LIFE+2010 Project Code: LIFE+10/ENV/IT/000321zeolifeenglish.weebly.com/uploads/1/0/3/0/10302063/... · 2019. 12. 7. · LIFE+2010 ‐ Project code: LIFE+10 ENV/IT/000321 ‐ Action

LIFE+2010 ‐ Project code: LIFE+10 ENV/IT/000321 ‐ Action 5a. – Partner 3 ‐ Deliverable

WATER POLLUTION REDUCTION AND WATER SAVING USING A

NATURAL ZEOLITITE CYCLE

Design, management and start up of ex‐situ (pilot plant model) and in‐situ test fields

Ex‐situ tests, pilot plant model

LIFE+2010 ‐ Project Code: LIFE+10/ENV/IT/000321

Deliverable product of the CRSA Med Ingegneria srl

Code of the associated Action: 3

Deadline: 15/03/2013.

Authors:

Tiziana Campisi

Federica Abbondanzi

Massimo Andretta


2

Index

1. INTRODUCTION 5

2. METHODS 6

2.1 GREENHOUSE INSTALLATION 6 2.2 FIRST EXPERIMENT (04/04/2012 – 16/07/2012) 7 2.2.1 SOIL TYPE 7 2.2.2 TREATMENTS 8 2.2.3 MONITORING MEASURES 11 2.3 SECOND EXPERIMENT (16/07/2012 – 20/09/2012) 11 2.3.1 SOIL TYPE 12 2.3.2 TREATMENT 13 2.3.3 MONITORING MEASURES 16

3. RESULTS 16

3.1 FIRST EXPERIMENT WITH CODIGORO SOIL 16 3.1.1 GERMIANTION MONITORING 17 3.1.2 LEACHETES MONITORING 20 3.1.3 EXPERIMENT RESULTS EVALUATION 22 3.2 SECOND EXPERIMENT WITH NITROGEN SUPPLYING REDUCTION 25 3.2.1 GERMIANTION MONITORING 25 3.2.2 LEACHETES MONITORING 28 3.2.3 EXPERIMENT RESULTS EVALUATION 31

4. CONCLUSION 43

5. TIME TABLE 44

6. INDICATORS OF PROGRESS 45


3

List of Figures

Figure 1 Greenhouse installation ............................................................................................. 7

Figure 2 Codigoro soil (on the left), natural zeolitite (sandy colour) and loaded zeolitite (brown

colour) ...................................................................................................................................... 8

Figure 3 Codigoro soil sieving operation and results. .............................................................. 8

Figure 4 Full pots in greenhouse pilot plent, sited following experimetnal design. ................ 10

Figure 5 Example of monitoring instrument of greenhouse condition. ................................... 11

Figure 6 Second experiment steps execution. ....................................................................... 13

Figure 7 Second experiment: urea and zeolitite addition before mixing and seeding. ........... 14

Figure 8 Second experiment: pots in greenhouse pilot plant. ................................................ 15

Figure 9 First experiment: height growth of sprouts. .............................................................. 19

Figure 10 First experiment: ammonia content in leaching water. .......................................... 20

Figure 11 First experiment: nitrates content in leaching water. ............................................. 21

Figure 12 First experiment: chlorides content in leaching water. ........................................... 21

Figure 13 First experiment: Comparison of 5 treatments in 4 replicates at final point (86 days). 22

Figure 14 Collection of the roots and drying in an oven at 70 ° C x 72h. ............................... 23

Figure 15 Effect of treatments on the production of aerial biomass at the end of cycle (dry weight).

Different circle indicate significant differences between the treatment (LSD p <0.05). .......... 23

Figure 16 Second experiment: Biomass growth in the 7 treatments. .................................... 27

Figure 17 Second experiment: Conductivity and Chlorides in 7 treatments. ......................... 29

Figure 18 Second experiment: Nitrogen contents (Ammonia and Nitrates) in 7 treatments. . 30

Figure 19 Effect of treatments on the production of aerial biomass at the end of cycle (dry weight).

Different letters indicate significant differences between the thesis (LSD p <0.05). .............. 31

Figure 20 Effect of treatments on the production of root biomass (fresh weight). Different letters

indicate significant differences between the thesis (LSD p <0.05) ........................................ 32

Figure 21 Example of apparatuses radicals, free from soil as the time of analysis, for each of the 7

treatments (by UNIVPM). ....................................................................................................... 33

Figure 22 Second experiement: overview of plant growth during UNIVPM measuments. .... 34

Figure 23 Example of UNIVPM measurements with instrument. ........................................... 35

Figure 24 Net photosynthesis in the comparison of treatments (by UNIVPM). ...................... 35

Figure 25 SPAD in the comparison of treatments (by UNIVPM). .......................................... 36

Figure 26 Analysis of macronutrients in the corn leaves (by CRSA) ..................................... 38

Figure 27 Analysis of micronutrients in the corn leaves (by CRSA) ...................................... 38

Figure 28 Growth rate of two maize productions in Codigoro soil ......................................... 39


4

Figure 29 Parameters trend for two maize productions in Codigoro soil ............................... 40

Figure 30 Damages to greenhouse for rainstorms ................................................................ 45

Figure 31 Example todamage to seeded pots ....................................................................... 45

Figure 32 Restoration greenhouse after rainstorms (in 10th April 2012). ............................... 45


5

1. Introduction

The main objective of this action is to realize a set of experiments in a pilot greenhouse in

order to assess the contribution of loaded zeolitite on soil and on crop growing. At CRSA

MED facilities, a pilot plant was designed and realized as a controlled greenhouse, in order

to study several process parameters, such as:

- - soil composition and particle size

- - selected seedlings growing (maize)

- - zeolitite amount

- - Nitrogen addition amount.

