growth Rate (RGR) was calculated according with the formula proposed by Hoffmann and Poorter (2002) using the following equation: (lnW2 − lnW1)/(t2 − t1) where ln = natural logarithm, W1 = dry weight of plant at time one (in grams), W2 = dry weight of plant at time two (in grams), t1 = time one (in days), t2 = time two (in days). Biomass Water Use Efficiency (WUE) was calculated as the ratio between total dry weight of plants and plants total water supply.

2.4. Leaf SPAD index, color and gas exchanges measurements Leaf chlorophyll content (e.g., SPAD index) of three randomly selected leaves of all plants in each experimental unit was measured with a chlorophyll meter (SPAD 502, Konica Minolta Sensing, Inc., Osaka, Japan). Leaf color was determined with a shot in the middle of the blade on three leaves of all plants of each treatment with a colorimeter (Minolta CR10, Konica Minolta Sensing, Inc., Osaka, Japan) that calculated the color coordinates (CIELAB): lightness (CL), tone (CA) and saturation (CB); CL varies from 0 (completely opaque or black) to 100 (completely transparent or white); CA ranges from positive (redness) to negative (greenness) values, as well as CB (positive is yellowness, negative is blueness). Leaf gas exchanges (net assimilation CO2 (ACO2 ) and stomatal conductance (gs )) were also measured using a portable photosynthesis system (LI-6200; LI-COR Inc., Lincoln, NE, USA). Measurements were made on most recent fully expanded leaves between 10:00 and 13:00 h on sunny day, using five replicate leaves per treatment. The LI6200 was equipped with a stirred leaf chamber with constant-area inserts and fitted with a variable intensity red source (leaf temperature chamber was 30 ± 2 ◦ C, leaf-air vapor pressure difference was 2.6 ± 0.3 ◦ C, and CO2 concentration was 365 ± 10 ␮l L−1 ).

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Table 1 Analytical data for SBS and Enersoil. SBS

pH

Volatile soilds (w/w%a )

C (w/w%a )

N (w/w%a )

C/N

MW (kDa)b

MW/MNb

DG CP Enersoilc –

10.5 10.1 10.0

71.5 ± 0.9 67.6 ± 0.2 60.0

43.1 ± 0.4 40.2 ± 0.1 30.0

6.67 ± 0.08 5.22 ± 0.05 0.20

6.5 7.7 150

164 75

1.9 1.5

Mineral elementsd

DG CP Enersoile

Si

Fe

Al

Mg

Ca

K

Na

Cu

Ni

Zn

Cr

Pb

P2 O5

0.25 0.51 0.22

0.52 0.83 0.10

0.10 0.12 0.14

0.27 0.43 0.38

2.08 2.59 0.39

1.59 1.79 4.92

0.19 0.20 3.15

262 269 2.98

24 62 1.30

361 307 2.56

15 24 1.30

46 94 0.71

1.14 1.44 0.54

C types and functional groupse concentration as mole fraction of total organic Cf

DG CP Enersoilg

Af

NR

OMe

OR

OCO

Ph

PhOH

PhOY

COOH

CON

CO

Af/Ar

LH

0.43 0.31 0.31

0.10 0.08 0.00

0.04 0.00 0.03

0.10 0.20 0.09

0.03 0.07 0.04

0.10 0.16 0.31

0.02 0.06 0.02

0.01 0.02 0.08

0.07 0.09 0.07

0.09 0.01 0.02

0.01 0.00 0.03

3.3 1.3 0.76

9.3 5.3 7.3

a Concentration values referred to dry matter: averages and standard deviation calculated over triplicates. b MW = weight average molecular weight; MN = number average molecular weight. c Vendor data (Intrachem Bio Italia, 2008). d Si, Fe, Al, Mg, Ca, K, N, P2 O5 , as % w/w; Cu, Ni, Zn, Cr, Pb, Hg as ppm. e Data obtained in this work. f Aliphatic (Af), ammine (NR), methoxy (OMe), alkoxy (OR), anomeric (OCO), aromatic (Ph), phenol (PhOH), phenoxy (PhOY, Y = alkyl or phenyl) carboxylic acid (COOH), amide (CON), ketone (C O) C atoms; Ar = Ph + PhOH + PhOY C;LH = liphophilic to hydrophilic C ratio; liphophilic C = Af + Ph + OMe + CON + NR + RO, + PhOY + OCOC atoms; hydrophilic C COOH + PhOH + C O C; relative standard deviations as% of mean values were within 10% of the reported mean values. g Data for precipitated product obtained in 15% yield by acidifying the products purchased (i.e., the dense liquid) at pH < 1.5 (see Section 3.1).