The pilot greenhouse was installed and set up at optimal controlled conditions

(temperature, humidity and light irradiation) for crop growing.

Two consecutive cycles, with several replicates of tests, were performed in order to assess

two main aims, as:

- Evaluation of the influence of NH4-loaded zeolitite on soil and on crop growth.

- Quantification of NH4-loaded zeolitite/soil ratio.

Many parameters were evaluated to understand what the best conditions are to be applied

during the field trials (action 5b):

• Height of shoots per unit of time, for a rough estimate of the speed of growth of the

plants

• The production and aerial roots of the plants grown, to estimate the capacity

• The photosynthetic activity of plants and the chlorophyll content of the leaves, to

evaluate the best conditions for plant growth

• Chemical analysis of percolation waters (pH, conductivity, nitrogen in various chemical

forms, chloride, phosphorus, potassium) to assess the possible loss of nutrients due to

leaching during the test

• Chemical analysis of macro and micronutrients in the leaves at the end of treatment to

assess the state of health of the plant

Considering the indicators of progress, the objectives were:


6

- Greenhouse pilot construction

- Experimental phase start up

- Quantification of NH4-loaded zeolitite/soil ratio achieved, with a first cycle expected

within 6 month

- Selection of crop for field tests

2. Methods

2.1 Greenhouse installation

For the ex-situ experiments, a greenhouse pilot plant was acquired and mounted in

09/03/2012 e 13/03/2012.

The greenhouse was placed in the garden of the CRSA Med Engineering, along the east-

west direction, to get the maximum irradiation.

Positioned on a base of stabilized base (h: 40 cm), the 60m3 greenhouse had a size of 6 x

2 x 1.90 meters, and had a door lock manually (Figure 1).

Inside the following measuring instruments were placed:

- luxmeter

- hygrometer

- Thermometer with data logger

- 1 socket for any heating systems, thermostatic


7

Figure 1 Greenhouse installation

Into the pilot greenhouse, two set of experiments were performed in simple plastic boxes

with almost 50 cm root elongation.

2.2 First experiment (04/04/2012 – 16/07/2012)

2.2.1 Soil type

The fist experiment was conducted using the soil coming from Codigoro site, where field

experiment (Action 5b) will be carried on.

Agricultural soil (Table 1), picked up along the first 20 cm of depth, was sieved with sieves

from 2 mm, in order to obtain a homogeneous substrate (ground end) for sowing maize

(Zea mays). In Figure 2 and Figure 3, the soil and the zeolitite (natural and NH4-loaded)

were shown, before experiment start.

Sieved soil samples were taken for analysis, conducted by laboratory of UNIFE (total N, P

similar, exchangeable K, TOC, pH, etc.).

Table 1. Physical and chemical characteristics of the soil used in the test (0 - 20 cm)

Type % sand % silt % clay Soil Class Total Nintrogen

(mg kg-1)

Clay- silty soil with dark organic

matter 19,16 41,87 38,97 Silty-clay 17.68


8

Figure 2 Codigoro soil (on the left), natural zeolitite (sandy colour) and loaded zeolitite

(brown colour)

Figure 3 Codigoro soil sieving operation and results.

2.2.2 Treatments

Twenty black PVC containers (5 treatments per 4 replicates) have been used to breed

corn plants. The dimensions of the container are h: 23.5 cm. Volume: 7 liters weight: 300

grams.

In each container a draining base about 3 inches high, with gravel was made, in order

to permit the leachate collection.

Each container was filled with 7.25 kg of sieved soil, to which urea and/or zeolitite had

been added according to the treatments described in Table 2.


9

Table 2 Treatment of First experiment

Treatment/ Variable

Blank (C) T1 T2 T3 T4

Colour code Green Yellow Orange Red White

Soil type* TdC TdC TdC TdC TdC

Zeolitite amount none 10 g/kg, loaded 20 g/kg, loaded 20 g/kg, loaded

20 g/kg, not loaded

(natural)

Nitrogen amount

Full fertilization

(100 %)

Fertilization up to 100%

Fertilization up to 100%

No fertilization No

fertilization *TdC: Codigoro soil

Simulating a high nitrogen fertilization of full field for corn, equal to about 370 kg/ha

(equivalent to about 800 kg/ha of urea) and a depth of distribution of the fertilizer along the

profile crop equal to 25 cm, urea have been added to the soil according to the different

treatments (table 2). In particular, for T1 and T2, urea was added compensating for the

amount of nitrogen already present in zeolitite loaded with the prototype of the project, and

measured by UNIMoRe.

The quantities of zeolitite natural and NH4-loaded additions have been calculated

based on the dry weight, after determining their moisture.