2.5. Experimental design and data analysis For the Euphorbia growth trials, a split-plot experimental design with product as the main plot and their application on plants as subplots was used. Each of the five treatments and the control was replicated three times, and each replication consisted of 24 potted plants (72 pots per treatment). Collected data were subjected to a two-way analysis of variance (ANOVA) and the treatment means were compared using Duncan’s Multiple Range Test (DMRT) at 5% of probability by using the package Statistica (Statsoft Inc., Tulsa, OK). 3. Results 3.1. Chemical and physical characteristics of SBS and Enersoil SBS and Enersoil chemical features are reported in Table 1 where it may be observed that the products contain both organic and mineral matter. The former is constituted by organic macromolecules containing aliphatic and aromatic C bonded to several acid and basic functional groups. The weight average molecular weight (MW), the number average molecular weight (MN), and the MW/MN ratio show that the SBS are composed by a mix of molecules with different molecular weight. It is also likely that the C types and functional groups listed in Table 1 were not homogeneously distributed over the macromolecular pool. Under these circumstances, the different chemical nature of the two SBS may be better appreciated comparing the aliphatic to aromatic C (Af/Ar) and the lipophilic to hydrophilic C (LH) ratios reported in this table. Based on the definition provided in Table 1 footnotes, LH is a measure of the product relative hydrophilicity, while Af/Ar is a measure of the relative aliphatic/aromatic nature of the product. The data show the CP organic matter is more hydrophilic and more aromatic than the DG one. The mineral fraction of the products contains several main and trace elements, presumably bonded to the organic functional groups. The organic and mineral fractions together contain all main plant nutrients. The products composition was found stable over one year storage. Data for longer aging time are not

available yet. By their chemical features and water solubility, the SBSare expected to provide an adequate easily available pool of nutrients for plant uptake. The data show that, compared to CP, the DGSBS has higher N content, but generally lower mineral content. By comparison, Enersoil is a dense liquid, described by the vendor (Intrachem Bio Italia, 2008) as natural organic ammendant extracted from Leonardite by alkaline hydrolysis with KOH containing 30% dry matter, 18% organic matter (10% being humic matter), 0.06% organic N, 9% organic C. A similar product, humic acid supplied by Adrich, was reported (Montoneri et al., 2009) to have molecular weight between 0.1 and 105 kDa, and to contain the same C types and functional groups as those listed in Table 1 for the CP and DGSBS. The humic fraction of Enersoil was precipitated at acid pH and was obtained in 15% yield, thus representing a substantial portion of the 18% organic matter content declared by the vendor. The Enersoil humic fraction, analyzed by microanalysis, yielded the following w/w% values: 67.6 C, 1.67 N, 40.6 C/N. The solid state 13 C NMR spectra of this product evidenced the same C type and functional groups as those of the SBS. Table 1 shows that, compared to the DG and CPSBS, the Enersoil humic matter contains relatively more aromatic C. The lower Af/Ar ratio for Enersoil is the likely results of the longer degradation time of the pristine Leonardite organic matter. The product hydrophilicity, as measured by the LH parameter, is in between the values for the two SBS. The lower 40.6 C/N ratio of the precipitated humic fraction relatively to the 150 C/N ratio declared by the vendor for the whole product suggests that the product contains also non humic N free organic matter. The Enersoil recommended dose by the vendor to apply varies from 5 to 20 kg ha−1 for soil and fertigation use, and diluted to 0.8–1% dry matter content for foliar spray use, each application to be repeated two–three times over the culture growth cycle. The collected data show that the SBS and Enersoil had very different chemical composition. The latter had much lower N content, as shown by the C/N ratio being 150 for Enersoil, and 6.5 and 7.7 for the DG and CPSBS, respectively. The lower N content of humic material extracted from fossil source, compared to SBS, has been evidenced also for the Aldrich humic acid material (Montoneri et al.,

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Table 2 Applied dose (g plant−1 ) of SBS and Enersoil by substrate drench (sd) or foliar spray (fs). Product/application mode

As supplied by the manufacturera

Dry matter

C

N

CPSBS/sd DGSBS/sd DGSBS/fs Enersoil/sd Enersoil/fs

5.0 3.1 1.5 6.2 3.1

4.55 3.09 1.54 1.88 0.94

1.8 1.3 0.65 0.56 0.28

0.24 0.21 0.10 0.0038 0.0019

a

Per plant applied amount of Enersoil dense liquid as purchased (see Section 3.1) and of solid SBS as received (see Section 2.1).

2009). The as purchased Enersoil dense liquid was analysed for mineral content. Compared to DG and CP, it was found to contain much more K and Na, and less Fe, Ca, P and trace metals (Table 1). Under these circumstances, to compare the three products for their effects on Euphorbia x lomi plants, the Enersoil was applied at the dose recommended by the vendor. The SBS were applied at the nearly equal dose of the as purchased Enersoil dense liquid. This choice was made based on the fact that, commercially, materials are evaluated for their benefits relatively to the cost per kg of the as purchased material. Table 2 reports the applied doses of the three products. Due to the intense black color, the CPSBS was applied only as substrate drench; in fact, foliar spray application of this product caused formation of brown spots on leaves which were expected to reduce plant photosynthetic activity (Morales and Warren, 2012). This effect was observed also by applying the same CP SBS dose as the DG SBS one. The latter product was less colored and did not present the same phenomenon as the former one. The doses chosen for comparing the different materials investigated in the work were based on the following criteria. First, from the commercial point of view products are rated based on their benefit/cost ratio, where cost is referred to the weight of the product as supplied by the vendor. Due to the different composition of the investigated products, the doses were worked out to perform two kinds of comparison: (i) to compare the SBS with Enersoil and (ii) to compare the two SBS, one with the other. Thus, as Enersoil is the reference commercial material, this product was applied at the doses recommended by the vendor. Table 2 shows that for the substrate drench (sd) application, we used 6.2 g of the as purchased Enersoil dense liquid versus 5.0 g of the solid CP as supplied by the manufacturer (see Section 2.1). This allowed the comparison of the CP SBS versus Enersoil at close doses. For comparing the two SBS, one with the other, we used the criterion of comparing them at the same applied N doses. It may be observed that while the dry matter doses range from 0.94 to 4.55 g plant−1 , the differences in the C and N applied doses by the different treatments vary over a wider range. Relatively to the lowest dose, the highest applied dose was 4.6 × higher for dry matter, 6.4 × higher for C and 126 higher for N. The CP and DG substrate drench applied N doses happened to be equal.

3.2. Plant growth and biomass yield Table 3 reports the plant biometric data as affected by the different treatments compared to the control plants. No significant differences were recorded for plant height by the different treatments compared to the control as an average value of 15.7 cm was recorded irrespective of the treatment. The other growth parameters were all affected by the treatments. The highest number of leaves was measured in the plants grown in the pots treated with CP by substrate drench (63.3 leaves plant−1 ), followed by those treated with DG by foliar spray and Enersoil by drench, i.e., 52.8 and 52.0 leaves plant−1 , respectively. The lowest production was observed (Table 3) in the control plants (32.5 leaves plant−1 ) (Table 3). Leaf area was highest in the same treatments characterized by the highest leaf number production; it was 686.3, 654.8 and 630.3 cm2 for CP by substrate drench, DG by foliar spray