The doses of 10 g/kg (T1) and of 20 g/kg (T2, T3 and T4) respectively correspond to

doses of 5 kg/m2 (50 t/ha) and 10 kg/m2 (100 t/ha), assuming a depth of homogeneous

distribution of zeolitite along the profile crop equal to 40 cm (depth of plowing).

The treatments were chosen in order to evaluate the best approach and select the best

zeolitite an nitrogen addition, as:

• Blank (C) : traditional agricultural practice (positive control)

• T1 : low dose of fine loaded zeolitite, with reduction of Nitrogen addiction

• T2 : high dose of fine loaded zeolitite, with reduction of Nitrogen addiction

• T3 : high dose of fine loaded zeolitite, without Nitrogen addiction

• T4: high dose of fine not loaded zeolitite, without Nitrogen addiction (negative

control)


10

Once prepared substrates for the different treatments, containers were filled and three

seeds of maize variety Cisko Class 300, per pot (Figure 4) were placed. Once the seeds

germinated, the seedlings were thinned to 2/pot.

The pots were then placed in the greenhouse and arranged in 4 blocks (A, B, C and D),

according to the experimental design to "completely randomized blocks".

They were then brought to field capacity and in each pot was added a thermometer of

maximum and minimum to measure the trend of night-day temperature of the substrate.

Inside the greenhouse a maximum and minimum thermometer with data logger is also

placed to measure changes in air temperature inside the greenhouse (Figure 4 and Figure

5).

Figure 4 Full pots in greenhouse pilot plant, sited following experimental design.


11

Figure 5 Example of monitoring instrument of greenhouse condition.

2.2.3 Monitoring measures

The measures that were carried out during the growth cycle were as follows:

1. The water collected in the drainage saucers, for each plant: pH, NH4-N and NO3-N,

conductivity, chlorides. Every 15 days;

2. Outside temperature and the soil in each pot, on a daily basis;

3. Plant height (from the neck to the basal area of the last leaf sound.) every 15 days,

approximately;

At the end of the vegetative cycle and production, the following steps were carried out:

1. Total biomass air, fresh weight and dry weight;

2. Root biomass, fresh weight and dry weight;

3. Production of grain: the total weight of kernels per plant, number of kernels per plant,

average weight per plant of the kernel;

2.3 Second experiment (16/07/2012 – 20/09/2012)


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2.3.1 Soil type

The second experiment was conducted using artificial standard soil (Std), in order to

evaluate the real potential of NH4-loaded zeolitite, minimizing the influence of agricultural

soil.

Po river sand and peat of northern European origin (Table 1, Figure 6a) were mixed with a

volume ratio of 1:1, to get the growing media (Figure 6b). The substrate was placed in

plastic containers from 6 litres of capacity. In the bottom of each container was placed a

layer of material "nonwoven fabric" (permeable to water), to prevent loss of substrate from

the drain holes of pots (Figure 6c and d).

Table 3. Chemical characteristics of the peat used in the test

Type % organic

carbon % organic nitrogen

pH

Natural organic soil (peat) 46 0,7 4

A: sand and peat

B: artificial soil


13

C: pots with permeable material

D: soil allocation in pot

Figure 6 Second experiment steps execution.

2.3.2 Treatment

Twenty-eight black PVC containers (7 treatments per 4 replicates) have been used to

breed corn plants, using 4 kg of substrate per pot, in which urea and/or zeolitite had been

added (Figure 7) according to the treatments described in Table 4.

Table 4 Treatment of Second experiment

Treatment/ Variable

Blank (C) T1 T2 T3 T4 T5 T6

Colour code Green Blue Brown Orange Red Yellow White

Soil type* Std Std Std Std Std TdC Std

Zeolitite amount none 10 g/kg,

loaded 10 g/kg, loaded

10 g/kg, loaded**

10 g/kg, NOT

loaded

10 g/kg, loaded and

residual from 1°

experiment

6 g/kg, loaded

Nitrogen amount

Full fertilization

(100 %)

Fertilization reduced to

70%


50%


30%


70%


50%

Complemented

fertilization

* Std; artificial standard soil; TdC: Codigoro soil ** 80% in s.s., fine as other treatment; 20% in s.s., very fine (<90µm), collected in prototype


14

A: urea addiction B: zeolitite addiction

Figure 7 Second experiment: urea and zeolitite addition before mixing and seeding.

Simulating a full range of nitrogen fertilization on maize compatible with the Nitrates Action

Programme Regional Emilia Romagna, 240 kg/ha of nitrogen (equivalent to about 522

kg/ha of urea) were provided to the theory of control, assuming a depth distribution of the

fertilizer along the profile crop equal to 25 cm.

The treatments T1, T2 and T3, with the same content of zeolitite, were supplied with a

reduction of 30, 50 and 70% urea nitrogen compared to the Control. In particular, in T3,

80% of the zeolitite is in coarse form (as in the other treatments), while the remaining 20%

is administered in the "fine" form, obtained by taking the fraction of undersize (<90 μ) in

output from prototype after the loading process. This fraction, as well as having a particle

size less than the zeolitite used in all theses, is enriched with phosphorus, in bound form,

missing from the zeolitite treated coarse.

The treatment T5 reused the soil of Codigoro, already used in treatment T1 of the first

experiment to evaluate a possible effect of residual nitrogen.