and Enersoil by drench, respectively, and lowest in the control (386.0 cm2 ). Flowers production was definitely highest in the plants grown in the pots treated with CP by substrate drench (4.0 flowers plant−1 ); the lowest production was observed in the control plants (1.2 flowers plant−1 ); the remaining treatments gave higher flower production than the control plants, but lower production than CP (Table 3). Shoot to root ratio (S/R) was highest in plants treated with CP applied by substrate drench (12.3); a lower value was measured for the DG substrate drench treatment (8.2); the control and the other treatments gave the lowest values. Moreover, a significant (p ≤ 0.05) interaction “product x application mode” was evidenced for S/R (Table 3). Overall, the CP substrate drench treatment gave the highest values for number of leaves per plant, leaf area, flower production per plant, and S/R. The measured values for these parameters were 2–3 times higher than the values measured for the control plants. The effects of the other treatments ranked in between the CP substrate drench treatment and the control. Fig. 1 reports the biomass production partition over the plant stem, leaves and root. Total biomass production was significantly affected by the treatment typology as higher total dry weight was measured in Euphorbia x lomi plants treated with CP by substrate drench (28.0 g), with respect to the other treatments (average 14.1 g). The lowest total dry weight was recorded for the control plants (6.5 g). The CP treatment therefore enhanced biomass production by a 2× factor relatively to the other treatment and by a 4× factor relatively to the control. This difference was essentially caused by the higher dry weight of leaves of the plants treated with CP applied by drench (17.7 g) relatively to the other treatments (average 7.8 g) and the control (3.8 g). Biomass partitioning over the other plant organs showed lower differences between CP by drench and the other treatments: i.e., for stems 4.2 g by CP versus 2.5 g by the other treatments, for root dry weight 6.1 g by CP versus 3.9 g by the other treatments, while the control plants always evidenced the lowest values, 1.8 and 0.9 g for stem and root dry weight, respectively (Fig. 1). Fig. 2 reports the plants Relative Growth Rate (RGR) for the control and the different treatments over the cultivation period, in August–October 2013. No significant differences were observed in August. In September, the treatments gave higher RGR compared to the control (p ≤ 0.05), with the CP treatment was characterized by the highest value. In October, the greater effect by the CP treatment was even more evident (p ≤ 0.01). The RGR of 3.3 g g−1 d−1 by the CP treatment at the end of the cultivation period in October was significantly higher by 1.3 × factor than the average value for the other treatments and by 1.8 × factor than the lowest 1.8 g g−1 d−1 RGR recorded for the control plants (Fig. 2). Fig. 3 reports the Water Use Efficiency (WUE) for the Euphorbias grown in the treated pots versus the control plants. It shows that the CP treatment gave the highest 1.3 g L−1 WUE value, followed by those for the other treatments running from 0.55 to 0.75 g L−1 and by the lowest 0.3 g L−1 value for the control plants. 3.3. Leaf SPAD index, color and gas exchanges Tables 4 and 5 report the data related to the plant photosynthetic activity. In particular, Table 4 shows that the leaf

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G. Fascella et al. / Scientia Horticulturae 197 (2015) 90–98

Table 3 Effect of product (P) and application mode (A) on plant height, leaves production, leaf area, flowers production and shoot/root ratio (S/R) of Euphorbia x lomi potted plants. Product/ application mode

Plant height (cm) a

Leaves (n. plant−1 )

Leaf area (cm2 )

Flowers (n. plant−1 )

Enersoil/fs Enersoil/sd DGSBS/fs DGSBS/sd CPSBS/sd Control

15.2 a 16.1 a 16.4 a 16.3 a 16.5 a 13.9 a

54.2 ab 52.0 ab 52.8 ab 47.2 b 63.3 a 32.5 c

562.8 b 630.3 ab 654.8 a 544.4 b 686.3 a 386.0 c

1.8 bc 2.4 b 1.7 bc 2.6 b 4.0 a 1.2 c

Significance Product Application mode PxA

ns ns ns

* ns ns

* * ns

* * ns

S/R 1.9 c 2.7 c 1.6 c 8.2 b 12.3 a 4.0 c * * *

*:significant; ns: not significant. a For each column, means followed by different letters are significantly different at p ≤ 0.05 (DMR test).

Fig. 1. Effect of product and application mode on dry biomass production of Euphorbia x lomi potted plants measured for each plant organ: i.e., stem, leaves and roots. Values are means ± standard error. For each color, columns with different letters indicate significantly different values at P ≤ 0.05 (DMR test).

Fig. 2. Effect of product and application mode on Relative Growth Rate (RGR, g g−1 day−1 ) of Euphorbia x lomi potted plants measured in August, September and October 2013 during the plant growth and production period. Values are means ± standard error; ns, *, ** indicate non-significant or significant different values at P ≤ 0.05 and 0.01, respectively (DMR test). Significant differences are as follows: in September, p < 0.05 between the five treatments and the control; in October, p < 0.01CP between SBS/sd and the control, p < 0.05 between CP SBS/sd and the other four treatments, p < 0.05 between the other four latter treatments and the control.