The treatment T6 provided a smaller amount of zeolitite (11.25 instead of 18.75 g / kg), with

the aim of matching the amount of nitrogen in the control considering all available content

in zeolitite, plus a minimum amount of urea nitrogen to reach 240 kg/ha.

The treatments were chosen in order to evaluate the best approach and select the best

zeolitite and nitrogen addition, as:

• Blank (C): traditional agricultural practice (positive control)

• T1: low dose of fine loaded zeolitite, with low reduction of Nitrogen addiction

• T2: low dose of fine loaded zeolitite, with medium reduction of Nitrogen addiction


15

• T3: low dose of fine and ultrafine loaded zeolitite, with high reduction of Nitrogen

addiction.

• T4: low dose of fine natural zeolitite, with low reduction of Nitrogen addiction

• T5: low dose of fine loaded zeolitite, residual from first experiment, using residual

Codigoro soil, with medium reduction of Nitrogen addiction (for simulating second

year of production)

• T6: high dose of fine loaded zeolitite, without Nitrogen addiction (regulation limit

test)

As for first experiment, the containers were filled and three seeds of maize variety

Cisko Class 300, per pot were placed. Once the seeds germinated, the seedlings were

thinned to 2/pot.

The 28 pots were then placed in the structure, protected by a network anti-hail, and

arranged in four blocks (A, B, C and D), according to the experimental design "completely

randomized blocks" (Figure 4 and Figure 8).

They were then brought to field capacity and in each pot was added a thermometer of

maximum and minimum to measure the trend of night-day temperature of the substrate.

Inside the greenhouse is also a maximum and minimum thermometer with data logger to

measure changes in air temperature inside the greenhouse.

Figure 8 Second experiment: pots in greenhouse pilot plant.


16

2.3.3 Monitoring measures

The measures that are being carried out during the growth cycle are as follows:

1. The water collected in the drainage saucers, for each plant: pH, NH4-N and NO3-N,

conductivity, chlorides. Approximately every 30 days;

2. Outside temperature, on a daily basis;

3. Plant height (from the neck to the basal area of the last leaf sound.) Every 15 days,

approximately;

4. Measurements of photosynthesis and fluorescence. At the end of experiment,

performed by Prof. Neri of University of Marche (UNIVPM).

At the end of the vegetative cycle and production, will perform the following steps:

1. Total biomass air, fresh weight and dry weight;

2. Root biomass, fresh weight and dry weight;

3. Production of grain: the total weight of kernels per plant, number of kernels per plant,

average weight per plant of the kernel;

4. Evaluating quantitative and qualitative morphological (relative growth rate,

density/appearance of the root);

5. Content of nitrogen in plant tissues;

6. Chemical analysis of the soil.

3. Results

3.1 First experiment with Codigoro soil The first experiment lasted 89 days and it was performed using the Codigoro soil that

presented the following chemical and microbiological content:

Codigoro soil u.m value Cation Exchange Capacity (CEC) meq/100 g 33.6

Exchangeable cation: Calcium mg/kg 5660

Exchangeable cation: Potassium mg/kg 582

Exchangeable Potassium (as K2O) mg/kg 701


17

Exchangeable cation: magnesium mg/kg 401

Exchangeable cation: sodium mg/kg 368

Total nitrogen mg/kg 2.7 -17.7 Assimilable phosphorous mg/kg 76.5

Assimilable phosphorous (as P2O5) mg/kg 175.3

Assimilable Iron mg/kg 62.4

Assimilable Manganese mg/kg 6.2

Assimilable Zinc mg/kg 1.9 Soluble boron mg/kg 1.61

copper mg/kg 42.8

Nitrifying bacteria (MPN/g) 3.80E+05

Nitrosating bacteria (MPN/g) 9.20E+04

These results confirmed the typical composition of an agricultural soil in Ferrara district,

with a medium-high nutrient content (ARPAV 2007- L’interpretazione delle analisi del

terreno - Strumento per la sostenibilità ambientale)

The quantities of loaded zeolitite additions have been calculated based on the dry weight,

after determining their moisture amounted (37% of the weight). The natural zeolitite

presented a negligible moisture value (5%).

The zeolitite chemical release test (saturation with NaCl substrate) resulted in the following

concentrations of ammonia nitrogen (by UniMoRE):

• Natural zeolitite : 41.64 mg / kg N-NH4

• Loaded Zeolitite : 9,980 mg / kg N-NH4 (9.98 mg / g)

3.1.1 Germination monitoring

In the following figures, the seed germination and plant growth is showed, as example of

daily control of all treatments.


18

at 15 days

at 23 days

at 36 days

at 54 days


19

at 84 days

The Figure 9 summarizes the overall performance of the experiment 1 from the point of

view of height growth of sprouts. Each point is the mean of 4 replicates.

Approximating the growth of plants to a linear path, it was possible to estimate the various

growth velocity (m as slope, and r2 as fitting) so as to compare the five treatments.

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 20 40 60 80 100 120Time (days)

Aver

age

heig

ht o

f the

spr

outs

(cm

) C T1

T2 T3

T4

Figure 9 First experiment: height growth of sprouts.