G. Fascella et al. / Scientia Horticulturae 197 (2015) 90–98

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Fig. 3. Effect of product and application mode on Water Use Efficiency (WUE, g L−1 ) of Euphorbia x lomi potted plants. Means are values ± standard error. Columns with different letters indicate significantly different values at P ≤ 0.05 (DMR test). Table 4 Effect of product (P) and application mode (A) on leaf chlorophyll content (SPAD index) and color coordinates (CL, CA and CB) of Euphorbia x lomipotted plants. Product/ application mode

Chlorophyll content (SPAD)

CL

CA

CB

Enersoil/fs Enersoil/sd DGSBS/fs DGSBS/sd CPSBS/sd Control

35.7a b 35.6 b 39.6 ab 36.0 b 42.0 a 31.6 c

37.4 bc 37.0 bc 40.1 b 39.0 b 32.4 c 47.2 a

−14.4 bc −14.0 bc −16.5 ab −15.6 ab −19.9 a −11.1 c

20.5 ab 19.7 ab 24.0 a 20.3 ab 26.4 a 17.6 b

Significance Product Application mode P×A

* ns ns

* ns ns

* ns ns

* ns ns

Color coordinates

*:significant; ns:not significant. a For each column, means followed by different letters are significantly different at p ≤ 0.05 (DMR test). Table 5 Effect of product (P) and application mode (A) on leaf gas exchanges – net assimilation CO2 (ACO2 ) and stomatal conductance (gs ) – of Euphorbia x lomi potted plants. Product/application mode

ACO2 (␮mol CO2 m−2 s−1 ) a

gs (mmol m−2 s−1 )

Enersoil/fs Enersoil/sd DGSBS/fs DGSBS/sd CPSBS/sd Control

2.74 d 3.45 c 4.54 b 5.28 b 6.19 a 1.73 e

0.010 cd 0.014 c 0.023 bc 0.031 b 0.043 a 0.005 d

Significance Product Application mode P×A

* * ns

* * *

*:significant; ns:not significant. a For each column, means followed by different letters are significantly different at p ≤ 0.05 (DMR test).

chlorophyll content was highest in the plants grown in the pots treated with CP by drench (42.0), followed by DG by spray (39.6), by the other treatments yielding values around 36, and the significantly lowest value of 31.6 for the control plant. Table 4 also reports the values for the three CIELAB color coordinates, namely the lightness (CL), the red/green (CA) and the yellow/blue (CB) coordinate (Datacolor, 2008). In this system, lower CL indicates less transparent color, more positive CA indicates

increasing redness, and more negative CA indicates increasing greeness, more positive CB indicates increasing yellowness, and more negative CB indicates increasing blueness. The data show that the leaves of the plants treated with CP have the lowest CL, the most negative CA and the most positive CB, while the opposite is true for the control plant leaves. These differences in the color coordinates of CP and control leaves correspond to more intense green color in the former perceived by the eye and, consequently, to a higher ornamental and commercial value. Table 5 shows the remarkably highest leaf gas exchanges of the plants grown in the pots treated by CP substrate drench. The net assimilation CO2 (ACO2 ) value for these plants was 6.19 ␮mol m−2 s−1 , followed by the significantly lower values of the plants treated by DG substrate drench and DG foliar spray, 5.28 and 4.54 ␮mol CO2 m−2 s−1 , respectively, by the plants treated with Enersoil by substrate drench (3.45), by the plants treated with Enersoil by foliar spray (2.74), and by the lowest 1.73 value for the control plants. A similar ranking order is observed for the stomatal conductance (gs ) as higher value was recorded in Euphorbias treated with CP by drench and lower gs was measured with Enersoil treatments and in the control plants (Table 5); a significant (p ≤ 0.05) interaction “product × application mode” was also evidenced.

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Table 6 Rankinga of treatmentsb in order of significantly decreasing effect on the different plant performance indicators. Enhancement factorc

Plant performance indicator

Ranking order

Plant height (Table 3) Leaves per plant (Table 3) Leaf area (Table 3) Flower per plant (Table 3) S/R (Table 3) Biomass production (Fig. 1) RGR (Fig. 2) WUE (Fig. 3) Chlorophyll content (Table 4) CL (Table 4) CA (Table 4) CB (Table 4) ACO2 (Table 5) gs (Table 5)

CP/sd = DG/sd = DG/fs = Enersoil/sd = Enersoil/fs = control CP/sd ≥ DG/fs = Enersoil/sd = Enersoil/fs3 DG/sd> control CP/sd ≥ DG/fs = Enersoil/sd3 Enersoil/fs = DG/sd> control CP/sd > DG/sd = Enersoil/sd3 Enersoil/fs = DG/fs3 control CP/sd > DG/sd > Enersoil/sd = Enersoil/fs = DG/fs = control CP/sd > DG/sd = Enersoil/sd = Enersoil/fs = DG/fs > control CP/sd > DG/fs = Enersoil/sd > Enersoil/fs = DG/sd > control CP/sd > DG/fs = Enersoil/sd3 Enersoil/fs = DG/sd3 control CP/sd = DG/fs3 DG/sd = Enersoil/fs = Enersoil/sd > control CP/sd = Enersoil/fs = Enersoil/sd3 DG/fs = DG/sd > control CP/sd = DG/fs = DG/sd3 Enersoil/fs = Enersoil/sd3 control CP/sd = DG/fs = DG/sd = Enersoil/fs = Enersoil/sd3 control CP/sd > DG/sd > DG/fs > Enersoil/sd > Enersoil/fs > control CP/sd > DG/sd3 DG/fs > Enersoil/sd = Enersoil/fs3 control

CP/control

CP/Enersoil

1.2 ns 1.9 1.7 3.3 3.1 4.0 1.8 4.3 1.3 0.69d 1.8 1.5 3.6 8.6

1.0 ns 1.1 ns 1.0 ns 1.7 4.5 2.0 1.2 1.6 1.2 0.88 ns 1.4 1.3 ns 1.8 3.1

a When there are no significant differences between treatment the “=” sign is used. The “≥” symbol is used when the adjacent treatments in the ranking do not differ, but they differ with those previously positioned in the ranking. For example, CPsd = DGfs = Enersoilsd = Enersoilfs ≥ DGsd > control means that there is no statistical difference among CPsd, DGfs , Enersoilsd and Enersoilfs , that DGsd is statistically equal to Enersoilfs , but it is significantly lower than CPsd, DGfs and Enersoilsd , and that the control is the lowest. b CPsd = treatment with CPSBS by substrate drench; DGsd and DGfs = treatment with DGSBS by substrate drench and foliar spray, respectively; Enersoilsd and Enersoilfs = treatment with Enersoil by substrate drench and foliar spray, respectively. c CP/control value ratio and CP/Enersoil best value ratio; ns = not significant. d Lower value indicates relatively higher color darkness.

Table 6 summarizes the ranking of the different treatments in order of decreasing effect on each plant performance indicator, based on the results reported in Tables 3–5 and Figs. 1–3. 4. Discussion It may be observed that there is no definite difference of effects as caused by foliar spray or substrate application mode. On most plant indicators the different application modes yield the same effect. The largest majority of effect differences arise from the different types of applied products. The CP substrate drench treatment ranks first in all cases. For seven plant performance indicators it yields the highest enhancement, sharing the first ranking position with no other treatment. The enhancement factor is reported in Table 6 as the ratio of the indicator value by the CP treatment to the value recorded for the control plants. It may be observed that this factor ranges from 1.3 for the Chlorophyll content indicator to 8.6 for the stomatal conductance (gs ) indicator. Particularly high are the enhancement factor values for the water use efficiency (4.3), biomass production (4.0), net assimilation CO2 (3.6), and the flower production (3.3). The data reported in Table 6 point out that all indicators, particularly the plant flower and biomass productivity, are well related to the plant photosynthetic activity as measured by the ACO2 parameter. The net assimilation CO2 is shown to be the most sensitive parameter toward the different treatments. This indicator determines the highest selectivity in the ranking order based on statistical significance; i.e., there are no two treatments yielding the same effect. The first two in the ranking order for the effect on ACO2 are the CP and the DG substrate drench treatments. These same treatments rank also first and second for their effect on the gs parameter. In this work the net assimilation CO2 is nicely related to the stomatal conductance by the following equation: ACO2 = a + bgs

(1)

where a = 1.58 ± 0.27, b = 115 ± 10.8, R (correlation coefficient) = 0.98. The other indicators are related to the net assimilation CO2 by a similar linear relationship, although with a lower correlation coefficient ranging from 0.70 to 0.78 over all measured indicators. The data show that the importance of enhancing the plant photosynthetic activity to enhance in turn plant growth and productivity.