In particular, in the Table 5, the various treatments in order of decreasing speed of growth

are represented. As can be seen, the control C, treatment with traditional practice, had a

growth rate higher than all other treatments. Considering the group of treatments with

loaded zeolitite, all treatments except T2 (high zeolitite amount and high reduction of

nitrogen) have a speed of growth similar to the control. It is important to notice that the high


20

nutrient contents of Codigoro soil could have an influence on crop growth because also T4

(with natural zeolitite) had a growth rate similar to control.

Table 5 First Experiment: growth rate in decreasing order

Treatment m r2

C 1.340 0.976

T1 1.295 0.968

T3 1.234 0.943

T4 1.225 0.928

T2 1.184 0.980

3.1.2 Leaching water monitoring

The monitoring of leaching water in the different treatments demonstrated that the content

of ammonia was related to urea addiction, and quickly reduced in all treatment after seed

germination (36 days).

Ammonia (N-NH4)

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 20 40 60 80 100Time (days)

Con

cent

ratio

n (m

g/l)

CT1T2T3T4

Figure 10 First experiment: ammonia content in leaching water.

The nitrates content in all treatments was higher than limit regulation (50 mg/l as NO3, and

11.3 mg/l as N-NO3), with a decreasing trend in 89 days (no significant difference between

treatments were observed).


21

At the final point, all the treatment expect for T1 reached a value below the regulation limit.

It is important to notice that the nitrates content (from 65 mg/l to 6.5 mg/l) was also high in

T4 with natural zeolitite, where no urea addiction was performed. This could confirm the

hypothesis of high original mineralization of Codigoro soil, considering also the high

chlorides content.

Nitrates (N-NO3)

020406080

100120140160180

0 20 40 60 80 100Time (days)

Conc

entra

tion

(mg/

l)

CT1T2T3T4

Figure 11 First experiment: nitrates content in leaching water.

Chlorides

050

100150200250300350400450500

0 20 40 60 80 100Time (days)

Conc

entra

tion

(mg/

l)

CT1T2T3T4

Figure 12 First experiment: chlorides content in leaching water.

For all the treatments, no significant difference in all measured chemical parameters were

observed.


22

3.1.3 Experiment results evaluation

At the end of experiment (89 days), the crop production was estimated collecting the

emerging biomass (plant) and the root, which were weighted before and after drying

(Figure 13 and 14).

Replicate A: from left – C, T1, T2, T3, T4 Replicate B: from left – T4, T3, T2, T1, C

Replicate C: from left – T4, T3, T2, T1, C Replicate D: from left – T4, T3, T2, T1, C

Figure 13 First experiment: Comparison of 5 treatments in 4 replicates at final point (86 days).


23

Figure 14 Collection of the roots and drying in an oven at 70 ° C x 72h.

In the production of aerial biomass (dry weight) measured at end of cycle, only T4 had

shown to produce less than C, T1 and T2 (p-level: 0.019), even if T3 appeared similar to

T4. Anyway no obvious other significant differences between treatments were detected

(Figure 15).

0.00

20.00

40.00

60.00

80.00

100.00

120.00

C T1 T2 T3 T4

aver

age

valu

e (g

)

biomass_dw (g)

Figure 15 Effect of treatments on the production of aerial biomass at the end of cycle (dry weight). Different circles indicate different groups between the treatment (LSD p <0.05).


24

The main final results of the first experiment was that the parameters of T1 treatment as 10

g/kg of loaded zeolitite and reduced fertilization were a good alternative solution to

traditional agricultural practice

Anyway, the use of Codigoro soil could have influenced the results, reducing the zeolitite

effect. So, in the second experiment, in order to observe the real potential of zeolitite, an

artificial standard soil has to be used.


25

3.2 Second experiment with nitrogen supplying reduction The second experiment lasted 73 days and it was performed using a standard artificial soil

for 5 treatments, and the Codigoro soil for only one treatment (T5) as the simulation of the

second year of production (T1 in first experiment).

3.2.1 Germination monitoring

In the following figures, the seed germination and plant growth are shown, as example of

daily control of all treatments.

at 0 days

at 8 days


26

at 18 days

at 35 days

at 42 days

Figure 16 summarizes the overall performance of the cycle 2 from the point of view of

height growth of sprouts. Each point is the mean of 4 replicates.


27

Approximating the growth of plants to a linear path, it was possible to estimate the various

growth rate (m as slope, and r2 as fitting) so as to compare the seven treatments.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 10 20 30 40 50 60 70 80Time (days)

Aver

age

heig

ht o

f the

spr

outs

(cm

)

C

T1

T2

T3

T4

T5

T6

Figure 16 Second experiment: Biomass growth in the 7 treatments.

In particular, in Table 6, the various treatments in order of decreasing speed of growth are

represented. As it can be observed, T5 (treatment with Codigoro soil) had a growth rate

significantly higher than all other treatments. Considering the group of treatments with sand

and peat, all treatments except T4 (not loaded zeolitite) had a speed of growth markedly

superior to the control C.

Table 6 Second Experiment: growth rate in decreasing order

Treatment m r2

T5 0,9345 0,9946

T3 0,7539 0,9820

T1 0,7298 0,9666

T2 0,6744 0,9897

T6 0,6055 0,9854

C 0,4823 0,9703

T4 0,4299 0,9726


28

3.2.2 Leachate monitoring

The monitoring of leachate in the different treatments considered many parameters, as.

• pH;

• conductivity;

• chlorides;

• ammonia nitrogen;

• nitrates;

• nitrous;

For the whole duration of the test, the pH was maintained at cost values for all treatments

(7.4 to 7.7). With regard to the conductivity measurements (Figure 17), only for the test T5

an increase was observed, while for all other treatments the conductivity remained constant

or, at most, decreases, testifying the stability of the system that does not washes salts. The

phenomenon observed in T5 was due to the leaching of chloride that occurred in the soil of

Codigoro.


29

Figure 17 Second experiment: Conductivity and Chlorides in the seven treatments.

As regards the ammonia nitrogen (Figure 18), already in the first 15 day of experiment,

when the request of plant nutrients is not yet at the maximum, it can be observed a

significant lower concentration in the leachate for all treatments compared to control.

With regard to nitrates (Figure 18), it was observed an increase in their concentration only

in the treatment T2. In the treatments T4, T5 and T6 a strong decrease comparable to the

control occurred, while for the treatment T3 but, especially for T1 and T2, the decrease was

more moderate.

The nitrates, present at day 15, were lower than regulation limit in many treatments (C, T3,

T4, T5 and T6) at the end of the experiment (70 days).


30

Figure 18 Second experiment: Nitrogen contents (Ammonia and Nitrates) in 7 treatments.

In particular, in Table 7, the various treatments in order of decreasing Nitrogen content in

water are represented. As can be seen, T6, treatment with lower zeolitite and nitrogen

addiction, had lower nitrogen content in water than all other treatments, mostly better than

control C.


31

Table 7 Second Experiment: Ammonia and nitrates in water, in decreasing order

Am

mon

ia

C

Treatment

Nitr

ates

Treatment

T6 T6

T3 T5

T5 T4

T1 C

T4 T3

T2 T1

C T2

3.2.3 Experiment results evaluation

At end of experiment, the production of aerial biomass (dry weight) was measured: T5 has

been shown to produce more than C, T2 and T4, while no significant differences between

treatments were present (Figure 19).

Figure 19 Effect of treatments on the production of aerial biomass at the end of cycle (dry weight). Different letters indicate significant differences between the thesis (LSD p <0.05).

Ciclo 2 ‐ Biomassa aerea

0,00

10,00

20,00

30,00

40,00

50,00

60,00

C T1 T2 T3 T4 T5 T6

Trattamento

Medie (g)

a

ab

b

a

a

ab ab


32

For the root biomass (fresh weight), measured at the end of test (Figure 20), T5 treatment

was the only one to determine a significant difference compared to all other treatments. In

fact, it had promoted the higher rot growth, with the exception of T2 which has instead

yielded an even greater effect compared to T5. However, no discernible other significant

differences between treatments were present.

Ciclo 2 ‐ Biomassa radicale

0,00

10,00

20,00

30,00

40,00

50,00

60,00

C T1 T2 T3 T4 T5 T6

Trattamento

Medie (g_fw)

a

b

a

a

a

a

ab

Figure 20 Effect of treatments on the production of root biomass (fresh weight). Different

letters indicate significant differences between the thesis (LSD p <0.05)

The measures of the roots carried out by UNIVPM involved the following variables as

shown in Figure 21:

• root biomass (dry weight);

• Morphological analysis: total length of roots

• Number of primary roots and absorbent; diameter radical

The T5 treatment has confirmed for determining most root biomass dry matter, followed by

T1 and T3. Other treatments have induced lower total production of roots.


33

Figure 21 Example of apparatuses radicals, free from soil as the time of analysis, for each of

the 7 treatments (by UNIVPM).

As written by UNIVPM, the treatment T5 showed an impetus to the development radical

already in the earliest stages of growth, at the growth stage to define the volume of the

primary structures. The impetus to growth was still maintained in the next stage of

production and continuous regeneration of the absorbent structures, for what concerns the

architecture and hierarchical organization structures, the T5 treatment showed again

features fully different from other thesis. The treatment had the fewest number of primary

roots in the first crown; roots showed however that the maximum diameters. Moreover, the

control treatment (C) presented reduced development in terms of accumulated biomass

and minimum roots diameters. A reduced number of primary branched especially from the

first crown resulted, too .

As for the treatments with sand-peat substrate, T4 and T6 had a decreased number of

roots to the first crown and above average diameters, showing a behavior similar to the

control, and submitting to some performance parameters even less. T1, T2, T3 instead

have induced an overall increase of the primary structures produced and root biomass.

Measurements of the photosynthetic activity and chlorophyll content of plants


34

Other measurements taken by UNIVPM, as photosynthetic activity (PN) and chlorophyll

content (SPAN), were carried out on all plants at the end of the crop cycle, before

destructive measures.

Figure 22 Second experiment: overview of plant growth during UNIVPM measuments.


35

Figure 23 Example of UNIVPM measurements with instrument.

The leaves of the treatment T1 and the control C showed a greater net photosynthesis

(PN), up to 30 mmol/m2/s CO2. The treatments T2 and T3 have presented values around

25, significantly lower than T1. The treatments T4 and T6 showed values of photosynthesis

almost halved. Finally T5 showed the lowest values ever, just over 10.

Figure 24 Net photosynthesis in the comparison of treatments (by UNIVPM).