Looking for a possible reason of the ranking order in Table 6, one could observe that several elements can be the cause of the remarkable highest effects of CP. These may be the highest supplied N per plant amount (Table 2), as well as the highest content of some mineral elements such as Fe, Ca and P, and of carboxylic, phenolic and amino groups, as shown in Table 1. These functionalities are likely to complex mineral ions, such Fe ions known to have an important role in the plant photosynthetic activity. Indeed, it may be observed from Table 1 that the sum of the NR, PhOH and COOH groups concentration values, expressed as mole fraction of total organic C moles, decreases from 0.23 for CP to 0.19 for DG, and further to 0.09 for Enersoil. The capacity of SBS to keep Fe ions in solution at circumneutral pH by its complexing acid and basic functional groups has been previously inferred responsible for the photosensitizing properties of SBS. As previously reported, by these properties, the SBS are capable to promote the mineralization of organic pollutants (Avetta et al., 2013; Gomis et al., 2014), and are also the likely cause of the enhanced photosynthetic activity observed in horticulture plants (Sortino et al., 2014). The availability of Fe ions, and other elements such as N, K, Ca, Mn, Zn, for enhancing the plant photosynthetic activity, and the leaf chlorophyll content and color (Netto et al., 2005; Dordas and Sioulas, 2008) has been claimed by several authors using commercial and non commercial products in the cultivation of several plant species, such as tomato (Sánchez Sánchez et al., 2009Siminis et al., 1998), onion seedlings (Bettoni et al., 2014), cowpea (Neri et al., 2002a), and chrysanthemum plants (Fan et al., 2014). Other authors have reported data showing the close relationship between leaf chlorophyll content, and plant growth and yield (Enriquez et al., 2004; Ciganda et al., 2009). Several others have reported enhanced plant growth and productivity, and/or photosynthesis, by application of commercial and non commercial humic substances to the growing substrate for the cultivation of tomato plants (Thi Lua and Böhme, 2001), pepper (Arancon et al., 2006), grapevine rootstocks (Zachariakis et al., 2001), olive trees (Tattini et al., 1990), and ornamental plants (Ahmad et al., 2013; Costa et al., 2008) as well as through foliar spray (Fernández-Escobar et al., 1999Neri et al., 2002b), with higher effectiveness of substrate drenching (Böhme, 1999Paunovic´ et al., 2013). The effectiveness of these products on improving plant growth also depends on the humate form and on the material used for the extraction (peat, coal), as well as on the

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concentration and frequency of treatments, as they are able to stimulate root growth in a manner similar to auxin. It is also suggested that these products may up-regulate genes responsible to plant organogenesis and flower development. Whereas the reason of the observed effects by SBS on the plant photosynthetic activity is certainly matter of scientific interest, from the commercial point of view the plant growth and productivity impact directly the economy of the plant production process and market value. At this regard, the water use efficiency (WUE) has both environmental and economic relevance. The management of water resources has become a key issue in many governmental policies. Basically, it has become clear that it is necessary to manage all forms of water use with greater precision and, in particular, to manage unmeasured uses of water including the impacts of plantation forestry, small farm dams, the capture of overland flows, reductions in return flows as a result of increases in irrigation efficiency and salinity interception (Young, 2010). The WUE issue is especially important in agriculture, as this sector of human activities is the major user of water, accounting for about 70% of the world’s freshwater withdrawals and over 40% of the Organisation for Economic Co-Operation and Development (OECD) countries’ total water withdrawals (Parris, 2010). The adoption of fertigation as used in this work is certainly in line with current water savings technologies and practices. However, the use of efficient plant growth biobased auxiliaries, such as CP, allows additional water savings. The WUE enhancement brought about by the treatments in this work, relatively to the control, could be due to a physiological process as suggested by Morard et al. (2010). In this process, the high molecular size fractions of the applied products could participate in plant water saving by slowing down its passage in roots. However, in the present work, CP has ranked first for its highest effects on WUE, biomass and flower per plant production, S/R, RGR, ACO2 and gs , with no other treatment matching its performance. The WUE value by the CP treated plants is certainly connected to the highest biomass production (Fig. 2). In turn, the enhancement of biomass and flower per plant production are most likely the results of the remarkable plant photosynthetic activity enhancement caused by CP. These relationships strongly point out how substances capable to enhance the plant photosynthetic activity can generate practical relevant economic and environmental benefits for agriculture and society. Based on the above cited literature, it is possible to observe that the same types of benefits can be obtained by using humic substances. Aside from the fact that this work shows much better performance of SBS compared to the commercial Enersoil humic material sourced from leonardite, the comparison of SBS and humic substances coming from fossil sources should account also for the source viability. The SBS are obtained from municipal biowastes; this material is the easiest worldwide most available source of renewable organic matter. Due to municipal collection, it is found in confined spaces, practically free of charge for potential users. For this reason, municipal biowaste has been defined a negative cost source of organic matter (Sheldon-Coulson, 2011). If properly exploited by processing it to added value products, it would become a viable biobased feedstock. Under these circumstances, the use of the SBS in agriculture, as well as in the chemical industry, would allow several economic and environmental benefits in different sectors of human activities. The replacement of humic substances and synthetic chemicals in the agriculture and chemical market would allow savings of fossil sources of humic substances, carbon and oil, and the consequent decrease of the emission of greenhouse gases. In order to evaluate the potential marketability of the SBS, one should consider that products marketability is rated based on benefits to cost ratio, where costs are referred to the weight of the product as marketed by the vendor. Form this point of view, the per plant applied amounts of the solid SBS products are lower than

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those of the as purchased Enersoil dense liquid (Table 2). Yet, the effects of the SBS are higher. This allow to forecast that the SBS could be allocated in the market at the same price per kg as Enersoil and be more competitive for their performance per kg of applied product as marketed. The results also show that the CP SBS applied by substrate drench is more effective than the DG SBS applied by substrate drench, although the amount of applied N is the same in both cases. The results therefore allow to conclude that both SBS are more effective than Enersoil, when compared for the ratio benefit/amount of applied product as received by the vendor or supplied by the manufacturer, and that the CP SBS is more effective than the DG SBS at equal N applied doses. The vis-à-vis performance comparison of SBS with the commercial Enersoil product reported in this work demonstrates that SBS could efficiently replace commercial humic products in the agriculture market. The current market value of these products ranges from 1.5 to 3.5 --C kg−1 (Ebay, 2015; Alibaba, 2015) for the solid product, and is even higher for liquid products. The Enersoil product can be purchased in 1 kg package for --C 7 kg−1 (Viscardi, 2015). Based on the 30% dry matter content, this price is equivalent to over 23 --C kg−1 dry matter. The SBS production cost has been estimated about 0.1–0.5 --C kg−1 (Montoneri et al., 2011). The figures prospect a cost effective production and successful allocation of SBS in the organic fertilizer market. To appreciate the full potential of SBS uses in agriculture, it should be taken in consideration the recent work published by Franzoso et al. (2015a,b,c). These authors have demonstrated that SBS reacted with polyethyleneco-polymers yield composites that can be manufactured as mulch films with enhanced mechanical strength. These findings offer the intriguing perspective that the same films could perform two functions, i.e., to provide protection for crops, and at the end of their product life to perform as auxiliaries for plant growth by releasing SBS to the soil. The multipurpose value of the SBS prospects a scenario where current municipal biowaste treatment plants integrated with SBS production facility were turned into cost-effective bio-refineries manufacturing added value products for use in other industrial sectors. In this fashionable scenario, a new sustainable business model could be generated. Based on the conversion of biowastes to bio-based product and viceversa, this business model could more safely operate into the eco-systems. It is obvious that the present manuscript does not report exhaustive results to answer all the many questions that can be posed about doses and reasons for the different effects which have been observed. However, it offers scope for further experimental plan aimed to compare the materials at the same C and N applied doses. The authors feel that such further investigation would have more value for its potential to assess the mode of action of the different materials and understand the reasons for the different performances. Nevertheless, the present results have value since they allow the evaluation of the SBS potential marketability through the comparison with the commercial Enersoil product. This is mostly important for potential process/product users in order to decide undertaking the risk of scaling up SBS production at commercial level. In this respect, the manuscript should be appraised for the real environmental and economic perspectives offered by the reported results for agriculture, the management of urban wastes, and the chemical industry.

5. Conclusion The SBS, obtained by the alkaline hydrolysis of municipal biowaste compost and digestate, particularly the CP SBO, have been proven to enhance the photosynthetic activity, the number of flowers per plant, the shoot to root ratio, the biomass production, the relative growth rate, aesthetic value, and water efficiency use of Euphorbhia x lomi potted plants, more than a market established

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