PN10 settembre 2012

0

5

10

15

20

25

30

35

40

controllo t1 t2 t3 t4 t5 t6

PN (c

o 2 μ

mol

/m2 s)

c

b

ab

b

cd

a

d


36

The SPAD index, which indicates the intensity of the green leaf area value related to the

presence of nitrogen and chlorophyll, was very low in treatments T4, T5 and T6 (Figure 25).

Especially the T5 has had a very low index close to 15, or less than half compared to the

treatments T1 and T3. In the treatment T5 the colour of the leaves was typically "chlorotic".

Figure 25 SPAD in the comparison of treatments (by UNIVPM).

The reduced transpiration and photosynthetic activity, as well as resulting in leaf chlorosis

induced by treatment T5, can be attributed to stress in plants whose root systems (as

described above) had already filled the volume of the container at the time of the survey,

resulting in the most developed.

It can supposed that plants in T5 had good availability of nitrogen from the beginning of the

cycle; reserve nitrogen which remained more than adequate for the needs of plants until

the end; it was more difficult to understand what role had the nitrogen released from loaded

zeolitite and what the urea nitrogen was. Tall plants growth in T5 compared to all other

treatments, even if nitrogen added with equal or greater in other treatments (eg. C, T1, T2).

However, it should be considered a possible effect of the Codigoro soil, relatively to the

availability of nutrients, as well as to an initial significant content of macro-and micro-

nutrients (as shown by chemical analysis), compared to sand-peat substrate,

constitutionally inert from the chemical point of view.

SPAD su mais in vaso10 settembre 2012

0

5

10

15

20

25

30

35

40

45

controllo t1 t2 t3 t4 t5 t6

indi

ce S

PAD

ab

a

b

a

bc bc

c


37

Focusing on the group of treatments based on sand-peat, T4 and T6 had produced a

smaller radical development and considerably more simplified by an architectural point of

view (therefore less efficient); measures of photosynthesis and SPAD index are in

agreement with this behavior, also confirmed by the reduced production of aerial biomass

and radical, at least for plants T4.

The negative effects on the physiology of plants produced by T4, could be partially

explained by a "locking" of ammonia nitrogen by natural zeolitites - and not re-sold to plants

in sufficient quantities during the initial step of crop cycle. It has to be noticed that the

nitrogen resulting from the hydrolysis of urea was in the ammonia form and it represented

the only source of this element in the substrate sand-peat for plants of maize (very

demanding).

The reduced performance of T6 could be explained by the lower concentration of loaded

zeolitites and the lower amount of urea nitrogen added to the substrate sand-peat (up to

10-20 times less compared to the other treatments).

Control, T1, T2 and T3 had maintained a good photosynthetic efficiency and chlorophyll

content even in the last days of the crop cycle; T1 presented the highest values. However,

plants of the control C, despite the full supply of urea, showed a significantly lower

production of biomass and a more simplified radical organization with respect to treatments

T1, T2 and T3: this can be probably related to the presence of loaded zeolitites into the

latter phase of crop cycle, and their role in increasing water retention and nutrients in a

naturally poor substrate.

Analysis of macro nutrients in leaves at the end of treatment Regarding the macronutrients (Figure 26), it can be observed that, while for phosphorus,

potassium, sulfur, calcium, magnesium and sodium, the concentrations of the control are

comparable to those found in the various treatments, as regards the nitrogen there is a

marked difference concentration in the treatments T1, T2, T3, containing loaded zeolitite

and with fertilization reduction. Moreover, the nitrogen content (almost 2.5%) in T1 and T2

led to suppose the possibility to increase the production, while for the other treatments the

nitrogen content less than 1% suggested a suffering situation, with limitation in plant

growth.


38

MACRO-nutrients in leaves

0

5000

10000

15000

20000

25000

30000

Total Nitrogen Phosphorous Sulfur Calcium Magnesium Potassium Sodium

mg/

kgss

CT1T2T3T4T5T6

Figure 26 Analysis of macronutrients in the corn leaves (by CRSA)

As regards the micronutrients (Figure 27), no significant differences between the control

and the various treatments are observed.

Figure 27 Analysis of micronutrients in the corn leaves (by CRSA)

Comparison Experiment 1 - Experiment 2 with Codigoro soil

2.5% =>

1% =>


39

Only one addiction of zeolitite in soil is sufficient to improve soil texture and maintain its

capability over time, thanks to zeolitite property.

In order to simulate the effect of zeolitite on plant growth for almost 2 agricultural cycles,

one treatment of first experiment (T1) was fertilized (reducing nitrogen addiction to 50%)

and seeding again in second experiment (T5). The fertilization with urea was due to low

content of residual nitrogen in the soil, after maize production in first experiment.

The comparison (Figure 28) showed a lower growth rates in second experiment than fist

one, due to higher consumption of nitrogen (not present in leachate).

Figure 28 Growth rate of two maize productions in Codigoro soil


40

0

100

200

300

400

500

600

C T1 (1° exp) T5 (2° exp)

Treatment

aver

age

prod

uctio

n (g

)

Emerging biomassRoots

Nitrates trend

0

20

40

60

80

100

120

140

0 25 50 75 100 125 150time (days)

conc

entra

tion

(mg/

l)

C

T1

starting second exp.

Ammonia trend

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 25 50 75 100 125 150time (days)

conc

entr

atio

n (m

g/l) C

T1starting second exp.

Figure 29 Parameters trend for two maize productions in Codigoro soil

Final Evaluation


41

In order to permit an overall evaluation of all analyzed parameters, a ranking approach was

carried out: a score at each parameter was given, for each treatment, considering that the

worst value as “1” score and the best one as “7” score. The sum of all scores gave the final

score for the treatment and permitted the selection of best treatment compared to Control

(32 point), to be performed in field (Action 5b).

Table 8 Final evaluation with score rate

TreatmentParameter

C T1 T2 T3 T4 T5 T6

Growth rate 2 5 4 6 1 7 3

Emerging biomass 2 4 3 6 1 7 5

Roots 3 5 4 6 1 7 2

Nitrates in water 4 2 1 3 5 6 7

Ammonia in water 1 4 2 6 3 5 7

Chlorides in water 6 2 5 7 4 1 3

Nitrogen in leaf 4 6 7 5 3 2 1

Photosynthesis 6 7 4 5 2 1 3

Chlophylla content 4 6 5 7 2 1 6

Final score Poor:9 – Excellent: 63 32 41 35 51 22 37 37

Following this approach, the best treatment with zeolitite resulted T2, standing for:

• 5 kg/m2 = 50 ton/ha of NH4-loaded zeolitite

• reduction of 50 % fertilization Considering the score of the treatment T5 with Codigoro soil, as real soil to be use in Action

5b, the previous selection was confirmed.


42

Even the treatment T4 resulted moderately lower than control, as the natural zeolitite had a

great potential in term of texture correction and soil improvement, the treatment T4 was

selected, standing for:

• 5 kg/m2 = 50 ton/ha of natural zeolitite

• Reduction of 30 % fertilizzation


43

4. Conclusion

A 60 m3 greenhouse (3.3 m x 9 m x h 2 m) was installed at CRSA facilities.

Maize was selected because of its high nitrogen requirements; easy to grow and

represents a typical crop in the farming system of the region (also related to animal

feeding).

Regarding the first experiment, 5 treatments per 4 replicates (20 tests) were conducted for

89 days of experiment. During the growth monitoring, 6 growth measures per 20 tests were

performed, collecting 120 data results. For water control, 6 chemical parameter

measurements per 20 tests per 5 times were analyzed, collecting a total of 600

measurements.

All these data permitted to select the best performance loaded zeolitite concentration,

concluding that the spreading of 50 ton/ha was the optimal technique, even if the already

enriched Codigoro soil influenced the evaluation.

For these reason, the second cycle of experiments (July-September 2012) was conducted

with an artificial soil (plus a treatment with already used Codigoro soil with zeolitite for

simulating second year of production in field).

The second experiment included 7 treatments per 4 replicates (total of 28 tests), lasting 73

days. During the growth monitoring, 4 growth measures per 28 tests were collected, for a

total of 112 data results. Moreover, as for first experiment, 5 chemical parameter

measurements per 28 tests per 2 times were analyzed, collecting 280 measurements.

The experiment was stopped at 73 days, thanks to information obtained with 2 additional

measurements for photosynthesis evaluation in plants (photosynthetic activity (PN) and

chlorophyll content (SPAD). The air temperature and the pot volume effect could effect the


44

real final evaluation, so the plants were collected and analyzed, also for macro and

micronutrients in leafs.

An overall evaluation was performed using a “ranking approach”, where each treatment

was classified in decreasing order, depending of measurement values and comparison

among all treatments.

In this way, it was possible to select also the best nitrogen supply concentration, selecting a

reduction of 30-50%.

Finally, four results were obtained, considering zeolitite concentration to be spread on soil

and nitrogen reduction:

NH4-loaded zeolitite (fine size, <3 mm))

Natural zeolitite (medium size 3-5 mm)

5 kg/m2 = 50 ton/ha 5 kg/m2 = 50 ton/ha

Reduction of 50 % fertilizzation Reduction of 30 % fertilizzation

5. Time Table

Realization steps of greenhouse experiments

Date Greenhouse experiment

12 March 2012 Greenhouse installation

04 April 2012 First experiment start

10 July 2012 First experiment end

16 July 2012 Second experiment start

27 September 2012 Second experiment end

On April 8, 2012, rainstorms with wind force 8 (gale moderate) have torn the greenhouse

and spilled many pots (Figure 30 and Figure 31). The experimental conditions were

restored (Figure 32) reinforcing the structure of the greenhouse and reproducing the

treatments and the experiment, started again into 10 April.


45

Figure 30 Damages to greenhouse for rainstorms

Figure 31 Example todamage to seeded pots

Figure 32 Restoration greenhouse after rainstorms (in 10th April 2012).

6. Indicators of progress


46

- Greenhouse pilot construction: within 1 month (31st January 2012)=> in 29/02/12.

Completed

Experimental phase start up: within 2 months (28th February 2012)=> in 31/03/12.

Completed

- Quantification of NH4-loaded zeolitite/soil ratio achieved after 12 month (31st

December 2012), with a first cycle expected within 6 months (30th June 2012). Completed

(Cycles 1 and 2) => Completed

- Selection of crop for field tests: within 12 months (15th March 2013) => Completed

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